Objective To investigate the underlying mechanism of abnormal specification of spermatogonial stem cells(SSCs)in male mice following Rb1 conditional knockout in mitotic-arrested prospermatogonia.Methods ① R language was used to analyze the single-cell RNA sequencing(scRNA-seq)data of prospermatogonia derived from postnatal day 0.5(P0.5)male mice through the gene expression omnibus(GEO)public database(accession number:GSE124904).② Mitoti-arrested prospermatogonia Rb1 conditional knockout(cKO)mice as well as Rb1 cKO mice with Id4-gfp transgene were generated using Vasa-Cre mice crossed with Rb1flox/flox or Id4-gfpTg;Rb1flox/flox mice.PCR was employed to detect the deletion of Rb1 in order to distinguish the control and cKO male mice.The testes of male mice(n=3～8)within a few days after birth were collected.After that,flow cytometry was applied to divide the ID4-GFP cells into 3 communities based on the GFP fluorescence intensity,and then detect the number of cells and cell cycle in each community.③ Germ cell proliferation(Ki67 positive,Ki67+),SSCs specification(FOXO1 nuclear transition),and germ cell differentiation(STRA8+)were detected with immunofluorescence staining.④ TUNEL staining was utilized to detect cell apoptosis.Results ① The results of scRNA-seq showed that in the two set clusters of prospermatogonia,the prospermatogonia that further specifies to generate SSCs had enriched genes that are associated with cell proliferation.② Germ cell proliferation assay indicated that the average ratio of Ki67+germ cells in the testicular cross-section of the cKO mice was significantly higher than that of the control mice at P2.5[(46.10±6.21)%vs(11.22±3.27)%,P<0.01].③ Flow cytometry revealed that,among the brightest community of ID4-GFP cells,the percentage of the cells at S phase was obviously higher in the testicular cells derived from the cKO mice when compared to the control mice at P2.5[(12.05±1.22)%vs(5.05±1.46)%,P<0.05].④ TUNEL staining displayed that cell apoptosis was detected in the testicular cross-section of cKO mice rather than that of the control mice.⑤ The results of SSCs specification exploration showed that statistical difference was observed in the percentage of cytoplasmic FOXO1 in the testicular cross-section between the control and cKO mice[(20.57±2.15)%vs(45.08±2.45)%,P<0.01].Conclusion Rb1 knockout in mitotic-arrested prospermatogonia disrupts their postnatal cell cycle re-enter and induces cell apoptosis,which further results in abnormal SSC specification.

Objective To explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.Methods Cynomolgus monkeys were randomly divided into positive and negative control groups(a total of 3 monkeys,with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group)and the subnormothermic perfusion group(n=3).The positive control group was directly sampled 1 hour after circulatory arrest,while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest.The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest.Before perfusion,cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys.After the start of perfusion,the levels of routine blood indicators in the perfusate were detected at 0,1,2,3,4,5 and 6 hours.Blood oxygen saturation was recorded,and the levels of Na+,K+,Ca2+,glucose and blood pH in the perfusate were measured,as well as the levels of IgG and IgM in the perfusate.After 6 hours of perfusion,the water content of the brain tissue was measured.Nissl staining was performed on the frontal cortex and hippocampal regions,and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein(GFAP),ionized calcium-binding adapter molecule 1(Iba1)and neuronal nuclear antigen(NEUN).Results The cross-matching results between the six genetically modified pig and the cynomolgus monkeys were negative.The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion,and the hemoglobin level showed a downward trend at 1,3,5 and 6 hours.The number of white blood cells and platelets decreased at all time points.The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%-98%,and the levels of blood oxygen saturation,Na+,Ca2+,glucose and pH were stable,while the K+level first increased and then decreased.There was no significant difference in the levels of IgG and IgM before and after perfusion.The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group(P<0.001).Nissl staining results showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity,with no significant increase in enlarged and deformed cells.In the hippocampal CA1 region,there was a slight increase in enlarged and deformed cells,and a few cells with undamaged structures showed reduced cell size.In the hippocampal dentate gyrus,fewer granule neurons had compromised structural integrity,with increased cell edema.NEUN immunofluorescence staining showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states,with clear axons.The granule cells in the hippocampal dentate gyrus were well preserved,but the nuclei were less well protected.GFAP immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons.Iba1 immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had thicker and fewer protrusions.Conclusions Compared with the positive control group,subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increases brain tissue edema in cynomolgus monkeys,but better preserves the morphological integrity of neurons and glial cells.The protective effects may be related to the continuous oxygen and energy supply,maintenance of ion homeostasis and perfusate pH,reduced rejection,and low metabolic state of the whole brain.

