Chlainomonas sp. on Upper Bagley Lake in the North Cascade Mountains, USA, to assess the spatiotemporal dynamics in bloom color intensity, community photophysiology, and community composition over eight weeks. We found that the algae biomass had a dynamic patchy distribution over space and time, which was decoupled from changes in community composition and life-cycle progress averaged across the bloom. The proportional representation of Chlainomonas sp. remained consistent throughout the study while the overall community composition shows a progression through the bloom. We found that community photophysiology, measured by the maximum quantum yield of PSII (Fv/Fm), decreased on average throughout the bloom. These findings suggest that the Chlainomonas sp. community on Bagley Lake is not simply an algal bloom with rapid increase in biomass followed by a population crash, as is often seen in aquatic systems, though there is a physiological trajectory and sensitivity to environmental stress. These results contribute to our understanding of the biology of Chlainomonas sp. and its response to environmental stress, specifically an extreme warming event.Snow algal blooms frequently occur throughout alpine and polar environments during spring and summer months; however, our understanding of bloom dynamics is limited. We tracked a recurrent bloom of  Chlainomonas sp., in a snow-on-lake progressed in asynchronous patches that showed a stress response via photophysiology measurments to an extreme heat event.A weekly charactrization of an 8-week bloom of the snow algae,  Snow algae, a group of closely related species in the phylum Chlorophyta, are adapted to live and bloom in snow habitats and grow and photosynthesize at near-freezing temperatures. Algal biomass can turn snow surfaces pink, red, orange, and green during bloom events due to the dominant pigments in the cells. These blooms are important in the cryosphere, because they reduce the reflectance of solar radiation off snow surfaces, which accelerates snowmelt in snowpacks and glaciers that are currently already melting rapidly due to climate warming  observation of color  on snow surfaces and (ii) characterization of the snow algal biology via microscopy, physiology, molecular tools, and other biological methodologies. Because snow algal blooms are so visible on snow surfaces, observation and quantification of color can be used as a measure of bloom area and can be correlated with snow algal biomass. Many kinds of remote sensing technologies have been used to observe snow algal blooms on snow surfaces, in an attempt to quantify bloom size and impact on melt , Chlainomonas, and Chloromonas that produce visible pink or red blooms in snow. Thus, biodiversity in snow is relatively simple compared to most aquatic systems. Our understanding of snow algae biodiversity has been improving recently with the use of gene-based surveys  reproduction that may impact how and when they bloom  on snow on top of Upper Bagley Lake in the Mt Baker National Forest, WA. The bloom can be well defined in terms spatially, occurring only on the snow-on-lake habitat, and the end of the bloom is determined by the seasonal loss of the snow habitat by mid-summer. This snow algal bloom is an early season bloom (begins with the first spring melts) and is accessible to sampling year-round.To address the dynamics of a snow algal bloom in situ under natural conditions, we studied a known recurrent pink snow algal bloom in the North Cascade Mountains . The North Cascade Range is a low-elevation mountain range and is the most glaciated mountain range in the continental US. The range runs north-south through Washington, with high snowpack accumulation and diverse geomorphology. The Cascades are known to host large and diverse snow algal blooms both above and below tree line, many of which were characterized in the 1970s Hoham . We haveChlainomonas sp. bloom a natural experimental design to observe the spatial and temporal changes during the bloom cycle from the start of visual surface biomass through the duration of habitable snow in the Bagley Lakes system. Our objective in this study was to characterize Chlainomonas sp. bloom dynamics by integrating measurements of environmental conditions  of the habitat, observations of community structure (using microscopy and amplicon metabarcoding), photophysiology, and snow surface color that marked the observable “bloom” on snow surfaces. Through this study design, we were able to address how the microbial community composition, algal physiology, and color intensity of snow surfaces as the bloom progressed over space and time.These combined characteristics give the Bagley Lake Upper Bagley Lake is in the North Cascades mountains of western Washington State. The lake (1277 m above sea level) sits 11.8 km northeast of Mt. Baker at 48.8632, −121.6793  and continues through mid-summer until the snowpack disappears from the surface of the lake. Snow algae often initially appear at the north side of the lake, suggesting they may be responding to the south facing light environment. Typically, by the end of the bloom on Bagley Lake, snow algae can be seen in the surrounding snow slopes, but not before, suggesting the snow-on-lake habitat is habitable before the snow over rocks or vegetation. We have observed this pattern of early blooms on the lake, which are dominated by o (Week 0) and was found outside the pre-determined grid. Sample locations B-H were then determined from the grid and sampled starting T1 when snow algae patches were found across the lake. This grid served as the scaffolding for all measurements and samples across the season. Samples were collected for light and electron microscopy, DNA extraction, and relative chlorophyll fluorescence analysis at each sample location.To capture the spatial variability of the bloom across the lake, we set up a sampling grid at 60 m intervals across the lake using ArcGIS Fig. . Point AWe monitored temperature and light conditions on the bloom from 21 May to 9 July 2021 using three HOBO loggers set across the lake at sample locations A, B, and G. Loggers were mounted flat, facing the sky, on white PVC pipes and took readings every five minutes. As the snowpack melted throughout the bloom, we repositioned the loggers each week to keep them as close to the snow as possible. Additionally, we gathered air temperatures from the surrounding area from the Northwest Avalanche Center NWAC  station At each site (n = 8 per week), we collected 15 ml of fresh pink snow from within a 5 m radius of the site pin and stored the samples in an insulated cooler packed with snow to keep cold without freezing. All samples were kept in the dark and remained as snow, and were imaged between 12 and 48 after collection. To image, 20–30 ul of liquid water from the sample that began to melt at ambient temperatures was added to a slide. We took 20 images from each of the eight samples at 400x total magnification , and visually assessed the cell structure and morphology. Samples for scanning electron microscopy (SEM) were collected from snow or meltwater. Biomass from samples dried on circular cover slips and coated with gold and palladium and imaged with a Tescan Vega 3 with Oxford EDS  SEM.oC before being filtered onto 25 mm 0.2 µm Supor filters . Prior to extraction, samples were washed three times with PBS buffer. Filters were cut with sterile scissors, transferred to bead beating tubes from Zymobiomics kit  DNA miniprep extractions kits and subjected to bead beating for 5 mins. Extractions proceeded from this step as following with the Zymobiomics DNA miniprep kit instructions.For DNA collections, ∼ 20 ml of snow water equivalent was added to a 50 mL conical vial and mixed into 20 ml RNA buffer preservative like RNAlater . Samples were stored at 4https://benjjneb.github.io/dada2/. The top 24 amplicons identified, which represent at least 90% of all sequence reads in the most samples, were then annotated with BLAST using nr from NCBI. Information of reference hits to all algae and chytrid taxa found in DNA was quantified using a Nannodrop to ensure extractions were successful. We submitted DNA to the University of Minnesota Genomics Center for amplicon library prep and sequencing of the V9 region of 18S SSU rRNA gene sequence using MiSeq Illumina 2 x 300 bp chemistry. UMGC prepared dual indexed Nextera XT DNA libraries following their improved protocol for library preparation which enables detection of taxonomic groups that often go undetected with existing methods  high enough for a successful reading. We used a saturation light pulse length of 1.5 s to achieve enough fluorescence for an accurate reading, which differed from previous research (Shreiber We measured the maximum quantum yield of PSII (Fv/Fm) of the bloom each week using an Opti-Sci OS1p field PAM fluorometer with a phytoplankton cuvette . We took all readings between 10:00 and 12:00. All samples had similar light histories from the time of collection to measurement. Following the preset sampling grid ((n=8 per week)), we collected 50 ml of pink snow in Falcon Tubes from each site within a 5 m radius of the site marker/GPS waypoint. We then allowed snow to partially melt into a slurry (10-15 minutes), allowing some algal cells to settle to the bottom of the tube. We collected algae from the bottom of the Falcon tube and transferred three 1.8 ml aliquots to the measuring tubes. Samples were loaded into the phytoplankton cuvette and dark adapted for 10 minutes. Following dark adaptation in the aquatic cuvette, we measured the algae's response to light according to the OS1p manual. We adjusted the modulation intensity of the actinic light prior to measurement to achieve a baseline fluorescence . We quantified cells from ten 1 x 1 mm boxes, from two prepared slides per preserved sample. The average number of cells per ml was calculated from the technical replicates.To quantify the intensity of color on the snow each week, five representative patches of algae were selected at each site and photographed for image analysis. To standardize imaging of the snow algal patches, we built an imaging stand out of PVC pipe with camera holder on top of a 0.5 x 0.5 m square quadrat shown in . The camWe quantified the extent of pink snow coverage in images via analysis of pixel intensity using FIJI-ImageJ Abràmoff . Each im2 grids  that marked a 6x6 120mChlainomonas sp. in the microbial assemblage in 18S SSU rRNA amplicon sequence data with a one-way ANOVA among weeks. Week 0 was kept in the graphical summary as a reference for the population at the onset of the bloom. Finally, we compared cell counts of samples used for fluorescence analysis with a one-way ANOVA on log-transformed cell counts (n = 8 per week). Any ANOVA that was found to be significantly different (P < 0.05) was followed by a Tukey post-hoc test to determine pairwise differences among the treatment levels. A correlation test was used