Suitable packaging material in combination with high-pressure processing (HPP) can retain nutritional and organoleptic qualities besides extending the product’s shelf life of food products. However, the selection of appropriate packaging materials suitable for HPP is tremendously important because harsh environments like high pressure and high temperature during the processing can result in deviation in the visual and functional properties of the packaging materials. Traditionally, fossil-based plastic packaging is preferred for the HPP of food products, but these materials are of serious concern to the environment. Therefore, bio-based packaging systems are proposed to be a promising alternative to fossil-based plastic packaging. Some studies have scrutinized the impact of HPP on the functional properties of biopolymer-based packaging materials. This review summarizes the HPP application on biopolymer-based film-forming solutions and pre-formed biopolymer-based films. The impact of HPP on the key packaging properties such as structural, mechanical, thermal, and barrier properties in addition to the migration of additives from the packaging material into food products were systemically analyzed. HPP can be applied either to the film-forming solution or preformed packages. Structural, mechanical, hydrophobic, barrier, and thermal characteristics of the films are enhanced when the film-forming solution is exposed to HPP overcoming the shortcomings of the native biopolymers-based film. Also, biopolymer-based packaging mostly PLA based when exposed to HPP at low temperature showed no significant deviation in packaging properties indicating the suitability of their applications. HPP may induce the migration of packaging additives and thus should be thoroughly studied. Overall, HPP can be one way to enhance the properties of biopolymer-based films and can also be used for packaging food materials intended for HPP. Currently, the consumer trend is towards a growing demand for minimally processed food with improved food safety, nutritional value, freshness, and natural flavors. In order to meet these demands, food industries have been using different processing technologies allowing reduced additives without compromising the sensory and nutritional qualities of the food material. Among different technologies, high-pressure processing (HPP) is one of the emerging noble technologies in the food industry that produce clean label foods free from chemical additives and causes minimal product quality loss along with extending shelf-life by inactivating microbes and enzymes ,2,3,4. HCommercially, HPP treatment is carried out in batch, semi-continuous, or continuous mode. Liquid products can be treated in a semi-continuous or continuous process whereas solid products are only treated in batch mode . It has In the food industry, fossil-based plastic is the ultimate choice for HPP of packaged food materials due to its excellent thermo-mechanical properties with high strength and flexibility, low cost, lightweight, shape versatility, high performance, and easy transportation. Among, others, polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), polyamide (PA), and ethylene-vinyl alcohol (EVOH) are used in food industries ,14,15. LBiopolymer-based compostable packaging is one of the alternatives to plastic packaging. Biopolymers like polysaccharides and proteins from plant or animal origins are being explored to develop packaging materials. Previous studies showed that biopolymer-based films have good film-forming capacity but this packaging system exerts certain shortcomings like low mechanical strength, high hydrophilicity, and poor barrier property as compared to synthetic plastics ,22,23,24Until now, HPP has been found to be effective in the modification of biopolymers like polysaccharides , proteinBio-based polymers such as polysaccharides, proteins, or their derivatives show good film-forming capacity and are believed to be futuristic replacements for synthetic plastics. Although these polymers form films, due to certain limitations in their pristine forms, some modifications are reported to enhance their packaging properties. Different physical and chemical modifications of biopolymers are being carried out. HPP is a novel technique that utilizes high pressure of more than 100 MPa and is primarily used for the preservation of food products in food processing industries. Recently, it has also been applied in the physical modification of biopolymer-based films ,33. For According to the literature, the surface roughness of the biopolymer-based film subjected to HPP largely depends on the level of pressure applied and the composition of the films. It is observed that FFS, when treated with HPP, mostly results in uniform and smooth surfaces. Wei, Zhang  studied 2 composite films demonstrated that HPP can diminish the transition layer between PVA and CHI  of polyvinyl alcohol and chitosan (PVA/CHI) loaded with nano-TiO and CHI b. Cross-pressure . The incpressure  films. Hpressure  reportedTensile strength (TS) and elongation at break (EAB) are critical mechanical properties associated with the performance of automatic packaging lines and also maintain food quality and integrity during handling, processing, shipping, and storage ,18. HPP In the case of starch, HPP treatment induces volume-driven transition like the transformation of starch crystalline structure from A- to B-type. Films prepared from HPP-treated FFS possess higher moisture content that helps in stabilizing the scattered amylopectin structure of A-type starches via van der Waals forces, resulting in the rearrangement of the helix structure leading to the formation of compact B-type structure ,38,39,40In the case of protein-based film, HPP is an effective factor that can induce the formation of hydrogen bonds, and disulfide bonds or may increase hydrophobic interaction between the components of the film, resulting in the formation of a more compact structure that leads to the considerable enhancement in the mechanical properties. Molinaro, Cruz-Romero  reported2 (0.10%) FFS when subjected to 200, 400, and 600 MPa increased the TS from 8.24 ± 0.27 MPa to 13.67 ± 0.41, 13.98 ± 0.33, and 17.15 ± 0.97 Mpa, respectively, and EAB also increased from 64.82 ± 1.10% to 68.48 ± 1.66, 68.12 ± 1.94, and 67.92 ± 2.73%, respectively, although the pressure level showed no significant impact. Similarly, TS of PLA-Ag-3% or PLA-Ag-5% when subjected to 200 and 400 MPa increased the TS but decreased the EAB as shown in HPP processing of FFS, containing nanoparticles also significantly influences the mechanical properties of the film. Lian, Zhang  showed tSolubility in water is one of the key features of biopolymer-based film. In food packaging applications, packaging material should be water-insoluble to protect the integrity of the packaged product, prevent moisture transport, and enhance shelf-life ,22. The −9 g/m s Pa) compared to film prepared by heating at 90 °C for 20 min, where the WVP was 3.10 × 10−9 g/m s Pa. This decrease in WVP may be attributed to the creation of the denser matrix with the application of HPP, which might have restricted the water movement. However, the impact of HPP (600 MPa) on the WVP of the TPS film was less evident as compared to the HP-treated BS film. This variation in WVP upon application of HPP at the same condition may be due to the structural differences between starches that might have influenced the extent of gelatinization. The degree of gelatinization is not only dependent on the starch type but on water concentration and processing parameters . Also, among the different types of starch , type A- is most sensitive to HPP influencing the film-forming properties ) increased to (1.59 ± 0.10) × 10−14 and (1.62 ± 0.13) × 10−14 (kg·m/[m2·s·Pa]) when subjected to 450 and 600 MPa   . Similar²·s·Pa)]  also repBesides pressure level, processing temperature also plays a vital role in the alteration of barrier properties of biopolymer-based films. It was observed that HP sterilization  of carrots using flexible PLA film was not suitable as the film was damaged during HP sterilization, as shown in Further, it was observed that the impact of HPP on the barrier properties of the biopolymer-based film is also dependent on the types of food materials being packed. As observed in PLASiOx-PLA film, application of 500 MPa at 50 °C for 15 min, OP increased by 31% when in contact with olive oil (fatty acid simulant) but OP was so large in case of the film contact with distilled water (aqueous simulant) that the film could not be used for food protection. Similar to the OP, WVP was also affected by the types of food being packed, for instance, WVP increased by 2170% when the film was in contact with distilled water and 71% when in contact with the olive oil compared to the control film. This could be due to the plasticizing effect of water on hydrophilic PLA leading to the swelling along with the creation of holes and cavities when subjected to high pressure as explained in 2 nanocomposite treated with 300 MPa [2 nanocomposite film significantly decreased when subjected to HPP (300 MPa) [Some other studies reported that HPP has no significant impact on the barrier properties of the film, for instance, 75-μm coextruded PLA film subjected to HPP  , PLA/Nan 300 MPa  as summa300 MPa) . Such anOverall, PLA film prepared by solvent casting method or by extrusion method and subjected to HPP increases the barrier property due to reduction in crystallinity of the film. Finally, it can be concluded that biopolymer-based films required serious modification for further enhancement for commercial applications.Among the biopolymers, PLA-based packaging is mostly used for the HPP of food materials at the commercial level. Only a few studies have shown the application of other biopolymers such as cellulose acetate and wheat gluten-based packaging systems for HPP. The mechanical properties of the biopolymer-based packaging system depend on the type of polymer matrix, different additives, and their interactions. It has been observed that HPP can lead to the modification of biopolymer-based packaging materials and is dependent on the processing conditions such as pressure level, temperature, and time of exposure. Only a few studies were found in the literature that reported the enhancement in mechanical properties of biopolymer-based