Objective To explore the protective effects of genetically modified porcine erythrocyte suspension as a subnormothermic normoxic mechanical perfusate on hypoxic-ischemic brain injury in cynomolgus monkeys caused by traumatic hemorrhage.Methods Cynomolgus monkeys were randomly divided into positive and negative control groups(a total of 3 monkeys,with 3 left cerebral hemispheres as the positive control group and 3 right cerebral hemispheres as the negative control group)and the subnormothermic perfusion group(n=3).The positive control group was directly sampled 1 hour after circulatory arrest,while the negative control group was placed at subnormothermic conditions for 6 hours after circulatory arrest.The subnormothermic perfusion group underwent 6 hours of subnormothermic normoxic mechanical perfusion of the bilateral common carotid arteries of the cynomolgus monkey hypoxic-ischemic brain injury model using genetically modified porcine erythrocyte suspension 1 hour after circulatory arrest.Before perfusion,cross-matching experiments were conducted between the six genetically modified pig and the cynomolgus monkeys.After the start of perfusion,the levels of routine blood indicators in the perfusate were detected at 0,1,2,3,4,5 and 6 hours.Blood oxygen saturation was recorded,and the levels of Na+,K+,Ca2+,glucose and blood pH in the perfusate were measured,as well as the levels of IgG and IgM in the perfusate.After 6 hours of perfusion,the water content of the brain tissue was measured.Nissl staining was performed on the frontal cortex and hippocampal regions,and immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein(GFAP),ionized calcium-binding adapter molecule 1(Iba1)and neuronal nuclear antigen(NEUN).Results The cross-matching results between the six genetically modified pig and the cynomolgus monkeys were negative.The number of red blood cells in the perfusate decreased significantly at 3 hours of perfusion,and the hemoglobin level showed a downward trend at 1,3,5 and 6 hours.The number of white blood cells and platelets decreased at all time points.The blood oxygen saturation in the subnormothermic perfusion group remained stable at 95%-98%,and the levels of blood oxygen saturation,Na+,Ca2+,glucose and pH were stable,while the K+level first increased and then decreased.There was no significant difference in the levels of IgG and IgM before and after perfusion.The water content of brain tissue at the end of perfusion in the subnormothermic perfusion group was significantly higher than that in the positive control group(P<0.001).Nissl staining results showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex of the subnormothermic perfusion group maintained better morphological integrity,with no significant increase in enlarged and deformed cells.In the hippocampal CA1 region,there was a slight increase in enlarged and deformed cells,and a few cells with undamaged structures showed reduced cell size.In the hippocampal dentate gyrus,fewer granule neurons had compromised structural integrity,with increased cell edema.NEUN immunofluorescence staining showed that compared with the positive control group,the pyramidal neurons in the prefrontal cortex and hippocampal CA1 region of the subnormothermic perfusion group had better morphological states,with clear axons.The granule cells in the hippocampal dentate gyrus were well preserved,but the nuclei were less well protected.GFAP immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had sparser protrusions that were more tightly associated with neurons.Iba1 immunofluorescence staining showed that compared with the positive control group,the subnormothermic perfusion group had thicker and fewer protrusions.Conclusions Compared with the positive control group,subnormothermic normoxic mechanical perfusion with genetically modified porcine erythrocyte perfusate increases brain tissue edema in cynomolgus monkeys,but better preserves the morphological integrity of neurons and glial cells.The protective effects may be related to the continuous oxygen and energy supply,maintenance of ion homeostasis and perfusate pH,reduced rejection,and low metabolic state of the whole brain.