to assess the relationship between Fv/Fm and integrated density where data was available from both measurements (n=6).We ran all statistical analyses in R ver. 3.3.0  experienced by the bloom fluctuated between 45 and 120 x103 LUX  has a 100% identity to full length Chlainomonas sp. that appears to be a new species in comparison to the described Chlainomonas species described from New Zealand, Japan, and Europe . All 4 amplicon sequence variants have the same BLAST hit results for reference sequences (Chlainomonas clade across the bloom. We recovered only a single ITS2 sequence (OP297764.1) that was annotated as Chlainomonas sp. across all samples. This sequence also supports the identification of the Bagley Chlaionmomons as a new species   and the next most abundant chytrid sequence's best match to a reference is only at 98.4%. Uncultured Chrysophytes (likely a single taxon represented by 3 ASVs) were also common in the amplicon samples in weeks 1-5, though were not observed commonly in microscopy samples. Chrysophytes likely have a higher DNA extraction efficiency from mixed community samples than Chlainomonas sp. that has an extremely thick cell wall that is hard to break in single cell PCR attempts and is difficult to penetrate with DAPI, a nuclear stain that is often easily penetrates cell walls (unpublished). This taxon has been observed in other years and can produce localized yellowish-brown blooms on Upper Bagely Lake, but did not bloom in 2021. Ciliates and Chrysophytes, and to some extent Chytrids, had a reduction in relative proportion during and following the heat dome in week 6.Patterns in other taxa can be also seen over the course of the bloom. Conifer DNA appears in the data early in the bloom season, ciliates are found mid-bloom, where populations may grow in response to increase algal biomass. Chytrid and fungal taxa  remain relatively stable through the bloom, except for some fungal dominated samples at the end of the bloom, but this was not consistent in every sample. Chytrid infection was observed thought the bloom in the microscopy data Fig.  and ampl7,45 = 22.63, P < 0.001)  and is consistent with the highest measurements for Fv/Fm that we measure for snow algae that we see in this region (Kodner unpublished data). Fv/Fm then declined to a plateau during week 4 of the study to value that indicate continued photosynthetic activity in the algae but some level of stress in PSII. This plateau was consistent through to the end of the study except for week 6, where we saw lower fluorescence values drop to their lowest levels .The mean Fv/Fm of the snow algal community in the Bagley Lake bloom ranged from a high of 0.62 during week one to a low of 0.20 during week 6 Fig. . The Fv/01) Fig. . Fv/Fm  increased significantly from beginning of bloom to the end  , coinciding with decreases in Fv/Fm that week, but not a decrease in cell density. The log of the Integrated density and Fv/Fm is are negatively correlated  suggesting a significant inverse relationship between these measurements during this bloom. However, this analysis included only a small number of measurements (n=6) and is suggestive rather than demonstrative.The cell density of algal samples collected from the same sampled used for relative chlorophyll fluorescence measurements were similar among weeks 2–6 Fig. . Cell de01) Fig. . IntegraThe observational transect survey of color across the survey area each week throughout the bloom showed heterogeneity with no clear visual progression among weeks Fig. . We obseChlainomonas sp. consistently dominated the eukaryotic community during the bloom where cell types were mixed throughout the patches. This suggests that life cycle development does not appear synchronized spatially or temporally either. However, the Fv/Fm of communities steadily decreased throughout the bloom season, suggesting some level of community-level stress that is independent of cell abundance or life-cycle but is inversely related to color intensity, which is a measure of bloom intensity on snow surfaces. The patterns we observed of Chlainomonas sp. blooms on Upper Bagley Lake indicate that, at least for this system, the broad-scale bloom dynamics are starkly different from those in well studied in marine environments  and generally sunny and warm conditions, compared to typical Pacific Northwest spring weather in which Bagley Lakes area experiences regular rain on snow events.Chlainomonas sp. in 2021 bloom suggests many small populations progressing through growth and development stages throughout the bloom and nutrient dynamics happen on a small scale in patchy blooms. Novis  suggesting that this sequence was derived from white snow, not pink snow. Tucker and Brown . If these algae are swimming up through the snowpack throughout the growing seasons, pinker snow later in the bloom could result from reproduction on snow surfaces and or continued vertical migration of cells. However, increased snow ablation as the season progresses  that suggested algae were stressed as the season progressed and responded to an excessive heat event. Additionally, the eukaryote community composition progressively changes over the bloom, suggesting ecological interactions between trophic levels and between algae and their parasites may be influenced by environmental stress like temperature. As our climate continues to warm, low-elevation snow ecosystems like Upper Bagley Lake will become more transient, reducing the growing season for these algae, which may impact the bloom dynamics and the phenology. In addition, extreme heat events likely to be more common, leading to increased photophysiological stress and unknown impacts on life cycle and may impact the ability of this species to bloom in the future. Systems like Upper Bagley Lake, where the bloom is dominated by one species and the start and end of a bloom can be monitored, provide an opportunity to study the complex factors that affect snow algae bloom events in a natural habitat. Further development of these types of studies will allow us to understand the mechanistic drivers of stress in these ecosystems and help predict how these taxa will respond to future warming.Our results characterize a geographically defined snow algal bloom, dominated by fiad106_Supplemental_FilesClick here for additional data file.


In January, 2023, the Science and Security Board of the Bulletin of the Atomic Scientists moved the hands of the Doomsday Clock forward to 90 s before midnight, reflecting the growing risk of nuclear war  commits each of the 190 participating nations”to pursue negotiations in good faith on effective measures relating to cessation of the nuclear arms race at an early date and to nuclear disarmament, and on a treaty on general and complete disarmament under strict and effective international control” UN, . ProgresAny use of nuclear weapons would be catastrophic for humanity. Even a “limited” nuclear war involving only 250 of the 13 000 nuclear weapons in the world could kill 120 million people outright and cause global climate disruption leading to a nuclear famine, putting 2 billion people at risk Bivens, . A largehttp://www.ippnw.org).The health community has had a crucial role in efforts to reduce the risk of nuclear war and must continue to do so in the future  Global Health Research Unit, is a member of the Independent Advisory Group, Collaboration for the Establishment of an African Population Cohort Consortium, and he was co‐chair of the InterAcademy Partnership, Climate Change and Health Working Group 2019–2022 and co‐chair of the Academy of Medical Sciences/Royal Society working group on “A healthy future—tackling climate change mitigation and human health together” 2020–2021 . I. H. reports honoraria for several speaking engagements, all donated to Back from the Brink, the International Physicians for the Prevention of Nuclear War, or Physicians for Social Responsibility; travel/meeting support for Nobel Peace Laureates' Summit, the World Federation of Public Health Associations World Congress, and the UN Human Rights Commission Youth Summit; and he is a member of the steering committee of Back from the Brink and the International Steering Group of the International Campaign to Abolish Nuclear Weapons, a Board member of the International Physicians for the Prevention of Nuclear War and Physicians for Social Responsibility, and a Trustee of the Phillips Exeter Academy . M. G. M. O. R. reports research grants from the Dutch Research Council, NOW (grant number COMPL.21COV.001) and from the Netherlands Organisation for Health Research ZonMw (grant number 09120012010063) and he is Chair of the Dutch guideline committee on cognitive impairments and dementia. T. R. reports a contract with the Institute for Energy and Environmental Research (USA) for papers addressing the health and environmental consequences of nuclear testing in multiple locations, including Australia, French Polynesia, central Pacific, and China; honorarium from The Choisun Ilbo media group in South Korea for a lecture on nuclear weapons in 2022 and for nuclear weapons presentations from Hyogo Medical Practitioners Association (Japan), Peace Boat (Japan) and the University of Sydney; he was an expert witness on radiation and health for Environmental Justice Australia acting for Mine‐Free Glenaladale regarding proposed Fingerboards Mineral Sands Mine to the Victorian Government Fingerboards Inquiry and Advisory Committee; he is a member of RV3 Rotavirus Vaccine Development Scientific Advisory Board, Murdoch Children's Research Institute/Royal Children's Hospital; he is a member of the Committee of International Campaign to Abolish Nuclear Weapons Australia; he is a member of the Internet Peace Prize Award Committee ; he was a member of the Victorian International Humanitarian Law Advisory Committee, Australian Red Cross; he is a Board member of the Initiative for Peacebuilding, Faculty of Arts, University of Melbourne; he is an At‐large Board member of the International Physicians for the Prevention of Nuclear War; he was co‐president of the International Physicians for the Prevention of Nuclear War 2012–23; and he is honorary principal fellow, Melbourne School of Population and Global Health, University of Melbourne. P. Y. reports grants from Atea Pharmaceuticals; honoraria for lectures, presentations, and educational events from bioMérieux and Pfizer Pharmaceuticals; fees for participation on an advisory board from Pfizer Pharmaceuticals; and he is a member of the Antimicrobial Stewardship Study Group Executive Committee (2022–2024) and the Clinical Practice Guideline Panel on Vaccinations in Immunocompromised hosts for the European Society of Clinical Microbiology and Infectious Diseases. C. Z. reports consulting fees for his role as senior adviser on the international journals project from the International Physicians for the Prevention of Nuclear War. The remaining authors declare no conflict of interest.