packaging systems when subjected to HPP. It either reduces the mechanical properties of the film or in some cases, the materials remain unaffected as summarized in The extent of modification of mechanical properties of the biopolymer-based film subjected to HPP is dependent on the level of pressure. It was observed that TS of untreated PLA-PEG-GO-2% film 50.80 ± 0.75 MPa decreased to 43.13 ± 6.64, 40.69 ± 0.77, and 40.14 ± 1.00 MPa when subjected to 300, 450, and 600 MPa, respectively. Similarly, EAB of the untreated nanocomposite film 25.31 ± 0.27% decreased to 20.32 ± 1.35, 17.98 ± 0.92, and 11.72 ± 1.35% when subjected to 300, 450, and 600 MPa, respectively . Such a Literature also reports that the extent of modification of flexible packaging materials subjected to HPP also depends on the types of food being packed. The application of HP into the film containing liquid food can result in plasticization of the material that weekends the structure leading to a reduction of mechanical properties. Notably, plasticization of polymers occurs in the amorphous region rather than the crystalline region. Such effect was evident in the low crystalline PLASiOx-PLA film, where the film in contact with olive oil (fatty acid simulant) experienced higher degradation in mechanical properties as compared to film contact with distilled water (aqueous simulant). PLASiOx-PLA film when subjected to 500 MPa at 50 °C for 15 min decreased TS and EAB by almost 25% and 32%, respectively when in contact with fatty acid simulant and 22% and 29%, respectively when in contact with aqueous simulant . Some studies have reported that HPP had a minor to no significant impact on the mechanical properties of the biopolymer-based films. Ahmed, Mulla  investigg, Tm, Tc, ΔHm, and ΔHc values of PLA-PEG-GO-1% GO nanocomposite film, showing the postponed thermal degradation compared to the untreated film. Such enhancement in the thermal stability could be attributed to the increase in the crystallinity of the nanocomposite due to HPP. Such enhancement in thermal stability was also observed for PLA film subjected to HP sterilization (700 MPa at 90 °C), due to crystallization of the amorphous external layer PLA matrix. Crystalline regions can act as mobility constraints for the macromolecules in the amorphous phase, thus enhancing thermal stability [As mentioned in tability . m value of the film in contact with oil simulant showed no significant difference but decreased when contact with water. Such variation was ascribed to the redistribution in the crystallite size when in contact with a liquid of a great affinity to PLA. Where the percentage crystallinity of the untreated film was 2.4% decreased to 1.7 and 1.9%, respectively when in contact with oil and aqueous simulant, respectively [g and Tm significantly decreased when exposed to 200 and 300 MPa for 5 and 10 min [g and Tc values of PLA-TiO2 nanocomposite film increased when subjected to HPP, but showed no significant variation in Tm value after HPP. Besides pressure and temperature, different foods/food simulants in contact with film subjected to HPP, significantly influence the thermal properties. As evident from PLASiOx-PLA film when subjected to HPP (500 MPa for 15 min at 50 °C), the Tectively . Some otd 10 min . In anotd 10 min  showed tg, and Tc, of the PLA-TiO2 nanocomposite film subjected to 300 MPa for 10 min showed no significant variation, however, % crystallinity increased significantly for the HP treated film. Similarly, no significant variation in Tm was observed for co-extruded PLA film subjected to HPP (600 MPa) [However, some studies have demonstrated that HPP had a minor no significant variation in the thermal properties of the film. For instance, PLA-PEG-cinnamon oil-4% film subjected to 300 MPa showed no significant variation in thermal properties, indicating PLA-PEG-cinnamon oil-4% was pressure resistant . Similar600 MPa) . Migration can be defined as the diffusion of substances from a region of greater concentration (the food-contact-surface) to one of a lower concentration . This process is often influenced by food-packaging interactions. The primary packaging material that comes in direct contact with food material interacts through mass transfer mechanisms like permeation, sorption, and migration ,64,65. A® from a PLA matrix and found that migration was so low that no variation in the initial concentration of the additives was detected in PLA film subjected to HPP (800 MPa at 20 to 40 °C for 5). The authors reported that approximately 0.03% w/w of Uvitex OB® was detected for the HP-treated PLA film in contact with four food stimulating liquids distilled water, 3% acetic acid, 15% ethanol, and olive oil, which is very low from a specific migration limit of 0.6 mg/kg. Interestingly, Mauricio-Iglesias, Peyron [Some of the studies have shown that HPP of the biopolymer-based film reduced the migration of packaging additives into food products as well as reduced the absorption capacity of the packaging materials. For instance, Mauricio-Iglesias, Jansana  investig, Peyron  reported2 in a 50% (v/v) ethanol solution as a food simulant. As shown in 2 increased with the increase in the concentration of nanoparticles (1 to 20 wt.%) and migration time (0 to 45 days). The highest concentration of TiO2 in the ethanol solution was found to be 0.43 ± 0.01 mg/kg for HP-treated nanocomposite film soaked for 45 days. Interestingly, Yang, Zhu [2 NPs from untreated PLA-TiO2 nanocomposite after 45 days of soaking in 50% (v/v) ethanol solution was 0.54 ± 0.04 mg/kg. The lower rate of migration of TiO2 NPs from HP-treated film could be due to the increase in the crystallinity of the composite film under the influence of HPP.However, some studies have also demonstrated that HPP may induce the migration of packaging additives. For example, Tang, Fan  investigang, Zhu  reportedSimilarly, Fan, Cui  examinedFrom the literature review, it has been observed that the application of HPP to the film-forming solution (FFS) can form much denser film and thus enhance the properties of the developed packaging materials. However, only a few studies have been carried out to investigate the impact of HPP on biopolymer-based films and most of the research is focused on the effect of HPP on the development of film from a single polymer. As stated, HPP may induce changes in the non-covalent bonding between the components of films resulting in the performance of the packaging materials. Therefore, composite packaging material such as a combination of polysaccharide-polysaccharide, protein-protein, polysaccharide-protein, and/or their combination with some functional compounds needs to be further examined. The petroleum-based packaging has been used for HPP of food products and its impact on the packaging properties is well established, while limited information is available on biopolymer-based packaging. Moreover, the results reported in the scientific literature show inconsistent results, and thus the understanding of the impact of HPP on biopolymer-based packaging material is rather limited. Therefore, further research is required to understand the post-performance of HP-treated biopolymer-based films. More research on packaging properties such as mechanical, thermal, barrier, and the integrity of the post-treated films should be performed in the lab and pilot-scale to optimize biopolymer-based packaging materials. Moreover, many additives such as nanoparticles or functional components are added to enhance the packaging properties and the application of HP may induce changes in the interaction between these components, and thus different migration rates into food may pose threat to human safety. Therefore, the migration of these novel materials from packaging material into food product needs to be evaluated and develop reliable models to predict its migration concentration during the course of processing, storage, and distribution. Furthermore, HPP and active packaging are suitable options for food packaging applications and the release kinetics of active compounds from biopolymer-based packaging materials under HPP is also an area to be explored.In this review, the effect of HPP on the functional properties of the biopolymer-based film was studied including the application of HP to the film-forming solution (FFS) prior to drying and HPP at low and high temperatures in foods or food simulants packaged in biopolymer-based film. The literature revealed that HPP of FFS, formed film with superior properties such as compact and uniform film, enhanced mechanical, thermal, and barrier properties compared to the untreated films. However, the application of HP treatment to the performed biopolymer-based packaging materials experiences irreversible structural damage causing a reduction in the functional properties of the packaging materials. HPP of biopolymer-based film at a relatively lower temperature (pasteurization) induces deviation in the packaging properties in the acceptable range. However, when exposed to HPP at high temperatures such as sterilization temperature, opacification, and embrittlement are observed in the film, and thus restrict their commercial applications.Currently, only a few studies have been carried out focusing on the effect of HPP on the structural, functional, and migration potential of biopolymer-based films. Moreover, the outcome of these studies is not homogeneous, which demands more technical advancement to achieve a deeper understanding of the real effects that HPP can cause on biopolymer-based films. Thus, industries can avail greater support in developing biopolymer-based packaging films having the desired properties for the HPP of foods and beverages.The information regarding HPP of synthetic plastics has already been consolidated to some extent, such as the desired properties of packaging materials for HPP of food products like flexibility, strength, dimensional stability, and heat seal integrity. This information can be taken into consideration in developing biopolymer-based film with the necessary requirements for HPP and a thorough study is required to evaluate their deviation during processing, storage, distribution, and consumption of processed food. Overall, the available limited information regarding biopolymer-based film demands complete studies to develop a biopolymer-based environmentally friendly packaging alternative for HPP.


