Preoperative anemia affects perioperative outcomes and often causes fatigue and psychological disorders. Therefore, anemia should be treated before a patient undergoes surgery. Ninjin’yoeito (NYT), a Japanese Kampo medicine composed of ginseng and Japanese angelica root with the other 10 herbs, is administered for anemia, fatigue and anxiety; however, there are a few reports that have prospectively examined the effects of NYT before surgery for gynecological diseases. Hence, we tended to investigate its efficacy and safety.In this open-label randomized trial, women with gynecological diseases accompanied by preoperative anemia (defined as < 11.0 g/dL Hemoglobin [Hb]) were randomly assigned (1:1) into the iron supplementation and NYT groups. Patients of the iron supplementation group and the NYT group received 100 mg/day iron supplementation with and without NYT (7.5 g/day) for at least 10 days before surgery. The primary endpoint was improvement in Hb levels before and after treatment, and Cancer Fatigue Scale (CFS) and Visual Analogue Scale for Anxiety (VAS-A) scores between groups. Statistical analyses were performed with Wilcoxon signed rank test, Wilcoxon rank sum test, and Fisher’s exact test as appropriate.P = 0.72). Contrarily, CFS (17.9 ± 10.2 vs. 8.1 ± 5.2) and VAS-A (56 mm (50–70) vs. 23 mm (6–48)) scores were significantly decreased only in the NYT group and these changes were greater in the NYT group . Liver dysfunction occurred in one patient of the NYT group.Forty patients were enrolled of whom 30 patients were finally analyzed after allocating 15 to each group. There was no difference in the characteristics between both groups. Hb significantly increased in both groups ; the difference in the elevations in Hb between both groups was statistically insignificant .The online version contains supplementary material available at 10.1186/s12905-022-01824-9. World Health Organization estimates that one-third of all women of reproductive age are anemic , and womNinjin'yoeito (NYT), a Japanese Kampo medicine, has been derived from the ancient Chinese medical literature and is composed of 12 herbs , namely,Therefore, this study explored the effectiveness and safety of administering NYT in addition to iron supplementation in patients with gynecological diseases with preoperative anemia. The study compared two groups of patients who were randomly assigned (1:1) to receive iron supplementation or iron supplementation with NYT.https://jrct.niph.go.jp/en-latest-detail/jRCT1051190012. The first participant was registered on 01/11/2017.This was a non-blinded, randomized controlled trial conducted at the Osaka University Hospital . Efficacy and safety were evaluated in two groups, one receiving only 100 mg/day iron supplementation (sodium ferrous citrate) and the other NYT group receiving 7.5 g/day of NYT extract granules along with iron supplementation (100 mg/day sodium ferrous citrate). This study was conducted in accordance with the Declaration of Helsinki  and the Women aged 20 or older who were scheduled for gynecological surgery at the Department of Obstetrics and Gynecology under the Osaka University Hospital between October 2017 and August 2021were enrolled into this study. All subjects had preoperative anemia based on the WHO criteria of hemoglobin (Hb) < 11.0 g/dL without other non-gynecological diseases that could cause anemia . The excAll details of NYT attachment, ingredients, and formulations of the constituent herbs are listed in STORK . Two treAfter written informed consent was obtained from each participant, peripheral blood was collected and complete blood count including red blood cell count, Hemoglobin (Hb), Hematocrit (Ht), serum iron, and ferritin were analyzed. Thereafter, the pre-decided interventions were prescribed to the patients on outpatient clinic visits. At admission for surgery, each laboratory parameter was re-analyzed and the change rates for each parameter was calculated. If patients required emergency surgeries and the treatments had been administered for less than 10 days, then those patients were excluded from this trial. Additionally, the participants were instructed to answer the questionnaires consisting of the Cancer Fatigue Scale (CFS), visual analogue scale for anxiety (VAS-A), and Pittsburgh sleep quality index (PSQI) at the entry and at admission. –43. CFS The primary outcomes were the changes in Hb, CFS, and VAS-A levels before and after treatment. The secondary outcomes were safety and changes in serum iron, ferritin, and PSQI before and after treatment.Referring to the previous reports , 38 compJMP 16.0.0  was used for statistical analysis. We compared the baseline characteristics of patients with the Wilcoxon rank sum test and Fisher’s exact test. The changes in Hb, CFS, and VAS-A values before and after treatment in each group were statistically analyzed with the Wilcoxon signed rank test. Wilcoxon rank sum test was performed when comparing the amount of change in those before and after treatment between control and intervention group. P-value of < 0.05 was considered for statistical significance. Normally distributed values are expressed as mean ± standard deviation and those with non-normal distributions are expressed as median and interquartile ranges.Forty patients enrolled into the study from October 2017 to August 2021, 6 patients withdrew their consent; 3 patients were excluded because their treatment period was < 10 days; 1 patient was excluded because of violation of eligibility criteria. Finally, 15 patients in the iron supplementation group and 15 patients in the NYT group were analyzed , while there were 5 patients with malignant disease (33%). In contrast, the NYT group had a mean age of 41.1 ± 8.3 years with a mean BMI of 22.6 ± 2.7 kg/m2, and length of administration was 39.9 ± 20.3 days. All patients were non-menopausal and 3 received hormone therapy during the study period, respectively. Uterine fibroid was again most common in the NYT group, as 5 patients had uterine fibroids (33%), while 4 patients had malignant disease (27%). None of these values showed significant between-group differences, neither of the baseline characteristics had significant differences between the groups  was 21.4 ± 2.6 kg/mComparing the outcomes in the iron supplementation group before and after treatment, significant increases were observed in Hb (9.9 ± 0.8 g/dL vs. 11.9 ± 1.6 g/dL) and Ht (32.3% ± 2.1% vs. 37.1% ± 4.3%), but no significant differences were observed in CFS and VAS-A scores and serum iron, ferritin, or PSQI values. Conversely, when the NYT group data from before and after treatment were compared, the Hb (9.8 ± 1.0 g/dL vs. 12.0 ± 1.0 g/dL), Ht (32.7% ± 2.1% vs. 38.2% ± 3.3%), and ferritin (median 4 ng/mL (3–10) compared with median of 19 ng/mL (10–26) significantly increased, while the CFS subscales  and VAS-A (56 (50–70) mm compared with median of 23 (6–48) mm) significantly decreased. Moreover, the serum iron or PSQI values from before to after treatment had no significant differences , affective fatigue (− 0.1 ± 1.9 vs. − 3.1 ± 4.4), and overall fatigue , hormones (e.g. corticoids) and cytokines , 48. ThiIn routine clinical practice, anti-anxiety drugs are considered as options for patients experiencing preoperative anxiety and other unspecified complaints. However, such drugs have side effects including dependence, drowsiness, dizziness, and lassitude , but theIn this study, one participant in NYT group presented live dysfunction. However, her liver dysfunction was very mild and she recovered soon after the discontinuation of the drugs she took. Since she took not only iron supplementation and NYT but also relugolix and loxoprofen sodium hydrate, it was difficult to determine which drug contributed to the elevated liver enzyme. A utilization study regarding the safety and effectiveness of ninjin'yoeito was reported . Among 8https://center6.umin.ac.jp/cgi-openbin/ctr/ctr_view.cgi?recptno=R000031244). However, with the enactment of the Clinical Trials Act stipulated by the Ministry of Health, Labour and Welfare in Japan on 01/04/2018, Japan Registry of Clinical Trials (jRCT) was newly established and the function of UMIN was transferred to jRCT. Hence, this study appears to be retrospectively registered. Forth, patients with a variety of gynecological diseases were included in this study. The level of fatigue and anxiety might differ between those with benign tumors and those with malignant tumors, and such potential observational bias could not be eliminated due to the small sample size. Therefore, a double-blind randomized controlled trial with a larger sample size needs to be conducted in the future. Further, although our analysis was limited to the preoperative therapeutic effects, it is thought that NYT may help alleviate the physical and psychological symptoms of patients after surgery. Therefore, in the future we need to investigate whether NYT can contribute to improving the postoperative outcomes.The limitations of this study include the lack of blinding with a placebo, since it was not possible to provide placebo compounds for ethical and financial reasons. Second, the sample size was small. Third, this study was registered initially at University hospital Medical Information (UMIN) locally available in Japan on 01/11/2017  indicate more severe fatigue [ fatigue .Additional file 2: Supplemental Table 2. Questionnaires of The Pittsburgh Sleep Quality Index. The Pittsburgh Sleep Quality Index is an 18-item questionnaire having 7 domains: sleep quality, sleep duration, sleep latency, sleep efficiency, sleep disturbances, use of sleeping medication, daytime sleepiness, and other dysfunctionality. Higher scores indicate more severe sleep disturbances [urbances .


