The Taiyangshan Wetland, a valuable wetland resource in the arid zone of central Ningxia, is critical for flood storage and drought resistance, climate regulation, and biodiversity protection. Nevertheless, the community structure and diversity of bacterioplankton in the Taiyangshan Wetland remains unclear. High-throughput sequencing was used to analyze the differences in bacterioplankton structure and major determinants in the Taiyangshan Wetland from April to October 2020. The composition and diversity of the bacterioplankton community varied significantly in different sampling periods but showed negligible differences across lake regions. Meanwhile, the relative abundances of bacterioplankton Bacteroidetes, Actinobacteria, Firmicutes, Chloroflexi, Tenericutes, Epsilonbacteraeota, and Patescibacteria were significantly different in different sampling periods, while the relative abundances of Cyanobacteria in different lake regions were quite different. Network analysis revealed that the topological attributes of co-occurrence pattern networks of bacterioplankton were high, and bacterioplankton community compositions were complicated in the month of July. A mantel test revealed that the bacterioplankton community in the entire wetland was affected by water temperature, electrical conductivity, dissolved oxygen, salinity, total nitrogen, ammonia nitrogen, chemical oxygen demand, fluoride, and sulfate. The bacterioplankton community structure was affected by ten environmental parameters  in April, while the bacterioplankton community was only related to 1~2 environmental parameters in July and October. The bacterioplankton community structure in Lake Region IV was related to seven environmental parameters, including dissolved oxygen, pH, total nitrogen, and chemical oxygen demand, whereas the bacterioplankton community structures in the other three lake regions were related to two environmental parameters. This study facilitates the understanding of the bacterioplankton community in wetlands in arid areas and provides references to the evaluation of aquatic ecological management of the Taiyangshan Wetland. Wetland plays a pivotal role in hydrological and biogeochemical cycles, biodiversity protection, culture, and tourism as a transition zone between terrestrial and aquatic systems ,2. ComplRecently, high-throughput sequencing technology has provided a new, cost-effective means for bacterial community analysis, completely changing the understanding of the bacterial community ,10,11,122, the wetland area is 1492.7 hm2, and the water area is 654.8 hm2. The terrain gradually rises from north to south, with altitudes ranging from 1330 to 1345 m. The wetland water area comprises permanent freshwater lakes, seasonal saltwater lakes, and inland salt marshes. The Taiyangshan Wetland is located at the intersection of many important ecological functional areas, including the hilly and gully soil and water conservation ecological function area of the Loess Plateau, the desertification prevention and ecological protection and control area of Ningxia, and the arid area in central Ningxia ecological improvement area. It is critical for flood storage and drought resistance, regulating microclimate, providing waterfowl breeding grounds, and protecting biodiversity in the central arid zone. It is a rare and unique wetland in the arid area of central Ningxia. The level of eutrophication in wetlands has increased, and bio-diversity has gradually decreased as a result of the dramatic impact of human activities and excessive utilization of water resources on the water area and water quality of wetland. Therefore, there is an urgent need to comprehensively evaluate the ecological status of Taiyangshan Wetland and promote wetland ecological management and ecological remediation. Understanding the wetland bacterioplankton community structure and its dynamic changes is important for systematically evaluating the aquatic ecosystem. However, the bacterioplankton community structure of the Taiyangshan Wetland and its driving factors remain unclear.The Ningxia Hui Autonomous Region is located in the northwest of inland China, typically the mid-latitude region of the northern hemisphere. The temperate continent has an arid and semi-arid climate. Located in the basin of the Kushui River in the arid area in central Ningxia, the Taiyangshan Wetland was identified as a national wetland park in 2018, with geographical coordinates of 106°32′01″~106°40′58″ E and 37°23′59″~37°29′17″ N. It is windy and rainy all year round, with strong evaporation. The total area of the Taiyangshan national wetland park is 2447.50 hmThe bacterioplankton community structures of the Taiyangshan wetland were examined in three sampling periods using high-throughput sequencing technology. This study has three objectives: (1) analyze the composition of the bacterioplankton community structure within wetlands and how it has changed over time; (2) compare the pattern of the bacterioplankton community between different sampling intervals and lake areas; and (3) identify the main environmental parameters that influence the composition of the bacterial community. The findings of this study will provide crucial information for assessing and managing the aquatic ecosystem of the Taiyangshan Wetland, as well as a scientific foundation for the preservation and effective management of wetland water ecological remediation and biodiversity.The Taiyangshan Wetland water area is divided into four lake regions based on the actual geographical pattern and water area characteristics, with 11 sampling sites S1~S11, . Lake ReEach sample (4 L) was mixed with surface water (at a depth of 0.5 m) and deep water (at a depth of 2.0 m) in equal amounts. After labeling, the water sample was placed in a polyethylene sampling bottle and stored in an incubator below 4 °C before being transported to the laboratory. The water samples for DNA analysis were filtered through 0.22 μm filter membrane within 24 h (under sterile conditions). The filter membranes were stored at −80 °C until DNA extraction. The sample pretreatment process was carried out in a sterile environment to avoid contamination of samples by external bacteria.−) were all measured using a YSI Pro-plus portable water quality analyzer. Fluoride (F−) was measured on-site using a HACH HQ40d portable water quality analyzer. Chlorophyll a (Chl a) was measured on-site by HACH Hydrolab DS5X. Total nitrogen (TN), total phosphorus (TP), available phosphorus (AP), ammonia nitrogen (NH4+-N), permanganate index (CODMn), and chemical oxygen demand (CODCr) were determined according to the method provided by the Water and Wastewater Detection and Analysis Method (4th edition) (Ministry of Ecology and Environment of the People’s Republic of China 2002). Sulfate (SO42−) was tested according to the water quality determination of the sulfate-Gravimetric method (GB 11899-89).Water temperature (WT), electrical conductivity (Cond), salinity , dissolved oxygen (DO), pH, total dissolved solids (TDS), and chloridion  following the manufacturer’s protocol. 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3’) were used as primer sets to 16S rDNA (V3 + V4) region for PCR amplification ,18. Therhttp://www.arb-silva.de, (accessed on 20 March 2021)) [The paired reads were joined using FLASH (version 1.2.11) , then thh 2021)) . The α dh 2021)) .p < 0.05). Due to the non-normal distribution of the data, the Dunn test is used for multiple comparisons between various sampling periods and lake areas, whereas the Kruskal–Wallis test is used for difference analysis. The Dunn test results are corrected by the Bonferroni method, and this process is completed in R by the R package “rstatix” (version 0.7) [In this study, 16 physical and chemical parameters related to the water environment are measured, and Kolmogorov–Smirnov normal distribution test is used to determine whether each water environment variable conforms to normal distribution. The results demonstrated that most water environment parameters did not comply with a normal distribution (ion 0.7) . The nonion 0.7)  in R.p-value less than 0.05 (Benjamini and Hochberg adjusted) as the standard. The co-occurrence pattern network of bacterioplankton is constructed, and calculated network topological attributes, which include node number (nodes), edges number (edges), average weighted degree (AWD), average path length (APL), average clustering coefficient (ACC), diameter, average degree (AD), and density. These parameters reflect the complexity of the composition of the bacterioplankton community to a certain extent. The co-occurrence pattern network is visualized using Gephi (version 0.9.2) [This study reveals the co-occurrence pattern of bacterioplankton via network analysis. In order to better display the results of network analysis, the low abundance and low-frequency groups (relative abundance less than 0.005 at least 5 samplings) are filtered in advance, and the paired Spearman correlation at the genus level is calculated using R”Hmisc” package . The corn 0.9.2) .A mantel test is used to determine the primary driving factors of the bacterioplankton community. In this study, the mantel test function in the R”linkET” package (version 0.0.3.5.) ,35 was u4+-N, AP, CODCr, F−, and Chl a were significantly different (p < 0.05). Nevertheless, according to the Kruskal–Wallis test, no significant differences existed in other parameters. In April, the contents of AP and Chl a were higher, and CODCr was much higher than in other months. In October, fluoride content was maximized.Cr, NH4+-N, and chlorophyll a, the Kruskal–Wallis test revealed that there are also significant differences in water environment parameters such as electrical conductivity, salinity, TDS, TN CODMn, Cl−, and SO42− among lake regions (p < 0.05). Electrical conductivity, salinity, and TDS in Lake Region IV are much higher than in other lake regions . The sequencing amount can cover the species in the sample