Skeletal muscle is a highly plastic tissue composed of a number of heterogeneous cell populations that, by interacting and communicating with each other, participate to the muscle homeostasis, and orchestrate regeneration and repair in healthy and diseased conditions. Although muscle regeneration relies on the activity of muscle stem cells (MuSCs), many other cellular players such as inflammatory, vascular and tissue-resident mesenchymal cells participate and communicate with MuSCs to sustain the regenerative process. Among them, Fibro-Adipogenic Progenitors (FAPs), a muscle interstitial stromal population, are crucial actors during muscle homeostasis and regeneration, interacting with MuSCs and other cellular players and dynamically producing and remodelling the extra-cellular matrix. Recent emerging single-cell omics technologies have resulted in the dissection of the heterogeneity of each cell populations within skeletal muscle. In this perspective we have reviewed the recent single-cell omics studies with a specific focus on FAPs in mouse and human muscle. More precisely, using the OutCyte prediction tool, we analysed the “virtual” secretome of FAPs, in resting and regenerating conditions, to highlight the potential of RNAseq data for the study of cellular communication. In order to do this, we extracted the FAPs RNAseq data from publicly available mouse and human skeletal muscle single-cell (sc) data sets , cd34 and Sca1 (Ly6a/e) . These c in vivo , they we in vivo  but they in vivo , muscle  in vivo . FAPs in in vivo . FAPs pr in vivo , and IL3 in vivo . To have in vivo .FAPs finally produce many of the ECM proteins, such as proteoglycans, collagens, laminins, fibronectin, to create the proper matrix environment for the ongoing regeneration. The balance between TGFbeta and TNFalpha (both secreted at least by macrophages) allows the proliferation of FAPs and ECM secretion, as well as their clearance by apoptosis in the late phase of regeneration. This clearance is a crucial event to prevent their differentiation toward adipose or fibrogenic phenotype, leading to permanent tissue scarring deposition characterized by an excessive ECM deposition, fatty infiltration  as well in vivo heterogeneity of FAPs, in mouse and human, during muscle homeostasis and regeneration. In mouse muscle, FAPs are generally clustered on the basis of Pdgfra (Ly6a/e—coding for Sca1-and Cd34) expression  have revealed the pression . In 2019pression . Two othpression  or Lum+ pression . Other dpression , many ofpression . During pression . Althougpression .COL4A1 and COL15A1 is restricted to LUM + FAPs, while COL14A1 is expressed in FBN1+ FAPs. To be noted, in mouse, Col4a1 and Col15a1 are also restricted to Lum + FAPs, while Col14a1, unlike in human, is expressed by both subpopulations. Moreover, TIE2 (COL1A1 and COL1A2), SERP4, SERPINE1, and CCL2; the second subpopulation expresses high levels of FBN1, and MFAP5 and CD55; finally, the third subpopulation has a gene expression profile similar to the adipocyte cluster, expressing, even if at lower levels, ADH1B, ABCA8, MYOC, SMOC2. Recently in 2021 CD90 accumulates in Type 2 diabetic skeletal muscle and is associated with enhanced degenerative remodelling of the ECM in those patients.Few studies have so far described human FAPs with scRNAseq technology . Rubenster, TIE2  is not eGSE138826;  and oneE143704; ). We opt muscles  and pote muscles ), or ext muscles . The gen muscles  function muscles  for huma muscles  as a locuscles . While secretome of each cluster would also be of interest , here we only looked at the overlap between the list of secreted proteins (SP and UPS) of each FAPs subcategory , and we identified 109 shared secreted proteins . 47% of in vitro in culture systems with proteomics approaches . Their complex orchestration during the process of muscle regeneration requires a well-defined and finely-tuned dialogue between all cell types so that each one can play its role at the right time and place. This dialogue is powered through a set of secreted molecules allowing co-regulation through cell to cell communication. Therefore, secretome studies on each cell type are deeply needed to decipher this orchestration. While the secretome of each cell type can be studied individually proaches , the valllenging . The higllenging  and Nichllenging  among otllenging ] that ne


Skeletal muscle satellite cells (SMSCs), which are multifunctional muscle-derived stem cells, can differentiate into adipocytes. Long-chain non-coding RNA (lncRNA) has diverse biological functions, including the regulation of gene expression, chromosome silencing, and nuclear transport. However, the regulatory roles and mechanism of lncRNA during adipogenic transdifferentiation in muscle cells have not been thoroughly investigated. Here, porcine SMSCs were isolated, cultured, and induced for adipogenic differentiation. The expressions of lncRNA and mRNA at different time points during transdifferentiation were analysed using RNA-seq analysis. In total, 1005 lncRNAs and 7671 mRNAs showed significant changes in expression at differential differentiation stages. Time-series expression analysis showed that the differentially expressed (DE) lncRNAs and mRNAs were clustered into 5 and 11 different profiles with different changes, respectively. GO, KEGG, and REACTOME enrichment analyses revealed that DE mRNAs with increased expressions during the trans-differentiation were mainly enriched in the pathways for lipid metabolism and fat cell differentiation. The genes with decreased expressions were mainly enriched in the regulation of cell cycle and genetic information processing. In addition, 1883 DE mRNAs were regulated by 193 DE lncRNAs, and these genes were related to the controlling in cell cycle mainly. Notably, three genes in the fatty acid binding protein (FABP) family significantly and continuously increased during trans-differentiation, and 15, 13, and 11 lncRNAs may target FABP3, FABP4, and FABP5 genes by cis- or trans-regulation, respectively. In conclusion, these studies identify a set of new potential regulator for adipogenesis and cell fate and help us in better understanding the molecular mechanisms of trans-differentiation. Trans-differentiation is a process through which the morphology and function of one type of cell change significantly to be transformed into another type of cell ,2,3. SkeDKK1 gene expression. Knocking out DKK1 significantly inhibits adipogenesis [Several studies have investigated the molecular mechanism of trans-differentiation from muscle cells to adipocytes ,11. An iogenesis . ClearlyNon-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNA (lncRNAs), and circRNAs, show complex and strong regulatory functions in the different physiological processes of cell proliferation, differentiation, and apoptosis ,14,15. IHowever, detailed studies to explore the role of ncRNAs in the adipogenic differentiation of muscle cells are sufficient. Our previous study indicated that miR-199a strongly regulates adipogenesis in C2C12 myoblasts by targeting FATP1 . MoreoveIn the present study, we utilize RNA-seq and gene temporal expression analysis to reflect the dynamic changes of lncRNAs and mRNAs during the process of adipogenesis in SMSCs to find the key driver molecules for the control of adipogenesis and cellular fate.3 muscle blocks were cut from the middle of the longissimus and semitendinosus muscles and washed with precooled PBS  three times. The connective tissue was cut in PBS and then quickly cut into 1 mm3 pieces and collected in a sterile centrifuge tube. Type I collagen 0.2%  was used to digest the tissue on a shaking table at 37 °C for 90 min. The samples were digested with 0.25% trypsin  for 30 min. The digestion was terminated with an equal volume of complete medium  and passed through 100 um and 40 um cell sieves. The cells were re-suspended and inoculated into a 60 mm cell culture dish. After incubation in a CO2 incubator for 2 h, the supernatant was aspirated and the cell pellet was transferred to a new Petri dish and recorded as P01. P01 cells were cultured for 24 h and then suspended into the supernatant, which was recorded as P02. P02 was purified from SMSCs. The medium was changed for the first time after 48 h of culture, and then the complete culture medium was changed every two days.The isolation, culture, and identification of SMSC were performed according to the Musarò’s method . RongChaSMSCs were inoculated into 24-well cell culture plates, and immunofluorescence staining of PAX 7 protein was performed when cell density reached 50%. After rinsing the cells with PBS three times, they were fixed with 4% paraformaldehyde for 30 min and rinsed with distilled water three times for 5 min each. Triton X-100 (0.1%) was allowed to permeate the cells for 30 min and then they were washed three times with distilled water for 5 min each time. 2% Goat serum  was used for blocking for 1 h. The primary antibody  was added to cover the bottom surface, and it was incubated at 4 °C overnight. After removing the primary antibody, the cells were washed with distilled water and incubated with the fluorescent secondary antibody  at room temperature in the dark for 1 h. After absorbing the secondary antibody, 250 μL DAPI solution  was added and observed under a fluorescence microscope.The adipogenic differentiation of SMSCs was based on our previous study, and improved adipogenic differentiation was induced at a cell density of 90%. First, the cells were cultured in DMEM-F12 medium  for 2 days (step 1). The cells were then cultured in medium containing insulin (850 nmol) and rosiglitazone (1 µmol/L) for another 2 days (Step 2). Steps 1 and 2 were recycled once, and the ordinary maintenance medium was replaced for culture cells for 2–4 days, and the solution was changed once every 2 days. The morphological changes and intracellular fat deposition of adipogenic differentiation cells were observed by Oil Red O staining solution . The cells were harvested at the undifferentiated (P), early (2 days), middle (4 days), and late (10 days) stages of lipogenic differentiation for RNA extraction. Three duplicate cell samples were collected from each time point.ftp://ftp.ncbi.nlm.nih.gov/genomes/Sus_scrofa, accessed on 13 April 2021). The number of perfect clean reads corresponding to each gene was calculated and normalized to the number of reads of Transcripts Per Million (TPM).Briefly, total RNA was extracted from different groups of cell samples using the Trizol reagent . After determining for total RNA integrity and DNA contamination, we prepared the final cDNA library in accordance with a strand-specific library preparation by dUTP method. Then, the Illumina HISeq 2500  platform was used to sequence the paired-end library. After removing low-quality reads, the clean reads were subjected to quality assessment. These included the classification of total and distinct reads and the assessment of their percentage in the library, analysis of saturation of the library, and correlation analysis of biological replicates. TopHat2 software was used to map the clean data to the porcine reference genome .The short time-series expression miner (STEM) clustering algorithm  was usedp adjust < 0.05 or q value < 0.05 were identified as statistically significant.Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and REACTOME were used to understand the regulatory functions of mRNAs in the 11 significantly enriched profiles, as previously described . The enrhttp://string-db.org, accessed on 28 April 2022) is a database for searching interactions that can show the direct physical interaction and the indirect functional correlation between proteins. We employed STRING database 25 to identify pairwise relationships among the DE mRNAs with up-regulated expressions in profile 1, 2, and 11 and the DE mRNAs with down-regulated expressions in profile 4 and 7, respectively, and then a protein–protein interaction (PPI) network was built.The Search Tool for the Retrieval of Interacting Genes (STRING)  was used to predict potential target genes of lncRNAs.Two regulation models (cis-regulation and trans-regulation) were used to analyze the target genes of DE lncRNAs. For cis-regulation analysis, we predicted the target coding genes within 10 kb upstream and downstream of lncRNA; for the trans-regulation analysis, the target genes were predicted based on the free energy that is needed to form secondary structures between lncRNAs and mRNA sequences. RNAplex software  in triplicate using the SYBR Premix Ex Taq II . The mean expression values were calculated relative to the mean expression level of the housekeeping gene GAPDH. All of the reactions were prepared while using three replicates and the expression levels of genes were expressed as fold change using the 2p < 0.05 was considered significant.The data were processed while using R, Microsoft Excel, and GraphPad Prism 8.5. Differences of gene expression among groups were analyzed using the Kruskal–Wallis test, and a false discovery rate (FDR) < 0.05 was considered to be statistically significant. In the case of qRT-PCR validation of the transcriptome, statistical analysis was carried out using GraphPad Prism 8.5 Software using one-way ANOVA. More than 90% of the cells obtained from the muscle of piglets were Pax7 positive, which is consistent with the identity of skeletal muscle satellite cells . These cImportantly, SMSCs also showed strong trans-differentiation abilities with the stimulation of adipogenic induction. A large number of lipid droplets began to be generated in the cells on the 2nd day of lipogenesis induction and formed large lipid droplets at the 10th day, which is shown by oil-red staining .The cells at the pre-differentiation , early differentiation , middle differentiation , and later differentiation  stages were collected and sequenced using the Illumina HiSeq 2000 sequencing platform. The average value for each sample was approximately 10.02 G. The Q20 of the sequencing data was more than 98%, and that of Q30 was more than 95%. After quality control of the original data and the removal of the reads compared to ribosomal RNA, an average of 95% of the clean reads was compared to the reference genome, of which the unique clean reads compared to the genome were more than 88% . These dA total of 6891 lncRNAs (6731 known genes and 160 new genes) were identified by sequencing, corresponding to 16,421 transcripts, with an average of 2.38 transcripts per gene. A total of 21,478 mRNA genes  were identified, corresponding to 57,592 transcripts, with an average of 2.68 transcripts per gene.As shown in The top 20 most highly expressed lncRNA genes in each group of cells are shown in Differences in the expressions of lncRNAs and mRNAs between the four groups of cells were analyzed using the Kruskal–Wallis test. A total of 1005 lncRNAs showed significant changes on the expression at four stages (FDR < 0.05). Similarly, 7671 mRNA were significantly differentially expressed with time (FDR < 0.05). The clustered heatmaps in padjust < 0.05. Modules with the same colour in We normalised the sequencing data to the samples in group P and then identified the temporal gene expression profiles using STEM. As shown in p < 0.05). Profiles 1 (133 genes), 2 (297 genes), 3 (482 genes), 10 (213 genes), and 11 (111 genes) showed that DE mRNAs were upregulated during lipogenic differentiation. Meanwhile, profiles 4 (258 genes), 5 (152 genes), 6 (191 genes), and 7 (1485 genes) showed that DE mRNAs were downregulated during the differentiation. Profile 8 revealed 518 DE genes with low expression at stage E, and profile 9 showed 425 DE mRNAs with upregulated expression in the E and M groups of cells. Notably, profile 1 and profile 5 showed that 133 and 155 DE mRNAs were continuously upregulated or downregulated at all four time points, respectively. These temporal expression analysis results provided effective information for further identifying the core factors driving adipogenesis in muscle cells.As shown in GO functional enrichment analysis was performed for the DE mRNAs in the 11 temporal gene expression profiles. The results were shown in In Next, the KEGG analysis showed metabolic pathway enrichment for the altered genes that revealed the changes in cell fate and cellular metabolism mediated by DE mRNAs. The top three pathways enriched by the upregulated mRNAs in profiles 1, 2, 3, 10, and 11 were PPAR signalling pathway, fatty acid elongation, regulation of lipolysis in adipocyte A. The geAs a free, open-source, and peer-reviewed knowledge-base of biomolecular pathway analysis methods, REACTOME provide bioinformatics tools for visualization, interpretation, and analysis of pathway knowledge to support basic research, genome analysis, modelling, and systems biology . REACTOMBy conducting GO, KEGG, and REACTOME enrichment analyses, lipid metabolism and cell cycle-related DE mRNAs were selected for protein–protein interaction (PPI) network analysis. The network was constructed using the STRING database. Out of the 7671 DE mRNAs, 1884 were regulated by 193 in the 1005 DE lncRNAs that were screened by target predication analysis. There were a total of 19,860 targeting relationships between DE lncRNAs and DE RNAs including 21 cis-regulations and 19,839 trans-regulations. Furthermore, the DE 1884 mRNAs were analysed by GO and KEGG to ascertain their functions and related pathways . The topAmong the mRNAs upregulated during the adipogenic differentiation of SMSCs, we particularly noticed fatty acid binding protein (FABP) family genes. Previous studies have reported that FABP genes  participate in the regulation of lipid synthesis and adipocyte differentiation , and theWe also screened DE lncRNAs for targeting the three FABP genes especially. 11, 13, and 15, lncRNAs were obtained by predicting the potentially regulated FABP3, FABP4, and FABP5, respectively . Among tNext, to validate RNA-seq results, three FABP genes and six other DE lncRNAs were selected for qRT-PCR. As shown in LncRNA is a type of non-coding RNA molecule with a transcript length of more than 200 nt. It widely exists in various tissues of organisms ,24,25. WAs muscle-derived pluripotent stem cells, SMSCs and muscle precursor cells can produce and store a large number of lipids and differentiate eventually into fat cells under special circumstances . This adp < 0.05, by STEM analysis. In different clustered profiles, lncRNAs and mRNAs showed significant increases or decreases in expression at different time stages. These altered genes play pivotal regulatory roles at the differential stages of trans-differentiation.During SMSC adipogenesis, the timing of gene expression is important, and gene expression at a single time point is insufficient to fully understand the dynamic characteristics of gene expression in the adipogenic differentiation of SMSCs. Therefore, in this study, we have analysed and compared the expression profiles of lncRNAs and mRNAs at different time points during adipogenesis using STEM temporal expression analysis to identify genes that are important for the regulation of trans-differentiation. We identified 1005 DE lncRNAs and 7671 DE mRNAs with altered expression during lipogenesis, in which DE 566 lncRNAs and 4265 DE mRNAs were clustered into 5 and 11 profiles, respectively, with The functions and related pathways of the altered genes were analyzed and contoured by bioinformatics tools, and we obtained some important information about the changes in genetic programming for shifting cell fate and cellular metabolism. The up-regulated mRNAs, especially hundreds of genes in profile 1, 2, and 11, were remarkably related to the control of lipid metabolism and fat cell differentiation, such as ABHD4, ACSL3, PIK3CB, and ACOX2. The down-regulated mRNAs in profile 4 and 7 were mainly enriched in the regulation for cell cycle, nucleic acid metabolism, and cell proliferation, such as FEN1, BARD1, CCNE1, RAD51C, and TIMELESS. Clearly, these genes may be responsible for changes in muscle cell fate primarily. By subsequent target gene prediction analysis, more than 20,000 cis- and trans-targeting relationships were found between DE lncRNAs and DE mRNAs. This also reflects the complexity of molecular networks involved in the control of trans-differentiation.In addition, we also analyzed the overall interaction between DE lncRNAs and DE mRNAs. Total 19,860 targeting relationships between DE lncRNAs and DE RNAs were found by target predication and the most significant changes on cell cycle and genetic information processing that are mediated by the lncRNA-mRNA interaction. This certainly reveals the important and complex roles of lncRNAs in trans-differentiation. Moreover, it is worth noting that the regulation of lncRNA showed obvious selection tendencies.FABP3, FABP4, and FABP5 were continuously expressed at high levels in the M and L stages of trans-differentiation. Additionally, the expression of FABP4 was higher than that of other FABP genes. This suggests that FABP genes, especially FABP4, can induce intracellular lipid droplet formation and accumulation at the late stages of SMSCs trans-differentiation. Meanwhile, we found that dozens of DE lncRNAs targeted three FABP genes through target gene prediction. These results provide useful information for the subsequent functional verification of candidate-regulated lncRNAs in muscle cells.Among the upregulated genes that regulate lipogenesis, we noted the FABP family genes, which are key regulators for lipids synthesis and fat cell differentiation. The FABP family was first found in rat intestinal mucosa in 1972, including FABP3, FABP4, FABP5, and FABP7 . ProteinIn conclusion, we gained insight into the time-series expression profiles of lncRNA and mRNA during the adipogenic transformation of porcine SMSCs through RNA sequencing. Thousands of lncRNAs and mRNAs showed significant changes on expressions during trans-differentiation. The key gene clusters that function for driving adipogenesis increased, and the genes in regulating cell cycle and DNA metabolism decreased, which was identified by using bioinformatics tools. In addition, we found that FABPs family genes, the key regulators of lipid synthesis and fat cell differentiation, have significant increases in expression levels during trans-differentiation, and these genes were controlled by dozens of lncRNAs. These findings provide a new set of potential drivers for muscle cell adipogenesis and also suggest that more attention should be directed to the evaluation of genetic programming and molecular interactions of trans-differentiation at different time stages.