Recently in the Journal, Amanda Williams described her experience of raising concerns about a group of trials with “untrustworthy data”. We were inspired by the work of Williams and colleagues to examine these and other trials by the same research group. Similar to Williams, we found that the patterns of reported data differed from the patterns expected to arise from valid randomisation. We also identified a high proportion of reported baseline p-values for categorial variables that differed from independently calculated p-values. We reported these findings to the affected journals but none of the concerns were addressed and no action will be taken about the majority. Despite the large number of unresolved concerns about these trials, readers will be unaware of the issues, which seems entirely unsatisfactory. We have used similar techniques in the past and so explored whether the observed distribution of baseline categorical variables was consistent with the expected distribution in 17 RCTs by Monticone and colleagues identified in a PubMed search that reported categorical baseline variables . The key finding was that the frequency counts were much more similar than expected.p-values differed from p-values calculated using the reported summary statistics. As the reported and calculated p-values should be identical (unless there are missing data), these incorrect p-values indicate impossible data. Collectively, these findings support and extend the earlier review by Williams and colleagues. A response to those concerns has failed to address the majority of the issues raised.The systematic review by Amanda Williams and colleaguesIn March 2023, we emailed the six publishers of the eight journals that published the 14 unretracted RCTs together with the editor and/or journal contact. At that time, three of the 10 RCTs assessed by Williams and colleagues had been retracted. We received no response from one publisher or its journal; for two publishers, we received no response from the publisher but a response from the journal; and for three publishers, we received an acknowledgement or response from the publisher and the journal. Of the responses, one indicated that the paper had been retracted and a notice would shortly appear, although no notice has been published 4 months later. The publication has 162 citations in google scholar including 12 in 2023. Two responses reiterated that they have previously investigated – one indicated the new information was insufficient to investigate or request raw data, and the other did not respond when new specific information was provided. One treated the email as a request for advice about submitting a publication and failed to respond to the subsequent clarification. One said that they would not investigate because their RCTs were not referenced in our email and then requested an affirmation that the trials were problematic before they would investigate. During the email exchange, it became clear that some important information in an editorial about this issue was out of date , but the editor said it would not be updated. One response noted that one of their RCTs had been retracted and that they would consider what to do about the others. Finally, one indicated the journal had passed the issue to the publisher.7 is similar to that of Williams. Despite the large number of unresolved concerns about the publication integrity of these RCTs, readers of the articles will be unaware of the issues, and potentially patients will be harmed if treatment decisions are based on unreliable trial results. This seems entirely unsatisfactory.Thus, more than 4 months after notifying the journals and publishers, we understand that no action will be taken about eight RCTs, one will be retracted, decisions might still be in process about four articles, and for two we have no information. None of the RCTs have attracted an Expression of Concern. Our experience of notifying concerns about publication integrity in this case and others the tentacles of unreliable research spread far and wide, and there seems little appetite to bring about the changes necessary to remove unreliable articles from the literature.As Williams lays out clearly in her editorial,


Nowadays, medium chain triglycerides (MCT) with special health benefits have been increasingly applied for fortifying food products. Therefore, the present work aimed to investigate the effects of MCT on traditional tea polyphenols-fortified cooked rice (TP-FCR). It was visualized by DSC, CLSM, XRD, FT-IR, and Raman spectroscopy. The higher content of starch-MCT complexes with an increase in the relative crystallinity and the generation of short-range ordered structures contributed to a more ordered and compact molecular arrangement, which can hinder the action of digestive enzymes on starch. SEM demonstrated that MCT transformed the microstructure of TP-FCR into a denser and firmer character, making it an essential component hindering the accessibility of digestive enzymes to starch granules and slowing the release of tea polyphenols in TP-FCR to attenuate starch digestion. Consequently, the addition of MCT reduced the polyphenol-regulated starch digestibility from 74.28% in cooked white rice to 64.43% in TP-FCR, and further down to 50.82%. Besides, MCT also reduced the adhesiveness and improved the whiteness of TP-FCR. The findings suggested that MCT incorporation could be a potential strategy in cooked rice production to achieve high sensory quality and low glycemic cooked rice. Tea products rich in polyphenols have functional properties such as hypoglycemia, antioxidants, obesity prevention, etc. . Adding To slow starch digestion, besides the interaction between tea polyphenols (TP) and starch, the interaction between lipids and starch is also important. Ai et al. proposed that lipids can interact with starch to reduce the sensitivity of starch to enzymatic hydrolysis and improve the anti-digestibility of starch . LuangsaWhat is more, many studies have proven that the type of lipid and its chain length and degree of unsaturation can affect complex formation, structure, and functional properties. Sun et al. proposed that maize starch interacting with different types of fatty acids (FAs) exhibited different V-type polymorphs, and the FAs with a chain length of 10–14 carbons showed stronger intermolecular interactions with MS and produced more ordered structures than other FAs . Zabar eMedium chain triglycerides (MCT) have high saturation and shorter carbon chain, are colorless and odorless at room temperature, and have low viscosity and good stability ,11, whicIn addition to the interaction between two components, such as starch and polyphenols, as well as starch and lipids, there are also three component interactions in the food system. Wang et al. reported that starch can interact with β-lactoglobulin and fatty acid (FA) to form ternary complexes, which had a larger short-range molecular order and higher relative crystallinity than binary starch-FA complexes . Lin et Therefore, this study aimed to investigate the effects of MCT on the digestive properties and quality of TP-FCR. The improvement of MCT on TP-FCR was evaluated by surface porosity, texture analysis, and color. Meanwhile, differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), Raman spectroscopy, confocal laser scanning microscope (CLSM), and scanning electron microscopy (SEM) were used to interpret the effects of MCT on TP-FCR in terms of the multi-scale structure. The effects of MCT on the digestive properties of TP-FCR were also characterized by dynamic modulus changes and fluorescence quenching spectrum during digestion.Golden Dragon Fish Northeast Pearl Rice (japonica rice) with 15.8% amylose content was purchased from Yihai Kerry Arawana Holdings Co., Ltd. . MCT was purchased from Shanghai HeYi Food Technology Co., Ltd. . The fatty acid composition of MCT is caprylic acid glyceride and decanoic acid glycerol ester, and the purity of MCT is 100%. Instant green tea powder (IGT) was purchased from Jiangsu Dehe Biotech Co., Ltd. . IGT is a water-soluble component extracted from green tea leaves, mainly consisting of tea polyphenols, flavonoids, alkaloids, theanine, and tea polysaccharides, with a tea polyphenol content of 18.36%, and free of carriers and other additives such as saccharides. Porcine pancreatic amylase (PPA) was purchased from Aladdin Reagent Co., Ltd. . DNS reagents and aspergillus niger amyloglucosidase (AAG 100 U/g) were purchased from Solarbio Technology Co., Ltd. . All other chemical solvents were analytical grade and purchased from Sinopharm Chemical Reagent Factory .2·h) to prevent moisture loss and cooled to room temperature.A total of 100 g of rice was rinsed three times and filtered; 150 mL of deionized water dissolved with 2 g of IGT was poured into the rinsed rice in a rice cooker . Then, different amounts of MCT  were added respectively and mixed well. Next, the rice was soaked for 30 min, cooked, and kept warm for 20 min. Subsequently, TP-FCR samples separated from the middle of the cooker were put into plastic bags with a moisture vapor transmission rate of approximately 0.84 mg/ at a vacuum degree of 25 Pa and a temperature of −45 °C for 48 h. Each lyophilized TP-FCR sample was divided into two portions, one of which was left intact, and the other was ground into powder for further detection. The TP-FCR without MCT was named as control.A whole pot of TP-FCR was placed on the Perfection V330 Photo Scanner  Co., Ltd., Beijing, China) without damaging the shape and then the image of the TP-FCR sample was taken. The surface porosity of TP-FCR was calculated using Image j 1.48u software .Texture properties of TP-FCR samples were tested using a texture analyzer  with cylindrical probe P/0.5, according to a modified method . TP-FCR The color of TP-FCR samples was measured using a Hunter-Lab colorimeter , and the results were expressed as the values of lightness (L*), redness/greenness (a*), and yellowness/blueness (b*) . The whiThe water absorption rate was determined according to the method of Wei et al. with slight modifications , TP-FCR 1 is the volume of rice (100 g) before cooking, V2 is the volume of the TP-FCR samples (10 g), and W is the weight of 100 g of rice after cooking.According to the method reported by Paesani and Gómez with some modifications , the drao), the peak temperature (Tp), the termination temperature (Tc) and the enthalpy change (ΔH) were recorded.Thermodynamic properties of TP-FCR were determined using differential scanning calorimetry . A total of 8 mg of the lyophilized powder of the sample was accurately weighed into an aluminum crucible, then sealed and balanced for 12 h before testing [Referring to the method described by Seo et al. , the XRD−1. The scanning resolution at 4 cm−1 and the cumulative number of scans at 32 were set.According to the method of Lv et al. , the lyo−1.According to a modified method of Sivam et al. , the lyow/v) was added to stain the starch. And 20 μL Nile Red  was added to stain the MCT [The lyophilized powder of TP-FCR was mixed with distilled water in the ratio of 1:7 and then 20 μL fluorescein isothiocyanate  at a ratio of 9:1, and added to the concentric cylinder for testing. The program scan time was 3 h, the frequency was 1 Hz, and the temperature was set at 37 °C. The samples without enzyme were used as control and the whole procedure was performed in the linear viscoelastic zone.The dynamic modulus change during digestion was determined by a rheometer  with reference to the method described by Xiao and Zhong with slight modifications . The conw/w) was added and maintained in a shaking water bath at 37 °C for 3 