according to the species accumulation curve, ACE, Chao 1, and Coverage .p < 0.0001; Chao1, p < 0.0001; Shannon, p = 0.0015). Observed_OTUs and Chao1 index were the highest in October, Shannon Index was the highest in July, and the three indexes were the lowest in April. Furthermore, Dunn-test (Bonferroni correction) results showed that each diversity indicator’s difference was not the same among the sampling periods. Observed_OTUs and Chao1 in October were significantly higher than in April and July. The Shannon Index was significantly higher in October than in April, but not significantly different from July , as shown in Observed_OTUs and Chao1 in the four lake regions were in descending order: Lake Region Ⅲ, Lake RegionⅠ, Lake Region II, and Lake Region IV, and Shannon was in the descending order as Lake Region II, Lake Region Ⅲ, Lake Region IV, and Lake Region Ⅰ. The indexes were not significantly different among the four lake regions , Bacteroidetes (17.17%), Actinobacteria (15.85%), Cyanobacteria (8.97%), Firmicutes (7.42%), Chloroflexi (3.44%), Verrucomicrobia (2.86%), Tenericutes (1.53%), Epsilonbacteraeota (0.95%), and Patescibacteria (0.94%), as shown in p = 0.001, p = 0.508, The NMDS with Bray–Curtis distance was used to assess the similarity of bacterioplankton communities across sampling periods and lake areas. ANOSIM statistical test showed that the composition of the bacterioplankton community of Taiyangshan Wetland differs significantly between sampling periods , dissolved oxygen , salinity ,TDS , CODMn , CODCr , F− , Cl− , SO42− , and Chl a  were found to be significantly correlated with the bacterioplankton community structure in the Taiyangshan Wetland. In July, TN  and TP  were significantly correlated with the bacterioplankton community. However, only TN was significantly correlated with the bacterioplankton community structure in October .A Mantel test was used to explore which water environment parameters are significantly related to the composition of the bacterioplankton community. As shown in p = 0.008) and F−  have a significant impact on the bacterioplankton community in Lake Region I. TN  and NH4+-N  are significantly correlated with bacterioplankton community of Lake Region II. The bacterioplankton community of Lake Region III is significantly correlated with pH  and SO42− . In Lake Region IV, dissolved oxygen , pH , TN , NH4+-N , AP , CODCr , F− , and Cl−  have a strong effect on bacterioplankton community  of Lake Region IV are much higher than those of other lake regions, which has become the primary source of water environment characteristic differences among the four lake regions. Because the nitrogen and phosphorus content in Lake Region Ⅲ is at a high level, it provides good conditions for algae growth where the content of chlorophyll a is high [The Taiyangshan Wetland is located in the arid area of central Ningxia, primarily formed by the Kushui River and groundwater supply. The Kushui River is the second first-class tributary of the Yellow River in Ningxia. It has a very high background mineralization degree and total hardness, with maximum values of 21,400 mg/L and 6488 mg/L, respectively. The farmland recession in the whole basin is large, and the water quality is poor. Our results show that under the direct influence of the background water quality, evapotranspiration, and lake structure of the Kushui River, the environmental parameters . The bacterial community structure is thought to be the result of a random distribution of external environmental factors and different habitats in flowing water areas . The watnd SO42− . The resommunity .4+-N, CODCr, F−, and SO42− were environmental parameters that influence the overall structure of the wetland bacterioplankton community. The driving factors were different between different sampling periods and lake areas. This study conducted a preliminary investigation into the bacterioplankton community structure in the Taiyangshan Wetland and its driving factors, but the precise effects of each environmental variable on the bacterioplankton community structure have not been established, and it should be encouraged in future research. Furthermore, the assembly mechanism of the bacterioplankton community in the river lake continuum generated by the Taiyangshan Wetland and the Kushui River should be studied to improve water ecological monitoring and management of the basin of the Kushui River.The first step in monitoring environmental and ecological conditions is to understand the structural and environmental driving factors of the bacterioplankton community. The present research shows that the bacterioplankton community in the Taiyangshan Wetland comprised 34 phyla of which Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Firmicutes, Chloroflexi, Verrucomicrobia, Tenericutes, Epsilonbacteraeota, and Patescibacteria were the main species. The bacterioplankton community structure changes significantly with the sampling period, but the difference between lake areas is insignificant. When the topological parameters of three sampling periods were compared, it was found that the bacterioplankton co-occurrence pattern networks have higher connectivity and cohesion in July, indicating that the bacterioplankton community structure is more complex. Water temperature, electrical conductivity, dissolved oxygen, salinity, TN, NH


High-quality wines in industrial winemaking frequently require a professional winemaker to make adjustments according to the wine of single-batch fermentation. Blending can improve the chemical composition and certain organoleptic properties of wine, promote copigmentation, and increase the complexity of the wine body and aroma. In this study, high-performance liquid chromatography (HPLC) and headspace solid-phase microextraction with gas chromatography coupled to tandem mass spectrometry (HS-SPME-GC-MS/MS) were used to study the effects of adding 20% of Merlot, Marselan, Syrah and Pinot Noir and different blending methods on the nutritional, taste, color and aroma components of Cabernet Sauvignon wine. The results showed that the highest total phenols and flavonoids, the greatest content of antioxidant characteristics, the optimal color according to the parameter of T, red% and blue% and the most abundant aroma were observed both in CGM (grape blending Cabernet Sauvignon and Merlot) and CGS (grape blending Cabernet Sauvignon and Marselan), thus indicating the higher quality and complexity of these wines. In addition, the co-grapes treatment afforded more color and hue value than co-wines, which indicates co-grapes had more stable and more varied colors than co-wines. Our findings provide theoretical support for improving wine quality and craftsmanship. Wine is a very complex mixture of compounds such as carbohydrates, polyphenols, organic acids, proteins, minerals and more . It can What is memorable about wine is its consummate quality, such as taste, nutrition, color and aroma, etc. . OrganicTherefore, it is very important to study the technology and application of wine brewing to improve the complexity and quality of wine. Polyphenols and volatile compounds, along with their interactions, are responsible for color and aroma, respectively . The conSome grape varieties may be rich in certain cofactors, while others may be deficient. Furthermore, not all grapes contain the same number of anthocyanins and polyphenols, so some varieties may benefit from the presence of other varieties that may contain an excess of these compounds. In addition, Escudero Gilete et al. demonstrate that blended wines from different grapes provide a more equilibrated ratio of anthocyanins/flavanols , which mThe Cabernet Sauvignon grape is in a dominant position in the market because of its easy planting, high tannin, deep color, abundant style and a strong sense of structure . HoweverThe wine grapes  used in this study were harvested from the Manas production area of Xinjiang in China on 3 September 2021 at optimum maturity. The physicochemical indexes of different grape varieties are shown in 2 and 50 mg/L of pectinase were added to the musts [2 [All wine samples were separately fermented at the same condition (as follows). CS, ML, MS, SY, PN, CGM, CGS, CGY and CGP musts were crushed and collected into 50 L stainless-steel containers. Then, 60 mg/L of SOhe musts . After bmusts [2 . The win2 in wine samples were tested according to the methods of the International Organization of Vine and Wine [The soluble solids content was measured by a digital refractometer . The pH of the wines was monitored by a digital pH meter . Titratable acidity, alcohol and free SOand Wine , Residuaand Wine .v/v) formic acid at a flow rate of 1 mL/min.The organic acids in wine samples were based on a previous method with some modifications . Each saTotal phenolic content of wine samples was determined by a spectrophotometer according to the Folin-Ciocalteu colorimetric method . The totHPLC analysis was performed based on the methods of Alejandro, Cáceres-Mella et al. with some revisions . Wines w2 = 0.9982), catechin , epicatechin , rutin , coumaric acid , procyanidin B1 , chlorogenic acid , vanillic acid , ferulic acid , quercetin  and kaempferol  standards, and phenolic compounds were quantified using a standard calibration curve. All qualitative and quantitative analyses of the phenolic composition were performed in triplicate.Phenolic compounds were identified by comparing retention times and chromatographic spectra of samples with gallic acid  according to previous reports with some modifications to the temperature program .The GC-MS system was an Agilent 7890B GC incorporated with an Agilent 5977 mass spectrometer . The column used was a 30 m × 0.25 mm HP-INNOWAX capillary with 0.25 μm film thickness . The carrier gas was helium, and the flow rate was 1 mL/min. The temperature program was as follows: 40 °C for 5 min, 50 to 86 °C at 3 °C/min, 86 to 90 °C at 0.8 °C/min, 90 to 180 °C at 2.5 °C/min, 180 °C for 3 