Low phosphorus (P) is one of the limiting factors in sustainable cotton production. However, little is known about the performance of contrasting low P tolerant cotton genotypes that might be a possible option to grow in low P condition. In the current study, we characterized the response of two cotton genotypes, Jimian169 a strong low P tolerant, and DES926 a weak low P tolerant genotypes under low and normal P conditions. The results showed that low P greatly inhibited growth, dry matter production, photosynthesis, and enzymatic activities related to antioxidant system and carbohydrate metabolism and the inhibition was more in DES926 as compared to Jimian169. In contrast, low P improved root morphology, carbohydrate accumulation, and P metabolism, especially in Jimian169, whereas the opposite responses were observed for DES926. The strong low P tolerance in Jimian169 is linked with a better root system and enhanced P and carbohydrate metabolism, suggesting that Jimian169 is a model genotype for cotton breeding. Results thus indicate that the Jimian169, compared with DES926, tolerates low P by enhancing carbohydrate metabolism and by inducing the activity of several enzymes related to P metabolism. This apparently causes rapid P turnover and enables the Jimian169 to use P more efficiently. Moreover, the transcript level of the key genes could provide useful information to study the molecular mechanism of low P tolerance in cotton.The online version contains supplementary material available at 10.1186/s12870-023-04100-6. Phosphorus (P) is one of the major elements required for all physiological processes, reproduction, and environmental adaptation that can maintain sustainable crop production . It is vGenotypes of the same species produced different yields under the same nutrient conditions . In termAmong various responses, modification in the root system is a well-documented response under low P condition . The greCarbohydrates such as sucrose and starch are important for coping with abiotic stresses by improving the osmotic pressure in leaves and roots . PhotosySimilarly, low P activates and regulates many enzymes related to the biosynthesis and degradation of carbohydrates (sucrose) such as sucrose phosphate synthase (SPS), fructose 1,6-biphosphatase (FBP), and sucrose synthase (SS) , 41. TheCotton is the leading fiber crop grown throughout the world, providing raw materials to the textile industry , 51. Chi− 1) in the soil is low and the cotton genotypes respond differently during the cotton growth period. Therefore, the current study is very important to understand the genotypic difference under low and normal P conditions. Moreover, the selection of the strong and weak low P tolerant cotton genotypes is the basis for the current study. Previously, we studied the agronomic performance and P use efficiency (PUE) of 384 cotton genotypes under low  and normal  P conditions, and based on dry biomass and PUE, 30 cotton genotypes were selected. The selected 30 cotton genotypes were again grown under low and normal P conditions in pot and hydroponic culture and finally two cotton genotypes  with contrasting low P tolerance were identified.Based on the multi-year P fertilizer experiments in the experimental farm of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences , we have found that the amount of available P (3 ± 0.5) mg kg2PO4) and normal (1 mM KH2PO4) P conditions. The seedlings were aerated with an electric pump and the solutions were renewed once a week. After obvious morphological variation, the 4 week old seedlings were harvested and various morphophysiological traits were measured.A greenhouse hydroponic experiment was conducted at CRI, CAAS, Anyang, China. According to the previous study, two cotton genotypes Jimian169 (strong tolerance to low P) and DES926 (weak tolerance to low P) were used in the experiment. The genotypes selected from the previous experiments were used in this study. The seeds of selected contrasting low P tolerant cotton genotypes were kindly provided by the ICR, CAAS, China. The selected seed permission was granted from the respective authority. Healthy seeds of both cotton genotypes were sown in sterilized sand in an incubator for 1 week. After germination, uniform healthy plants were selected and transplanted in a plastic container (7 L) in a growth condition of 16/8 h light/dark cycle, 28 °C temperature, and 60% relative humidity. Half concentration Hoagland solution was applied during the first week followed by full strength as mentioned in our previous study [At the same time, a pot experiment was conducted in the greenhouse at the CRI, CAAS, Anyang, Henan province, China. The soil for the pot experiment was collected from 0 to 20 cm low fertile arable soil from the cotton field. The two contrasting low P tolerant cotton genotypes Jimian169 and DES926 were sown in the plastic bucket  having soil collected from the cotton field under low and normal P conditions. The other nutrients were used at the recommended levels. All the management practices were kept the same for all the buckets except P levels.From each treatment, six plants were randomly selected and the shoot length was measured with the help of calibrated scale . After h2 (mol air)− 1, and the light intensity was set as 1000 μmol photon m− 2 s− 1. About 50 mg of fresh leaf sample was used to measure chlorophyll and carotenoid contents. The collected samples were cut into small pieces and incubated overnight in acetone: ethanol (1:1) solution for 48 h at 25 °C. The absorbance values for chlorophyll and carotenoid contents were measured according to our previous study [The photosynthetic traits were measured from the third fully expanded leaf by using the photosynthetic machine  from 9:00 to 11:00 a.m. . Carbon us study .2SO4-H2O2, and the final P concentration was analyzed using the Bran + Luebbe Continuous-Flow Auto Analyzer III. The various PUE related definitions were measured according to our previous study [P concentration in root and shoot tissues were measured according to Kjeldahl method . The grous study .(1) Total P accumulation (TPA) calculated as the P concentration x total plant dry matter;(2) P utilization efficiency (PUtE) calculated as the total plant dry matter divided by P concentration;(3) P uptake efficiency (PUpE) calculated as TPA divided by root dry matter.2.6 Determination of carbohydrate contents and enzymatic activities related to phosphorus and carbohydrate metabolism.− 1) and α-glucoamylase , and water. The final values of glucose, fructose, sucrose, and starch were recorded at 505 nm, 480 nm, 480 nm, and 620 nm, respectively. The glucose content was expressed as μmol g− 1 FW, whereas fructose, sucrose, and starch as mg g− 1 FW.The carbohydrates contents like glucose, fructose, sucrose, and starch were measured according to the instructions provided by the company. A fresh sample of 0.1 g from root and shoot was added in a 5 ml 80% ethanol solution. The extract was centrifuged at 8000×g for 10 minutes and the supernatant collected was stored at 4 °C for the next analysis. Further, the supernatant was incubated for 48 h at 37 °C in acetate buffer (4.5 mmol·L− 1 g− 1.The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), and phosphofructokinase (PFK) were measured according to the protocols provided by the company . The root and shoot fresh weight was homogenized with sodium acetate buffer (0.1 M) and centrifuged at 8000 g at 4 °C. The extracts were incubated under dark condition for 30 minutes at room temperature. The absorbance of ACP, ALP, and PFK was measured at 405 nm, 510 nm, and 340 nm. The activities of APA, ALP, and PFK were expressed as μmol min− 1 g− 1 FW.The sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities were determined by using the commercially available kits provided by the manufacturer. The fresh samples of about 0.1 g from root and shoot were homogenized in 1 ml extraction buffer and were centrifuged. The enzyme reaction was measured according to the protocol and the final values were recorded at 480 nm using a spectrophotometer. The activities of SS and SPS were expressed as μg min2, 2 mM benzamidine and 2 mM e-aminon-caproic acid. Further, the solution was kept at 80 °C until the analysis of PEPC. The reaction was started by adding 3.25 mmol phosphoenolpyruvate at 30 °C. The absorbance was measured at 340 nm and expressed as nmol min− 1 g− 1 FW.Phosphoenolpyruvate carboxylase (PEPC) was extracted and measured according to the commercial chemical kits in accordance with the manufacturer. About 0.1 g fresh root and shoot samples were halogenated in 1 ml buffer (50 mM Hepes-KOH), 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM DTT, 12 mM MgCl2, 10 mM β-mercaptoethanol, 0.25 mM fructose 1,6-biphosphate, 10 mM KF, and 100 μL of extract. After that, the solution was kept in dark for 10 minutes at 25 °C followed by adding the trichloroacetic acid (30%). The Pi released was measured according to the instructions provided by the company and the final values were obtained at 340 nm. FBP activity was expressed as nmol min− 1 g− 1 FW.Fructose 1,6-biphosphatase (FBP) was determined using the commercially available kits from Suzhou Comin Biotechnology, China. The root and shoot samples (0.1 g) were mixed in 50 mM Hepes-HCl (pH 7.6), 5 mM MgClThe malonaldehyde (MDA) content in root and shoot was measured according to the standard protocol .− 1 min− 1 FW [For the determination of antioxidant enzymes activities, 0.2 g fresh samples was obtained by removing the midrib portion. The sample was washed, dried, powdered, and homogenized in 5 mL chilled sodium phosphate buffer . The sample was centrifuged at 12,000×g for 20 min at 4 °C. The supernatant was used to measure superoxide dismutase, peroxidase, and catalase enzymes activities, and results were expressed as U mgin− 1 FW , 67.2 (0.1 mM), 0.3 mL NBT (750 mM), 0.3 mL riboflavin (20 M), and 0.3 mL distilled water. The reaction mixture tubes were put in 15 W lamps light for 10 min and then transferred to dark for 15 min, and absorbance was recorded at 560 nm using the UV spectrophotometer.Superoxide dismutase (SOD) activity was determined by measuring the photoreduction of nitroblue tetrazolium (NBT) at 560 nm. About 20 μL of enzyme extract was added to the reaction mixture of 0.3 mL methionine (13 mM), 1.5 mL phosphate buffer , 0.3 mL EDTA-Na2O2, POD catalyzes the conversion of guaiacol to tetra-guaiacol. Amounts of 0.1 mL H2O2 (300 mM), 2.7 mL potassium phosphate buffer , 0.1 mL guaiacol (1.5% v/v), 2 mM EDTA solution, and 0.1 mL enzyme extract made up the reaction mixture. A spectrophotometer was used to measure the absorbance at 470 nm every 30 s for up to 2 min.Peroxidase (POD) activity was determined at 25 °C guaiacol. In the presence of H2O2 absorption at 240 nm. The reaction buffer contained 15 mM hydrogen peroxide (H2O2) and 50 mM potassium phosphate buffer at a pH of 7.0. Next, 100 μL of enzyme extract was added to the reaction mixture for the reaction initiation. The extinction coefficient of 40 mM− 1 cm− 1 was used to determine the quantity of H2O2 in the reaction mixture after 1 min, indicating the activity of CAT.The activity of the catalase (CAT) enzyme was determined using a previously described method that involves calculating the reduction in Hwww.ncbi.nlm.nih), and are provided in Supplementary Table S-ΔCt.Based on the transcriptomic data (data not published), essential members of P metabolism  and carbohydrate metabolism  were selected for quantitative real-time PCR (qRT-PCR) analysis. The samples collected at the end of the hydroponic experiment from both cotton genotypes under low and normal P conditions were used for qRT-PCR. Total RNA from each sample was extracted using the Trizol method. About 0.5 μg of total RNA was reverse transcribed into single-stranded cDNA using the PrimeScript RT Master Mix, and then the genomic DNA was digested with DNA remover as suggested in the protocol. The gene-specific primers and the housekeeping histone3.3 (ATG09810) were designed using primer-blast  . All the figures expressed as mean ± standard error were drawn in Graphpad Prism 8.In comparison with Jimian169, the shoot length, root dry matter, shoot dry matter, total plant dry matter, and single leaf area of DES926 decreased under low P by 32.7, 15.9, 9.2, 10.3, and 37.5% in hydroponic culture, while 22.6, 10.6, 4.1, 5.2, and 20.9% in pot culture, respectively , shoot SS (32.2 and 39.1%), root SPS 33.5 and 42.4%), and shoot SPS (22.5 and 24.2%) activities were greatly inhibited by low P than normal P , shoot SOD (26.3 and 27.5%), root POD (49.7 and 44.7%), shoot POD (46.7 and 47.4%), root CAT (27.4 and 29.6%), and shoot CAT (26.1 and 25.4%) were greatly inhibited under low P as compared to normal P in both hydroponic and pot cultures  was performed by using the various studied traits to identify the key traits. In hydroponic culture, the PC1 contributed 78.1% and was associated with P conditions, while cotton genotypes were associated with PC2 and shared a variation of 19.8%. Root dry matter, shoot dry matter, total dry matter, chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, transpiration rate, root P accumulation, shoot P accumulation, total P accumulation, root SOD, root POD, shoot POD, and root MDA were the key traits contributing to PC1 and root morphology, enzymes related to P metabolism, and carbohydrate contents mainly contributed to PC2  and chlorophyll traits, PUE traits, and enzymes related to antioxidant system and carbohydrate metabolism. In addition, root morphology (except root diameter), P metabolizing enzymes, and carbohydrate contents were strong positively correlated with each other . Based on various morphological and physiological indicators, the cotton genotypes Jimian169 and DES926 were identified as strong low P tolerant and weak low P tolerant genotypes, respectively. The current study highlighted that the two contrasting low P tolerant cotton genotypes differed in morphology, physiology, and biochemical adaptation to low P stress. Low P significantly reduced plant morphological traits, however, the reduction was more in DES926 as compared to Jimian169  in the shoots than roots under low P in both hydroponic and pot cultures. However, genotype Jimian169 has comparatively higher carbohydrate contents than DES926 Fig. , which igaris L. . The incgaris L. , higher garis L. , a reducgaris L. , and a dgaris L. . It may garis L. . In contgaris L. , 90. Morgaris L. . These rSucrose is the end product of photosynthesis that is responsible for energy metabolism and the synthesis of complex carbohydrates . It can P transporter (PHT) genes were up-regulated and the transcript levels in the root and shoot of Jimian169 were comparatively higher Fig. , indicatLow P inhibited shoot growth, leaf photosynthesis, and enzymes related to carbohydrate metabolism and antioxidant system. However, low P significantly improved root morphology, P metabolism, and carbohydrate accumulation, particularly in Jimian169. The increase in PUE of Jimian169 might be attributed to its better root system that can uptake P efficiently under low P condition. The multivariate analysis showed that root-related traits, carbohydrate contents, and enzymes related to P metabolism were the most affected by genotype. Further, the variation among the tissue physiological traits was more in roots than in the shoot, suggesting the importance of root over shoot in low P tolerance. Based on these results, the mechanism of low-P tolerance in Jimian169 involved two major stratigies: (1) increasing the ability to use P efficiently by inducing P metabolizing enzymatic activities under low P; and (2) enhancing carbohydrates metabolism and subsequent partitioning into the roots under low P condition Fig. . The traAdditional file 1.