h. Fluorescence spectra of the supernatant of TP-FCR samples digested for 0, 20, 40, 60, 90, 120, 150, and 180 min were performed with a fluorescence spectrophotometer  by referring to the method of Chen et al. [A 0.2 g of TP-FCR lyophilized powder was added to sodium acetate buffer (pH 5.2), and the pH was adjusted to 6.5 with 1 mol/L and 0.1 mol/L NaOH, then the pre-activated mixed enzyme solution  was added and maintained in a shaking water bath at 37 °C for 0, 20, 40, 60, 90, 120, 150, 180 min, respectively. Anhydrous ethanol was then added to inactivate the enzyme. The mixture was then centrifuged and separated the supernatant. The glucose content at different times was measured using the DNS method and the contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were calculated based on the glucose content. The RDS, SDS, and RS contents were calculated according to the following formulas:20 and G120 are the glucose content released within 20 min and 120 min, respectively, FG is the content of free glucose before enzymatic hydrolysis, and TS is total starch content.For the in vitro digestion of cooked white rice and TP-FCR samples, the test method of Fu et al. with slight modification was referred to . A 0.8 gt is the percentage of digested starch at a given time, C∞ is the estimated percentage of starch digested at the end of the reaction, “t” is the digestion time, and “k” is the digestion rate constant.The digestion rate constant (k) was obtained by fitting the digestion rate curve of starch. The starch digestion data have often been fitted to a first-order equation :Ct=C∞1−esample is the area under the hydrolysis curve of sample and AUCfresh white bread is the area under the hydrolysis curve of the reference sample (fresh white bread).The hydrolysis index (HI) and predicted glycemic index (pGI) were calculated using the following formulas :HI/%=AUCAll test data were obtained by at least three tests and expressed as the mean ± standard deviation. The data was analyzed by the one-way ANOVA and Duncan’s multiple range test at a significance level of 0.05 using SPSS 20.0 software, and all figures were drawn using Origin 2021.The results of the surface porosity in The TPA experiment, also known as the twice-chewed test, mainly involves compressing the sample twice by simulating the chewing motion of the human mouth . The texColor is an important indicator for evaluating the quality of TP-FCR. The increase in whiteness and brightness of the cooked rice is critical for higher consumer acceptance . It was It has been reported that the starch can interact with other substances during gelatinization, to affect the water absorption, dispersion, expansion, and solubility of starch . Gerits 1,A2, the peak area and peak temperature of peak I of TP-FCR decreased with the increase of MCT, while the peak area and peak temperature of peak II increased with the increase of MCT. This indicated that the dissociation enthalpy of incomplete and low-ordered crystal structures decreased with increasing MCT, while the dissociation enthalpy of the complexes increased with increasing MCT, which was consistent with the results in To investigate the interactions between MCT and TP-FCR, the thermodynamic properties of TP-FCR were determined using DSC. As shown in o, Tp, and Tc) reflected the thermal stability of the microcrystals, while the ΔH value was related to the number of crystals (double helix or single helix structure) [p values, and the heat absorption peaks range (To–Tc) of peak I, while increasing the dissociation enthalpy, Tp values, and the heat absorption peaks range of peak II. Jakobek proposed hydrophobic interactions and hydrogen bond formation between polyphenols and carbohydrates or lipids [p value of peak I may be due to the interaction between starch, polyphenols, and MCT, which reduced the incomplete and low-ordered crystal structure in starch, and thus the energy required for microcrystalline melting was decreased. The increase in the dissociation enthalpy and Tp value of peak II can be attributed to the formation of more starch-MCT and starch-TP complexes, resulting in more stable crystals in TP-FCR that required more energy to dissociate. The formation of complexes with ordered structures can resist the hydrolysis of digestive enzymes, and the steric hindrance of MCT may reduce the accessibility of digestive enzymes to starch in TP-FCR, which was consistent with the results of in vitro digestion of starch.The melting temperatures (Tructure) ,43. It cr lipids . The intr lipids . The decThe native rice starches displayed an A-type crystalline structure with diffraction peaks at 15°, 17°, 18°, and 23° 2θ, whereas V-type complexes generally showed diffraction peaks at 13° and 20° 2θ ,46. Lipi−1, which was due to the stretching vibration of O-H in TP and starch. The broad peak intensity of TP-FCR at 3500–3200 cm–1 decreased with the increase of MCT, which was caused by the increase of MCT and the protective effect of MCT on TP that resulted in more MCT and linear hydrophobic chains of TP being encapsulated in the helical lumen of the starch through hydrophobic interactions and thus interfered with the formation of hydrogen bonds between starch molecules. Similar conclusions that the intensity of broad peaks of starch-TP complexes at 3500–3200 cm−1 decreased with the increasing addition of TP were reported by Li et al. [FT-IR spectrograms  revealedi et al. .−1 and (1020/995) cm−1 can be used to reflect the short-range orderliness and the helical structure of starch [−1 and 2850 cm−1 compared with the control TP-FCR. The absorption peak at 1740 cm−1 was the C=O stretching vibration peak of MCT and the peak at 2850 cm−1 was the stretching vibration peak of -CH2 of MCT [−1 increased as the amount of MCT increased, indicating that the structure of TP-FCR was more short-range ordered after the addition of MCT, because the larger the ratio was, the more short-range ordered. Starch interacted with MCT and TP via hydrophobic interactions to form V-type complexes, so that the amorphous amylose and the amorphous long side chain of amylopectin that did not form a double helix could form a single helix structure, thus increasing the degree of short-range order of molecules. The addition of MCT reduced the ratio of (1020/995) cm−1, suggesting that MCT interacted with amylose and affected the structure of the amorphous region.The IR-ratios of (1045/1020) cmf starch ,51. As c2 of MCT . As show–1 is sensitive to changes in the short-range molecular order of starch, and the FWHM at this location is often used to characterize the degree of starch polymerization [−1 decreased with increasing MCT, indicating an increase in the short-range ordering of starch in TP-FCR, which was consistent with the FTIR results. As shown in −1 of the spectrum were fitted to the split peaks, and the peak areas at 1130 cm−1 and 1340 cm−1 were recorded. The characteristic band of 800 to 1500 cm−1 was mainly generated by the vibration of glucose monomer, and there were many heterogeneous peaks in this region, which were formed by the accumulation of functional groups and the high overlap of chemical bonds [−1 and 1340 cm−1 were closely related to the single helix structure of the starch [−1 and 1340 cm−1 increased with the increase of MCT addition, which indicated that the double helix would be deconvoluted into single-helix structure when MCT and TP were compounded with starch, increasing the single-helix structure.The half peak width (FWHM) of the Raman spectrum at 480 cmrization ,54. A smrization ,55. As sal bonds . Among te starch . As show3 was caused by the interaction of starch with endogenous lipids in TP-FCR to form starch-lipid complexes, where some tiny red dots represented the unbound lipids present in the TP-FCR. The increase in the yellow fluorescence area in 3–F3 suggested that more starch-MCT complexes were formed with increasing MCT. The small red dots appearing in 3–F3 indicate that the MCT attached to the starch surface and the large red dots may be free MCT. It has been reported that the increased resistance of starch to enzymatic hydrolysis through the addition of lipids was directly related to the formation and structure of starch-lipid complexes [Single fluorescent labeling of CLSM and multi-channel overlay images were used to observe the binding of starch and MCT in TP-FCR. Among them, the starch in TP-FCR showed a green color when stained with FITC, MCT appeared red in color when stained with Nile Red, and the yellow fluorescence fraction represented the binding matrix formed by starch and lipids ,46,58. Aomplexes ,59 and t1,A3 that the cross-section and surface of the control TP-FCR at 100× exhibited a flat structure, which had small, dense pores and thin pore walls. The cross-section of the control TP-FCR at 500× showed a uniform and dense three-dimensional network structure. However, as can be seen from 1–F1,B2–F2,B3–F3,B4–F4, the number and density of pores in the cross-section and the surface of TP-FCR decreased gradually with the increasing addition of MCT, and compared with the control TP-FCR, the cross-section of TP-FCR with MCT was uneven and the pore walls thickened. When the addition of MCT was 4% and 5%, the cross-sectional layers of TP-FCR appeared a densely arranged structure.As shown in Li et al. revealed that hydroxyl groups in TP can interact with water to retain a large number of water molecules , and at The control TP-FCR had a uniform and dense network structure as well as thin pore walls, which made it more accessible to digestive enzymes and thus more susceptible to hydrolysis. The formation of starch-MCT complexes caused it to form a dense and compact structure, providing a physical barrier for the diffusion of digestive enzymes into the starch granules and slowing the release of tea polyphenols in TP-FCR, which was conducive to improving the anti-digestibility of TP-FCR.Dynamic modulus changes during digestion can reflect the rate of TP-FCR digestion. As can be seen in TP-FCR contained TP, and the free TP in the system exhibited a quenching effect on tryptophan and tyrosine, which were the main residues of the intrinsic fluorescence of proteins ,65. The 2 values ranged from 0.990 to 0.995, indicating that all fitted the first-order rate equation well [According to the above results, the addition of MCT can inhibit the digestion of TP-FCR, so the in vitro digestibility properties of TP-FCR were measured. The starch fraction and kinetic parameters obtained by fitting the starch hydrolysis rates with a first-order kinetics model were summarized in ion well ,69. The ion well ,70. Simuion well ,47. TP-FThis work suggested that the addition of MCT affected the adhesiveness, texture, color, and digestive properties of TP-FCR. Results revealed that MCT significantly improved the anti-digestibility of the TP-FCR. SEM, CLSM, DSC, XRD, FT-IR, and Raman spectroscopy demonstrated the important role of MCT addition in reducing the starch digestibility of TP-FCR from the perspective of multi-scale structural changes. Meanwhile, it can be concluded that the high anti-digestibility of TP-FCR with MCT can be attributed to structural changes caused by the formation of starch-lipid complexes and the inhibitory effect of released polyphenols on amylolytic enzymes. In addition, MCT reduced the tendency of TP-FCR grains to stick to each other and the bottom of the pot and improved the color of TP-FCR. This study provides a reference value for applying MCT as a quality improver to starchy foods with high glycemic index. However, in vivo animal studies and clinical trials are needed to further investigate the mechanisms involved in starch metabolism of TP-FCR with MCT.