min, then at 25 °C/min, up to 230 °C, with a final holding time of 5 min.The mass spectra of the sample volatile compounds were compared with those in the NIST library, and the volatile aromatic compounds were identified by comparing the retention time and mass spectra of the sample volatile compounds with the standard display . UtiliziWine sensory analysis was conducted according to Peng et al. . The welThe one-way analysis of variance (ANOVA) with a Duncan test of the SPSS 19.0 software was used to examine the variation between samples . The effects of different samples of the aroma profiles were evaluated by principal component analysis (PCA) using SIMCA 14.1 . Origin (2021) was used to generate histograms, radar charts and overlays. For each sample group, three parallel samples were analyzed.2 in wine (13–29 mg/L) was sufficient to protect wine from further oxidation and microbial contamination [2 (<100 mg/L) content were under the legal limit established for wines [mination . Of noteor wines ,18.The organic acids have a great influence on the flavor balance of the wine, as well as on the chemical stability and pH, thus affecting the quality of wine . In addiRegarding the varietal factor, it can be summarized that the MS and PN wines showed higher tartaric and malic acids than the other wine samples. Citric acid slows down yeast growth and participates in biochemical and metabolic processes , up to 1p < 0.05). Before fermentation, MS wine had the highest total phenol concentration of 1272 mg/L . The total alcohol content of CS and ML was significantly higher in all wines than the others, and the co-grape treatment had a higher total alcohol content compared to the co-wines . The upsp < 0.05). The content of 1-hexanol in CY wine is the highest, reaching 85.73 mg/L, followed by CS wine, 51.05 mg/L. The blending treatment reduces the n-hexanol content to two to three times the original, which suggests that blending results in a significant reduction in the green character of the wine [Alcohol may be derived from yeast fermentation. C-6 alcohol is a kind of common plant volatile with a typical “green” odor . They arthe wine ,15. In athe wine . These cthe wine .n-octanol compared with other wines, with pleasant rosy, citrus aromas. Additionally, the highest levels of citronellol, 6-Nonen-1-ol and 3-Methylthiopropanol were found in blends of Cabernet Sauvignon and Syrah varieties. In the blended wine, it was observed that the content of higher alcohol was significantly affected by the blending method (p < 0.05). Compared with the co-wine treatment, the content of 1-heptanol, phenylethyl alcohol, isoamylol and ethyl alcohol was significantly higher in the co-grape treatment, which may be related to the maceration effect in the early stage of the co-grape treatment, which is consistent with the research of Sánchez-Palomo, who found that the concentration of higher alcohol is significantly correlated with immersion time [Wines blended with Pinot Noir and Cabernet Sauvignon grapes had significantly the highest concentrations of benzyl alcohol and ion time .Esters have long been considered an important contributor to wine aroma as they are the main source of fruity aromas . Most esThe production of fatty acids is related to the initial composition of the must and fermentation conditions. These compounds have been described as fruity, fatty cheese, and rancid notes . VolatilPrincipal Component Analysis (PCA) was used to assess the effect of parameters (different blended varieties and blending methods) on the aroma profile of the analyzed wines . The firn-propyl benzoate and 3-hexenyl isobutyrate. They were negatively related to PC1 and were specific for PN and ML.The projection of the wine samples on PC1 showed a clear separation according to the grape variety A. TherefRegarding PC2, further differences in wine samples were related to the method of blending. Therefore, as shown in Of the 53 compounds analyzed and quantified, only 9 had OAVs greater than or equal to 1.00 in wine . The intThe floral series is the most intense main aroma series of all treatments. The main contributors to this series are leaf salicylate, citronellolformate, phenethyl acetate, ethyl laurate, 1-octanol and 1-decanol. The effect of different varietal blends on this aroma family is markedly different. Leaf salicylate, citronellolformate and phenethyl acetate reached the highest levels in CGW wines. It is worth noting that the concentration of floral series in the blended wine samples is distinguished higher than the blended wine.The fruit series is another main aroma series in this study, dominated by ethyl octanoate, ethyl hexanoate, ethyl laurate, ethyl 9-decenoate, isoamyl acetate and phenethyl acetate. The highest concentration of fruit aromas was found in CGP wine samples, followed by CGW wines. Similarly, the concentration of fruit series in the blended wine samples is about twice that of the blended wine.Fats and nuts were the other two main aroma families in this study. The fats series was dominated by five compounds , and the nuts series was dominated by four compounds . The highest fat and nut concentration was found in CGY wines, followed by CWY. Interestingly, the fat series by the co-wines treatment is significantly higher than co-grapes.The results of the sensorial analysis are shown in The bitterness of CS and SY was higher than other wines B, but thThis work provides a better knowledge of the nutritional, taste, color and volatile composition of Cabernet Sauvignon wines elaborated by the blending technique. The results showed that the wines brewed by the blending technique present a more complex chemical characteristic and aroma complexity than monovarietal wines. High total phenols, total flavonoids and antioxidant activity were found in CGM, CGP and CWP wines. In terms of color parameters, CGM and CGS have a higher red tint, and CGM has a higher blue tint. These wines show better colors. In addition, the wines made by the CGP combination performed best in the fruity characteristic due to its high ester concentrations. The CGY and CWY wines had a high content of ester, alcohol and aldehyde compounds, showing the characteristics of nutty and fatty aromas. Notably, the co-grape treatment had higher antioxidant properties and aroma complexity compared to the co-wine treatment. Our research provides a feasible alternative to traditional methods to improve the chemical and organoleptic characteristics of Cabernet Sauvignon wines.


S. aureus 28.1%; Str. agalactiae 7.3%; M. bovis 3%), which primarily affect milk nutritional components decreasing mainly milk fat content (range 1%–2.5%), but did not show them to be associated to an increase of the risk of antimicrobial residues. These latter ones were recovered only in 7/331 samples at concentrations far below official MLRs. The results support currently active surveillance programs’ efficacy in reducing AR risks, which may be further improved by prioritizing them based on geographical area characteristics. Mastitis is one of the major diseases of dairy cows that affects milk quality and quantity and increases the potential risk for the presence of antimicrobial residues (AR) in milk, which could lead to the development of antimicrobial resistance (AMR) among human pathogens. Even if the presence of AR in milk and milk products is low in many countries, the threat is not negligible and cannot be ignored. These problems may be investigated by applying a One Health approach, and this prevalence study aimed to estimate the risks for human health related to milk production applied to dairy herds in Lombardy. Three hundred thirty-one bulk tank milk samples were randomly collected and analyzed by CombiFoss 7 and MilkoScan 7 , an HPLC system coupled to a Q-Exactive Orbitrap (AR), and qPCR (contagious pathogens). The data were analyzed by a generalized linear model. The results showed a relatively high prevalence of contagious pathogens ( Milk production represents a significant food source for human consumption and an important part of the gross national product in many countries, including Italy. Moreover, many people are directly or indirectly occupied in the milk production chain. Besides the positive economic and social values, the milk production chain may also represent a potential risk to human health. Indeed, as described by ref.  in a OneAntimicrobial residues (AR) are a primary concern worldwide, being related to the development of antimicrobial resistance (AMR) among human pathogens ,13,14. TStreptococcus agalactiae, Staphylococcus aureus, and Mycoplasma bovis) are considered within the major threats for milk safety and quality [Str. agalactiae has been considered for a long time as a zoonotic agent [S. aureus is also considered a zoonotic agent, mainly when Methicillin-resistant S. aureus (MRSA) strains are considered [Among mastitis pathogens, contagious ones , and the human-animal interface is quite wide.About 45% of Italian milk is produced in Lombardy , a regioThe presence of AR in bulk milk is checked with high frequency by dairies following current EU regulations. This guarantees consumers that the milk and milk products do not contain an amount of AR potentially dangerous for human health. However, this does not mean that AR are absent from the products, and this may represent a potential risk of the development of AMR in humans. These residues may be identified nowadays even when their concentrations are much lower than the official maximum residue limits (MRL) .The study aimed to estimate the presence of hazards to human health related to milk production through a prevalence study on dairy herds in Lombardy by applying a One Health approach. More specifically, we investigated the presence of hazards as contagious bacteria in bulk tank milk (BTM), the relationship of their presence with milk quality, and the presence of AR in BTM with molecular and chromatographic methods.The study was designed as a prevalence study and the