Real‐world evidence data on the use of granulocyte colony‐stimulating factor (G‐CSF) in patients with non‐small cell lung cancer (NSCLC) are limited. MONITOR‐GCSF is a pan‐European, multicentre, prospective, non‐interventional study designed to describe patient characteristics, treatment patterns and clinical outcomes in patients receiving biosimilar filgrastim in the prophylaxis of chemotherapy‐induced neutropaenia (CIN) and febrile neutropaenia (FN).In this subanalysis, patient characteristics, treatment patterns, and outcomes are described for 345 patients with stage 3 or 4 NSCLC, receiving up to six chemotherapy cycles. Patients were treated with biosimilar filgrastim as per their treating physician's best judgement.CIN (any grade) occurred in 13.6% of patients in Cycle 1 and in 36.5% of patients in all cycles. FN occurred in 1.4% of patients in Cycle 1 and in 5.2% of patients in all cycles. Grade 3–4 FN occurred in 1.2% of patients in Cycle 1 and in 3.8% of patients in all cycles.Results show that in real‐life practice in patients with NSCLC, biosimilar filgrastim has similar effectiveness and safety to the known effectiveness and safety profile of reference filgrastim, supporting the use of biosimilar filgrastim for the real‐world treatment of neutropaenia in patients with NSCLC. Treatment patterns and outcomes associated with CIN/FN prophylaxis with biosimilar filgrastim in 1,447 patients with solid or haematologic malignancies have previously been reported . Data were summarised overall and according to age ≥65 years and ≥70 years at baseline. Here we present results for patients with NSCLC only.33.1n = 240, 69.6%) were men. Mean body weight was 72.3 kg (range: 40.9–123 kg). Mean age was 62.9 years (range: 40–86 years), and overall, 142 patients (41.2%) were aged ≥65 years and 81 patients (23.5%) were aged ≥70 years.A total of 345 patients with NSCLC were included in MONITOR‐GCSF. Of these, 101 patients (29.3%) had stage III disease, and 241 (69.9%) had stage IV disease. Most patients (Monotherapy regimens used by patients included docetaxel in 14 patients (4.1%) and topotecan in 11 patients (3.2%) , followed by carboplatin/etoposide  and carboplatin/paclitaxel . Carboplatin/etoposide was the most commonly prescribed chemotherapy regimen in patients aged ≥70 years , followed by cisplatin/etoposide  and cisplatin/gemcitabine or carboplatin/paclitaxel .3.2Data on biosimilar filgrastim prophylaxis were available for 341 patients with NSCLC, including 142 aged ≥65 years and 81 aged ≥70 years  patients experienced one or more CIN (any grade) episode and 18  patients had FN (any grade) throughout the duration of the study. In Cycle 1, CIN (any grade) occurred in 47  patients and FN occurred in 5  patients. Grade 3 or 4 FN occurred in four patients  in Cycle 1 and in 13 patients  in all cycles.Changes to the chemotherapy regimen are detailed in Table 3.4Adverse events reported in patients with NSCLC included arthralgia, bone pain, cough, gastroenteritis, and myalgia  experienced changes to their chemotherapy regimens—in addition to reasons such as lack of efficacy, tolerability concerns, and disease progression, chemoresistance may have been responsible for this finding since this is a widely reported limitation of cisplatin . This is a lower level than in the overall population where 53.7% of patients experienced AEs . MA: Amgen , Helsinn Healthcare , Hospira , Teva , Merck KGaA (advisory role), Merck (advisory role), Sandoz , Pierre Fabre Medicament , Vifor Pharma , Tesaro , Novartis , Roche (speakers bureau), Johnson & Johnson (speakers bureau). NH: at the time of manuscript development Hexal AG (employment). PG: Sandoz (speakers bureau).


The effect of baseline hypertension status on the BMI–mortality association is still unclear. We aimed to explore the relationships of body mass index (BMI) and waist circumference (WC) with all-cause mortality among older hypertensive and normotensive Chinese individuals.This retrospective cohort study was conducted in Xinzheng, Henan Province, Central China. The data came from the residents’ electronic health records of the Xinzheng Hospital Information System. A total of 77,295 participants  aged ≥ 60 years were included from January 2011 to November 2019. Cox proportional hazard regression model was used to examine the relationships.2, the hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of BMI < 18.5, 24–28 and ≥ 28 kg/m2 for mortality in hypertensive participants were 1.074 (0.927–1.244), 0.881 (0.834–0.931) and 0.856 (0.790–0.929), respectively, and 1.444 (1.267–1.646), 0.884 (0.822–0.949) and 0.912 (0.792–1.051), respectively, in normotensive participants. Compared with normal waist circumference, the adjusted HRs and 95% CIs of central obesity for mortality were 0.880 (0.832–0.931) in hypertensive participants and 0.918 (0.846–0.996) in normotensive participants. A sensitivity analysis showed similar associations for both hypertensive and normotensive participants.During a mean follow-up of 5.3 years, 10,755 deaths were identified . In adjusted models, compared with a BMI of 18.5–24 kg/mLow BMI and WC were associated with a higher risk of all-cause mortality regardless of hypertension status in older Chinese individuals. The lowest risk of death associated with BMI was in the overweight group in normotensive participants and in the obesity group in hypertensive participants.The online version contains supplementary material available at 10.1186/s12877-022-03057-9. A total of 9,144 participants who were followed for less than 2 years were then excluded to execute a sensitivity analysis. The data screening flow chart is presented in Fig. The subjects of this study were older people in Xinzheng, Henan Province, Central China. The data came from the residents' electronic health records of the Xinzheng Hospital Information System from January 2011 to November 2019. Doctors set up health records for each resident at their first hospital visit or health examination, and participants aged 60 or older could receive free annual health examinations. At the start of the study, 87,686 older adults were eligible for the study. We excluded participants with the following conditions: (1) missing information for drinking, smoking, exercise, resting heart rate (RHR), systolic blood pressure (SBP) or diastolic blood pressure (DBP) (2), normal (18.5–24 kg/m2), overweight (24–28 kg/m2), and obesity (≥ 28 kg/m2) [2), and 3) as a continuous variable. WC was measured at the midpoint of the distance between the lowest costal ridge and the upper border of the iliac crest and was analyzed in the following 2 ways: 1) as a binary variable  [Demographic and clinical information was collected at each health checkup for participants. Demographic information included sex , age, marital status, current drinking (yes/no), current smoking (yes/no), and regular exercise (yes/no). Marital status was defined as married or not, including unmarried, widowed and divorced. Current smoking was defined as having smoked more than 100 cigarettes during their life and still smoking now. Current drinking was defined as drinking occasionally, often or daily compared to never drinking. The definition of regular exercise was 30 min of moderate-intensity exercise more than three times per week. Clinical data included anthropometric measurements, laboratory investigations, and self-reported disease history. Participants wearing light clothing took off their shoes, and then their weight and height were gauged. BMI was calculated as the weight in kilograms divided by the square of the height in meters. BMI was analyzed by the following 3 methods: 1) as four groups according to the BMI classification standard for Chinese individuals: underweight (BMI < 18.5 kg/m8 kg/m2) , 2) as en males)  and 2) aThe outcome was all-cause death from January 2011 to November 2019. Information on deaths came from the Centers for Disease Control’s cause of death reporting system in Xinzheng.2 or < 24 kg/m2) and WC  was explored. The proportional hazards assumption was verified with graphical methods and with models including time-by-covariate interactions. Statistical analyses were performed using SPSS V 21 and R V 4.0.3. P < 0.05 with two-sided tests was considered statistically significant.Continuous variables were described as means and standard deviations (SDs). Categorical variables were presented as numbers and proportions. The chi-square test for categorical variables and the t-test for continuous variables were used to compare the difference between two groups defined by blood pressure. The associations of BMI and WC with all-cause mortality among normotensive or hypertensive participants were analyzed by a Cox proportional hazard regression model, and HRs with 95% CIs of BMI and WC in categories and continuous variables were expressed in separate models. Model 1 adjusted for age and sex. Model 2 adjusted for confounders including age, sex, marital status, regular exercise, current drinking, current smoking, RHR to estimate the relationship across rising BMI categories. The potentially nonlinear relationship of continuous BMI and WC with all-cause mortality was explored by restricted cubic spline models with four knots. In addition, stratified analysis was performed by subgroups of sex, smoking, alcohol consumption, and exercise status using a Cox regression model to test the consistency of these relationships. Finally, the combined effect of BMI (≥ 24 kg/mP < 0.001). The mean  follow-up was 5.3 (2.3) years. In 41,357 hypertensive participants, during 229,630 person-years of follow-up, 6,377 (15.4%) all-cause deaths occurred. In 35,938 normotensive participants, during 178,735 person-years of follow-up, 4,378 (12.2%) all-cause deaths occurred.Hypertensive participants were older and had higher BMI, WC and RHR than normotensive participants. The proportion of female subjects was higher in participants with hypertension Table . The mea2, the HRs (95% CIs) of BMI < 18, 18–20, 20–22, 24–26, 26–28, 28–30 and ≥ 30 kg/m2 for all-cause mortality were 1.099 (0.913–1.323), 1.145 (1.036–1.267), 1.092 (1.014–1.175), 0.932 (0.868–1.002), 0.902 (0.830–0.980), 0.864 (0.775–0.963) and 0.935 (0.831–1.053), respectively. Each SD increase in WC was associated with a 5.4% (3.1%-7.8%) lower risk of all-cause mortality in Model 2. As a binary variable, the HR (95% CI) of central obesity in the risk of all-cause mortality was 0.880 (0.832–0.931) compared with normal waist.In normotensive participants, after adjusting for other covariates, the HRs (95% CIs) of BMI < 18, 18–20, 20–22, 24–26, 26–28, 28–30 and ≥ 30 kg/m2 for the risk of all-cause mortality were 1.516 (1.287–1.785), 1.278 (1.153–1.416), 1.084 (1.001–1.174), 0.992 (0.906–1.085), 0.824 (0.729–0.932), 0.992 (0.833–1.181) and 0.915 (0.718–1.166), respectively, compared with a BMI of 22–24 kg/m2. Each SD increase in BMI was associated with a 10.9% (7.7%-13.9%) lower risk of all-cause mortality. Each SD increase in WC was associated with a 5.2% (1.9%-8.4%) lower risk of all-cause mortality in Model 2. As a binary variable, the HR (95% CI) of central obesity in the risk of death was 0.918 (0.846–0.996) compared with normal waist. After excluding subjects with a follow-up of less than two years, a sensitivity analysis was conducted to check the robustness of the associations (Supplementary Table The associations of BMI and WC with all-cause mortality are presented in Table BMI had a nonlinear association with all-cause mortality among all hypertensive participants and female and male subgroups based on the adjusted Cox model Fig. . These aBMI had a nonlinear association with all-cause mortality among all normotensive participants and male subgroups, and the risk increased among those with very low BMI values Fig. . The femThe combined effect of BMI and WC on all-cause mortality in hypertensive and normotensive participants is shown in Fig. 2 in normotensive participants and BMI 28–30 kg/m2 in hypertensive participants, according to eight groups of BMI models. Reverse J-shaped curves were discovered in hypertensive participants, but the curves were almost horizontal with a BMI of ≥ 25 in normotensive participants. The risk of mortality decreased with an increase in WC regardless of hypertension status, and the protective effect of central obesity against death in hypertensive participants was higher than that in normotensive participants.In this study, we found that compared with normal weight, the underweight group was associated with a higher risk of all-cause mortality, overweight was associated with a lower risk of death in normotensive participants, and overweight and obesity were associated with a lower risk of death in hypertensive participants. The nadir of risk is BMI 26–28 kg/m2 in normotensive participants, similar to the geographically distant USA in NHANES [2 in participants aged ≥ 65 and 24.0–24.9 kg/m2 in participants aged < 65. A cohort study conducted in Taipei [2. A Korean