COVID-19 pandemic has caused disruption in higher medical education and healthcare worldwide. To thrive in times of uncertainty, medical higher education institutions have to adapt to the post-COVID-19 era and innovate its international activities. To make a difference in societies locally, nationally and internationally, they will have to enhance their global presence. Internationalization is the best way to the exchanging of knowledge, enhancement of the medical curriculum, and mobilization of talent and resources for research and teaching. To remain competitive, universities will need to expand their international activities. This paper highlights several suggestions to enhance internationalization of medical higher education institutions in the post-COVID-19 era. Internationalization is the best way to the exchanging of knowledge, enhancement of the medical curriculum, and mobilization of talent and resources for research and teaching. To remain competitive, universities will need to expand their international activities. To thrive in times of uncertainty, they have to adapt to the post-COVID-19 era and innovate its international activities. To make a difference in societies locally, nationally and internationally, they will have to enhance their global presence.Challenges: Physical recruitment activities suffered due to COVID-19, and some economies, hit hard by the virus, may witness a decline in numbers of outbound students. While China will remain the largest student recruitment market in the near future, its overall weak growth was evident even before pandemic [Opportunities: There is a need and an opportunity for diversification of recruitment portfolios going beyond high-value regions and countries , with a focus on ‘potential growth regions’ such as Sub-Saharan Africa, Central Asia, and others. Decision support systems go beyond clinical practice [Universities will be able to recruit more outstanding and diverse international students by strengthening existing partnerships, working closely with their alumni, targeting carefully selected new geographic regions, and taking advantage of emerging big data analytics. practice  and new practice .Challenges: Ecological problems exemplified by climate change will affect international travel. There will be a need to make digital interaction simple and natural. Opportunities: A positive consequence of digitisation of classes during the pandemic has been the flexibility that it has given students. This can be an opportunity to cater to the needs of students who are working, commuting or traveling.Universities will have to provide students with diverse mobility opportunities rendering benefits for individuals and communities at a local, regional and global level. Challenges: Some faculty members, especially in countries where the English language is not commonly spoken, may be resistant to change and comprehensive internationalization (partly because of language barrier). However, the survey by the International Association of Universities suggests just the opposite: internationalization gives a unique opportunity for professional, personal growth and modernization of the education process [Universities will have to empower staff members to be facilitators and advocates for comprehensive internationalization and create an environment and a culture to nurture creativity and achievement, with a focus on quality and excellence. There is a need to identify and invest in opportunities that promote global mobility for staff so that they become more internationally knowledgeable and interculturally skilled.  process .Challenges: Internationalization of the curriculum (IoC) has been emphasized in many universities for a period of time, and might receive less attention from the staff in the post-COVID −19 era. However, IoC has been defined as an ongoing process and opportunity for the incorporation of international, intercultural and global dimensions into the content of the curriculum, as well as learning outcomes, assessment tasks, teaching methods and support services of the study programme [The internationalized curriculum will ensure that all our students are exposed to international and intercultural perspectives. Having international partnerships as part of a degree shall give more legitimacy to the program and shall make the students adapt to the globalized world. Online learning innovation may be important driving force for an internationalized curriculum; however, the curriculum should be engaging, and communication should be interactive to provide results that are similar to an in-class experience. rogramme .Challenges: There are limited funding opportunities for global health and related topics. Opportunities: Universities have to invest in young researchers to develop international excellence and to support career development that spans the postgraduate, early career, and international leadership stages. The internationalized curriculum shall cover SDGs.The future research collaborations have to tackle important global problems outlined in UN Sustainable Development Goals (SDGs) . AlthougChallenges: There is growing competition among HEIs for a place in the best partnerships and networks. Partnerships will allow to embrace opportunities not otherwise available.In post COVID −19 era partnerships and networks have to bring diverse viewpoints, inspiring discussions, additional resources, and rewarding activities to address global issues. Alumni around the world has to play an important role in establishing new partnerships and networks. Challenges: There is a need to be sensitive to understandings and attitudes towards internationalization, and miscommunication between stakeholders. Financial restrictions may also pose difficulties. Opportunities: Such a review process will help to strengthen competence, confidence, and credibility.Universities will have to have an inclusive system for monitoring, reflecting upon, evaluating and reviewing internationalization progress. Below we highlight several suggestions to enhance internationalization of medical Higher Education Institutions (HEI) in the post-COVID-19 era:Practical solutions for internationalization have to be tailored to individual HEI needs and recourses and be flexible and adaptable.


Therefore, this study aims at the above-mentioned problems. Here, we found that the waxy corn starch diet resulted in significantly higher expression levels of fat-synthesis-related genes than lipolysis-related genes, leading to abdominal fat deposition in broilers. Increasing the level of dLys in the diet increased the protein content in muscle by promoting protein synthesis and inhibiting protein degradation and also promoted the expression of lipolysis-related genes, thereby degrading the generation of abdominal fat in broilers. In conclusion, our findings signify that increasing the dLys level to 1.32% when using the waxy corn starch diet could improve carcass traits.In recent years, affected by the international trade situation, China’s corn import volume and import price have risen sharply, which has increased the production cost of poultry farming. Poultry nutritionists have been encouraged to use different grains instead of corn. However, it is unclear whether feeding broiler chickens with different starch sources affects the utilization of digestible lysine (dLys) in different dietary starch sources on liver lipid metabolism and breast muscle protein metabolism in broiler chickens. The experimental design was a 3 × 3 two-factor completely randomized design. A total of 702 one-day-old male Arbor Acres Plus broilers were randomly divided into nine treatments of six replicate cages with thirteen birds each. The treatments consisted of three different starch sources  with three different dLys levels . The trial lasted from 1 to 21 days. Carcass traits, serum metabolites, breast muscle protein and liver lipid metabolism were evaluated. A significant interaction effect (p < 0.05) for dietary starch sources and dLys levels was noted in the percentage of abdominal fat and gene expression related to breast muscle protein metabolism throughout the experimental period. The waxy corn starch diet and a 1.08% dLys level in the diet increased both the percentage of abdominal fat (p < 0.01) and blood total cholesterol (p < 0.05) in the broilers. The waxy corn starch diet significantly upregulated the mRNA expressions of Eif4E, AMPK, FABP1, ACC and CPT1 (p < 0.05). The 1.32% dLys level significantly upregulated the mRNA expressions of mTOR, S6K1, Eif4E, AMPK and PPARα (p < 0.05) and significantly downregulated the mRNA expressions of MuRF and Atrogin-1 (p < 0.05). In summary, the waxy corn starch diet resulted in significantly higher expression levels of fat-synthesis-related genes than lipolysis-related genes, leading to abdominal fat deposition in broilers. Increasing the level of dLys in the diet increased the protein content in muscle by promoting protein synthesis and inhibiting protein degradation and also promoted the expression of lipolysis-related genes, thereby degrading the generation of abdominal fat in broilers. Our findings signify that increasing the dLys level to 1.32% when using the waxy corn starch diet could improve carcass traits.This study investigated the effects of digestible lysine ( AM) and amylopectin (AP). Previous reports have demonstrated that AP is easier to digest than AM [RDS, a low AM/AP starch) is consumed, glucose is rapidly released in the small intestine, a process that may not be able to continuously meet the normal energy requirements of broilers. In such cases, more amino acids may be oxidized to provide the energy needed by the body, resulting in a decrease in amino acid utilization [In recent years, affected by the international trade situation, corn import volume and import price have risen sharply in China, which has increased the production cost of poultry farming . Therefo than AM , and sta than AM . Additio than AM . When ralization .Lys) is defined as a basic essential amino acid whose carbon skeleton cannot be synthesized by poultry and therefore must be met through the diet for the maintenance and growth of poultry [Lysine  [mTOR promotes the synthesis of new proteins by directly phosphorylating the expression of translation-related downstream proteins, such as the signaling molecules ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein-1 (Eif4E) [UPP) and autophagy–lysosome pathway [The synthesis of new muscle proteins is highly regulated by multiple signals integrated by the mammalian target of rapamycin (mTOR) . In fact (Eif4E) ,14. In a pathway . The UPP pathway .AMPK) signaling pathway. The activation of AMPK inhibits lipogenesis and deposition while increasing fatty acid oxidation, affecting cholesterol and triglyceride synthesis, thereby regulating cellular energy balance [PPARα), carbohydrate-responsive element-binding protein (ChREBP), sterol regulatory element-binding protein-1c (SREBP-1c), malic enzyme (ME) and acetyl CoA carboxylase (ACC) participate in hepatic lipid metabolism [The liver is the center of glycogen synthesis, gluconeogenesis and energy metabolism in poultry. It acts as a central regulator of lipid homeostasis and is responsible for coordinating the synthesis, export and utilization of fatty acids as energy substrates ,18. Lipi balance . A priortabolism ,20,21.It is unclear whether feeding broiler chickens with different dietary starch sources would affect the utilization of dLys in their bodies, thereby affecting broiler breast muscle protein and liver lipid metabolism, due to differences in the digestion rates of these different starch sources. Therefore, the purpose of this study was to investigate the effects of different dLys levels on broiler carcass traits, serum metabolites and postprandial glucose and insulin changes under different dietary starch sources and to explore the responses of genes related to breast muscle protein and liver lipid metabolism to different starch sources and different dLys levels.All animal procedures were conducted in accordance with the Beijing Regulations of Laboratory Animals  and were approved by the Laboratory