Background: Breast carcinoma is well recognized to be having the highest global occurrence rate among all cancers, being the leading cause of cancer mortality in females. The aim of this study was to elucidate breast cancer at the genomic and transcriptomic levels in different subtypes so that we can develop more personalized treatments and precision medicine to obtain better outcomes.Method: In this study, an expression profiling dataset downloaded from the Gene Expression Omnibus database, GSE45827, was re-analyzed to compare the expression profiles of breast cancer samples in the different subtypes. Using the GEO2R tool, different expression genes were identified. Using the STRING online tool, the protein–protein interaction networks were conducted. Using the Cytoscape software, we found modules, seed genes, and hub genes and performed pathway enrichment analysis. The Kaplan–Meier plotter was used to analyze the overall survival. MicroRNAs and transcription factors targeted different expression genes and were predicted by the Enrichr web server.Result: The analysis of these elements implied that the carcinogenesis and development of triple-negative breast cancer were the most important and complicated in breast carcinoma, occupying the most different expression genes, modules, seed genes, hub genes, and the most complex protein–protein interaction network and signal pathway. In addition, the luminal A subtype might occur in a completely different way from the other three subtypes as the pathways enriched in the luminal A subtype did not overlap with the others. We identified 16 hub genes that were related to good prognosis in triple-negative breast cancer. Moreover, SRSF1 was negatively correlated with overall survival in the Her2 subtype, while in the luminal A subtype, it showed the opposite relationship. Also, in the luminal B subtype, CCNB1 and KIF23 were associated with poor prognosis. Furthermore, new transcription factors and microRNAs were introduced to breast cancer which would shed light upon breast cancer in a new way and provide a novel therapeutic strategy.Conclusion: We preliminarily delved into the potentially comprehensive molecular mechanisms of breast cancer by creating a holistic view at the genomic and transcriptomic levels in different subtypes using computational tools. We also introduced new prognosis-related genes and novel therapeutic strategies and cast new light upon breast cancer. Breast carcinoma is well recognized to be having the highest global occurrence rate among all types of cancers, being the leading cause of cancer mortality in females worldwide . In the Currently, the clinical approach to treating breast cancer has been mainly composed of surgery, radiotherapy, chemotherapy, endocrine treatment, and targeted therapy . AlthougPrevious studies have mostly analyzed the molecular mechanisms by comparing the difference between tumor and normal tissues of the breast or focused only on one subtype of breast cancer . ActuallSRSF1, BUB1B, KIF23, HNRNPF, and ELAVL1 and obtained an exact result in our study. We re-analyzed the dataset deposited by In this study, we preliminarily delved into the potentially comprehensive molecular mechanisms of breast cancer by creating a holistic view at the genomic and transcriptomic level in four different subtypes using computational tools. To the best of our knowledge, this is the first time such a systematic biological study was performed on breast cancer according to its subtypes and at the genomic and transcriptomic level. We also first explored genes such as http://www.ncbi.nlm.nih.gov/geo/geo2r/) is an online tool that can be used to screen DEGs across different groups. Using GEO2R of GEO, groups  were compared to identify DEGs of the four subtypes. Benjamini–Hochberg false discovery was used for p-value adjustment. Genes were declared as DEGs when |lgFC|≥3 and the adjusted p-value (adj.p) < 0.01. The heat map was performed by SangerBox online tool version 3.0 (http://www.sangerbox.com/tool), and the volcano plot was drawn by GraphPad Prism for Windows .A microarray dataset with accession number “GSE45827” from the GEO database was downloaded . This dahttps://string-db.org/)  . DEGs wedb.org/) . CytoHubdb.org/) . The crip-value, and signal pathways with adj.p ≤ 0.05 were recognized.Genes clustered with MCODE were analyzed by the Cytoscape ClueGO plugin , choosing Reactome and KEGG databases to retrieve pathways . Bonferrhttps://kmplot.com/analysis/), an online database, was used to analyze the overall survival (OS) with hazard ratios (HRs), 95% confidence intervals (95% CIs), and logrank p-value. The JetSet best probe set was selected as gene probes. During the prognosis analysis, patients were split into two groups in accord with the auto-select best cutoff. The logrank p-value <0.05 was considered to show a statistical significance. The forest plot was drawn using Xiantao scholar (https://www.xiantao.love/), another online platform for data analysis.The Kaplan–Meier plotter mRNA breast cancer database  was used to evaluate the functions of prognosis-related hub genes according to several bioinformatics methods, such as co-expression, physical interaction, prediction, co-localization, and shared protein domains and pathways  . MiRNAs The microarray dataset “GSE45827” which includes primary invasive breast carcinoma  and 11 normal tissues has been analyzed. Using the GEO2R tool, we found 1170, 1058, 733, and 854 DEGs which are significantly variably expressed between TNBC vs. normal, Her2 vs normal, luminal A vs. normal, and luminal B vs. normal, respectively. Most of the DEGs overlapped between the four molecular subtypes . The heaMCODE identified groups of genes with a similar function, and each module has the most effective genes, called seed genes. Similarly, the TNBC subtype has the most modules and seed genes. Interestingly, these sets of seed genes in different subtypes exhibited few overlaps  are in PPI networks TNBC vs. normal, Her2 vs. normal, luminal A vs. normal, and luminal B vs. normal, respectively . The topoverlaps . Network subtype . The hubvia a MAD2 inhibitory signal, and resolution of sister chromatid cohesion. Furthermore, there were also some pathways that are unique to specific subtypes, for example, the ERBB4 pathway in TNBC and the NOTCH4 pathway in luminal B.The pathway enrichment analysis was executed based on genes identified by MCODE. We reached 51, 25, 10, and 15 pathways by performing the pathway enrichment analysis from 163, 112, 53, and 88 genes, respectively . The genCDC6, NDC80, BUB1B, FOXM1, NUF2, MCM4, CDC20, BUB1, MCM2, CCNB2, ASPM, PRC1, PLK1, HNRNPF, CCNA2, and KIF2C were related to good prognosis in TNBC  in the Her2 subtype, while in the luminal A subtype, it showed the opposite relationship , chromosome segregation (FDR=1.68e-24), microtubule cytoskeleton organization involved in mitosis (FDR=8.79e-20), spindle (FDR=8.94e-18), mitotic cell cycle checkpoint (FDR=9.00e-17), negative regulation of the mitotic cell cycle (FDR=3.42e-14), and metaphase/anaphase transition of the mitotic cell cycle (FDR=3.00e-17) .The miRNAs and TFs, as important regulators of DEGs, were predicted using the Enrichr web server. It is worth noting that the luminal A subtype had the most TFs, while it had the least hub genes among the four molecular subtypes. Seven TFs exerted their function in all four subtypes . TFs thaIn the present study, bioinformatic approaches were carried out to show the DEGs, modules, seed genes, PPI, and hub genes in each subtype . The topological clusters which have high-density regions in the network, also called modules, find in MCODE-identified groups of genes with a similar function. Genes in the highly interconnected subnetwork modules are expected to be involved in the same pathways or in roles with related biological functions. Each module has a most effective gene which has high centrality, named seed genes. The nodes in the PPI network represent genes, while the edges indicate both functional and physical protein associations. Nodes with high degree, betweenness, closeness, and MCC are significant for the network and are called hub genes which can serve as targets. The analysis of these elements implied that the carcinogenesis and development of TNBC were the most important and complicated processes in breast carcinoma, occupying the most DEGs, modules, seed genes, hub genes, and the most complex PPI network.RACGAP1 is supposed to be not only a strong poor prognostic marker in luminal-like breast cancer but might also be a predictor of response to treatment with tamoxifen and adjuvant chemotherapy (CDK1 and CCNA2 have been previously revealed to be considerably higher in breast cancer tissues than those in normal tissues, and these genes lead to breast cancer development and are related to poor prognosis (CDC20 was related to worse prognosis only in the TNBC subtype, and we think that the different datasets that we analyzed result in the inconsistency. Other genes such as BUB1, NUF2, CDC20, ASPM, KIF2C, and PRC1 have biological relevance to breast cancer progression, and PLK1, NDC80, and CCNB2 only to TNBC progression, and these genes predict worse prognosis (AURKB, CDC6, and ECT2 suggest a poor prognosis for breast cancer (FOXM1 to impede advanced relapse and treat endocrine resistance (SRSF1, BUB1B, KIF23, and HNRNPF also play an important role in breast cancer, but so far, there is few reports on the association between these genes and the treatment or prognosis of breast cancer in the previous studies (RPA1 and CDC5L in TNBC, but their mechanism remains to be discovered with exploratory studies.The role of some of those hub genes that we identified in our study has been verified in breast cancer, such as UBE2C whose overexpression plays a critical role in the incidence and development of breast cancer, and such a therapeutic strategy that combines palbociclib with tamoxifen might be promising in patients with HR+/HER2-breast cancer overexpressing UBE2C . Otherwiotherapy . In addirognosis ; howeverrognosis ; in contrognosis ; likewist cancer , whereassistance . In addi studies ; inspiriIn terms of the pathway enrichment analysis, the nodes represent the pathway, while the edges mean that there is a functional similarity between the two pathways. As was consistent with the abovementioned analysis, the pathway enrichment analysis also showed that TNBC is the most complex subtype in breast cancer. In addition, there is an overlap of the pathways in the TNBC, Her2, and luminal B subtypes, while the pathways enriched in the luminal A subtype were unique. This suggested that the luminal A subtype occurs in a completely different way. If so, the treatment of the luminal A subtype should be different from the other three subtypes, especially the postsurgical adjuvant therapy, or in other words, there should be a specific treatment strategy to be formulated just for the luminal A subtype compared with the others. Of course, pathways about DNA replication and DNA repair are only included in TNBC, which means studies focused on these pathways can help shed light upon TNBC and develop treatments that are only indicated for TNBC. Also, for the same reason, patients of TNBC, Her2, and luminal B subtypes may benefit from studies on mitosis in the future. Otherwise, in addition to pathways related to DNA replication, DNA repair, and mitosis, the ERBB4 signal was only enriched in TNBC, while the NOTCH4 signal was only in the Her2 subtype, and chemokine and kinesins in both of them, compared with the luminal B subtype. ERBB4, a member of the human epidermal growth factor receptor family, has been previously reported to be a valuable prognostic marker when united with the pathologic stage in TNBC and may be helpful in predicting the therapeutic efficacy for TNBC . HoweverAccording to the result of TF analysis, although most of the TFs have been reported to be associated with breast cancer, there were still TFs such as EOMES, POU3F2, NR3C1, and RUNX1 that were less reported in breast cancer. Studies focused on these TFs may shed light upon breast cancer in a new way and provide a novel therapeutic strategy. For microRNA analysis, the miR-875 family has been reported to serve as a marker for detection and prognosis in breast cancer . Others,The present research also includes some limitations. First, our study only looks at data in one dataset, and the GPL platform used in the dataset is now not universally applicable. Second, most of the clinicopathologic features are not included in the dataset, and we cannot rule out that these factors might have influenced our results. Third, we just predicted the TFs and miRNAs through DEGs and did not calculate the relationship between them or carry out experiments to validate it, but this is just what we are doing now.In this study, we preliminarily delved into the potentially comprehensive molecular mechanisms of breast cancer by creating a holistic view at the genomic and transcriptomic levels in different subtypes using computational tools. Our study introduced a network of genes, pathways, prognosis-related genes, TFs, and miRNAs which are possibly associated with a different subtype of breast cancer, and they can be good candidates for further analysis and provide novel approaches to treat breast cancer.