study [2 in both subgroups aged ≥ 65 and < 65. This difference may be because the study participants were mainly volunteers, which causes a bias. A meta-analysis conducted in older white adults found that the lowest risk of death was at a BMI of 27–27.9 kg/m2, which is higher than that in younger people [Our results found that the lowest risk of mortality in the BMI group was for a BMI of 26–28 kg/mn NHANES , 17. A pn NHANES  that hadn Taipei  found than study  found than study –23 have an study . Adiposean study . A study2, which was higher than the 26–28 kg/m2 in hypertensive participants; obesity in hypertensive participants had a significant protective effect, which was not found in normotensive participants. Several studies that focused on the BMI-mortality relationship in hypertensive participants concluded similar results. Due to the high correlation between BMI and WC, we found a kind of waist paradox, the protective effect of central obesity against death in hypertensive participants was higher than that in normotensive participants.Meanwhile, we found that the lowest risk in BMI in hypertensive participants was 28–30 kg/mIn a cohort study in Jiangsu and Anhui , Yang etThis phenomenon may be due to the following reasons. First, obese people were more willing to check their own physical condition, so diseases could be found and treated earlier, and they also have better compliance with doctors' treatment plans , 32. Sec2 relative to those with a BMI of 24–26 kg/m2, which presented a U-shaped curve in hypertensive people. This may be because of the small sample size of this study, with a total of 2535 subjects enrolled. In contrast to our findings, McAuley et al. [2, 25.0–29.9 kg/m2 did not have a statistically significant effect on death. The potential reason may be that the study was underrepresented, as most of the participants came from an upper or middle social status and excluded those whose BMI was < 18.5 kg/m2.Some studies produced different results. For example, Li et al.  found thy et al.  found thSome studies have shown that poor antihypertensive medication adherence leads to a higher risk of death , 42. TheThere are some advantages of our study. First, the data of this cohort study were obtained from a large-scale periodical health check in Henan, China. Demographic and laboratory data were collected, and the sample size and statistical power were adequate. Second, few studies have analyzed WC, which can reflect abdominal adipose tissue, and it was analyzed in our study. Third, height and weight were objectively measured rather than self-reported. Finally, we did not adjusted for SBP, DBP, previous history of diabetes, cancer, coronary heart disease and stroke, which are influenced by excess body fat, so we to some extent avoided the overadjustment problem .However, some limitations of this study should be noted. Because the subjects were all older than 60, these relationships should be generalized cautiously to people younger than 60. We adjusted for confounders including age, sex, marital status, exercise, drinking, smoking, RHR, but some potential factors may exist that we did not adjust for, such as antihypertensive medication.Overall, this study found that the lowest risk of death relating to BMI was in the overweight group in normotensive older adults and in the obese group in hypertensive older adults. There was a reverse J-shaped relationship between BMI and all-cause mortality in hypertensive older adults, and in normotensive older adults, the risk of all-cause mortality was higher with a low BMI. Participants with central obesity had a lower risk of all-cause mortality regardless of hypertension status. More research is needed to explore the optimal BMI range for hypertensive and normotensive older people to decrease their risk of death.Additional file 1:Supplementary Table 1. Sensitive analysis of BMI and WC with All-cause mortality. Supplementary Table 2. Hazard ratios of All-cause mortality according to BMI or WC for various subgroups.


Background: Type 2 diabetes (T2D) is considered as one of the most significant metabolic syndromes worldwide, and the long-term use of the drugs already on the market for T2D often gives rise to some side effects. The mulberry leaf (ML), Morus alba L., has advantages in terms of its comprehensive therapeutic efficacy, which are characterized as multicomponent, multitarget, multipathway, and matching with the complex pathological mechanisms of diabetes.Methods: T2D rats were established by a high-fat diet combined with an intraperitoneal injection of streptozotocin; an evaluation of the hypoglycemic effects of the ML in combination with fasting blood glucose and other indicators, in addition to the utilization of metabolomics technology, was performed to analysis the metabolite changes in serum of rats.Results: MLs significantly reduced the fasting blood glucose of T2D rats, while improving the symptoms of polyphagia and polyuria. ML treatment altered the levels of various metabolites in the serum of T2D rats, which are involved in multiple metabolic pathways , played a role in antioxidative stress and anti-inflammation, modulated immune and gluconeogenesis processes, and improved obesity as well as insulin resistance (IR).Conclusion: The ML contains a variety of chemical components, and metabolomic results have shown that MLs regulate multiple metabolic pathways to exert hypoglycemic effects, suggesting that MLs may have great promise in the development of new hypoglycemic drugs. With the development of economies and the improvement of peoples’ living standards, the incidence of diabetes is increasing year by year. In 2021, the 10th edition of the Diabetes Map of the International Diabetes Federation showed that there were about 537 million diabetics in the world, and it is estimated that, by 2045, the number of diabetics may increase to 784 million , among which type 2 diabetes (T2D) is the majority, accounting for more than 90% of the total number of diabetic patients. T2D is a chronic metabolic disease characterized by disorders in the metabolism of glucose and lipids ; the long-term use of the drugs already on the market for T2D often gives rise to some side effects .Morus alba L., is a commonly used medicine in traditional Chinese medicine, and it has various pharmacological effects. In studies, it has been shown that the ML can resist oxidation and regulate lipid metabolism  has a long history of treating diabetes in clinical practices in China , which atabolism  as well tabolism , and itstabolism . HoweverThe pathogenesis of T2D is complex and not yet fully understood, and it is generally believed that the pathogenesis of T2D is related to pancreatic islet cell damage, reduced insulin secretion, and insulin resistance (IR). Small-molecule metabolites and metabolic pathways are disturbed in diabetic patients, leading to abnormal body indicators and the development of diabetic complications . DisordeThe development of omics techniques, including genomics, transcriptomics, proteomics, and metabolomics, has made it possible to explore TCM’s mechanisms in the treatment of T2D . MetabolIn order to fully understand the chemical composition and hypoglycemic mechanisms of MLs, UPLC-Q-Exactive Orbitrap-MS was used to explore the chemical composition of MLs, and a metabolomics technique was used to explore the abundance of changes in the metabolites in the serum of rats after treatment with MLs. This study provides a new idea for the prevention and treatment of T2D in addition to a new vision for the development of new hypoglycemic herbal medicines based on MLs.Mulberry leaf , metformin , streptozotocin , citrate buffer solution, sodium pentobarbital , 10% neutral formalin fixation solution , methanol, acetonitrile, formic acid , ammonium acetate, and ammonia .Isoquercitrin (no. 111809-201804), 1-deoxynojirimycin (no. 112008-201801), caffeic acid (no. 110885-201703), 7-hydroxycoumarin (no. 111739-200501), β-sitosterol (no. 110851-201909), threonine (no. 140682-201302), phenylalanine (no. 140676-201706), vitamin B1 (no. 100390-201806), resveratrol (no. 111535-201703), and scopolamine lactone (no. 110768-200504); all of the above were purchased from the China Academy of Food and Drug Inspection and Quarantine.A high-sugar and high-fat feed  was purchased from Beijing Huafucang Biotechnology Co., Ltd.A centrifuge , blood glucose meter, blood glucose test paper , rotary evaporator , constant-temperature water bath pot , ultrahigh-performance liquid chromatograph , quadrupole orbital ion mass spectrometer , desktop high-speed cryocentrifuge , freeze dryer , scroll oscillator , and a reversed-phase T3 chromatographic column .Preparation of ML powder: the MLs (2.0 kg) were soaked in deionized water (20 L) for 30 min and extracted at 85–95°C for 2.5 h. The concentrated extracts were placed into a vacuum oven and dried into powder. The powder was weighed and the yield was calculated (yield: 29.55%).Sample pretreatment of MLs of LC-MS: 0.5 g of the ML powder was weighed and placed in a volumetric flask, 50 ml of methanol was added, it was sonicated for 30 min, it was cooled down and the lost weight was made up for with methanol, it was filtered with a 0.22 μm microporous filter membrane, and then the continued filtrate was taken.Sample pretreatment of standard compounds for LC-MS:10 mg of each compound was weighed in a 10 ml volumetric flask, dissolved with methanol, and shaken well to prepare a solution with a concentration of 1 mg/ml. Of each compound solution, 1 ml was taken into a 10 ml volumetric flask, mixed into a mixed standard solution containing 10 compounds, and filtered through a 0.22 μm microporous membrane to obtain the filtrate.2O), and the gradient elution, as well as the gradient elution conditions, were as follows: 0–3 min (5% A), 3–45 min (5–85% A), 45–45.1 min (85–5% A), 45.1–50 min (5% A).The chemical composition of MLs was determined according to the optimized liquid chromatography and mass spectrometry method. A UPLC column , with a column temperature of 45°C, a flow rate of 0.3 ml/min, and an injection volume of 3 μL was used; the mobile phase of solution A (acetonitrile), solution B (0.1% formic acid in H2 (data-dependent acquisition), and a parent ion list, PIL-MS2. Various strategies, such as CID fragmentation, were applied, and the scan range was 120.00–1800.00 Da. The mass spectrometry conditions of positive ion detection modes were as follows: The spray and capillary voltages were set to 4.0 KV and 35.0 V, respectively. The tube lens voltage was 55 V, and the ion source temperature was set to 400°C. Nitrogen (purity >99.99%) was used as both the sheath gas (35 arb) and the auxiliary gas (10 arb) The mass spectrometry conditions of negative ion detection modes were as follows: The spray and capillary voltages set to 3.0 kV and 35.0 V, respectively. The tube lens was set to 55 V, and the ion source temperature was set to 300°C. Nitrogen (purity >99.99%) was used as both the sheath gas (35 arb) and the auxiliary gas (10 arb). The sample assay profiles were compared with the standard profiles, compound database, and the contents of reports in the literature to infer the structures of the measured compounds. They were also combined with a multivariate statistical analysis to determine the chemical composition of MLs.The mass spectrometry was performed with a heated electrospray ion source (HESI), positive and negative ion detection modes, a Fourier transform high-resolution full scan , a data-dependent ddMSHealthy male SD rats (body weight of 200–220 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. and raised in a specific pathogen-free animal laboratory of the Beijing University of Chinese Medicine . After adaptive feeding for 1 week, rats were randomly divided into control and T2D groups. The control rats were fed with a regular diet, and the T2D rats were fed with a high-sugar and -fat diet. After 4 weeks, the T2D rats had fasted for 12 h, and a 1% STZ citrate buffer solution  was injected intraperitoneally with a dose of 35 mg/kg. The control rats were intraperitoneally injected with the same amount of the citrate buffer solution. On the seventh and eighth day after injection, the FBG was measured by tail vein blood sampling. The T2D rats were established successfully when the FBG ≥12 mmol/L; the unqualified rats were excluded. The T2D rats were randomly divided into a T2D group, a metformin (0.2 g/kg) group, and an ML treatment (4.0 g/kg) group according to their FBG and body weight (10 rats in each group). The rats in each group were given an intragastric administration once a day for 12 consecutive weeks; the rats in the control and T2D groups were given the same amount of distilled water.After 12 weeks of administration, the rats were anesthetized by an intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg). Blood was collected from the abdominal aorta, placed at room temperature for 2 h, and centrifuged at 3500 rpm for 