Animal Ethical Committee of China Agricultural University (Protocol Number: AW03602202-1-3).A total of 702 one-day-old male Arbor Acres Plus broiler chickens  were randomly divided into 9 treatment groups based on a 3 × 3 two-factor experimental design. The treatments consisted of 3 different starch sources  with 3 different dLys levels . Each group included 6 replicate cages with 13 birds each. The experimental period was 21 days. The formula and nutritional level of the experimental diets are shown in Arbor Acres Plus broilers. All birds had access to feed and water ad libitum in crumble-pellet form and via nipple drinkers, respectively.Bird management was based on the guide for On day 19, the birds from the dietary 1.20% dLys groups of different starch sources and the cassava starch groups of different dLys levels were selected. After fasting for 4 h, feeding for 0.5 h (set as zero) and at 0.5, 1.0, 1.5 and 2.0 h after feeding, blood samples were collected from birds’ wing veins in each group and kept in sterile serum collection tubes. Blood was collected once from each bird to avoid the influence of stress on the experimental result.On day 21, six birds close to the average weight (one bird/replicate) in each treatment were selected for sample collection. The blood samples were drawn from birds’ wing veins and kept in sterile serum collection tubes. The birds were euthanized after intravenous injection of sodium pentobarbital (30 mg/kg). Six small pieces of the pectoralis and liver samples were washed with saline solution and collected into 1.5mL sterile Eppendorf tubes, and then snap-frozen in liquid nitrogen immediately and stored at −80 ℃ for mRNA analysis.On day 21, six birds close to the average weight (one bird/replicate) in each treatment were selected for slaughter, after which the breast muscle (%) and abdominal fat (%) were determined.g at 4 °C for 10 min, and the obtained serum samples were stored at −80 °C for later testing of glucose, cholesterol, triglycerides, uric acid and blood urea nitrogen. The serum glucose, insulin, urea nitrogen, uric acid, total cholesterol and triglycerides were analyzed using an XH-6080 radioimmunoassay analyzer.The collected blood samples were centrifuged at 5000× The total RNA was extracted from the pectoralis and liver tissues using an RNAiso plus  according to the manufacturer’s recommendations. The nucleic acid concentration was determined using a Nanodrop 2000 spectrophotometer , and the RNA purity was verified using 1.5% denaturing agarose gels. Next, cDNA was synthesized by using a high-capacity cDNA reverse transcription kit  and stored at −20 °C. All quantitative real-time PCR (qRT-PCR) assays were performed using an SYBR Premix ExTap kit  in a 7500-fluorescence detection system . The primer sequences for qRT-PCR are listed in GLM) of SPSS 20.0 statistical software  was used to conduct a two-factor analysis of variance. The statistical model for this study is shown below. Differences between the treatment groups were considered statistically different at p < 0.05. All experimental data  were based on one bird in each cage as the experimental unit. The different starch sources and different levels of digestible lysine were used as independent variables. The carcass characteristics, blood biochemistry, breast muscle protein and liver metabolic genes were used as dependent variables.The data were first tested for homogeneity of variances, after which the general linear model  is the interaction effect of the ith and jth treatments and ijkε is the error associated with ijk.YHere, p > 0.05). However, there was a significant interaction effect between dietary starch sources and dLys levels on the percentage of abdominal fat in broilers (p < 0.01). At the 1.08% and 1.32% dLys levels, waxy corn starch significantly increased the percentage of abdominal fat in the broiler chickens compared with corn starch (p < 0.01). At the 1.20% dLys level, different dietary starch sources had no significant effect on the abdominal fat percentage of the broilers (p > 0.05). In addition, increasing the level of dLys in waxy corn starch could also significantly reduce the percentage of abdominal fat in the broilers (p < 0.01).As shown in p < 0.05). At 0.5 h after feeding, the insulin response level of the cassava and waxy corn starch diets reached their peaks, while the insulin response level of the corn starch diet increased steadily over time and finally remained at a stable level after 1.0 h.The different starch sources and dLys levels affected the glucose and insulin responses in the broilers 2 h after feeding . At 0.5 The glucose levels in broilers subjected to 1.08% and 1.32% dLys peaked at 0.5 h after ingestion, but the glucose levels in broilers subjected to 1.32% dLys were relatively stable, and the glucose levels in broilers subjected to 1.08% dLys decreased rapidly, while the glucose levels in the broilers subjected to 1.20% dLys increased with time. In broilers subjected to 1.08% and 1.20% dLys, the insulin response levels reached a peak at 0.5 h after feeding, while the insulin response levels of broilers subjected to 1.32% dLys reached a peak at 1.0 h.p > 0.05) (p < 0.05). Compared with those consuming the corn starch diet, the serum total cholesterol levels of broilers consuming the cassava starch and waxy corn starch diets were significantly increased (p < 0.05). Compared with broilers subjected to 1.32% dLys, the serum total cholesterol levels in broilers in the 1.08% and 1.20% dLys groups were significantly increased (p < 0.01). However, the dietary starch sources and dLys levels had no significant effect on serum urea nitrogen, uric acid and triglyceride (p > 0.05).The dietary starch sources and dLys levels had no significant interactions on serum urea nitrogen, uric acid, total cholesterol or triglyceride levels in the broilers ( > 0.05) . The dieS6K1, Eif4E, MuRF, CathepsinB, Atrogin-1 and M-calpain in broiler breast muscle (p < 0.05) (Eif4E in breast muscle compared with corn starch and waxy corn starch (p < 0.05), and corn starch significantly increased the mRNA expressions of MuRF, CathepsinB, Atrogin-1 and M-calpain in breast muscle compared with waxy corn starch (p < 0.05). At 1.20% dLys, cassava starch and waxy corn starch significantly increased the mRNA expression of S6K1 and Eif4E in breast muscle compared with corn starch (p < 0.05), and waxy corn starch significantly increased the mRNA expression of MuRF, CathepsinB, Atrogin-1 and M-calpain compared with cassava starch (p < 0.05). At the 1.32% dLys level, corn starch and waxy corn starch significantly increased the mRNA expression of S6K1 and Eif4E in breast muscle compared with cassava starch (p < 0.05). DLys levels significantly affect the mRNA expression of mTOR: compared with 1.08% dLys, 1.32% dLys significantly increased the mRNA expression of mTOR (p < 0.05).There were significant interactions between dietary starch sources and dLys levels on the mRNA expressions of  < 0.05) . At the p > 0.05) : the waxy corn starch diet significantly increased the mRNA expressions of AMPKα1, FABP1, ACC and CPT1 in broiler livers compared with the cassava and corn starch diets (p < 0.05). DLys levels significantly affect the mRNA expressions of AMPKα1 and PPARα in the broiler liver (p < 0.01). Compared with 1.08% dLys, 1.20% and 1.32% dLys significantly increased the mRNA expressions of AMPKα1 and PPARα (p < 0.01).Dietary starch sources and dLys levels showed no significant interactions with regard to the expression of genes related to lipid metabolism in the broiler liver ( > 0.05) . DietaryPrevious research found that Lys not only affected the process of body protein deposition and promoted the development of muscles  , but alsUric acid is the end product of amino acid catabolism, and any amino acid not required as a building block during protein synthesis is broken down. During amino acid catabolism, the amino terminus of each amino acid must be removed. Because nitrogen compounds are not utilized in energy transduction pathways, the accumulation of amino groups may have adverse effects on the animal. Currently, researchers believe that serum uric acid levels can be used as a measure of amino acid availability  and, becThe amount of native starch hydrolyzed by amylase in the animal gut is negatively correlated with the dietary AM content . CurrentmTOR signal passage and affected skeletal muscle protein synthesis. Similarly, Zheng et al. [mTOR signaling pathway, thereby enhancing the phosphorylation expression of its downstream signaling molecules S6K1 and Eif4E and finally promoting protein synthesis in dairy cow skeletal muscle. S6K1 mainly accelerates protein synthesis by enhancing translation initiation factors [Eif4E promotes protein synthesis by stimulating the translation of a subset of mRNAs with 5`-terminal pyrimidine motifs [mTOR, S6K1 and Eif4E, which confirmed our speculation that increases in muscle protein synthesis caused by Lys are also mediated by enhancing the translation initiation of its related mRNAs. In addition, this study also found that a 1.32% dLys level could down-regulate the expression of protein-degradation-related genes such as muscle RING finger protein (MuRF) and muscle atrophy factor 1 (Atrogin-1). Atrogin-1, muscle atrophy F-box protein (MAFbx) and MuRF are the most representative E3 ubiquitin ligases in skeletal muscle, which mediate the polyubiquitination of proteins and target them to the 26S proteasome for degradation [MAFbx, MuRF and Atrogin-1 has been found to be significantly upregulated in various conditions that lead to muscle degradation [Amino acids play a key role in regulating protein synthesis or degradation in various tissues, including skeletal muscle. A study by Watanabe et al.  confirmeg et al.  also fou factors , while Ee motifs . Therefoe motifs . In the radation . Currentradation . AlthougAMPK signaling pathway, PPARα and SREBP-1c have been shown to play key roles in liver lipid metabolism [AMPK and PPARα in the liver tissue of broilers. PPARα activation induces the transcriptional upregulation of fatty acid transporters and downregulation of fatty acid synthesis [PPARα enhanced fatty acid β-oxidation by regulating the expression of PPARα target genes, thereby reducing the accumulation of lipids in vivo. In summary, increasing dietary dLys levels could activate the AMPK signaling pathway, thereby promoting the transcription level of PPARα, and finally inhibiting the accumulation of abdominal fat in broilers. Furthermore, the intake of a high AP diet can rapidly elevate blood glucose content in broilers, and blood glucose can change the expression of related transcription factors in a series of tissues through the lipid synthesis pathway, thus directly affecting the expression of lipogenic genes [AMPK, CPT1, FABP1 and ACC transcription factors. Although the genes related to lipid synthesis and decomposition were significantly upregulated in the broilers fed with the waxy corn starch diet, combined with the previous phenotypic results, we judge that the rate of lipid synthesis was significantly higher than the rate of lipid decomposition, which eventually led to abdominal fat deposition in the broilers. Our findings further support the results of Yang et al. [As the main transcription factors in the tabolism . In thisynthesis . Similarynthesis  also shoic genes . In the g et al.  and Yin g et al. .In the current study, the waxy corn starch diet and a 1.08% dLys level in the diet increased both the percentage of abdominal fat and serum total cholesterol in broilers. The waxy corn starch diet resulted in significantly higher expression levels of fat-synthesis-related genes than lipolysis-related genes, leading to abdominal fat deposition in the broilers. Increasing dLys in the diet increased protein content in the muscles of the broilers by promoting protein synthesis and inhibiting protein degradation and also promoted the expression of lipolysis-related genes, thereby degrading the generation of abdominal fat in the broilers. In summary, our findings signify that increasing dLys levels to 1.32% when using the waxy corn starch diet could improve carcass traits.