sample size was calculated assuming a prevalence of contagious pathogens of 30% on a total dairy herd population of 4931 herds . The totThree hundred thirty-one raw bovine milk samples were randomly collected among the 931 dairy herds, each divided into two bottles of 500 mL, one delivered refrigerated for molecular and quality assays and the other one frozen and stored at −20 °C until being analyzed for antimicrobial residues.10 transformed for further analysis.After collection, milk samples were placed at 4 °C immediately and analyzed within 24 h. Milk fat and protein were measured using MilkoScan 7 , while somatic cell counts (SCC) were determined by Fossomatic 7DC (according to ISO 13366-2/IDF 148-2:2006 standards). The SCC was LogS. aureus, Str. agalactiae M. Bovis, and Prototheca spp. This latter pathogen, being of environmental origin, was not considered further in this paper. The reaction conditions of qPCR were as follows: 95 °C for 1 min, 40 amplification cycles at 95 °C for 5 s and 60 °C for 25 s. The cycle threshold (Ct) values were considered positive when the values were ≤37, as suggested by the manufacturer. The qPCR reactions were performed on Stratagene Mx3005P .Milk samples were analyzed using qPCR through a commercial diagnostic kit  according to the manufacturer’s instructions. This technique showed sensitivity and specificity, respectively, of ≥0.95 and ≥0.99 for the contagious pathogens ,31,32. T−1 with the internal standard (IS), added of 100 μL, 20% w/v of Tri-chloroacetic acid for protein precipitation, and extracted with 5 mL of McIlvaine buffer (pH 4.0). After sonication for 10 min and centrifugation , the sample was defatted with hexane (2 × 3mL), and then purified by Oasis HLB SPE. The SPE was pre-conditioned with 3 mL of methanol and 3 mL of Milli-Q water; the extract was loaded and then washed with 2 × 3 mL methanol:water (5:95 v/v) before the final elution with 5 mL of methanol. The eluate was dried and reconstituted in 200 μL of methanol: 0.1%formic acid (10:90 v/v).The sample extraction for the antibiotic confirmatory method was performed following the analytical protocol of Chiesa et al. ,33. BrieInstrumental analyses were performed by an HPLC system coupled to a Q-Exactive Orbitrap . A Synergi Hydro-RP reverse-phase column , with a C18 guard column  was used and the mobile phase consisted of a binary mixture of Phase A (aqueous HCOOH 0.1%) and B (MeOH). The chromatographic gradient and the mass parameter set for the full scan acquisition combined with an independent data acquisition for the confirmatory response were well described in the previous work of Chiesa et al. .The HPLC-HRMS method was fully validated according to Commission Decision 2002/657/EC guidelines and SANCO/2004/2726 revision 4, as reported in Chiesa et al. . The recAll solvents of analytical LC-MS grade were provided by Merck . Sixty-six antibiotics belonging to different classes: Enrofloxacin, Difloxacin, Danofloxacin, Levofloxacin, Lomefloxacin, Marbofloxacin, Norfloxacin, Enoxacin, Flumequine, Nadifloxacin, Oxolinic acid, Nalidixicacid, Amoxicillin, Ampicillin, Phe-noxymethylpenicillin, Benzylpenicillin, Cefadroxil, Cefalexin, Cefalonium, Cefalothin, Cefazolin, Cefoperazone, Cefquinome, Cefapirin, Ceftiofur, Desfuroylceftiofur, Cloxacillin, Dicloxacillin, Benethamine penicillin, Nafcillin, Oxacillin, Piperacillin, Tylosin, Tilmicosin, Oleandomycin, Spiramycin, Neospiramycin, Kitasamycin, Josamycin, Tu-lathromycin, Erythromicyn A, Rifaximin, Sulfadiazine, Sulfadimethoxine, Sulfadimidine, Sulfamerazine, Sulfamethoxazole, Sulfamonomethoxine, Sulfapirydine, Sulfatiazole, Tri-methoprim, Chlorotetracycline, Oxytetracycline, Tetracycline, Doxyicline, Lincomycin, Chloramphenicol, Tiamphenicol, Florfenicol, Florfenicol amine, Tiamulin, Valnemulin, Dimetridazole, Ronidazole, Tinidazole), and Enrofloxacin-d5  were bought from Merck .The Solid Phase extraction cartridges SPE Oasis HLB  were provided by Waters .Str. agalactiae results ; Sj = effect of S. aureus results ; Mw = effect of M. bovis results ; (Aj + Sk + Mw) × Di = effect of contagious pathogens nested in the district; (Aj + Sk + Mw) × Vz = effect of contagious pathogens nested in t in herd size, eijkwz = residual error. Data descriptions were calculated by XLSTAT 2022 1.2 . Data were also analyzed by ANOVA using a generalized linear model applying the GLM procedure of SAS 9.4 . The model was:Overall, 331 bulk milk (BTM) samples from an equal number of dairy herds were collected in 12 provinces of Lombardy and classified into the three major geographical areas . The alpine area grouped the provinces of Como, Lecco, and Sondrio; the Po valley area included the provinces of Cremona, Lodi, Milano, Mantova, Monza, and Pavia; the semi-alpine area included the provinces of Bergamo, Brescia, and Varese.The number of herds for each area, and herd size, are reported in The herd size showed large variability (6–1150 cows/herd) with an average of 191 cows/herd, close to the average of the two areas with the largest cow population , as expected.The yearly milk production also showed large variability, with the lowest amount in the alpine area, where the frequency of small herds is higher, and the highest in herds located in the Po Valley where there is the largest frequency of large herds . IndividThe average SCC and total bacteria count (TBC) are reported in 10 cells/mL as the results of the mean for the two area with the largest number of herds, while in the Alpine area the SCC mean values were lower. The values observed were largely below the current threshold defined by the EU .The SCC values showed an average of 5.34 ± 0.22 Log10 5.00 cells/mL).The TBC values were also relatively close in the range 4.01–4.21 CFU/mL with the lowest values in the Alpine area and the highest in the Semi-alpine one. Additionally, in this case, the values are quite low compared with the EU thresholds , while Str. agalactiae was recovered from 7.2% (24 herds), and M. bovis only in 3.0% of the herds (10 herds). The frequencies of the three pathogens were different at χ2 test (p < 0.001) The highest prevalence for S. aureus was in the Alpine area, whereas Str. agalactiae and M. bovis prevalences were higher in the Semi-alpine area. The risk prevalence associated with the production area was assessed using the Po Valley prevalence as reference, and the result showed that the only significant risk ratio was related to S. aureus in the Alpine area (risk ratio 1.6 and confidence limits 1.0–1.8). In the other cases the risk ratio was >1, but not significant at χ2 test or Fisher exact test (α = 0.05).When contagious pathogens prevalence was considered, the overall prevalence of All 331 raw bovine milk samples were analyzed by the HPLC-HRMS method. Str. agalactiae and its interaction with herd size, respectively. Fat (%) showed to be the parameter most influenced by the factors considered  variance. The effects due to the presence of contagious pathogens were estimated by a general linear model as described in the material and methods. Herd TBC was uninfluenced by the presence of the pathogens and their interaction with area and herd size. SCC and protein (%) were significantly influenced by nsidered  and, amoThe risk for the presence of AR related to the health status (presence or absence of contagious pathogens), assessed by calculating the prevalence ratio between the positive samples in herds with contagious pathogens (3/127) and the positive samples in contagious-negative herds (4/204), was numerically >1 , but not significant at the Fisher exact test (α = 0.05).The One Health approach is generally associated with severe disease control/prevention or major zoonosis such as tuberculosis and antimicrobial resistance ,36. HoweTherefore, the improvement of food safety in cow milk production, through the improvement of animal health, and the resulting enhancement of human health may represent a good example of the One Health approach. Milk should be produced in herds that guarantee both the health and welfare of the animals, but milk could be a passive vector of zoonotic bacteria, AMR bacteria, and AR, particularly when cow health does not have a proper health level . BesidesMycobacterium bovis and Brucella abortus).To assess the presence of some of these problems and the related potential risks, a prevalence study was designed and applied to dairy farms in Lombardy, where >40% of Italian milk is produced. The study was focused on milk quality and AR, since the region is free from the major zoonotic bacteria , which influence both milk quality and quantity . Indeed,S. aureus, Str. agalactiae, M. bovis) suggests a potential risk for human health. This aspect may be negligible if the results of recent studies [Str. agalactiae from humans to cows should also be considered [The presence of a relatively high frequency of herds with contagious pathogens  and for a decrease in milk quality (fat and protein proportion), the results showed some unexpected results. Indeed,  results . Indeed, results ,43,44. HThe assessment of the risk for the presence of AR residues showed only 7 positive samples out of 331 with a concentration largely below the official MRL defined by EU relations. Other studies showed an association between SCC and AR in milk , and theThis study attempted to apply in practice the One Health approach to milk production by taking in consideration both the presence of hazards and their consequences on human and potentially environmental health. The prevalence study conducted in Lombardy dairy herds showed that the One Health approach is suitable for this application. The results showed a relatively high prevalence of contagious pathogens, which primarily affect milk nutritional components and herd sustainability, but do not increase the risk of antimicrobial residues. The positive results support currently active surveillance programs’ efficacy, which may be further improved by prioritizing them based on geographical area characteristics.