This work proposes an interpretable radiomics approach to differentiate between malignant and benign focal liver lesions (FLLs) on contrast-enhanced ultrasound (CEUS). Although CEUS has shown promise for differential FLLs diagnosis, current clinical assessment is performed only by qualitative analysis of the contrast enhancement patterns. Quantitative analysis is often hampered by the unavoidable presence of motion artifacts and by the complex, spatiotemporal nature of liver contrast enhancement, consisting of multiple, overlapping vascular phases. To fully exploit the wealth of information in CEUS, while coping with these challenges, here we propose combining features extracted by the temporal and spatiotemporal analysis in the arterial phase enhancement with spatial features extracted by texture analysis at different time points. Using the extracted features as input, several machine learning classifiers are optimized to achieve semiautomatic FLLs characterization, for which there is no need for motion compensation and the only manual input required is the location of a suspicious lesion. Clinical validation on 87 FLLs from 72 patients at risk for hepatocellular carcinoma (HCC) showed promising performance, achieving a balanced accuracy of 0.84 in the distinction between benign and malignant lesions. Analysis of feature relevance demonstrates that a combination of spatiotemporal and texture features is needed to achieve the best performance. Interpretation of the most relevant features suggests that aspects related to microvascular perfusion and the microvascular architecture, together with the spatial enhancement characteristics at wash-in and peak enhancement, are important to aid the accurate characterization of FLLs. I.Liver cancer is the third leading cause of cancer-related death globally, accounting for about 906 000 new cases and 830 000 deaths worldwide . The incImaging plays an important role in the diagnosis and management of liver cancer –11]. As. As11]. Differently from CT and MRI, contrast agents used for CEUS are purely intravascular and allow for real-time assessment of the vascular enhancement patterns without the use of ionizing radiation at higher spatial and temporal resolution –11]. Mo. Mo11]. Current assessment of FLLs with CEUS is performed by purely qualitative evaluation of the vascular enhancement patterns, with emphasis on presence, type, and degree of arterial phase enhancement of the FLL, as well as presence, timing, and degree of contrast wash-out , 11], , .Besides requiring expertise in both acquisition and interpretation , 13], c, c13], cA number of methods have been proposed to quantitatively analyze CEUS images for computer-aided FLLs characterization , 20]–2–220]–2. Initial0–2. Init0–2. Initet al. , ,  and the Recently, a method based on convolutional neural networks was also proposed to achieve end-to-end lesion classification, avoiding the need for hand-engineered feature extraction . The metOne major limitation of previous studies is the lack of analysis and interpretation of the importance of each feature in producing the output prediction, which could offer useful clinical insights and provide a better understanding of the decision process, possibly allowing for debugging and improvements at all steps of the acquisition and processing chain. Moreover, most of the methods require a motion compensation step , 20]–2–220]–[20–[222], 0–20–22, 25]. M. M25]. M. M. M25]In this work, we propose an interpretable machine learning approach to differentiate benign and malignant FLLs on CEUS, requiring minimal manual input and avoiding the need for motion compensation. Our approach is based on the extraction of features that are relatively robust to motion. Similar to previous work, this is achieved by spatial and spatiotemporal analysis of the CEUS loops; however, our method differs from existing work in a number of aspects. First, we intentionally avoid the use of model fitting for TIC analysis, as the presence of motion and the overlap between the arterial and portal phase generally make model fitting unreliable. Second, we propose for the first time in the liver a set of CEUS-based spatiotemporal features to capture simultaneously TIC characteristics in time and space , 30]. T. T30]. TII.A.This HIPAA-compliant study was approved by the Institutional Review Board of all participating institutions. Informed consent was obtained from each patient prior to data collection. All data were deidentified and pseudonymized prior to analysis.The study included 72 patients at risk of HCC who were examined for evaluation of FLLs at the Thomas Jefferson University Hospital  or the Stanford University Medical Center . CEUS was performed by injection of a bolus of Lumason, according to the ACR CEUS LI-RADS Working Group . RecordiB.1)2 to 8.24 × 8.24 cm2, depending on the actual lesion size in centimeters.For each lesion, a certified radiologist indicated the location of the lesion on one frame of the US cine, where the lesion was well visualized, as shown, for example, in 2)CEUS is an established modality for assessment of microvascular perfusion , 17]. F. F17]. FTo go beyond temporal TIC analysis, we also performed a spatiotemporal similarity analysis by comparing each pixel TIC with neighboring TICs in a ring kernel , 30], w, w30], w3 in volume , after which the formation of a vascular network is required to permit further tumor growth, a concept known as angiogenesis switch . L. L3 in vs switch . As descs switch , prior ts switch , and aga3)mean) was calculated on the CEUS loop by averaging over all pixels in the manually segmented lesion at the reference frame. A straight line was fit to the TICmean wash-in, in the interval defined by the time points at which the TICmean intensity is between 5% and 50% of the intensity at peak, using only valid frames. Similarly, a straight line was fit to the wash-out in the second half of the time window defined from the appearance time to the end of the recording N = 8), wash-in texture features (N = 43), peak texture features (N = 43), and wash-out texture features (N = 43) , , , 24], , . By our Comparing the obtained performance with similar methods for computer-aided diagnosis of FLLs, accuracies ranging from 85.8% to 91.8% have been reported –26], , 6, 22], . In addiCurrent clinical assessment of CEUS by LI-RADS assigns each lesion to the following risk groups: benign (LR-1), probably benign (LR-2), intermediate risk for HCC (LR-3), probably HCC (LR-4), definite HCC (LR-5), and probably of definite malignant, not HCC-specific (LR-M). While LR-4 and LR-5 have shown to be highly predictive for HCC, the diagnosis of LR-3 and LR-4 lesions remains challenging. In fact, in our dataset, about 40% of LR-3 and 21% of LR-4 lesions were actually benign see . SimilarOne of the main limitations of this study resides in the datasets, which was relatively small, including only 72 patients for a total of 87 FLLs, and more importantly very imbalanced, consisting of only 13 benign lesions compared to 74 malignant lesions. Of these, 71 resulted to be HCC and 3 only ICC; thus, any influence on the performance of the different CEUS enhancement patterns observed for different lesion types cannot be evaluated in this study. In addition, the ratio of malignant to benign cases does not reflect the actual prevalence of malignant cancers in the American population, which is estimated at 50%–57% , 56]. H. H56]. Hk-fold cross validation, as this method has been shown to be robust toward overfitting while providing a good estimation of the diagnostic performance . I. I58]. IV.An interpretable radiomics approach was proposed for characterization of FLLs by CEUS by combining features extracted by spatiotemporal analysis and features extracted by texture analysis at different fiducial time points. The proposed method requires minimal manual input, with no need for motion compensation or the use of dedicated equipment . Promising results were obtained on a multicenter clinical validation, including 72 patients and 84 FLLs, achieving bACC of 84%. The analysis of feature importance shows that aspects related to perfusion (peak time and wash-in time), the microvascular architecture , and the spatial characteristics of contrast enhancement at wash-in  and peak (GLCM Energy) are particularly relevant to aid FLLs diagnosis.supp1-3161719


Narcolepsy patients were observed improvements in their academic performance during the COVID-19 home quarantine. Therefore, we aim to investigate the influence of sleep behavioral changes on school/work performance in narcolepsy patients during the home quarantine.Patients admitted to Shandong Provincial Qianfoshan Hospital from Jan 1, 2017 to Jan 1, 2021 who were diagnosed with narcolepsy were studied by online questionnaires in two different periods (during and 1 year after the COVID-19 home quarantine), including five aspects: (1) changes in school/work performance ; (2) daytime functions; (3) clinical symptoms; (4) psychological moods; (5) medication situations.P < 0.001), longer total sleep time , better sleep quality , and lower anxiety level (P = 0.005). Their school/work performance improved parallelly [with better percentile ranking (P = 0.001) and lower SDS1 scores (P = 0.002)]. The results of multiple linear stepwise regression analysis showed a linear regression relationship between TST [efficient (95%) −7.356 (−13.570 to 1.143)], SDS1 score  and the percentile ranking after adjusting for potential effects. Both the improvements of sleep behavior and school/work performance disappeared after the end of COVID-19 home quarantine. No similar fluctuation was found in the control group.A total of 46 narcolepsy patients 34 (73.9%) narcolepsy type 1, 12 (26.1%) narcolepsy type 2 with average age of 20.76 ± 8.99 years and an equal number of age and gender matched control subjects were enrolled. During the COVID-19 home quarantine, the narcolepsy patients were found that they altered sleep patterns, including later get up time (Changes in sleep pattern during the COVID-19 home quarantine, such as longer sleep time and later wake-up time, can reduce the degree of daytime sleepiness and increase the degree of daytime wakefulness of narcolepsy patients, which can alleviate the impact of the disease on school/work performance. However, the concentration of hypocretin in CSF of narcolepsy type 2 is not low, and the positive rate of HLA-DQB1*0602 in the blood is significantly lower than that of narcolepsy type 1  With other diseases which cause sleepiness at the same time, such as sleep apnea respiratory syndrome, sleep deprivation syndrome and acute cerebral infarction, according to the ICSD-3; (2) Discharged patients who were unable to be contacted; (3) Patients with key data missing.via the class of mock exams which was information from school registers. The formula is as follows:*(Percentile ranking) = class rankings/the class size × 100%. The “work performance” of workers is expressed by self-evaluation of the impact of quarantine on work (SDS1).We used the percentage of the number of people who are ahead of them in class to evaluate the school performance of these students. This index was calculated All the data were obtained by telephone interviews. The