10 min; the upper serum was taken and stored at −80°C.Of the serum, 200 μL was put into a 1.5 ml centrifuge tube, and 800 μL of a chromatographic-grade precooled methanol/acetonitrile solution (v:v = 1:1) was added, which was mixed by a rapid vortex for 30 s, stood at 4°C for 30 min, and centrifuged at 12000 rpm at 4°C for 20 min. The supernatant, with a volume of 600 μL, was transferred to a new 1.5 ml centrifuge tube and freeze-dried. The freeze-dried sample was redissolved in 300 μL of a precooled acetonitrile aqueous solution (v:v = 1:1), and then was fully vortexed–oscillated. Samples were centrifuged at 12000 rpm at 4°C for 10 min, and 200 μL of the supernatant was transferred to a liquid bottle for a nontargeted LC-MS metabolomics analysis. Each sample, with a volume of 20 μL, was mixed into a 1.5 ml liquid vial to form a quality control (QC) sample.The samples were separated by ThermoFisher Scientific U3000 rapid liquid chromatography. Serum samples were detected with a reversed-phase T3 chromatographic column, the metabolites in samples with a weak polarity were eluted, and as much of the metabolite information in the samples as possible was collected.Samples were analyzed by a Waters ACQITY UPLC BEN, T3, 2.1 × 100 mm, 1.7 μm column. Mobile phase A is water containing 0.1% formic acid, and mobile phase B is pure acetonitrile. The sample injection volume was 3 μL, the flow rate was controlled as 0.3 ml/min, and the mass spectrum scanning mode was as follows: positive and negative ion scanning, the range was 80–1200 m/z, the capillary voltage and tube lens voltage were −110 V, the capillary temperature was 350°C, and the multistage mass spectrum collision normalized energy was 40 V, 20 V, and 10 V, respectively. Data dependency scan mode. The procedures of the liquid mass gradient in positive and negative ion modes were as follows: 0–1 min (95% A), 1–9 min (95–60% A), 9–19 min (60–10% A), 19–19.1 min (10–95% A), and 19.1–24 min (95% A). During the whole analysis process, samples were placed in an automatic sampler at 4°C. The QC samples were continuously injected on five occasions to balance the liquid mass system and then tested in sequence. For every five samples tested, a QC test was performed, and the stability of the system was evaluated based on the results of the QC samples.6, the cycle time was 50 ms, and the scanning range was 80–1200 m/z.The mass spectrometer adopted a quadrupole orbital ion trap mass spectrometer with a thermoelectric spray ion source. The ion source voltage was 3.7 kV, the capillary heating temperature was 320°C, the volumetric heating and evaporation temperatures were 300°C, the sheath gas and auxiliary gas were nitrogen, the sheath gas pressure was 30 psi, the auxiliary gas pressure was 10 psi, and the collision gas was nitrogen. The pressure was 1.5 mTorr, and the first-stage full scanning parameters were the following: the resolution was 70000, the automatic gain control target was 1×10The original data were imported into Progenesis QI (Waters) software for data preprocessing, peak alignment, deconvolution, peak extraction, multivariate statistical analysis, and identification. After the raw data were imported, the automatic mode was selected for data processing, the mass score correction was performed through Lockmass, and the QC samples were used as the reference group for peak alignment.http://www.HMDB.ca), KEGG (http://www.genome.jp/), and NIST (https://chemdata.nist.gov), and the quality errors used in searching the databases were 0.005 Da (ms1) and 15 ppm (ms2)  less than 30%, a fold change >1.2, a VIP >1, and a p-value < 0.05 were selected as differential metabolites , and SIMCA 14.1 software was used to make a volcano map of differential metabolites. Metabolic pathways were analyzed by using the Metabolites Biological Role (http://csbg.cnb.csic.es/mbrole2/) website.All of the metabolites were identified by a comprehensive scoring of their “score”, “fragmentation score”, “mass error (PPM)”, and “isotope similarity” according to databases. The databases were HMDB (pm (ms2) . The prop-values of <0.05 (*), <0.01 (**) were adopted for statistical significance; fasting blood glucose, body weight, water intake, and food intake data were analyzed using one-way ANOVA.Experimental data were expressed as mean ± standard deviation (SD), and the statistical analysis of the data was performed using SPSS 22.0 software. The MLs were detected using UPLC-Q-Exactive Orbitrap-MS, and the total ion chromatography (TIC) in positive and negative ion modes was obtained as shown in A total of 16 flavonoids were identified in MLs, including rutin  (1), que21H20O12 (ppm = −1.73), which shows its molecular ion peak in a HESI negative ion mode at m/z 463.08899, and produces m/z 301.03 plasma fragments in MS2, suggesting that isoquercitrin breaks the glycosidic bond at the C-ring 3-position hydroxyl group, generating m/z 301 [M-H-C6H11O5]- fragments, followed by m/z 271 [M-H-C6H11O5-CO]- fragments, and the cleavage behavior is shown in The elemental composition of isoquercitrin (standard compound) is C+ of compound 2 is m/z 303.05032, presumably with an elemental composition of C15H10O7 (ppm = -1.32). The secondary mass spectrum [M-H]- yielded fragment ions m/z 229.05 [M + H-2CO-HO2]+ and m/z 153.02 [M + H-2C8H6O3]+; together with the literature , pr9H8O4 (ppm = 0.06), and its molecular ion peak in a HESI negative ion mode shows m/z 179.03497, which produces m/z 135.05 fragments in MS2, suggesting that it drops a carboxyl group to produce 135.03 [M-H-COOH]- fragments, and the specific cleavage process is shown in The elemental composition of caffeic acid (standard compound) is C- of compound 17 was m/z 167.03462, presumably of elemental composition C8H8O4 (ppm = 2.16), which yielded m/z 123.04523 [M-H-OCH4]- as well as m/z 108.02171 [M-H-OCH4]- fragment ions in MS/MS; combined with the literature, compound 17 was presumed to be isovanillic acid, and the cleavage pattern is shown in The molecular ion peak [M-H]d-glucopyranoside (45), and 6-gingerol (46). The molecular ion peak [M-H]- of compound 44 was m/z 567.17242, and the secondary mass spectrum showed a high abundance of m/z 405.12036 [M-H-162]-, 243.06654, and other fragment ions, which may be oxidized in a similar manner as resveratrol; combined with chemical professional databases and the related literature , syringaresinol  (39), moterature , it is p6H13NO4 (ppm = −0.67), which shows its molecular ion peak in a HESI positive ion mode as m/z 164.09184, producing fragments of m/z 146.08, 82.07, and 69.03 in MS2, suggesting that 1-deoxynojirimycin first drops a hydroxyl group to generate a 146.08 [M + H-OH]+ fragment, followed by a 96.05 [M + H-(OH)3]+ fragment; meanwhile, 1-deoxynojirimycin may drop a CH6O4 to generate a 82.07 [M + H-CH6O4]+ fragment, followed by a 69.03 [M + H-CH6O4-CH]+ fragment , trigonelline (48), and liriodendrin (49). The elemental composition of 1-deoxynojirimycin (standard compound) is Cfragment . The cle9H6O3 (ppm = -0.12), showed its molecular ion peak in a HESI negative ion mode at m/z 161.02443, producing almost no fragmentation in MS2, and a small peak in the second mass spectrum with the molecular ion peak as the base peak, producing a small peak of 133.03 [M-H-CO]- fragmentation; the cleavage pattern is shown in A total of three coumarins were identified, including 5,7-dihydroxycoumarin (50), skimmetin (51), and scopoletin (52). The 7-hydroxycoumarin (standard compound), as a simple coumarin molecule with an elemental composition of Cl (+)-rhamnose monohydrate (56), linamarin (57), guanosine (58), ligustilide (59), phytosphingosine (60), and psychosine (61).A total of nine other classes of compounds were also identified, including cinnamyl alcohol  (53), ciOur previous study showed that MLs significantly improve the oral glucose tolerance test (OGTT), the insulin tolerance test (ITT), serum insulin, and other indexes in T2D rats, regulate blood glucose metabolism, improve glucose tolerance, enhance pancreatic islet function, improve insulin resistance, reduce blood lipid level, inhibit the expression of inflammatory factors, and improve T2D symptoms . TherefoIn this study, PCA was used to observe the overall change trend in the data of each group. The PCA results showed that all of the samples were distributed within a 95% confidence interval, indicating that the PCA model was reliable. The PCA score showed that the samples of the control and T2D groups could be separated significantly under positive and negative ions, suggesting that the metabolite level in the serum of T2D rats had significant changes and that the physiological metabolism in the serum was disturbed. The metabolites in the serum of the rats in the ML group were significantly different from those in the T2D group, and tended to recover to those of the control group, suggesting that MLs regulate the metabolic process and improve the metabolic disorder in T2D rats .p-value < 0.05), and look for metabolites whose metabolic levels are reversed by MLs. Finally, 88 endogenous differential metabolites were obtained in the serum, of which 62 were upregulated and 26 downregulated (Table S6).Then, a supervised OPLS-DA model was used to compare each group in pairs . All of A heat map analysis of the potential differential metabolites in the serum of rats was conducted. The results showed that the levels of metabolites in the serum were significantly different between the control and T2D rats, and that the levels in the ML group tended to recover to those of the control group, suggesting that MLs could correct the abnormal levels of serum metabolites in T2D rats, as shown in p value), which is also represented along the vertical axis. The horizontal axis represents the enrichment factor; the greater the enrichment factor, the higher the enrichment level of the differential metabolites in this pathway. The pathway on the upper right of the figure is the significantly affected differential pathway.Pathway enrichment was carried out for the metabolites with significant changes in the serum of rats, as shown in l-phenylalanine, l-tyrosine, l-threonine, and l-valine were significantly downregulated in T2D rats, and that the l-histidine, 4-imidazolone-5-propionoate, carbamoyl phosphate, N-acetylornithine, l-proline, serotonin, 5-hydroxyindoleacetaldehyde, creatine, and l-lysine contents in the serum were upregulated (The results showed that, after treatment with MLs, the levels of citrulline, creatine, L-histidinol, pyruvic acid, cis-aconitic acid, egulated . The metThe incidence of T2D is increasing year by year, and it is characterized by its long duration and susceptibility to diabetic complications, which place a heavy burden on society and families . T2D is MLs are the dry old leaves of Morus alba L., and are highly effective in the treatment of type 2 diabetes , but theMeanwhile, we evaluated the hypoglycemic efficacy of ML extracts in T2D rats; the results showed that ML extracts can significantly reduce FBG and improve the symptoms of diabetes in T2D rats . The serFinally, 88 differential metabolites were found in the serum. As shown in Combined with the results of the differential metabolite pathway enrichment and the references, the results show that ML extracts increased the content of histidine in the body by regulating histidine synthesis and degradation, alleviating oxidative stress, improving chronic inflammation, and alleviating the symptoms of T2D . ML extrThe experimental results showed that various lipid metabolites were significantly up-regulated in the serum of T2D rats, such as glycerol phosphocholine, plant sphingosine, and dihydroxyacetone phosphate. An abnormal lipid metabolism is beneficial to the development of T2D . DisordeIn conclusion, ML extracts contain a variety of chemical components and were able to significantly reduce fasting blood glucose in T2D rats. ML extracts may regulate multiple metabolic pathways, such as carbohydrate, amino acid, and lipid metabolism, in the body, regulate the immune state of the body, reduce oxidative stress and inflammation in the body, regulate gluconeogenesis, and increase the efficiency of fat mobilization as well as BAT thermogenesis to achieve the effect of lowering glucose and improving the symptoms of T2D.In this article, the chemical composition of ML was analyzed by LC-MS and a total of 61 compounds were identified, including flavonoids, organic acids, phenolic compounds, alkaloids and coumarins, etc. The efficacy evaluation results showed that ML extracts could significantly reduce fasting blood glucose in T2D rats and improve other symptoms of diabetes. The serum metabolomics results can conclude that ML extracts improve the symptoms of T2D by regulating lipid metabolism, carbohydrate metabolism, and amino acid metabolism, regulating immune status, reducing oxidative stress and inflammation, regulating glucose metabolism, and increasing fat mobilization as well as BAT heat production.