To prospectively evaluate the diagnostic accuracy of magnetic resonance imaging (MRI) for the planning of surgical treatment of deep pelvic endometriosis.From January 2020 to December 2021, we evaluated 72 patients with symptoms characteristic of endometriosis to plan appropriate surgical treatment. Sensitivity (Se), specificity (Sp), positive and negative predictive values (VPP/VPN), and the accuracy of MRI for the detection of deep pelvic endometriosis were calculated.Seventy-two patients  suspected of having pelvic endometriosis were recruited. Pelvic endometriosis was confirmed at pathologic examination in 56 (77.7%) of 72 patients. A total of 22 (39.3%) of 56 patients were subjected to video laparoscopy (VLS), and 16 (72.2%) of 22 were treated by surgery. Se, Sp, VPP, and VPN in intestinal endometriosis diagnosis were, respectively, 100%, 93.3%, 100%, and 87.5%, and diagnostic accuracy was 95.4%. MRI Se in ureteral endometriosis diagnosis was 50%, Sp 100%, VPP 100%, VPN 78%, and diagnostic accuracy 82%. MRI Se in endometrioma diagnosis was 92.3%, Sp 100%, VPP 100%, VPN 90%, and diagnostic accuracy 95.4%. MRI Se in rectum-vaginal septum (SRV) endometriosis diagnosis was 80%, Sp 100%, VPP 100% VPN 85.7%, and diagnostic accuracy 91%. The MRI Se in the diagnosis of endometriosis involving ULS was 100%, Sp 92.8%, VPP 89%, VPN 100%, and diagnostic accuracy 95.4%. Complete concordance results in a 100% accuracy for all calculated values in diagnosing bladder endometriosis localizations.MR imaging demonstrates high accuracy in detecting deep pelvic endometriosis in specific locations. It allows the localization of deep pelvic lesions with highly fibrotic components that are hardly recognizable with other imaging methods and not visible with VLS. Endometriosis is a chronic inflammatory disease characterized by the presence and proliferation of endometrial stroma and glands in abnormal locations with a predilection for the ovaries, the pelvic viscera, and the peritoneum of the pelvic excavation , 2. LikeFrom January 2020 to December 2021, we evaluated 72 patients with symptoms characteristic of endometriosis to plan an appropriate surgical treatment. All patients recruited in the study had, to varying degrees, distinct symptomatology for endometriosis  and, in some cases, strongly suspect symptoms for intestinal endometriosis .N-Joscina butyl bromide , with the following sequences: turbo spin-eco (TSE) T2 weighted oriented in the sagittal plane, TSE T2 coronal, and T1 TSE axial. The T2 arrangements oriented in the para-axial plane, perpendicular to the long axis of the cervix, provided a circumferential view of the cervix and the upper floor of the vagina, which helped evaluate the involvement of the uterine-sacral ligaments. The second phase, Clisma-RM-CE, with a total duration of about 9 min, involved the distension of the colon, so the patients were placed in the left lateral decubitus on the table of the MRI to introduce a catheter of Foley (20 F) in the rectal and infuse 1,500 ml of PEG solution at 37 4 4 4, after pharmacological intestinal hypotonization by further administration of 10 mg iv of Joscina’s N-butyl bromide .Given the symptoms and the objective examination, after a preliminary transvaginal ultrasound imaging, the patients were subjected to diagnostic completion with MRI to assess the presence and location of any endometriosis lesions to plan a proper therapeutic approach. MRI study predicts that on the day before the examination, patients make a preparation with a diet without slag and polyethyleneglycol (three sachets of Isocolan) dissolved in 1,500 ml of water . MRI tests were performed with a 1.5 Tesla magnet  using a four-channel surface coil (SENSE-body coil). The examination protocol included two phases: the first involved a resonance of the pelvis at high resolution (RM-HR), of a total duration of about 17 min, performed after distension of the vagina with 50–60 ml of sterile gel and administration of 10 mg intravenous Once the bowel was fully distended, an intravenous injection of 0.1 ml/kg contrast agent paramagnetic Gadobutrol  was administered. During post-processing, MIP reconstructions of urographic sequences  were performed in all MRI investigations. MRI was well tolerated by all patients who completed the entire imaging protocol. In all cases, the images were adequate for diagnosis. The intestinal preparation was always sufficiently good. Out of 72 MRI patients, 22 were subjected to VLS. In the case of intestinal endometriosis involvement, the lesion’s presence, localization, and extent were evaluated. On the suspicion of pelvic ureter involvement, the localization, degree of involvement, ureteral dislocation (traction), and secondary extrinsic or intrinsic compression by the reactive fibrotic tissue were evaluated with MRI for the possible presence of hydronephrosis. Finally, other pelvic localizations of endometriosis  were also investigated. We calculated sensitivity (Se), specificity (Sp), positive predictive (VPP), and negative predictive (VPN) for the detection of deep pelvic endometriosis located in a different location.Seventy-two patients  were examined using MRI upon the clinical suspicion of endometriosis. A total of 56 patients (56/72) were found positive for endometriosis localization. Two patients (22/56) were subjected to VLP, and 16 (16/22) were treated by surgery. More precisely, 1 (1/16) patient was subjected exclusively to vitreolysis, 7 (7/16) patients with intestinal involvement were subjected, respectively, to colic resection (3/7), adhesives (2/7), and nodulectomy (2/7); 8 (8/16) patients with urethral participation underwent bilateral ureterolysis (3/8), monolateral ureterolysis (4/8), and urethral resection (1/8), respectively. We did not find lesions in the parametrium.The MRI investigation identified eight intestinal lesions of endometriosis in eight patients, five in the rectum, two in the sigmoid-rectal passage, and one in the sigmoid. The laparoscopic investigation confirmed the presence of 7 intestinal endometriosis lesions in 7 patients treated with intestinal resection of the sigma-rectal tract in 3/7 cases, nodulectomy at the level of the rectum in 2/7 cases, and adhesiolysis in 2/7. One (1/8) distal sigmoid lesion reported to MRI was not confirmed to VLS .MRI Se in intestinal endometriosis diagnosis was 100%, Sp 93.3%, VPP 87.5%, VPN 100%, and diagnostic accuracy 95.4% .The MRI investigation identified 5 ureteral lesions in 4 patients, the VLS 11 lesions in 8 patients, in 4/8, a monolateral ureterolysis was performed , and in 3/8, a bilateral ureterolysis was performed . Finally, one (1/8) patient was subjected to ureteral resection and T-T anastomosis for intrinsic ureteral endometriosis. In all these cases, the MRI examination showed the late phase; in particular, in 2/4 positive points, MRI showed hydro-ureteronefrosis, and in the other two cases, ureteral dislocation angle without dilation was evident. The remaining 4 cases of ureteral involvement documented at VLS were not detected at MRI; of the three instances where VLS involvement was bilateral, in one case, MRI saw the participation of the left ureter, and in another place, there was a correct diagnosis of bilateral dislocation, and the other finally turned out a false negative .MRI Se in ureteral endometriosis diagnosis was 50%, Sp was 100%, VPP was 100%, VPN was 78%, and diagnostic accuracy was 82% .Ovarian localizations of deep pelvic endometriosis were identified, including 17 ovarian endometriomas at MRI  in 12 patients, at VLS, 17 endometriomas in 12 patients , only in 2/12. Patients were subjected to annessiectomy in a bilateral case and only one left . MRI Se The MRI also identified 8 nodules of the rectum-vaginal septum in 8 patients, surgical treatment, in this case, was excision of the nodule .MRI Se in SRV endometriosis diagnosis was 80%, Sp 100%, VPP 100%, VPN 85.7%, and diagnostic accuracy 91% .The MRI identified 15 lesions of the uterine-sacral ligaments in 9 patients, VLS 11 lesions in 8 patients; in this case, the removal of the nodules proceeded. . The MRIThe MRI finally identified two bladder lesions: in one case, it was a nodule of the upper bladder wall of 2 cm removed by partial cystectomy ; in the Complete concordance resulted in a 100% accuracy for all calculated values in diagnosing bladder endometriosis localizations .Several studies have shown that MRI is the second, after ultrasound, best non-invasive method for analyzing endometriosis, with an overall sensitivity, for different disease sites of 90% .Particularly in the evaluation of ULS by MRI, Bazot et al., in a 2011 study, showed an increase in diagnostic accuracy compared with conventional MRI using an additional thin-layer para-axial T2 sequence (3 mm), which has a higher spatial resolution and better follows the anatomical course of the ULS  allows a better “detection” of intestinal lesions, increasing diagnostic accuracy compared with pelvic MRI without distension, as reported in a study by Scardapane et al. .Clisma-RM-CE, however, requires more execution time and maybe less tolerated by patients. Therefore, it is considered a diagnostic completion of pelvic RM-HR by these authors, which remains the preferred examination in the diagnosis of DPE, reserving colic distension to patients with a clinical suspicion of intestinal involvement or who, in the first stage of the examination, have severe deep pelvic lesions .In this regard, from our experience, thanks to accurate informed consent and a comfortable clinical environment, all patients have been able to complete the entire examination protocol.Vimeracti et al., in a 2012 study, also performed very well in evaluating intestinal and ureteral endometriosis with RM-CE enema with values of Se, Sp, VPP, and VPN of 100% .According to Flaxman et al., for the surgical plan of endometriosis, the 3D reconstruction of 2D dimensional images taken with MRI will be the new way for surgeons to improve anatomical comprehension with a better vision of structural orientation, but unfortunately, in our study, we did not use this handy tool .In our series were included 22 patients subjected to enema-RM-CE for the diagnosis of deep pelvic endometriosis and compared with the VLS, considered the gold-standard reference. As for the intestinal lesions, we evaluated the presence, the seat, and the extent but not the degree of trans-parietal infiltration. The RM-CE enema examination in our personal experience showed sensitivity and specificity values in the diagnosis of intestinal endometriosis of 100% and 93%, respectively, 3% of which were sufficiently consistent with those obtained in similar studies of Scardapane et al. corresponding to 94% and 97%, respectively . The MRIIn our case study, 4/8 cases of ureteral involvement were identified, while the remaining 4 cases were falsely negative. As all these were subjected to ureterolysis and only one to colectomy in the absence of hydro-ureteronefrosis, it can be hypothesized that the lack of diagnosis was due to the presence of an extrinsic involvement that did not determine pathological conditions recognizable to MRI.The Se and Sp values obtained were 50% and 100%, respectively, which for sensitivity was at variance with the literature whose values were 75% and in perfect agreement with the Sp (100%) . Our stuAs for the other localizations affected by deep pelvic endometriosis, there is no doubt that the MRI results are more accurate than CT in their detection.Proof of this is that Biscaldi et al., in a 2014 study, compared the two methods (MDTC-e and RM-e) in reference only to the rectum localizations. The authors justify this choice by stating that the comparison of the identification of the other deep pelvic localizations is not feasible, because MRI, in this context, represents the “gold standard.” CT is not used for this purpose because of the low contrast resolution inherent in the method, which generates poor diagnostic accuracy for pelvic localizations of the disease . A compaComparing our case studies and with the data reported in the literature, it was found that the sensitivity and specificity in the detection of endometriosis lesions at the rectovaginal septum were perfectly average: If 80% vs. 44.4–84%, Sp 89% vs. 77.8–100% , 21–23. In this context, the thin-layer T2 para-axial sequences that better support the anatomy of the ligaments have been able to achieve a significant improvement in diagnostic accuracy.Our results in the study of the bladder were 100% sensitivity and specificity; in particular, the latter was in line with the results of other studies (Sp 94.7%–100%), while the sensitivity was significantly higher than that found in the literature (23.1%–88%). However, the low incidence of bladder involvement in our case studies (2/22 cases) , 20–24 iThe MRI is the most accurate method in the study of pelvic endometriosis. It is considered the most critical examination in the planning of patients to undergo laparoscopic or laparotomic surgery.MRI, in fact, non-invasively and without the use of ionizing radiation, provides a panoramic evaluation of the pelvis and, thanks to the high contrast resolution, allows to characterize deep pelvic lesions with a high fibrotic component hardly recognizable with other imaging methods and not visible to the VLS as sub-peritoneal. The completion of the examination with MRI-enema, moreover, allows to reach high values of diagnostic accuracy for lesions involving the intestine; in fact, for a correct localization/measurement of colic lesions, the water distension of the colon is necessary. It is essential to have the excretion phase accompanied by MIP reconstructions in evaluating the ureters. However, our study in this field shows some limits, especially for the diagnosis of extrinsic lesions (adhesions) that do not affect hydro-ureteronefrosis. Another limitation was the absence of 3D reconstruction. With good inter-operator reproducibility, RM-CE enema investigation is proposed in preoperative planning to ensure the best treatment approach and address the surgical choice between nodulectomy or segmental colic resection, ureterolysis, or ureteral resection. However, the proposed protocol requires intestinal preparation and implies a longer investigation time to reduce patient compliance. In this regard, with our experience in the study of deep pelvic endometriosis, we conclude that multidisciplinary assessment, a comfortable clinical environment, a fully informed consent regarding the modalities of the MRI investigation, and the diagnostic advantages of this approach form the basis of a full collaboration of patients and have allowed obtaining excellent diagnostic results.