Mycobacterium tuberculosis complex (MTBC) organisms, affects a range of humans and animals globally. Mycobacterial pathogenesis involves manipulation of the host immune system, partially through antigen presentation. Epitope sequences across the MTBC are evolutionarily hyperconserved, suggesting their recognition is advantageous for the bacterium. Mycobacterium tuberculosis var. bovis (MBO) strain Ravenel is an isolate known to provoke a robust immune response in cattle, but typically fails to produce lesions and persist. Unlike attenuated MBO BCG strains that lack the critical RD1 genomic region, Ravenel is classic-type MBO structurally, suggesting genetic variation is responsible for defective pathogenesis. This work explores variation in epitope sequences in MBO Ravenel by whole genome sequencing, and contrasts such variation against a fully virulent clinical isolate, MBO strain 10-7428. Validated MTBC epitopes (n = 4818) from the Immune Epitope Database were compared to their sequences in MBO Ravenel and MBO 10-7428. Ravenel yielded 3 modified T cell epitopes, in genes rpfB, argC, and rpoA. These modifications were predicted to have little effect on protein stability. In contrast, T cells epitopes in 10-7428 were all WT. Considering T cell epitope hyperconservation across MTBC variants, these altered MBO Ravenel epitopes support their potential contribution to overall strain attenuation. The affected genes may provide clues on basic pathogenesis, and if so, be feasible targets for reverse vaccinology.Tuberculosis, caused by  M. tuberculosis variant bovis (MBO) strain BCG began, tuberculosis remains the deadliest single infectious agent in the world for humans. Our understanding of Mycobacterium tuberculosis complex (MTBC) pathogenesis remains lacking, both in humans and in the myriad non-human hosts MTBC variants attack. An underlying mechanism behind this is the subversion and misdirection of the host immune response, achieved both directly by targeted alteration of host kinase signaling cascades, and indirectly by intentional presentation of conserved antigens that drive specific immune feedback4. In a background of mycobacterial antigenic hyperconservation, this research asked what level of epitope variation can be detected in each an attenuated and a virulent MBO strain, and if variation may signal loss of virulence through dysregulation of host immune control.Over 100 years since vaccination by 7. Over the course of the infection, immunity develops, targeting of specific antigens arises, the infection is suppressed, and re-infection by the same pathogen later hindered. On the other hand, immune responses to mycobacterial pathogens like M. tuberculosis are well-known to be more nuanced and skewed towards a cell-mediated immune response9. Early research suggested a limited role of B cells or antibodies in protection, and although these subsets do have beneficial roles11, it is known that a successful immune response against MTB infection absolutely requires CD4+ and CD8+ T cell activity12. Subsequent work has focused heavily on a process of initial engulfment of MTB by macrophages, subsequent intracellular MTB replication and cell signaling that leads to a Type IV hypersensitivity response wherein T cells contribute towards granuloma formation to seal in the antigen provoking the response4. In most infections, this successful response drives the granulomatous encasement of MTB, not its sterilization13. While most patients with latent TB infection (LTBI) do not progress again to active disease, MTB is able to survive indefinitely within the confines of the granuloma, and in reactivation, MTB drives caseation of the granuloma core and subsequent spillage of bacilli from the granuloma into the lung, allowing dissemination and respiratory transmission to others14. This reactivation is again believed to involve MTB host manipulation by antigen presentation in a hypersensitized host state, and is not associated with an increased bacterial load, indicating some degree of bacterial control over the host immune response by antigen presentation drives disease14. Amidst this backdrop, it has been observed by multiple groups that T cell epitopes in M. tuberculosis are hyperconserved, equivalent to that seen in the most essential MTB genes15. This supports a model wherein T cell recognition of MTB epitopes is essential to bacterial survival15. After millennia of coevolution in human hosts, MTB has developed a strategy of immune subversion not rooted in antigenic obfuscation, but in the intentional presentation of conserved targets that manipulate the host immune system. This triggers unproductive immune responses that, at least at a population-scale, confer fitness benefits for the pathogen16. In short, T cell responses are necessary to control tuberculosis, but MTB has also evolved to exploit these same responses, provoking specific immune responses that can ultimately benefit MTB and perpetuate disease17.At a simplistic level, a host’s immune response to infection—including recognition of pathogen markers, response by innate and adaptive immunity, and ultimate pathogen clearance—depends on the receipt of signals differentiating self and foreign molecules. Antigens here generically refer to such markers that a host can react to in mounting a pathogen-specific immune response. Under the classic model, after initial infection, pathogen antigens recognized by the immune system are under selective pressure to change, and the host in return faces selective pressure to maintain recognition of changing targets in a process long referred to as an evolutionary arms raceGiven the specialization of pathogenic mycobacteria into manipulation of the host by intentional presentation of specific, hyperconserved T cell epitopes, this work posits that evidence of attenuation and adaptation can be found in epitope variation. Changes observed in known T cell epitopes in attenuated strains may represent a contributor to such attenuation, and identification of such epitopes would therefore be informative for pathways necessary for mycobacterial subversion of host immune responses. While most vaccine strategies continue to target immunodominant antigens like ESAT-6 or the Antigen 85 complex, the identification of other antigens where variation is associated with dysregulation of host immune manipulation could lead to selection of better, more protective targets.M. tuberculosis variant bovis (MBO) strains, with a hypothesis that variation in T cell epitopes will be more frequent in attenuated strains, and that these variant epitopes may contribute to observed attenuation. To explore this topic, polymorphisms were extracted from the recently sequenced genomes of MBO strain Ravenel, a naturally attenuated cattle strain that does not carry the causative genomic lesions—large, contiguous deletions of genetic material like RD-1—of BCG strains, and MBO strain 10-7248, a fully virulent cattle clinical isolate20. For additional comparison, the MBO strain BCG-1 (Russia) vaccine strain believed closest to the now-lost ancestral BCG strain22 and the MBO strain AF2122/97 reference 24 were analyzed, the former to assess epitope variation in a truly dysfunctional MBO strain, and the latter to exclude variation fixed in the MBO genetic background for any epitopes characterized only in non-MBO complex members. Validated MTBC epitopes collected from the Immune Epitope Database (IEDB) were compared against SNPs in the genomes of the four selected strains, with a focus on 1–3 amino acid changes to represent variation through point mutations, and to limit both nonspecific hits on other epitopes and the analysis of more extensive variations known to exist in PE/PPE genes contrary to the overall pattern of epitope conservation7. The analysis is represented by the schematic in Fig. This work sought to analyze variation of known MTBC epitopes between a subset of virulent and attenuated M. tuberculosis var. tuberculosis-derived, and additionally, one epitope can have multiple known variants in the database—thus, degenerate mismatches—and so each mismatch was then interrogated for a perfect match against other variant epitope sequences, as well as against the AF2122/97 epitope hits to reduce the chance variants were simply fixed in the MBO background. Of the 303 Ravenel single amino acid hits against the epitope dataset, 188 mismatches were found to have a 100% match against a different variant epitope in the dataset and were therefore excluded, leaving 115 epitopes in Ravenel that differed by 1 amino acid from the IEDB sequences. Comparing these 115 epitopes to the output for the epitope workflow for AF2122/97 yielded 100% matches for 110, leaving just 5 epitopes in Ravenel that were mismatches from known sequences, and different from the same epitope sequence in the MBO reference strain AF2122/97  was also unreliable, mapping to an unknown PE/PPE family gene on an unplaced contig. These excluded sequences are listed in Supplemental File esxJ hits could be an assembly error  observed in epitope 229352 was earlier reported as a SNP20. The large, polar, charge-bearing glutamic acid to a small, flexible, non-polar glycine is a major alteration, and across homologues, position 263 is almost always either a glutamic acid, or an aspartic acid (the COG3583 consensus sequence residue). RpfB in Ravenel is unique in NCBI’s Identical Protein Groups database, and TBLASTN searches of the NCBI NR database and WGS database filtered by Mycobacterium tuberculosis complex (taxid: 77643) showed no hits for this substitution outside Ravenel. Per a partial crystal structure by Ruggiero et al., residue 263  in Paraguay. Like epitope 595988, RpoA’s T cell epitope 597585 has been validated in a bovine interferon gamma release assay38. The substitution is not one believed associated with drug resistance, and while the physiochemical differences between aspartic and glutamic acids are minimal, this change may influence immune recognition, or even still incur minor fitness costs given the fundamental role RpoA plays. Regardless, it seems unlikely this epitope would be a sole