flowchart is as follows . The queT-tests and the nonparametric Wilcoxon signed-rank test were used to compare the same groups during and after the COVID-19 home quarantine, independent sample T-tests and Mann–Whitney U-tests were used for comparisons between different groups during and after the home quarantine. Correlation analysis was conducted for differences in the changes in academic performance of the two groups and various sleep behavior indicators. Pearson correlation analysis was used for the data in line with a normal distribution. The Spearman rank test was used for data that did not conform to a normal distribution. Finally, linear regression analysis of the percentile ranking and various sleep behavior indicators in the narcolepsy group were performed. Univariate linear regression analysis of percentile ranking and changes in sleep patterns in the patients and healthy controls respectively. We performed further multiple linear stepwise regression analysis between the percentile ranking and SDS, TST, PSQI score, number of naps, PHQ score in patients after adjusting some potential effects, including age and BMI, grade, different medicine used. Because of the multiple comparisons, FDR (Benjamini–Hochberg procedure) was used to correct P values and control false positive rates.IBM SPSS Statistics, version 26  was used for statistical analysis. Categorical variables were expressed by percentage (%). McNemar's Chi-squared test was used to compare the same groups during and after the COVID-19 home quarantine. Pearson's chi-square test was used to compare different groups during and after the home quarantine. Continuous variables data were represented as the mean ± SD. Paired P = 0.461). There were 30 males (65.2%) and 16 females (34.8%), with a male to female ratio of 1.88 to 1. There was a significant difference in body mass index  between narcolepsy patients and healthy controls (P < 0.001). Among patients, 35 (76.1%) had received medication, at the same time, different adverse reactions were reported. Some patients voluntarily stopped pharmaceutical treatment before or during the home quarantine, and only 10 (21.7%) insisted on the pharmaceutical treatment regularly during the COVID-19 home quarantine. Further baseline characteristics were presented in A total of 46 narcolepsy patients and 46 healthy controls were included in our study , faster sleep latency , more anxiety , more depression , and relatively lower academic performance (the difference was not significant).Compared to healthy controls, narcolepsy patients had more severe EDS , longer TST , longer sleep latency  and longer nap duration , better sleep quality , and lower anxiety level . Meanwhile, an improved class ranking  and a lower effect of illness on self-rated school/work  were observed in students. In addition, the number of daytime naps increased, EDS decreased (ESS score), and clinical symptoms of narcolepsy decreased (UNS score). Meanwhile, the TST of workers was also prolonged , and the sleep quality was better . However, there was no significant difference in daytime naps, EDS, UNS, anxiety and depression in workers. Details was presented in During the home quarantine, students had the later get up time , experienced more severe EDS , delayed get up time , longer TST , and worse sleep quality . At the same time, we observed that during the pandemic period, their academic performance was lower, latency for falling asleep was prolonged, while anxiety and depression were aggravated, though there were no significant differences in these aspects.As shown in r = 0.422, P = 0.012), TST , sleep quality , number of daily naps , and degree of depression . After the home quarantine, the percentile ranking of narcolepsy patients was correlated with the degree of SDS1 , bedtime , and number of daily naps . There was no significant correlation between the percentile ranking of healthy controls during the home quarantine and any sleep pattern indices. After the home quarantine, it was correlated with sleep latency  and TST . As show in r = 6.811; 95%CI: 2.351, 11.271; P = 0.04), better sleep quality , decreased number of naps  and prolonged TST .As shown in P = 0.004], TST , PSQI score , number of naps , PHQ score  in patients. After FDR corrected P values and controlled the false positive rate, only SDS1 had a linear regression relationship with the percentile ranking. We performed further multiple linear stepwise regression analysis, there was a linear regression relationship between the percentile ranking and TST  after categorization for potential effects. As shown in There was a univariate linear regression relationship between tthe percentile ranking and SDS [6.811 (2.351–11.271), As a lifelong disease, narcolepsy was reported to destroy the stability of the cortex and induced a decline in attention, reaction, executive ability and cognitive function –22. As aEarly observations consistently indicated that the fragmentation of nocturnal sleep in patients with narcolepsy affects the quality and brings pain to patients . InocentDuring the home quarantine, compared with the control group, the sleep latency of patients was significantly shorter. At the same time, their degree of daytime sleepiness was heavier and more prone to emotional changes, such as depression. The apparent reduction of sleep latency and the disorder of sleep rhythm are the characteristics of narcolepsy patients. To further study their sleep patterns during quarantine, we studied the different sleep patterns of narcolepsy patients and healthy controls during and after the COVID-19 home quarantine. We divided the patient group into students who took online courses at home and people who worked from home. During quarantine, the sleep latency of narcolepsy students increased by 2.5 ± 3.9 min, the time to get up was delayed by nearly 1 h, the TST increased by 1.5 h, and their sleep quality improved. However, narcolepsy workers only showed an increase in TST (0.7 ± 0.5 h) and an improvement in sleep quality during the home quarantine. Consistent with Wu's study , no signvia the class of mock exams which was information from school registers. The percentile ranking of students during the home quarantine was better than that after the quarantine, which was related to the influence of the decreased degree of illness on learning (r = 0.422), the increase of TST (r = −0.359), the improvement of sleep quality (r = 0.488), the decrease of daily naps number (r = 0.361), and the decrease of depression (r = 0.390). It is suggested that the “behavioral therapy” of home quarantine can promote the change of sleep pattern to some extent, thereby improving the study performance of narcolepsy patients. Contrary to narcolepsy patients, the school/work performance of healthy controls were believed to have been more affected during the home quarantine, and their grade percentage decreased, however, there was no significant correlation between the percentile ranking and various sleep pattern indices, which was considered to be related to a variety of factors, among which the increased anxiety and depression of healthy people affected academic performance, which was consistent with the literature report .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Enterobacterales (CRE) in the Emirate of Abu Dhabi is dominated by highly resistant Klebsiella pneumoniae clones ST14, ST231, and CC147, respectively. In the absence of continuous, molecular typing-based surveillance, it remained unknown whether they lately emerged and rapidly became dominant, or they had been present from the early years of the endemic. Therefore, antibiotic resistance, the presence of carbapenemase and 16S methylase genes, and the sequence types of CRE strains collected between 2009 and 2015 were compared with those collected between 2018 and 2019. It was found that members of these three clones, particularly those of the most prevalent ST14, started dominating already in the very early years of the CRE outbreak. Furthermore, while severely impacting the overall antibiotic resistance patterns, the effect of these clones was not exclusive: for example, increasing trends of colistin or decreasing rates of tigecycline resistance were also observed among nonclonal isolates. The gradually increasing prevalence of few major, currently dominating clones raises the possibility that timely, systematic, molecular typing-based surveillance could have provided tools to public health authorities for an early interference with the escalation of the local CRE epidemic.Recent studies showed that the current endemic of carbapenem-resistant  Enterobacterales resistant to carbapenems due to the production of various carbapenemases had already been sporadically encountered during the last decade of the 20th century, but the threat they represent became global toward the end of the first decade of the 21st century [Clinical isolates of  century , most of century ,16,17,18Enterobacterales (CRE). Consequently, the emergence and spread of clones of the unfolding CRE epidemic have not been closely followed. Only as recently as in 2018, a relatively small-scale research project revealed in Dubai the dominance of the highly resistant Klebsiella pneumoniae sequence type (ST) 14 strains [K. pneumoniae ST147 and, in two regions, of K. pneumoniae ST231 strains.In the United Arab Emirates, just as well as in other countries of the region, the methodology needed for such molecular typing has not been broadly applied by public health laboratories for the surveillance of carbapenem-resistant  strains . This waThese prevalence reports, however, did not provide any insight on the timely changes of the local CRE epidemiology, time of emergence, and dynamics of spread of the clones currently dominating the local CRE scene. Answering these questions, even retrospectively, however, could provide helpful information to plan early preventive strategies to mitigate future epidemics and to highlight the importance of early, molecular typing-based, continuous surveillance to interfere even with the initial spread of highly resistant pathogens. In the current, retrospective study, we analyzed CRE strains collected in the hospitals of the Abu Dhabi Emirate in 2009–2015, i.e., during the early years of the local CRE epidemic. The data obtained were compared with those extracted from a recent, nationwide study [K. pneumoniae was the most common species encountered (82.2%) followed by Escherichia coli (11.4%), Enterobacter sp. (3.8%), and other Enterobacterales (2.5%). Urine was the most common sample type (24.2%) followed by respiratory (21.1%), wound and tissue (17.5%), blood (16.6%), and rectal or skin screening specimen (20.6%). The overall coverage rate of CRE encountered in hospitals A–E reporting to Abu Dhabi Antimicrobial Resistance Surveillance Program was 11.7%, exhibiting some variation between hospitals . The carriage rate of the blaOXA-48-like gene exceeded that of blaNDM. Nevertheless, the high frequency of the latter one, together with the strains carrying both genes and with a few isolates with blaVIM, resulted in a high rate (40.4%) of MBL producers. Nearly 50% of the isolates carried at least one 16S methylase gene  values of >8 mg/L, and 11.7% were either extremely