Subgroup analyses showed physical activity of different intensities significantly impacted on improving college students’ depression and anxiety, but physical activity of 6 < 9 Mets intensity had a greater effect on anxiety than on depression. Interventions of eight weeks or less performed better than those of over eight weeks while interventions less than four times per week had a significant effect on improving the situation. The overall effect of a single intervention of 30 min was more effective than one of over 60 min. (4) Conclusion: Physical activities can effectively improve the situation of anxiety and depression for college students during the COVID-19 pandemic. However, a higher quality RCT experiment is needed to prove it.(1) Background: Although physical activity has been widely recognized as an effective way to improve anxiety and depression, we lack a systematic summary of research on improving anxiety and depression during the COVID-19 pandemic. The study aims to systematically analyze how physical activity impacts on this situation in college students during COVID-19. (2) Methods: Both Chinese and English databases  were analyzed. All the randomized controlled trials (RCTs) about physical activity intervention for this were included. We received eight eligible RCT experiments before the retrieval time (4 October 2022) in the meta-analysis. (3) Results: Physical activity benefits for college students with significant anxiety were (SMD = −0.50; 95% CI = −0.83 to −0.17; I COVID-19 is a contagious respiratory disease caused by a new type of coronavirus called Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) ,4,5,6,7,Early in the COVID-19 pandemic, researchers reported that restricted range of motion and physical activity in quarantined residential areas could lead to the incidence of additional psychological problems such as depression, anxiety, suicidal tendency, and intentional self-harm ,18,19. ISeveral pieces of research have shown that physical exercise can improve the physical and mental health of individuals of different ages ,48,49,50It can be seen that the specific impacts of physical activity on the mental health  of university students during the COVID-19 prevention and control period have not been clearly demonstrated. Therefore, this study seeks to answer a research question: How did physical activity impact the mental health  of university students during the COVID-19 prevention and control period? In order to answer this question, we conducted a meta-analysis to evaluate the effects of physical activity. Consequently, it is organized as follows. First, we address the materials and methods used for the meta-analysis. Second, we present the results of the analyses. Third, we discuss the results and link them to previous studies. Fourth, we address the limitations of the study and then conclude.This systematic review followed the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA statement). The literature search was conducted in Chinese and English databases including PubMed the Cochrane Library, EMBASE, Web of Science, Scopus, Chinese National Knowledge Infrastructure, and Wanfang Data, with PICOS (participants interventions comparisons outcomes) as a retrieval reference . DatabasThe inclusion and exclusion criteria of the literature are detailed in Two reviewers (L.Q. and Z.P.) independently performed the literature review, data extraction, and crosschecking. If the results of the chosen article were elaborated in a graphical format, efforts were made to request their authors for numerical data. Extracted data on the following variables were: 1. basic information of the studies ; 2. basic characteristics of study participants ; 3. intervention measures ; 4. tools for assessing mental health; and 5. outcome measures . Inconsistencies were resolved by a third independent reviewer and group discussion, as required (X.M.).Two authors (Q.L. and P.Z.) utilized a tool offered by the Cochrane Collaboration (Cochrane RoB1.0) . We assep < 0.05 was taken as the significant level and I2 statistic was used to assess heterogeneity. I2 (0%), I2 (≥25%), I2 (≥50%), and I2 (≥75%), respectively, indicated no heterogeneity, mild heterogeneity, moderate heterogeneity, and high heterogeneity. When I2 ≥ 50%, the random-effects model was used to combine the data. When I2 < 50%, the fixed-effects model was used. If a crossover design was found in the articles, the results in the primary stage before the crossover design were selected. If there was more than one physical activity group studied in the articles, the groups were combined to avoid the loss of data and potential analysis [Stata 17.0 was used for heterogeneity test, data merging, forest plot, and risk of bias assessment. The outcomes in the literature review were continuous outcome variables, which were inconsistent with the units of the outcome indicators in the original design. For continuous outcomes, we calculated the standardized mean difference (SMD) and 95% confidence intervals (95% CI). -SMD- < 0.1 was a small effect size, 0.10 ≤ -SMD- ≤ 0.34 was a small effect size, 0.35 ≤ -SMD- ≤ 0.64 was a medium effect size, 0.65 ≤ -SMD- ≤ 1.19 was a large effect size, and -SMD- ≥ 1.20 was a very large effect size . The hetanalysis .n = 127), and unrelated studies (n = 2480). The remaining 102 publications were further screened through full text reading and only 8 were singled out for meta-analysis, as the other 94 studies or experiments were not related to the design of the study due to such factors as lack of physical activity for intervention (n = 43), the time of the experiment before the outbreak of COVID-19 (n = 4), and not targeting university students as the study objects (n = 47). Detailed analysis and characteristics about the meta-analysis of the eight studies are shown, respectively, in In the database, we retrieved 2811 publications in total. In the preliminary screening, we eliminated 2709 studies by removing duplicate publications and reading titles and abstracts, including duplicate publications  [2 > 50%, the random model was used for analysis. In eight experiments, the depression and anxiety state after physical activity intervention were measured. Compared with the control group without physical activity intervention, physical activity brought about a statistically significant improvement of medium effect [2 = 84%; p < 0.001; Z = 2.98, 2 = 80.7%; p < 0.001; Z = 3.27, Meta analysis included a total of eight experiments ( = 1196) ,63,64,65 = 1196) . The eff = 1196) . When inm effect ,63,64,65p = 0.091), weekly physical activity frequency , and daily physical activity time , and duration . For depression, Met intensity , for weekly physical activity frequency , for daily physical activity time , and for duration weeks .Covariates  can be influencing factors for relieving depression and anxiety through physical activity. It has been pointed out that in the same period, university students in different countries had almost the same Met values . Met canIn order to further discuss the impact of physical activity intervention on university students’ anxiety and depression during the epidemic prevention and control period, we divided the subjects into four subgroups according to the Met intensity, the number of physical activity sessions per week, the duration of physical activity, and the number of weeks of physical activity in all the included literature, considering the differences in the research characteristics of each experiment .The systematic review and meta-analysis of this study aim to sort and analyze the related experiments on physical activity for university students during the prevention and control of COVID-19, and to understand the mechanism and effects of physical activity on university students’ mental illness during the prevention and control of COVID-19, so as to provide an exercise prescription for relieving university students’ mental health during COVID-19. The results show that physical activity is beneficial to alleviate the mental health disorders of university students during the COVID-19 prevention and control period, and physical activity of different intensities can improve the mental illness of university students to varying degrees  physical activity is superior to Moderate (3 < 6 Mets) physical activity in improving university students’ anxiety and depression, and this finding is consistent with the research results during COVID-19 , but conty level . This maty level , thus ofIn the study of physical activity during COVID-19 on mental health, it was pointed out that physical activity can improve negative emotions such as long-term home isolation, fear of being infected by viruses, and students’ anxiety about learning caused by COVID-19 ,95. The In general, this research summarizes the different degrees of improvement of physical activity for university students’ mental health during the control and prevention of COVID-19, and also confirms the important role of physical activity for university students during the period. However, we need more randomized controlled experiments to strengthen this hypothesis.Although the studies included in this systematic meta-analysis were conducted in different countries such as China, the US, Belgium, Turkey, and Saudi Arabia, one third of the experiments were conducted in China. According to some research, the prevalence of anxiety and depression are in line with the pandemic, but it remains stable in countries with great epidemic prevention and control. Countries with good epidemic prevention and control have low risks of infection, as well as incidents of anxiety and depression, and so do university students ,103. TheThe meta-analysis in this research indicates that physical activity has a significant impact on mental health improvement among university students with anxiety and depression during the control and prevention of COVID-19. Specifically, physical activity with an intensity of 3 < 6 Mets and 6 < 9 Mets can significantly improve university students’ anxiety and depression, but physical activity with an intensity of 6 < 9 Mets can bring about better improvement than physical activity with an intensity of 3 < 6 Mets. The effect of intervention for less than eight weeks will be greater than that of intervention for more than eight weeks, and with the increase in the number of intervention weeks, the relief effect on anxiety will be significantly reduced. The improvement effect of less than four interventions in a week on anxiety and depression showed a significant state, while more than four times did not show a significant state. Physical activity intervention of ≤30 min showed significant improvement in anxiety and depression, but ≥60 min physical activity intervention time only improved depression, not anxiety. It is necessary to note that emotion regulation and emotional reactivity are two essential elements for mental health, and that higher levels of emotion regulation and emotional reactivity, which are closely related to physical activity, can bring better mental health to individuals. The findings of this study theoretically enrich the dimension of emotion regulation theory, namely the mediating mechanism of physical activity between emotion regulation levels and emotion reactivity, and further support the idea that physical activity can improve emotion regulation levels, which has realistic significance for improving the mental health of college students during COVID-19.It should be noted that the specific impact of the internal mechanism of the human body and the follow-up results after the intervention have not been obtained in this study. In addition, the quality and quantity of the methodology included in the test limit our ability to make conclusions about its effectiveness. In the future, larger range and higher quality RCT experiments are needed to verify the current research results.