Researchers have proved that simple renal cysts (SRCs) might be correlated with renal dysfunction, but it is still controversial. Thus, we conducted clinical research study with large sample size and long-term follow-up to clarify the relationship between SRCs and renal dysfunction.A total of 571 SRCs patients in outpatients of nephrology department were included, we investigated the clinical characteristics of growth SRCs compared with non-growth SRCs, evaluated the incidence of renal dysfunction in SRCs and explored the risk factors of renal dysfunction in growth SRCs.. 2.4%). Renal dysfunction was significantly associated with age, female, total cholesterol, diastolic blood pressure, final maximum diameter and yearly change in maximum diameter in growth SRCs.The mean baseline age was 51.31 ± 14.37 years in the whole cohort, ranging from 19 to 79 years, and 57.6% of them were male. The median follow-up duration was 3 years, ranging from 1 to 10 years. In addition, the final maximum diameter increased 1 mm (2.74%) per year. Patients in growth SRCs group tented to have higher percentage of hypertension, hematuria, large cyst and multiple cysts compared with non-growth SRCs group. The prevalence of renal dysfunction was 15.6% after the follow-up, and the prevalence of renal dysfunction was about 10 times higher in growth SRCs group than non-growth SRCs group (23.3% vsSRCs were closely related to the decline of renal function, we recommend close follow-up for growth SRCs. Kidneys play a vital role in maintaining fluid, electrolyte, and acid–base balance. The loss of renal function could lead to internal environmental disorders and develop into cardiovascular events. Moreover, hypertension, obesity, diabetes and dyslipidemia might lead to the coexistence of cardiovascular disease and renal dysfunction . ClinicaSRCs are the most common renal cystic diseases in adults, the prevalence of SRCs was reported to be 7.2–10.5% in Chinese cohorts, and higher in males and elders , 4, 6. I2; (6) no severe complications, such as malignant diseases, autoimmune diseases, heart failure and severe liver diseases. We also excluded diabetes patients, because they might progress to diabetic nephropathy, which would significantly affect renal function. A total of 571 patients were included in the current study  18–79 years old at the initial visit; (2) diagnosed with SRCs by ultrasound and reviewed at least once per year; (3) no surgical intervention; (4) no hereditary nephropathy, such as polycystic kidney disease and tuberous sclerosis; (5) no renal dysfunction, baseline eGFR > 60 mL/min/1.73 mThe following data were collected: age, gender, systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), follow-up period, complications , hypercholesterolemia, hypertriglyceridemia, nephrolithiasis and hematuria) and laboratory examinations , total cholesterol (TC), triglyceride (TG)). The ethics committee of Guangdong Provincial Hospital of Chinese Medicine approved the study (YE2019-197-01).2). According to MDRD formula, eGFR (mL/min/1.73 m2) = 175 × (serum creatinine/88.4)−1.234 × age−0.179 [× 0.79 ]  , we defit test or Mann–Whitney U test for continuous variables, and χ2 test for categorical variables to compare the differences in demographic and clinical variables between different groups. We used multivariable logistic regression analysis to estimate risk factors associated with renal dysfunction in growth SRCs group. SPSS version 23.0 was used for all statistical analysis with two tailed P values of 0.05. Kaplan–Meier and log-rank test were used for prevalence of renal dysfunction between growth SRCs group and non-growth SRCs group, using GraphPad Prism 8.0.2.Data were presented as mean ± standard or median (interquartile range) for continuous variables, and proportions for categorical variables. We used independent-samples P < 0.001), hematuria , large cyst  and multiple cysts  compared with non-growth SRCs group. Age, SBP, DBP and final maximum diameter were significantly different between growth SRCs group and non-growth SRCs group .Among 571 patients, the mean baseline age was 51.31 ± 14.37 years, ranging from 19 to 79 years, and 329 (57.6%) of them were male. The median follow-up duration was 3 years, ranging from 1 to 10 years. Follow-up characteristics of the cohort stratified according to the development of SRCs are shown in Table 2 in the whole cohort. As shown in Table P < 0.001). The mean serum creatinine was 97.39 ± 20.90 μmol/L in growth SRCs group, and 79.82 ± 15.24 μmol/L in non-growth SRCs group. The mean eGFR was 73.47 ± 19.62 mL/min/1.73 m2 in growth SRCs group, and 93.10 ± 23.32 mL/min/1.73 m2 in non-growth SRCs group. Serum creatinine and eGFR were significantly different between growth SRCs group and non-growth SRCs group (both P < 0.001). The probability of free survival of renal dysfunction based on the development of SRCs in the Kaplan–Meier analysis showed significant differences, as shown in Fig. The mean baseline serum creatinine was 78.57 ± 16.23 μmol/L, and the mean baseline eGFR was 96.42 ± 23.63 mL/min/1.73 mP < 0.05). After adjusted for the confounding factors, using multivariable logistic regression analysis, renal dysfunction was associated with age , female , TC , DBP , final maximum diameter  and yearly change in maximum diameter   . Such diThe reliability of diagnosing SRCs by ultrasound is approximately 100% , the curKidney cortical volume would decrease and the number and size of SRCs would increase with healthy aging, accompanied with decline of nephron number and decreased renal function . TherefoThere was a significantly increased proportion of hypertension in growth SRCs, and renal dysfunction was correlated with blood pressure level in our study. Kim et al.  analyzedThe subjects of the study were all outpatients from nephrology department, which may lead to unavoidable selection bias. In addition, the current retrospective study did not exclude the effects of drugs on the growth of SRCs and it could not express the natural process of SRCs. The existence of SRCs was often overlooked due to its rare progression to significant complications. However, our research found that SRCs were closely related to the decline of renal function and the prevalence of renal dysfunction was about 10 times higher in growth SRCs group than non-growth SRCs group. We recommend close follow-up for growth SRCs.