The movement of the eyebrows primarily depends on four muscles: the frontalis, procerus, orbicularis oculi, and corrugator supercilii. As the face ages, the strength of these four brow muscles gradually becomes unbalanced, the fat pads shift, and skin elasticity decreases, often resulting in brow ptosis and periorbital wrinkles. Due to the individual differences in the distribution of eyebrow muscles and fat pads, clinicians need to master the anatomical structure of the eyebrow before designing a personalized treatment for patients to achieve the ideal eyebrow shape and perieyebrow status. This article elaborated on the anatomy of the eyebrows, analyzed and summarized the mechanisms behind the formation of brow ptosis and periorbital wrinkles, and discussed treatment method to guide clinicians in selecting more precise and less invasive brow cosmetic techniques to achieve the ideal eyebrow shape and peri-eyebrow condition.

OBJECTIVE To study the effects of Tongxie yaofang reducing colon hypermotility in irritable bowel syndrome with diarrhea （IBS-D） rats by regulating gut microbiota-enteric nervous system （ENS）-muscularis macrophages （MM） crosstalk. METHODS Forty newborn male SD rats were randomly divided into control group， model group， TCM group [Tongxie yaofang 2.68 g/（kg·d）， calculated by raw material]， and positive control group [Live combined bifidobacterium and lactobacillus tablets 0.27 g/（kg·d）]， with 10 rats in each group. Except for the control group， the IBS-D model of liver stagnation and spleen deficiency syndrome was induced in the other 3 groups with the method of mother-child separation+chronic restraint+Folium Sennae- induced diarrhea. After modeling， the administration groups were given relevant drug liquid intragastrically， once a day， for consecutive 2 weeks. At the end of modeling and after administration， the fecal properties （the incidence and the rate of loose stools）， colonic motility （colon emptying time）， and visceral sensitivity [abdominal withdrawal reflex （AWR） scores under different pressures] of rats were observed in each group. The concentration of lipopolysaccharide （LPS） in serum was detected after medication， and the expressions and distribution of bone morphogenetic protein 2 （BMP2）， colony stimulating factor 1 （CSF1） and Toll-like receptor 4 （TLR4） in colon tissue were detected； the diversity， species composition and differences of gut microbiota were also determined. RESULTS At the end of modeling， compared with the control group， all rats of the other three groups suffered from loose stools （100%）， the rate of loose stools and AWR scores at different pressures increased significantly， and colon emptying time was shortened significantly （P＜0.01 or P＜0.05）. The incidences of loose stools were 20% in TCM group and 80% in the positive control group； the rate of loose stools and AWR scores at different pressures， serum concentration of LPS and protein expressions of CSF1 and TLR4 in muscle layer of colon tissue in TCM group significantly decreased， compared with the model group； colon emptying time， the average optical density of BMP2 protein in muscle layer of colon tissue， and the indexes of Chao 1， Shannon and Faith’s PD and Simpson E-mail：772699670@qq.com index of rats in TCM group were all prolonged or increased significantly， compared with the model group （P＜0.01 or P＜ E-mail：aiwangzi312@163.com 0.05）. The relative abundance ratio of Firmicutes/Bacteroidetes， from low to high， was in the order of TCM group， control group， positive control group and model group； the species composition of gut microbiota in TCM group was closer to control group， with dominant bacterial genera including Prevotella and Blautia. CONCLUSIONS Tongxie yaofang can regulate the expressions of BMP2 and CSF1， the key proteins of gut microbiota-ENS-MM crosstalk， by changing the gut microbiota， thus alleviating the abnormal hyperfunction of colon motility in IBS-D rats.

The movement of the eyebrows primarily depends on four muscles: the frontalis, procerus, orbicularis oculi, and corrugator supercilii. As the face ages, the strength of these four brow muscles gradually becomes unbalanced, the fat pads shift, and skin elasticity decreases, often resulting in brow ptosis and periorbital wrinkles. Due to the individual differences in the distribution of eyebrow muscles and fat pads, clinicians need to master the anatomical structure of the eyebrow before designing a personalized treatment for patients to achieve the ideal eyebrow shape and perieyebrow status. This article elaborated on the anatomy of the eyebrows, analyzed and summarized the mechanisms behind the formation of brow ptosis and periorbital wrinkles, and discussed treatment method to guide clinicians in selecting more precise and less invasive brow cosmetic techniques to achieve the ideal eyebrow shape and peri-eyebrow condition.