Introduction: In hepatocellular carcinoma (HCC), alternative splicing (AS) is related to tumor invasion and progression.Methods: We used HCC data from a public database to identify AS subtypes by unsupervised clustering. Through feature analysis of different splicing subtypes and acquisition of the differential alternative splicing events (DASEs) combined with enrichment analysis, the differences in several subtypes were explored, cell function studies have also demonstrated that it plays an important role in HCC.Results: Finally, in keeping with the differences between these subtypes, DASEs identified survival-related AS times, and were used to construct risk proportional regression models. AS was found to be useful for the classification of HCC subtypes, which changed the activity of tumor-related pathways through differential splicing effects, affected the tumor microenvironment, and participated in immune reprogramming.Conclusion: In this study, we described the clinical and molecular characteristics providing a new approach for the personalized treatment of HCC patients. Hepatocellular carcinoma (HCC) has become the second leading cause of cancer-related deaths worldwide, with more than 800,000 deaths each year . SurgicaAberrant alternative splicing (AS) is the result of splicing regulatory sequence mutations or ectopic RNA binding protein regulation. It plays an indispensable role in cancer and many other diseases . AlthougAll splicing data for liver cancer were downloaded from the cancer genome atlas (TCGA) SpliceSeq database including AS data, expression data, phenotype data, and survival data .We also downloaded the human genome sequence from the TCGA database , the humWe used the R package Rtsne (v0.15), which based on the t-distributed stochastic neighbor embedding (t-SNE) method, to cluster the samples according to their PSI values . BecauseTo further detect the differences in the distribution of age, sex, grade, pathologic T stage, alcohol, hepatitis B, and hepatitis C groups in the AS subtypes, Fisher test was applied .p < 0.05); and 2) Chi-squared test based on the median PSI reaching a significant level (p < 0.05). After DASEs were obtained, those whose average PSI of cancer samples were greater than the average PSI of normal samples were regarded as upregulated, and those whose PSI were less were regarded as downregulated. Next, DASEs between samples of different subtypes and normal samples were collected in the same way, and the number of different AS types in the relevant subtypes was counted, with the results included in a histogram. After obtaining the DASEs between subtypes, the overlap similarity of the upregulated and downregulated DASEs between subtypes was calculated as follows: Overlapping similarity = intersection of two sets/minimum value of the two sets. According to the obtained DASEs, analysis of variance was used to screen differential AS events between the two subtypes, with a threshold of p < 0.05, and then the intersection was taken for subsequent analyses.DASEs of cancer samples and normal samples were called according to the PSI value of AS. DASEs met two conditions: 1) Wilcoxon rank-sum test between groups reached a significant level ) was first used to obtain the reference sequence of each chromosome from the reference genome , and theAll gene sets were downloaded from the MSigDB database , and theThe correlation between the differential gene sets within the subtypes and the AS events was further calculated. First, the AS event was selected according to a PSI interquartile range of greater than 0.05 as the threshold in the samples, and then the screening results were used to calculate the Spearman’s correlation coefficient (coef) for the differential gene set. Alternative splicing-related pathways (SPs) in the MSigDB database were searched using “splice”, “splicing”, and “spliceosome” as keywords, and protein-coding genes in related pathways were used as splicing factors (SFs). After that, Spearman’s correlation coefficients of AS events and SPs and SFs were further calculated, and then the largest |coef. of SP| and |coef. of SF| corresponding to each AS event was selected to construct a scatter plot. Because coef. of SP and coef. of SF had the greatest number of AS events greater than 0.5 at the same time, the relevant AS events were selected for further PSI display, as well as the strongly correlated SP enrichment score of each subtype and the strongly correlated SF differential expression [edgeR (v3.28.1)] . The R pTo predict whether an immunosuppressive agent has a therapeutic effect on different AS subtypes, SubMap was used to map different AS subtype samples to samples with inhibitor processing information , as well50 values, and then the differences in the IC50 value of the samples of different AS subtypes was further counted using Kruskal’s algorithm to detect the significant differences. Next adj.p < 0.05 was used to screen for significantly different drugs, and the IC50 values of bosutinib, dasatinib, midostaurin, elesclomol, pazopanib, bortezomib, sorafenib, docetaxel, and gefitinib were plotted in box plots.The R package pRRophetic (v 0.5) was then to predict the sample’s response to 138 drugs , generatp < 0.05 was used as a threshold to screen significantly related AS events for subsequent analyses.Cancer samples were collected according to the PSI value of differential AS in the previous step combined with OS data, and batch Cox one-way regression analysis was performed on differential AS. After regression analysis, Lasso regression was further performed on the single-factor Cox regression results and a risk scoring model was built. This process mainly relied on the R package glmnet (v4.0–2). In the glmnet function , Y is Sup-value of <0.05 indicating that the difference between the high and low risk groups was significant.Where PSI represents the PSI value of the corresponding AS, β represents the regression coef. of the corresponding gene in the lasso regression result, and RScore represents the PSI value of the significantly related AS event in each sample multiplied by the corresponding AS event. The coef was then calculated, where i is the sample and j is the AS event. On the basis of the risk score of the sample, the high and low risk groups were divided by the median as the node and combined with the overall survival (OS) and disease-free interval (DFI) data to generate a Kaplan–Meier curve, with a Paired tumoral and adjacent normal samples were from patients diagnosed with HCC and accepted surgery at the Department of Transplantation, Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine  after the written informed consent. All these samples were kept and processed as previously described. RT-PCR was implemented using transcripts specific primers by 2 × Green PCR Mix . Splicing specific transcripts were distinguished using agarose gel electrophoresis and grayscale-measured using software ImageJ . PSI of each lane was calculated by the greyscale of the longer transcript divided by the sum greyscale of the longer and the shorter transcripts.HCC AS data  was downloaded from TCGA SpliceSeq database, and 11,179 AS events were obtained, corresponding to 4423 genes, which included 568 AAs, 469 ADs, 968 alternate promoters (APs), 6346 alternate terminators (ATs), 1992 ESs, 29 mutually exclusive exons (MEs), and 807 retained introns (RIs). At the same time, relevant HCC expression data and clinical data were downloaded from the UCSC Xena database, and 370 cancer samples and 50 normal samples were obtained after integration.According to the t-SNE method, the PSI values of all samples were displayed in clusters, and a scatter plot revealed that the cancer samples could be clearly distinguished from the normal samples . Next, uThe survival analysis of the three subtypes was further based on OS, and a Kaplan–Meier curve was generated. The results showed that the survival difference of the three subtypes was significant, and the survival curve of cluster 4 samples dropped faster .The distribution differences of age, sex, grade, pathological T stage, alcohol consumption, hepatitis B, and hepatitis C groupings in AS subtypes were further examined . The resOn the basis of the PSI value of AS, the DASEs of cancer samples and normal samples were retrieved. After threshold screening, 1,777 DASEs were obtained, of which 977 were upregulated and 800 were downregulated, corresponding to 1,005 genes . AccordiNext, the DASEs between the three subtypes and the normal samples were obtained. Cluster 1-normal had 1,681 DASEs, including 948 that were upregulated and 733 that were downregulated; cluster 3-normal had 2,545 DASEs, including 1,275 that were upregulated and 1,270 that were downregulated; and cluster 4-normal had 2,862 DASEs, including 1,565 that were upregulated and 1,297 that were downgraded. . After oThe AS score, GC content, and AS fragment length of the overall DASEs were further analyzed. For the length of AS fragments, the AS length of downregulated AAs was significantly higher than that of upregulated AAs and unchanged AAs, while the AS length of downregulated ADs was significantly lower than that of unchanged ADs, ESs, and RIs. There were no significant differences among the three groups .For GC content, the GC content of downregulated AAs was significantly higher than that of upregulated AAs, and the GC content of downregulated ESs was significantly higher than that of upregulated ESs and unchanged ESs. The GC content of upregulated RIs was significantly higher than that of upregulated RIs .For the AS score, the score of the first 5′ site of upregulated ADs was significantly higher than the score of the first 5′ site of unchanged ADs , and theNext, we analyzed the total DASEs corresponding to 1,005 genes, and drew heat maps based on the expression of related genes. The heat map showed that the overall gene expression of cluster 3 samples was high, while the overall gene expression of cluster 4 samples was low .A total of 32,284 gene sets was downloaded from the MSigDB database. The enrichment score of each sample for all gene sets was obtained and the cumulative distribution curve of GSVA scores according to different subtypes was generated. The score curve showed that compared with normal samples, the GSVA score distribution of samples of other subtypes was relatively. The score distribution of cluster 1 samples among subtypes was the most concentrated, while