A series of nonpharmaceutical interventions (NPIs) was launched in Beijing, China, on January 24, 2020, to control coronavirus disease 2019. To reveal the roles of NPIs on the respiratory syncytial virus (RSV), respiratory specimens collected from children with acute respiratory tract infection between July 2017 and Dec 2021 in Beijing were screened by capillary electrophoresis‐based multiplex PCR (CEMP) assay. Specimens positive for RSV were subjected to a polymerase chain reaction (PCR) and genotyped by G gene sequencing and phylogenetic analysis using iqtree v1.6.12. The parallel and fixed (paraFix) mutations were analyzed with the R package sitePath. Clinical data were compared using SPSS 22.0 software. Before NPIs launched, each RSV endemic season started from October/November to February/March of the next year in Beijing. After that, the RSV positive rate abruptly dropped from 31.93% in January to 4.39% in February 2020; then, a dormant state with RSV positive rates ≤1% from March to September, a nearly dormant state in October (2.85%) and November (2.98%) and a delayed endemic season in 2020, and abnormal RSV positive rates remaining at approximately 10% in summer until September 2021 were detected. Finally, an endemic RSV season returned in October 2021. There was a game between Subtypes A and B, and RSV‐A replaced RSV‐B in July 2021 to become the dominant subtype. Six RSV‐A and eight RSV‐B paraFix mutations were identified on G. The percentage of severe pneumonia patients decreased to 40.51% after NPIs launched. NPIs launched in Beijing seriously interfered with the endemic season of RSV. To control SARS‐CoV‐2 transmission, a series of nonpharmaceutical interventions (NPIs), including social withdrawal, school closures, wearing masks, travel restrictions, personal hygiene improvements, and border closures, have been implemented and are widespread in different countries.These NPI measures have also affected the community transmission of endemic diseases and the seasonal circulation patterns of other respiratory viruses,RSV is the most important viral cause of acute respiratory tract infections (ARTI) in children and is a major pathogen leading to infant death worldwide.To investigate the changes in endemic patterns of RSV infection in pediatric patients under the pressure of NPIs of COVID‐19 in Beijing, launched on January 24, 2020, clinical specimens from children with ARTI in Beijing from July 2017 to December 2021 were retrospectively analyzed for epidemic characteristics and demographic and clinical features of RSV infection, focused on the data comparison before and after NPIs launched.22.1g, 10 min). Partial supernatant was used for viral nucleic acid extraction, and the remaining supernatant was stored at −80°C for future use.Clinical respiratory specimens were collected from children with ARTI visiting the affiliated Children's Hospital, Capital Institute of Pediatrics (Beijing) from July 1, 2017, to December 31, 2021, for respiratory pathogen screening using a capillary electrophoresis‐based multiplex PCR (CEMP)‐compatible assay‐Respiratory Pathogen Multiplex Detection Kit (Ningbo HEALTH Gene Technologies Ltd.).2.2Total nucleic acid (DNA and RNA) was extracted from 140 µl supernatant of each collected specimen using the QIAamp MinElute Virus Spin Kit (Qiagen GmbH) according to the manufacturer's instructions.2.32, and buffer were included. Nucleic acid extracts from clinical specimens were amplified and then subjected to capillary electrophoresis on a GeXP capillary electrophoresis system (Sciex). Signals of the 15 labeled PCR products were measured by fluorescence and separated into 14 kinds of size fragments, as well as human DNA and human RNA: Flu A 105 nt , Flu B 212.7 nt, AdV 110.2/113.9 nt (representing different subtypes), human bocavirus (HBoV) 121.6 nt, Rh 129.6 nt, human parainfluenza virus 181.6 nt, chlamydia (Ch) genus 190.5 nt, hMPV 202.8 nt, Mycoplasma pneumoniae (Mp) 217 nt, human coronavirus (HCoV) 265.1 nt, and RSV 280.3 nt.According to the manufacturer's instructions in the CEMP Assay Kit for multiplex polymerase chain reactions (PCRs), deoxynucleoside triphosphates (dNTPs), MgCl2.4The extracted nucleic acid of RSV‐positive specimens was used as a template to synthesize complementary DNA (cDNA) by a conventional two‐step reverse transcription reaction with random primers according to the manufacturer's instructions. The moloney murine leukemia virus (M‐MLV) reverse transcriptase (200 U/μl) was used . Ribonuclease inhibitor  and dNTP  were added separately to mix.For RSV subtyping, cDNA obtained by reverse transcription was used as the PCR template. Forward (P4: 5′‐TGGGACACTCTTAATCAT‐3′) and reverse primers  were used.To amplify the full‐length RSV G gene for phylogenetic analysis, nested PCR was used. Primers S4298 (5′‐TGGCCYTAYTTTACACTAATAC‐3′) and F164 (5′‐GTTATGACACTGGTATACCAAC‐3′) in the first round produced a 1489 bp fragment. Primers S4373 (5′‐ATCTCCATCATGATTGCAAT‐3′) and F5763 (5′‐ATAGCCTTTGCTAACTGCAC‐3′) were used in the second round, producing a 1390 bp fragment.2.5For all the sequences of RSV Subtypes A and B, CD‐hit was introduced to trim the sequences with high similarity (identity >97.5%).2.6When a mutation has been fixed in a population and occurs independently in a new strain many times, reducing the likelihood that the mutation was caused by epistatic effects, parallel and fixed (paraFix) mutations occurred. The detection of paraFix mutations was performed with the R package sitePath.2.7To analyze the clinical features of RSV infection, hospitalized children with a single RSV infection and detailed clinical data were enrolled. Clinical data, such as age, sex, oxygen saturation, respiratory rate (RR), body temperature, and chest radiography or computerized tomography (CT), were collected. The severity was evaluated according to the “Guidelines for the Diagnosis and Treatment of Community‐acquired Pneumonia in Children (2019 edition)” issued by the “China National Health Commission.” Severe community‐acquired pneumonia cases were defined as meeting any of the following criteria: (1) poor general condition, (2) disturbance of consciousness, (3) hypoxemia: cyanosis, rapid respiration, RR ≥ 70 breaths/min (infant), RR ≥ 50 breaths/min (1‐year‐old or older), assisted breathing , intermittent apnea, oxygen saturation <92%, (4) hyperthermia, persistent hyperthermia for more than 5 days, (5) dehydration or food refusal, and (6) chest radiograph or CT ≥ 2/3 side lung infiltration, lobar lung infiltration, pleural effusion, pneumothorax, atelectasis, lung necrosis, and lung abscess.2.8x¯ ± s, while those with a skewed distribution are presented as the median and interquartile range. χ2 and rank sum tests were used for statistical analysis. Statistical significance was set at p < 0.05.January 24, 2020 was set as the boundary before and after the NPIs launched in Beijing. SPSS Statistics  software was used to process the data. In the descriptive analysis, measurement data with a normal distribution are presented as 33.1From July 1, 2017 to December 31, 2021, a total of 10 588 specimens from children with ARTI were collected, and 9198 were finally included after excluding those with short‐term repeated testing and incomplete common clinical information. There were 5400 males and 3798 females, with a mean age of 3.229 (0.001–17.855) years. The positive rates of RSV were 13.56% (1247/9198), hMPV, 2.76% (254/9198), AdV, 3.53% (325/9198), Rh, 16.46% (1514/9198), HBoV, 6.87% (632/9198), HCoV, 0.91% (84/9198), Flu A, 1.64% (151/9198) and Flu B, 0.87% (80/9198), and PIV, 8.12% (747/9198). The positive rates of Mp and Ch were 11.82% (1088/9198) and 0.57% (52/9198), respectively.By using January 24, 2020, as the dividing line Figure , when LeThere were 890 cases among 1247 positive for RSV with a single RSV pathogen, including 521 males and 369 females, with a median age of 0.60 (0.17–1.88) years. In addition, 357 cases were infected with multiple pathogens, 215 males and 142 females, with a median age of 1.19 (0.42–3.11) years. Among these cases, RSV and Rh coinfection accounted for 31.09% (111/357), RSV and HBoV accounted for 13.17% (47/357), and RSV and AdV accounted for 9.80% (35/357). There were 48 cases of triple infection and 13 cases of quadruple infection or above.3.2By subtyping PCR for 1247 RSV‐positive specimens, subtypes of RSV were identified among 1150 specimens Figure , in whic3.3Full‐length G gene sequences of 302 RSV‐A and 348 RSV‐B were obtained by reverse transcription and nested PCR assays. Before NPIs launched, there were 174 for RSV‐A and 108 for RSV‐B, while after NPIs launched, there were 128 for RSV‐A and 240 for RSV‐B. Phylogenetic analysis revealed that all RSV‐A sequences in the study belonged to the ON1 genotype Figure , which c3.4Sequences of 61397  and 86 673  were used as references before the NPIs launched to analyze amino acid mutants on glycoprotein G. For RSV‐A, 155 and 105 sites and for RSV‐B, 103 and 157 sites were mutated before or after the NPIs launched, respectively. Loci with mutation rates greater than 10% are listed separately and presented in Table Sequences of RSV‐A and RSV‐B were aligned and compared with all genotypes in the evolution of viruses. Specific mutations of ON1 in Beijing were T113I, V131D, N178G, H258Q, and H266L. No specific mutation of BA9 was found in Beijing. Furthermore, mutations in parallel and fixed (paraFix) patterns were found by identifying the polymorphism clades in each phylogenetic pathway in global circulation . In view of the developmental status and growing environment of children, the five groups were gradually distinguished by age. The highest number of RSV infections was observed among children aged 28 days to 1 year , and the lowest was observed among children aged >6 years . From before to after NPIs launched, the distribution of RSV decreased from 69.83% (250/358) to 43.04% (102/237) among those aged 28 days to 1 year (p < 0.001), while it increased from 13.69% (49/358) to 28.27% (67/237) (p < 0.001) and 8.38% (30/358) to 24.05% (57/237) (p < 0.001) among children aged >1–3 years and >3–6 years, respectively.Among 890 cases with a single RSV infection determined in the study, 595 cases were inpatients with detailed clinical data. There were 360 males and 235 females, with a median age of 0.48 (0.16–1.81) years. The clinical characteristics of RSV‐infected children in the hospital before and after NPIs launched were compared on the basis of sex, age, and severity of pneumonia, the criteria for grouping . Furthermore, we observed changes in sex and age in patients with different severities of pneumonia  and 61.22% (30/49) to 37.31% (25/67) (p = 0.0108). In detail, hospitalization duration, intensive care unit(ICU) admission, and clinical outcome were counted to evaluate the severity of ARTI (Table p = 0.1200) from before to after NPIs launched, while the proportion of ICU admission also dropped from 73.6% (176/239) to 59.4% (57/96) (p = 0.0103), and 4.2% (4/96) more children were cured during the hospital stay after NPIs launched.Notably, the percentage of severe pneumonia patients also changed from 66.76% 239/358) to 40.51% (96/237)  and November (2.98%) under the Level II or III NPIs. It has been confirmed that droplets and aerosols are common ways of transmitting respiratory viruses, such as SARS‐CoV‐2 and RSV, while direct contact is another common route of RSV transmission. Isaacs's et al.However, the monthly positive rates of RSV then increased quickly to 22.34% in December 2020 and 23.71% in January 2021 with the relaxation of NPIs in Beijing and then decreased to 12.03% in February and 10.40% in March 2021. Abnormally, the positive rates of RSV increased again in April  and May 2021  and then stayed at approximately 10% during the summer from June to September 2021, which was followed by a normal epidemic season of RSV from October 2021, with the highest positive rate in December 2021 , although far below the corresponding rates  in 2017, 2018, and 2019. This is consistent with the delayed surge of RSV in other areas.From 2017 to 2021, the alternating predominant‐subtyping epidemic model in Beijing of RSV was AABBA Figure . In 2021Full‐length sequencing of the G protein was performed in our experiment. We referenced the earlier ON1 and BA1 sequences as templates to analyze the changes in the overall mutation rate of G protein amino acid sites. We found that the overall mutation number of the Subtype A virus decreased from 155 to 105 after NPIs launched, while the mutation number of the Subtype B virus increased from 103 to 157, showing the completely opposite trend. This is a very interesting result. We speculate that this may be related to the alternate epidemic of Subtypes A and B to a certain extent. It was reported that the pre‐existing antibodies in the population have great pressure on the virus variation.For the specific amino acid sites, such as T113I, V131D, L230P/T, V236I, K244R, F280Y, K286E, and K320T in RSV‐A and A131T, T137I, P201T, Y213D, P217L, G241D, V269A, P284L, T288I, and P301A in RSV‐B, the replacement of the previous loci was basically completed. Nevertheless, similar important mutation sites have been reported in the other provinces of China and other countries, which also indicates that samples from different regions may vary to a certain extent. To further verify the adaptability of these mutations, paraFix analysis was performed. The paraFix mutations occur when a mutated site has been fixed in a population, reducing the likelihood caused by epistatic effects. This method has been validated in the presence of important mutant strains of SARS‐CoV‐2.In addition, clinical characteristics were also influenced by the NPIs. The most prominent age group of RSV infection was 28‐days to 1‐year‐old children. After NPIs launched, the distribution of RSV decreased from 69.83% to 43.04%. This may be due to the decrease in RSV exposure caused by various NPIs during the period of COVID‐19 in Beijing and the accumulation of RSV‐susceptible people resulting in the delay or even outbreak of the epidemic. According to the data on hospitalization duration, ICU admission, and clinical outcome, we found that the severity of ARTI caused by RSV decreased significantly, which was similar to the results in Shanghai, China.In this study, we summarized the epidemiology and clinical phenotype changes of RSV from 2017 to 2021 in Beijing. The pressure of NPIs of COVID‐19 affected the transmission pattern of RSV, which led to the delay of epidemic seasons in 2020 and the abnormal increase in RSV‐positive rates in the summer of 2021. The replacement of subtypes was surveilled from B to A in the summer first. Furthermore, the paraFix mutations of important amino acid sites are of great significance to the development of drugs and vaccines under the new situation. The age distribution of RSV infection was changed due to the NPIs of COVID‐19, and the clinical data showed the lighter phenotype of ARTI. The surveillance of respiratory‐associated viruses should be maintained under the COVID‐19 strategy to avoid unexpected outbreaks of other diseases.Ming‐Li Jiang, Yan‐Peng Xu, Hui Wu, and Hang‐Yu Zhou performed the experiments. Lin‐Qing Zhao conceived and designed the experiments. Ru‐Nan Zhu, Yu Sun, Dong‐Mei Chen, Fang Wang, Yu‐Tong Zhou, Qi Guo, Aiping Wu, and Yuan Qian contributed reagents/materials/analysis tools. Yan‐Peng Xu and Lin‐Qing Zhao wrote the paper. Aiping Wu, Yuan Qian, Hang‐Yu Zhou, and Lin‐Qing Zhao reviewed the manuscript. Lin‐Qing Zhao is the critical reviewer for this manuscript and made the final decision on the manuscript. All authors have read and approved the final manuscript.The authors declare no conflict of interest.The original study was approved by the Ethics Committee of the Capital Institute of Pediatrics  and was a retrospective and prospective study. The ethical committee voted that written informed consent was not required for respiratory syncytial virus RNA‐positive specimens collected retrospectively.Supplementary information.Click here for additional data file.