or pandrug-resistant . Overall, strains were nonsusceptible to 11.28 ± 2.17 antibiotics of the 17 tested. Eighty-four percent of the strains carried a carbapenemase gene (data not shown). Significantly more carbapenemase producers (67.0%) exhibited meropenem MIC values >8 mg/L than their nonproducing counterparts (6.3%) . Sequence types ST38, ST69, ST156, ST131, ST167, ST410, ST1196, and ST1284 were identified, but none of them qualified by our arbitrary definition (present with >10 members) as a major clone. Representatives of international clones ST38, ST69, and ST131 expressed a variety of carbapenem resistance mechanisms. Two of the ST38 strains produced OXA-48, one expressed NDM-1, the single ST69 isolate carried OXA-232bla, two ST131 did not produce carbapenemase, and one expressed NDM-1.The 45 K. pneumoniae strains were assigned to 45 pulsotypes (data not shown). The results of the MLST analysis revealed ST14, ST231, clonal complex (CC) 147 (including single-locus variants (SLVs) ST273 and 392), ST11, ST15, ST29, ST45, ST101, ST188, ST307, ST471, ST711, ST2098, and ST2150 correspondingly, of which the first three qualified as major clones. The three major clones together represented 58.6% of the species and almost half (48.2%) of the CRE encountered until 2015. Each of the major clones was present in 5 out of 6 hospitals studied  followed by a moderate decrease thereafter. The latest major clone appearing in Abu Dhabi was the K. pneumoniae ST231 in 2014. Importantly, the rate of neither of the three clones has significantly changed after 2014–2015 when compared with more recent data of 2018–2019 [Since their first encounter during the first study period, the combined rates of the three major K. pneumoniae clones represented an increasing proportion within the species reaching a peak in 2014–2015. Of them, ST14 strains were the first to be encountered in Abu Dhabi as early as 2011. After that, with the exception of 2012, representatives of this clone have been present all throughout the study period reaching over 35% of blaOXA-48-like carrying strains exceeded that of isolates with blaNDM. However, when all MBL producers were considered  with the exception of 2014–2015, i.e., the period with the highest rate of OXA producers, their rate exceeded those of carrying only a class D carbapenemase gene , double carbapenemase producers have been present at a rate of over 10%. The rate of CRE not carrying any of the five carbapenemase genes tested and not exhibiting any carbapenemase activity sharply dropped after 2012–2013 and has remained low since, below 15%. The opposite trend was seen with 16S methylase genes. Their absence among the few isolates of the first collection period was followed by a sharp increase taking their rates above 50% in 2014–2015 followed by a moderate decrease only, which did not reach the level of statistical significance .K. pneumoniae strains affected the most the rate of non-carbapenemase producers, as this has always been rare among members of the major clones. It was noteworthy that while the most considerable increase in the rate of 16S methylases was observed among members of the major K. pneumoniae clones, a similar, albeit less pronounced trend was also seen among sporadic strains of the same species , and a high rate of double carbapenemase producers and MBLs [In countries of the Arabian Peninsula, and specifically in the UAE, most publications on CRE either reported the first encounters with specific carbapenemase producing imeframe ,16,17,18and MBLs ,20,21 arThe current study clearly showed that the ratios of these three clones, albeit at different speed, increased over time practically from the very early years of the outbreak, and by as early as 2014–2015, their combined rate exceeded 60% with a slight, nonsignificant decline subsequently. This increasing trend was the most pronounced regarding ST14 isolates , i.e., tK. pneumoniae and among other species as well   . The dece clones . Considee clones ) during sistance .Despite some variations in the rates of strains producing different carbapenemase  all throKlebsiella CRE, whereas it has increased among clonal and sporadic K. pneumoniae strains mirroring the trends regarding the presence of 16S methylase genes among them , UAE University, offered a service to detect the genes of the five most common carbapenemases in CRE strains isolated at Abu Dhabi hospitals. Laboratories could submit nonrepeat Enterobacterales isolates exhibiting nonsusceptibility to any of the carbapenems by any of the methods used. Strains were obtained from 5 major government hospitals , managed by the Abu Dhabi Health Service Company (SEHA), and also from another nonprivate hospital   .Although, in 2013, the Health Authority of Abu Dhabi  issued a circular advising laboratories to use this service , hospitaStrains were included in the study irrespective of whether they were isolated from clinical or patients’ screening samples but not from environmental specimens. No samples were collected for the sake of the study, all patient identifiers were masked, and we did not have access to databases containing patients’ data. To estimate the coverage of CRE encountered in Abu Dhabi, the number of isolates studied was compared with that of nonrepeat CRE isolates reported to the Abu Dhabi Antimicrobial Resistance Surveillance Program (AD ARS) covering all but one hospital  since 2010 (Jens Thomsen–unpublished).When analyzing changes in time, the period between 2010 and 2015 was divided into a single 3-year-long and two 2-year-long periods . These retrospective data were also compared with more recent (2018–2019) figures from Abu Dhabi extracted from the first nationwide CRE surveillance study [Strains were stored at −80 °C in Tryptic Soy Broth  containing 10% glycerol.Antibiotic susceptibility testing. The methods to determine quantitative susceptibilities to ceftazidime, ceftazidime–avibactam, cefotaxime, imipenem, meropenem, ertapenem, aztreonam, aztreonam–avibactam, trimethoprim–sulfamethoxazole, ciprofloxacin, gentamicin, amikacin, tetracycline, chloramphenicol, fosfomycin, colistin, and tigecycline, as well as the interpretation and criteria labeling of strains as pan- or extremely drug-resistant , were previously described [escribed . A “resiescribed .Detection of the antibiotic resistance genes. The detection of blaNDM, blaOXA-48-like, blaVIM, blaIMP, and blaKPC; determination of the alleles of blaNDM, blaOXA-48-like, and blaVIM; and detection of 16S methylase genes armA, rmtA, rmtB, rmtC, and rmtD were carried out as previously described [rmtE and rmtF were detected according to Davis et al. [escribed . Carriags et al.  and Hidas et al. , respectMolecular typing of the isolates. XbaI digested genomes were separated by pulsed field gel electrophoresis (PFGE) as described [E. coli and K. pneumoniae clones with high prevalence were identified by the following arbitrary criteria: PFGE clusters exhibiting ≥80% pattern similarities and containing more than 4 members were further divided into subclusters exhibiting ≥90% pattern similarities. In each subcluster, representatives of each unique carbapenem resistance mechanisms  were randomly selected and subjected to MLST analysis [escribed ,31. E. canalysis ,33. A clSequencing. PCR amplicons were sequenced for allele determination and for MLST using the Big Dye Cycle Terminator V.3.1 on a 3130X analyzer .Statistical analysis. The level of association of features with groups of strains was assessed by the chi-squared test, whereas differences in particular rates of features were analyzed by Student’s unpaired t-test .As the data presented in this study are not based on case-control observations, any possible effect of a timely, systemic, molecular typing-based surveillance of MDR strains on the increasing trends of the local CRE prevalence remains speculative. Nevertheless, the current study showed that members of the major clones currently dominating in the region had already been present, albeit undetected, during the early years of the CRE epidemic. This raises the possibility that timely investigations leading to the early identification of their sources and common routes of their transmission could have a mitigating effect on the escalation of the outbreak. In our views, such real-time surveillance work could also be highly beneficial in the future to alleviate the nationwide spread of other emerging multiresistant pathogens. In countries where public health laboratory systems with the proper methodological capacities are still rudimentary or nonexistent, their establishment could considerably improve surveillance including that of MDR pathogens.


Background: The County of San Diego Health and Human Services (SDHHSA) established a goal to vaccinate 1.9 million residents as quickly as possible to attain vaccine induced herd immunity. This strategy would minimize the emergence of more transmissible variants, to which some vaccines may be less effective. With this strategy in mind, UC San Diego Health (UCSDH) collaborated with the local health authorities and the San Diego Padres to build a superstation in downtown San Diego in the parking lot of a baseball stadium. Methods: Building on the experience of rapidly vaccinating the UCSDH workforce in mid-December 2020, UCSDH and SDHHSA partnered to more efficiently distribute SARS-CoV-2 vaccine in San Diego County by building a vaccine superstation. The San Diego Padres offered their parking lot as the site; it was centrally located, easily accessible, quick to set up, and semipermanent. They also provided infrastructure support, event coordination, and internet capability. Occupying a space of ~6.5 acres, the superstation included 12 lanes serving 12 cars each, with ~3 cycles every hour, as well as a pedestrian walk-up station. Altogether, the site had the capacity for >5,000 vaccinations daily. This effort required coordination among administration, healthcare providers, IT specialists, and support staff—a daily workforce of >300 persons. The workforce needs were met using a multipronged approach, including flexible staffing, coordination of volunteers, and recruitment of previously retired providers. The private–public partnership enabled the superstation to be up and running in 5 days. Results: The operation was quickly ramped up to provide >6,000 vaccines daily. Initially only open to healthcare workers, on January 17 the superstation was expanded to persons aged >75 years, with further expansion to those aged ≥65 years on January 23. From January 11 to February 5, >100,000 individuals received their first dose of vaccine at the superstation, corresponding to ~31% of all San Diego county vaccinations. Conclusions: Vaccination of as many people as quickly as possible is essential to controlling the pandemic. Unchecked replication of SARS-CoV-2 allows increases the chance that the virus may develop mutations that render vaccines and therapeutics less effective. Our model vaccine superstation was replicated at 3 more sites around the county, the limited allocation of vaccine has been the only barrier to further expansion. A force of 10 superstations could administer a first dose to the remaining 1.6 million county residents within 32 days.Funding: NoDisclosures: None