cluster 3 samples had more scores of less than 0, and cluster 4 had more scores of greater than 0 . After oCorrelations between differential gene sets within subtypes and AS events were further calculated. According to the interquartile range of the sample PSI, 3,662 AS events were screened, and strong correlations were screened from each subtype according to a Spearman’s correlation coefficient greater than 0.6. Eighty-eight splicing events and 326 gene sets in cluster 1 samples had a strong correlation, 519 splicing events in cluster 3 samples and 1,103 gene sets had a strong correlation, and 320 splicing events and 1,187 gene sets in cluster 4 samples were strongly correlated . The abop < 0.01).Twenty-four SPs and 370 SFs were identified from the MSigDB database. In further calculations of the Spearman’s correlation coefficients of AS events and SPs and SFs, a scatter plot showed that the coef. of SP and coef. of SF at the same time were greater than 0.5 AS events, including 138 AS events in each subtype. A heat map of the median of related events showed that the PSI value of cluster 3 samples was higher than that of the other subtypes, and the enrichment score of SPs of cluster 3 samples was higher. For SFs, in the cluster 4 samples, some SFs were significantly downregulated . A Venn p < 0.01). The difference in PSI of PQBP1 was not significant (p = 0.081), but the trend was consistent with the other three SFs. Therefore, the upregulation of PSI in HCC may be responsible for the upregulation of SFs such as PABPN1, CCDC12, ISY1 and PQBP1. Overall, the heterogeneity and homogeneity of splicing changes in HCC-related pathways may suggest a distinct role for alternative splicing in tumorigenesis and maintenance of cancer cell survival. Irregular splicing may regulate isoform switching of genes in cancer biological pathways and mRNA expression to promote HCC infiltration and invasion.The longer spliced isoforms of these SFs were significantly overexpressed in all HCC subtypes and validated by RT-PCR in HCC tissues . These mWe further explored the immune status of AS subtypes. A heat map showed that the StromalScore, ImmuneScore, and ESTIMATEScore of the cluster 3 samples were slightly lower than the other two subtypes. From median data, the tumor purity of cancer samples was significantly higher than that of normal samples, and the immune-related scores were significantly lower than normal samples. There was no significant difference in the expression of immune checkpoints and the enrichment scores of related pathways in each subtype. For most immune cells, the immune cell score of normal samples was significantly higher than that of cancer samples. In addition, cluster 4 samples had a higher activated CD4 T cell enrichment score and a lower activated CD8 T cell enrichment score. For G3–4 and hepatitis B patients, the related differences were also obvious .Kaplan-Meier analysis was further performed within the group for grade grouping and hepatitis B grouping. This showed that for the G3–4 samples, the Kaplan–Meier curves between the internal AS subtypes were significantly different, and for the samples with hepatitis B, the Kaplan–Meier curves between the internal AS subtypes were significantly different . The aboUsing SubMap to predict the possible therapeutic effects of related immunosuppressive agents on different AS subtypes, the results showed that the similarity between cluster 4 samples and CTLA4 response samples reached a significant level, suggesting that CTLA4 inhibitors may have a better effect on cluster 4 samples. .We further predicted the samples’ responses to 138 drugs to obtain predicted IC50 values. The results showed that there were significant differences in the degree of response of 111 drugs among the different subtypes . The IC5According to the obtained PSI values of DASEs, the differential AS events between subtypes were screened. There were 1,109 DASEs between cluster 1 and cluster 3, 1,154 DASEs between cluster 3 and cluster 4, and 1,177 DASEs between cluster 1 and cluster 4. Intersection of the three clusters resulted in a total of 455 DASEs for subsequent analysis . Using cLasso regression was then performed on 111 survival-related DASEs, and 20 AS events were obtained to construct a risk model. Regression coefficients and PSI values were analyzed to obtain the following risk scores: PSI39967 * (−5.5748) + PSI64018 * (−2.0928) + PSI46796 * (−0.5633) + PSI83140 * (−0.3544) + PSI44266 * (0.3162) + PSI85919 * (−0.3101) + PSI50488 * (−0.2602) + PSI19307 * (−0.1502) + PSI17008 * (−0.0950) + PSI19309 * (1.0474) E−13) + PSI50489 * 0.0014 + PSI85920 * 0.0023 + PSI85601 * 0.0223 + PSI58889 * 0.5534 + PSI1730 * 0.6888 + PSI24866 * 1.0308 + PSI61665 * 1.0423 + PSI 82016 * 1.6102 + PSI18599 * 40.6609 + PSI24760 * 51.4456 .According to the risk score of the samples, the high and low risk groups were divided by the median as the node, and a Kaplan–Meier curve was drawn on the basis of the OS data and DFI data. The results showed that the difference between the high and low risk groups was significant (OS, p < 0.0001; DFI, p = 0.0083; HCC is the most common primary liver cancer. Liver cancer is the sixth most common cancer and the second leading cause of cancer-related deaths worldwide. In the past few decades, the incidence of liver cancer and liver cancer-related deaths has increased in many parts of the world, including China . SorafenAlternative splicing can be regulated by many different mechanisms, such as histone modification and DNA methylation, which are usually associated with specific SFs or transcriptional elongation . The latFuture research aims to determine the molecular drivers of the transition from cluster 1 to cluster 4 subtypes. These changes may be triggered by changes in the genome or by epigenetic or transcriptional regulators that have been shown to drive splicing factor changes in other tumor types. Understanding these mechanisms will allow us to determine the development of AS-based HCC treatments. AS research is moving in the direction of making full use of the potential of AS in precision medicine.In this analysis, we identified AS subtypes through unsupervised clustering, analyzed the characteristics of different spliced subtypes, obtained DASEs, and combined the findings with GSVA enrichment analysis to explore the differences in the subtypes. Finally, on the basis of the DASEs of different subtypes, the survival-related AS time was identified, and the PSI value was used to construct a risk proportional regression model to guide prognosis. We systematically described clinical, splicing, transcriptomic, genomic, and immunological characteristics, and identified the underlying regulatory mechanisms of AS in HCC subtypes .Our research shows that the splicing regulation of SFs may play a role in the transformation and survival of HCC cancer cells. We studied AS comprehensively and systematically and used TCGA data to explore possible non-classical regulatory mechanisms in HCC. The data sample size of our research was sufficient, and the results of the verification data are good and have strong statistical significance, covering a wide range of fields. Our findings may provide the foundation for more in-depth research in the future, such as studies of the splicing regulation mechanism, cancer biomarker design, targeted drug screening, and other clinical applications.


To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state 19F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes.The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A  The lateral pressure profile has been found to be a key physical property of biomembranes. It plays an important role in the function of membrane proteins, as it can influence their conformation and structure ,2. PromiLateral pressure has been defined by Cantor and others by extending the concept of surface tension to arbitrary layers in the membrane parallel and to the bilayer plane ,13. ThisUsually, the lipidic component of biomembranes is considered the major determinant of the lateral pressure profile across the bilayer. Often, lipids are characterized by their spontaneous curvature, or an envelope shape, resulting from the conformational space consumption of the lipid headgroup compared to that of the lipid chains ,15,16. FIn this study, we explored the use of peptides instead of lipids to control the lateral pressure profile. In this way, on the one hand, insight into the physical aspects of how membrane-active peptides interact with membranes and might influence membrane constituents, such as membrane proteins, can be gained. On the other hand, peptides could also be a tool for studying the influence of lateral pressure, as they may allow a better tuning of the lateral pressure profile change compared to lipids, due to the versatility of peptide sequence design. Peptides inserting into the interface region of the bilayer have been suggested to modulate the lateral pressure in a similar to lipids possessing a positive spontaneous curvature ,18,19. B19F-NMR based approach 3), MAP (KLALKLALKALKAALKLA), magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS) were synthesized using standard Fmoc solid phase synthesis [D/L 4-trifluoromethyl-phenylglycine  in position 13 , and separating the diastereomeric mixture by HPLC. eviously ; and graynthesis ,47, follynthesis ,26,48. Avol:vol) chloroform/methanol, with amounts adjusted to yield the desired molar ratios. The solution of each sample was spread on 10–15 glass slides , which were dried under vacuum, stacked, and covered with an empty glass slide. The stacks were incubated at 97% relative humidity and 48 °C for 24 h to hydrate the bilayers, and sealed in parafilm and clinch foil. This sample preparation procedure led to stacks of ~103 hydrated bilayers with reconstituted peptides. Successful reconstitution following this protocol has been demonstrated in numerous studies, e.g., for the alignment of antimicrobial peptides [For oriented samples, 0.3–0.5 mg of labeled PGLa, one of the other peptides and dimyristoyl-phosphatidylcholine  were co-dissolved in a 1:1  operating at 470.7 MHz 19F resonance frequency, using a double resonance 19F/1H probe equipped with a turnable flat coil resonator, to fit the oriented samples from Doty Scientific . For each spectrum, approximately 2000 scans of 10 ms free induction decay under ~15 kHz 1H-decoupling with a recycle delay of 2 s were acquired.