major driving factor of observed attenuation in Ravenel and might instead be a contributor among a constellation of changes.RpoA, or the DNA-directed RNA polymerase subunit alpha, is a core subunit of the RNA polymerase (RNAP) complex conserved across bacteria. It is the target of the antibiotic rifampicin, and while this critical enzyme is known to be intensely conserved, in MTB in particular, changes in RpoA-RpoC are known to be associated with rifampicin resistance, especially with changes in RpoBA 4th single amino acid mismatch in Ravenel, in epitope 163423, was found to be in a fragmented, assembled sequence of EsxJ. This epitope mismatch was discarded as unreliable. Subsequently, the last 1aa mismatch (epitope 163642) was found to involve this same gene and was also discarded. Finally, the 3aa mismatch in Ravenel was determined to fall in a partial PPE family protein (WP_152345480.1) that aligns ambiguously with multiple possible PE/PPE genes. Due to the unreliable nature of these genes in sequencing and assembly, this match was disregarded.43 and is one of the selected antigens in the ID93 subunit vaccine45. It is suggested that variation in some PE/PPE genes, unlike in most other antigens, may actually be beneficial to MTB in avoiding a productive, Th1-dominant host immune response44. In strain 10-7428, epitope 10022, one of three recognized in PPE42, presented with Asn232Asp. This modification is seen in only one other sequenced isolate. Interestingly, 10022 is a validated B cell epitope unlike the exclusive T cell epitopes changes seen in Ravenel. In contrast with other PE/PPE family genes analyzed in this work, PPE42 is a relatively distinct gene and sequencing, and placement of this substitution is clean in the Ravenel assembly. However, as no in vitro crystal structure has been obtained for this protein and PE/PPE genes are often poorly understood regardless, any interpretation of this finding must remain limited absent further investigation in vitro.Only one unique single epitope change was observed in the virulent strain 10-7428, a modification of PPE42 (Mb2640 in MBO). This protein is known to be highly immunogenic and stimulates a strong humoral response in human patientsesxJ, and this epitope was discarded as an artifact.In Ravenel, an additional epitope change was reported that also arose from an atypical assembly pattern around 47. Antigen 85B within the MTBC is strongly conserved, with more variation across NTM but noted antibody cross-reactivity between all variants48. In BCG-1 Russia, a single substitution was observed, Phe140Leu, that resulted in a change that appears fixed across all BCG strains, with very few non-BCG examples from lab or clinical strains. This single amino acid change modifies four characterized epitopes  that all overlap this position.The Antigen 85B complex has been known for decades to be a dominant secretion product and immune stimulator49. Loss of MarP or its catalytic residues leave the bacterium severely impaired for pathogenesis in vitro and in a murine model of infection51. In BCG-1 Russia, epitope 600022 of MarP carried Asn165Thr change, a mutation in the flexible linker region between transmembrane anchor and protease domain. Like the Ag85B mutation, this substitution is also fixed across BCG strains and appears again only in the same small number of lab and clinical isolates, raising the possibility these isolates may be evolutionarily similar to BCG, or cases of BCG-osis.MarP is a serine protease involved in maintenance of intracellular pH during phagocytosis through the processing of peptidoglycan through its interactions with RipA52. T cell epitope 20707 presented with Thr46Pro. At a molecular level, the sequence change is another significant structural alteration, though whether this affects the function of this protein or its immune recognition in BCG-1 are beyond the scope of this work.Mce  family proteins are a class of protein involved with entry into host cells and interference with host cell signaling pathways, particularly through the ERK1 and ERK2 MAPK pathway for inducing cytokine production PE/PPE family changes were numerous in BCG-1, but as before, they are difficult to rely on and are reported only with the caveat that their true sequence and placement in the assembly are unclear.53. The use of these tools may also be complicated by the specific portions of molecules being studied—surface residue substitutions have been shown previously to be more poorly predicted than changes to residues buried within the protein54, and epitopes herein were exposed residues. Despite this, a message remains in these contradictory findings: no consensus changes of a magnitude > 0.5 kcal/mol in either a stabilizing or destabilizing direction was measured for any affected protein in Ravenel or 10-7428, and it is therefore less likely that any observed substitutions are sufficient to modify an affected protein enough to ablate functionality. This investigation is premised on the idea that changes to epitope recognition can lead to dysregulation of host manipulation by Mycobacterium species. Indeed, if mutant proteins retain their molecular functionality but still alter virulence, modified epitope recognition is a means of describing this contradiction.Finally, in silico predictions for ΔΔG were variable and conflicting Table , a knownM. tuberculosis var. tuberculosis, and after sequencing a uniquely attenuated M. tuberculosis var. bovis genome in strain Ravenel, the haystack of 4130 epitope alignments yielded 3 needles of epitopes with non-synonymous changes in characterized epitopes. Two of these epitopes have been validated in bovine IFN-γ release assays, and one in a murine model showing IFN-γ and TNF-α release. As recent work has demonstrated, MBO Ravenel provokes potent cell-mediated immune responses in the bovine host, but fails to produce granulomatous lesions in nearly all cases, and the process of an infection leading to granuloma formation is known to be tightly associated with the precise presentation of specific T cell epitopes at specific times20. In contrast to these, an isolate from a dairy cattle outbreak—MBO 10-7428—was found to contain no T cell epitope variation, and instead the only change fell in a B cell epitope in a gene where variation is associated with increased virulence. These results warrant the further investigations into how these changes in T cell epitopes may be an indicator of attenuation in MBO Ravenel and other strains, an investigation of their role in pathogenesis, and whether any of these epitopes may prove useful as subunits for vaccine development, as has already been proposed for RpfB34. An essential next step towards validating the biological implications of this work would be in the testing of wild-type vs. altered epitopes in a bovine T cell assay utilizing samples from previously infected cattle. Detectable differences, positively or negatively, in T cell activation between these epitopes would support a role in attenuation and merit closer scrutiny into the immunological effects. The analytic process described here is simple, and with limited development could be readily incorporated into standard comparative genomics analysis for MTBC organisms, increasing the amount of information researchers can extract from each experiment.Using epitope data gleaned largely from 55. The IEDB (accessed September 2022) was used to collect all epitopes validated in the Mycobacterium tuberculosis complex , primarily from Mycobacterium tuberculosis var. tuberculosis (n = 4111) and M. tuberculosis var. bovis (n = 783), including 356 M. tuberculosis var. bovis BCG strains55. These sequences include non-peptide targets like LAM which were removed (n = 46), B cell-specific discontinuous peptide sequences (n = 2), as well as peptides that had undergone post-translational modifications (n = 28), both because they are unable to be processed through tblastn and because the presence or absence of these modifications could definitionally not be verified by screening against whole genome sequences.The US National Institutes of Health and the Department of Health and Human Services jointly support and operate the Immune Epitope and Analysis Database (IEDB), a comprehensive database of over 1.5 million experimentally validated epitopes from a range of species and diseases57. Two of these strains of M. tuberculosis var. bovis were sequenced and analyzed by our lab: attenuated Ravenel (GCF_018305025.1) and virulent 10-7428 (GCF_018305045.1)19. From existing databases, the attenuated reference BCG-1 (Russia) (GCF_001483905.1), and virulent reference AF2122/97 (GCF_000195835.3) were selected. The BLAST + package (v2.11.0)57, preinstalled on the MSU HPCC, was loaded and the makeblastdb command used with default parameters to generate local databases based on each of the four genomes mentioned. All tblastn searches were with default BLAST parameters, with the filtered list of 4818 epitope sequences searched against each genome database57. Some highly repetitive epitopes—particularly those from PE/PPE genes—were caught by filtering, Karlin-Altschul parameters were not calculated by the BLAST algorithm, and they were subsequently excluded from analysis. The tblastn output was produced twice, once in pairwise-alignment format, and once in tabular format. After processing below, epitopes that were unique to our dataset were also TBLASTN searched against the broader NCBI WGS database to determine their presence or absence across all sequenced strains.The remaining 4818 sequences were converted to FASTA format and uploaded to the High-Performance Computing Center at Michigan State University —DDGun3D, PremPS, and INPS—as well as an additional recently released tool, DynaMut2, using in vitro crystal structures preferentially and DeepMind’s AlphaFold in silico predictions when in vitro structures were not available29.Unique epitopes in Ravenel, 10-7428, and BCG-1 Russia were queried through UniProt and NCBI, and effects on protein stability examined by three high-performing software tools benchmarked by Pancotti et alSupplementary Information.