The main objective was to characterize behavioral changes in metritic primiparous cows treated with chitosan microparticles (CM) or ceftiofur (CEF). A secondary objective was to compare behavioral patterns of metritic cows with nonmetritic (NMET) cows. Nulliparous Holstein cows (n = 311) were fitted with a neck-mounted automated health-monitoring device (AHMD) from −21 to 60 d relative to calving. Cows diagnosed with metritis (d 0), characterized by watery, fetid, red-brownish uterine discharge within 21 d in milk were assigned randomly to CM (n = 45), intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water on d 0, 2, and 4; CEF (n = 47), subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid on d 0 and 3; and control , no treatment. For comparison, NMET cows (n = 180) were matched with metritic cows according to age at calving and calving date. Postdiagnosis, there was an effect of treatment and an interaction between treatment and time on rumination and activity. The interaction showed that CM had lesser rumination than CEF from d 1 to 11, d 18, and d 20; CM had lesser rumination than CON from d 2 to 8; and CEF was not different from CON. The interaction showed that CM had lesser activity than CON on d 2, from d 6 to 11, and d 13 to 14; CM was not different from CEF; and CEF had lesser activity than CON on d 8, 9, 13, and 14. Prediagnosis, cows in CM, CEF, and CON had lesser rumination and activity than cows in NMET. Postdiagnosis, cows in CM, CEF, and CON had lesser rumination than NMET from d 0 to 2 and had lesser activity than NMET from d 0 to 5. In summary, CM decreased rumination and activity compared with CON, which indicates a negative systemic effect of CM. This may be associated with exacerbated inflammation in the uterus. Additionally, metritic cows had decreased rumination and activity prediagnosis, which may allow for the use of AHMD for metritis diagnosis. AHMD), which have been considered an alternative to identification of sick animals  for the treatment of metritis and observed detrimental effects on milk yield, fertility, and survival when compared with ceftiofur-treated or no treatment. The decrease in survival was mostly a consequence of increased culling due to failure to cure metritis, pelvic inflammation, peritonitis, and mass in the pelvis, suggesting that CM resulted in exacerbated inflammation in the uterus.A derivate of chitin, chitosan is a biopolymer part of the exoskeleton of arthropods and cell walls of fungi and yeast that has broad-spectrum antimicrobial activity . de OlivNMET) cows. Our objective was to characterize the rumination and activity of metritic cows treated with CM and ceftiofur (CEF) and cows left untreated (CON), using AHMD. A secondary objective was to compare behavioral patterns of metritic cows with NMET cows.Based on the negative effects observed by Cows used in this study were a subset of those enrolled in a larger experiment that evaluated the effect of different treatments on cure of metritis . All aniCows were examined for diagnosis of metritis at 5, 7, and 9 DIM as previously described . Cows wiOn d 12, all cows in the CM, CEF, and CON treatments were re-examined to determine cure of metritis. Treatment success and cure occurred when the vaginal discharge on d 12 was mucoid and not fetid and cows did not receive antimicrobial escape therapy. All metritic cows were eligible to receive non-antimicrobial and antimicrobial escape therapy after d 0 if they exhibited signs of dehydration, anorexia, and weakness, or persistent rectal temperature ≥39.5°C. Antimicrobial escape therapy was determined by farm personnel and could include penicillin G procaine , ampicillin trihydrate , or ceftiofur hydrochloride .P > 0.10) existed before treatment assignment. The models included the fixed effects of treatment, time (day relative to diagnosis), and the interaction between treatment and time. The effect of treatment on rumination and activity postdiagnosis was analyzed using similar models, but with the addition of rumination and activity data on d −1 as a covariate. When a main effect of treatment was observed, multiple comparisons were performed using Tukey adjustments. When an interaction between treatment and time was observed, multiple comparisons within day were performed using the SLICE option. The effect of treatment on the proportion of cows receiving escape therapy was analyzed by chi-squared test using the FREQ procedure of SAS. A separate analysis was performed to compare the treatments (CM vs. CEF vs. CON) with the NMET group as reference. When an effect of group was observed, multiple comparisons were performed using Dunnett adjustments, with the NMET group as reference. Statistical significance was defined as P ≤ 0.05 and statistical tendencies as 0.05 < P ≤ 0.10.Rumination and activity data, pre- (d −7 to −1) and post- (d 0 to 21) diagnosis were analyzed separately, excluding prepartum data. The association between treatment (CM vs. CEF vs. CON) and rumination and activity prediagnosis was analyzed by ANOVA for repeated measures using the MIXED procedure of SAS version 9.4 , and no differences (P ≤ 0.05) for CM than CEF and CON (80.0 vs. 61.7 vs. 59.0%). Administration of escape therapy that included antimicrobials was greater for CM than CEF , but there was no difference between CM and CON . There was no difference (P = 0.59) in the interval from diagnosis to receiving antimicrobial escape therapy between treatments, and the mean (±SE) was 6.5 ± 0.4, 7.7 ± 0.3, and 5.8 ± 0.3 d for CM, CEF, and CON, respectively.The percentage of cows diagnosed with metritis that were diagnosed as cured 12 d after enrollment were 47% (21/45), 70% (33/47), and 56% (22/39) for CM, CEF, and CON, respectively. Administration of escape therapy with any therapy was greater (P < 0.01) on rumination and activity. The effect of treatment on rumination showed that cows in CM had (P < 0.01) lesser rumination (432 ± 11 min/d) than CEF (484 ± 10 min/d), and tended to have (P = 0.06) lesser rumination than CON (469 ± 12 min/d). There was no difference (P = 0.63) in rumination postdiagnosis between CEF and CON. There was an interaction (P < 0.05) between treatment and time on rumination postdiagnosis, and the interaction showed that CM had lesser (P < 0.05) rumination than CEF from d 1 to 11, d 18, and d 20; CM had lesser (P ≤ 0.05) rumination than CON from d 2 to 8, and CEF was not different from CON (P < 0.05) lesser activity (628 ± 15 arbitrary units) than CON (684 ± 15 arbitrary units). There was no difference (P = 0.60) in activity postdiagnosis between CM and CEF. There was an interaction (P < 0.05) between treatment and time on activity, and the interaction showed that CM had lesser (P ≤ 0.05) activity than CON on d 2, from d 6 to 11, and d 13 to 14; CM was not different from CEF; and CEF had lesser (P ≤ 0.05) activity than CON on d 8, 9, 13, and 14  and prediagnosis rumination and activity. Cows in CM, CEF, and CON had lesser (P ≤ 0.05) rumination and activity than NMET cows. Rumination for CM, CEF, CON, and NMET was 308 ± 14, 314 ± 13, 275 ± 15, and 356 ± 7 min/d. Activity for CM, CEF, CON, and NMET was 694 ± 21, 659 ± 19, 650 ± 22, and 749 ± 12 arbitrary units, respectively. Prediagnosis, there was no interaction between group and time (P > 0.10) on activity and rumination. Postdiagnosis, there was a main effect of group (P < 0.01) on rumination and activity. Cows in CM and CON had lesser (P ≤ 0.05) rumination than cows in NMET (492 ± 5 min/d), and NMET was not different from CEF. There was an interaction (P < 0.01) between groups and time on rumination postdiagnosis, and the interaction showed that CM had lesser (P ≤ 0.05) rumination than NMET from d 0 to 13, and from d 15 to 20; CEF had lesser (P ≤ 0.05) rumination than NMET from d 0 to 2, and d 10 to 11; and CON had lesser (P ≤ 0.05) rumination than NMET from d 0 to 4, and from d 17 to 21 (P ≤ 0.05) activity than cows in NMET (695 ± 8 arbitrary units), and NMET was not different (P = 0.34) from CON. There was an interaction (P < 0.01) between group and time on activity postdiagnosis, and the interaction showed that CM had lesser (P ≤ 0.05) activity than NMET from d 0 to 14; CEF had lesser (P ≤ 0.05) activity than NMET from d 0 to 5, and d 14 to 15, and CON had lesser (P ≤ 0.05) activity than NMET from d 0 to 5, and d 19 (According to the model in which CM, CEF, CON, and NMET were included, there was an association between group (17 to 21 . Cows inand d 19 .In the current study, we compared the rumination and activity in metritic cows treated with CM or CEF with untreated cows using a subset of cows from a previous study . As expeRumination is stimulated by mild tactile stimulation and low to moderate distension of the rumen by the ruminal contents on the tension receptors in the luminal surface of the reticulorumen . TherefoInterestingly, cows in CEF had similar rumination postdiagnosis compared with CON, despite CEF having been reported to have greater cure rate  and incrSimilar to the results for rumination, CM had decreased activity compared with CON. Interestingly, CON had a faster recovery in activity than CEF, despite the reported increase in cure rate and milk yield for CEF compared with CON . We explOur results also revealed that cows diagnosed with metritis had decreased rumination and activity compared with cows not diagnosed with metritis, particularly before diagnosis. Similar to our findings, others have also observed reduced rumination and activity before and after metritis diagnosis .In conclusion, CM used as described herein decreased rumination and activity compared with CON, which indicates a negative systemic effect of CM. This corroborates and complements our previous observations in the larger study evaluating cure and performance. Additionally, metritic cows had decreased rumination and activity prediagnosis, which may allow for the use of AHMD for diagnosing metritis.