Abstract: Objective　To understand the epidemiological characteristics of the migratory bird population with hypertension in Hainan and to provide a basis for the health management of the migratory bird population. Methods　One hundred and eighty migratory birds with hypertension who had lived in in the Mangrove Bay community of Chengmai, Hainan for 3 months or more were selected as the study population. Demographic data, including gender, age, ethnicity, occupation, medication use, family history of hypertension, hometown living environment, Hainan living environment (presence of water area and greenbelt around the residence, type of water area and greenbelt), smoking and alcohol consumption, and height and weight were collected by questionnaire and physical examination to investigate the epidemiological characteristics of the study subjects, and unconditional dichotomous logistic regression analysis was used to identify factors affecting the reduction of blood pressure among the hypertensive migratory bird population before and after taking medication in Hainan. Results　A total of 180 study subjects were included, including 77 (42.8%) males and 103 (57.2%) females, with 78 (43.3%) subjects aged ≤65. The hypertension levels were: 87 (48.3%) had normal high blood pressure, 56 (31.1%) had grade 1 hypertension and 37 (20.6%) had grade 2 hypertension. The higher the blood pressure classification, the lower the percentage of migratory individuals whose blood pressure dropped during their stay in Hainan. Age, hypertension classification, dietary habits, living environment, body mass index (BMI), and alcohol consumption were associated with a decrease in blood pressure during Hainan among the study participants who took hypertension medication regularly. Salty diet (OR=2.778, 95%CI:1.070-7.213, P=0.036) and alcohol consumption (OR=2.686, 95%CI:1.042-6.925, P=0.041) were unfavorable factors for lower diastolic blood pressure before taking medication; overweight (OR=3.487, 95%CI:1.306-9.310, P=0.013) was an unfavorable factor in the reduction of diastolic blood pressure after taking medication. Conclusion　The blood pressure reduction is more significant in migratory individuals in migratory individuals under 65 years old, with a light diet, no alcohol consumption and normal BMI. Environmental factors, age, dietary habits and lifestyle are the main causes of blood pressure, which can provide a reference basis for the health management of the migratory birds with hypertension in Hainan.

Crizotinib is a multi-target small molecule tyrosine kinase inhibitor, which can act on anaplastic lymphoma kinase (ALK), cellular-mesenchymal to epithelial transition factor and c-ros oncogene 1 (ROS1). It is mainly used in patients with ALK/ROS1-positive advanced non-small cell lung cancer. Liver injury is one of the most common adverse reactions of crizotinib. The typical symptom of crizotinib-induced liver injury is asymptomatic aminotransferase elevation, and fatal liver failure can occur in very few patients. The mechanism is mainly related to mitochondrial-mediated apoptosis, excessive autophagy, oxidative stress, and anaphylaxis. The risk factors involved are history of liver diseases, hepatitis B virus infection, combined use of immune checkpoint inhibitors or H 2-antagonist/proton pump inhibitor, gene polymorphisms of signal transducer and activator of transcription 1 and cytochrome P450. It is very important to assess liver function comprehensively before treatment, and strengthen the therapeutic drug monitoring during treatment. Measures such as temporary (reduce the dose when re-use) or permanent discontinuation should be taken when necessary.

Crizotinib is a multi-target small molecule tyrosine kinase inhibitor, which can act on anaplastic lymphoma kinase (ALK), cellular-mesenchymal to epithelial transition factor and c-ros oncogene 1 (ROS1). It is mainly used in patients with ALK/ROS1-positive advanced non-small cell lung cancer. Liver injury is one of the most common adverse reactions of crizotinib. The typical symptom of crizotinib-induced liver injury is asymptomatic aminotransferase elevation, and fatal liver failure can occur in very few patients. The mechanism is mainly related to mitochondrial-mediated apoptosis, excessive autophagy, oxidative stress, and anaphylaxis. The risk factors involved are history of liver diseases, hepatitis B virus infection, combined use of immune checkpoint inhibitors or H 2-antagonist/proton pump inhibitor, gene polymorphisms of signal transducer and activator of transcription 1 and cytochrome P450. It is very important to assess liver function comprehensively before treatment, and strengthen the therapeutic drug monitoring during treatment. Measures such as temporary (reduce the dose when re-use) or permanent discontinuation should be taken when necessary.