Objective:To establish a GC method for the determination of related substances in myristyl alcohol.Methods:Use a column packed with polyethylene glycol as the stationary phase(HP-INNOWAX,0.25 mm×30 m,0.25 μm).Maintain the column temperature at 90℃,raise the temperature to 180℃by 5℃per minutes,maintain for 25 minutes.The temperatures of injection port is 270℃.The temperatures of detector is 280℃.Cal-culate the content by normalization method.Result:Lauryl alcohol,myristyl alcohol,pentadecanol,cetyl alcohol,stearyl alcohol and oleyl alcohol showed good linear relationships within the ranges of 6.2-123.8,4.2-134.2,5.2-104.2,4.5-90.3,4.8-96.0,4.8-95.2 μg·mL-1,respectively,with the correlation coefficient above 0.999 8.The limit of detection of aforementioned fatty alcohols were 0.2,0.2,0.2,0.3,0.5 and 0.5 μg·mL-1,which were more sensitive than the method in USP monotherapy Myristyl Alcohol.The content of lauryl alcohol was 0.2%-0.6%and that of cetyl alcohol was 0.1%-0.9%in 5 batches of samples.Sum of re-lated fatty alcohols and unspecified impurities was 0.7%-2.5%.Conclusion:The method established in this study has good separation effect,high specificity and high sensitivity,and solved the problem of difficult identifica-tion of related fatty alcohols caused by high noise at the baseline of the method in USP monotherapy myristyl alco-hol.It can be used for the the determination of related substances in myristyl alcohol.It also provides reference and guidance for the quality management and control of other fatty alcohols.

Objective To observe prenatal ultrasonic findings of fetal malformation of cortical development(MCD).Methods Totally 37 singleton fetuses with MCD diagnosed by fetal cranial MRI and/or genetic examination were retrospectively enrolled,and prenatal ultrasonic manifestations were observed.Results Among 37 fetuses,29(29/37,78.38％)were found with abnormal Sylvian fissure,26(26/37,70.27％)with widened/asymmetrical lateral ventricles,22(22/37,59.46％)with abnormal surface sulci,14(14/37,37.84％)with abnormal cavity of septum pellucidum(CSP)/corpus callosum(CC),11(11/37,29.73％)with midline deviation/curvature,11(11/37,29.73％)with widened subarachnoid space,10(10/37,27.03％)with head circumference＜2 standard deviation below mean value,9(9/37,24.32％)with abnormalities in the cerebellar hemisphere/vermis,7(7/37,18.92％)with abnormal echoes in the brain parenchyma,7(7/37,18.92％)with midline cysts and 2(2/37,5.41％)with enlarged ganglion eminence.Conclusion Fetal MCD mainly presented as abnormal Sylvian fissure,widened/asymmetrical lateral ventricles and abnormal surface sulci,which might accompanied by abnormal CSP/CC,midline deviation/curvature,widened subarachnoid space and abnormal head circumference in prenatal ultrasound.

Objective·To determine hyaluronan(HA)expression in the endocrine-resistant microenvironment of estrogen receptor-positive(ER+)breast cancer and elucidate its impact on the acquired resistance.Methods·Chemiluminescent immunoassay was used to quantify HA levels in the culture supernatants of fulvestrant-resistant breast cancer cells.An immunofluorescence(IF)assay was performed to visualize the colocalization of CD44 and HA in MCF7/FulR cells.Using an established adaptive endocrine-resistant breast cancer mouse model,HA expression in resistant breast cancer tissues was assessed by immunohistochemistry(IHC)assay.Single-cell RNA sequencing(scRNA-seq)and RNA sequencing(RNA-seq)were conducted to examine transcriptomic profiles and alterations in HA-related genes in resistant breast cancer cells.Flow cytometry(FCM)was utilized to measure the proportion of CD44+CD24-cells in MCF7/FulR.The correlation between HA synthesis genes and cell stemness was investigated in clinical ER+breast cancers from GEO data sets.Hyaluronidase(HAase)treatment was applied to remove the"HA coat",and RT-qPCR and Western blotting analysis were carried out to monitor changes in stemness-related molecules.CCK-8 assays,flow cytometry(FCM),and Hoechst 33258 staining were performed to determine changes in apoptosis and fulvestrant efficiency after HAase treatment.Results·IF results revealed that compared with MCF7 cells,the"HA coat"on the surface of MCF7/FulR cells was significantly thickened.IHC demonstrated markedly increased HA retention in fulvestrant-resistant mouse breast cancer tissues.ScRNA-seq and RNA-seq analyses indicated elevated expression of stemness-related genes and HA synthesis-associated genes in fulvestrant-resistant breast cancer cells.Correlation analysis revealed a positive association between HA synthesis and cancer stemness in ER+breast cancer.IF and RT-qPCR results demonstrated that removing the HA coating from the surface of MCF7/FulR cells led to a significant reduction in the expression of stemness-related molecules;concurrently,CCK-8 assays,FCM analysis,and Hoechst 33258 staining revealed that"HA coat"clearance reduced MCF7/FulR'tolerance to fulvestrant and increased apoptosis.Conclusion·Endocrine-resistant breast cancer cells develop an enriched"HA coat",which promotes stemness in fulvestrant-resistant tumors.Disruption of this HA coat through HAase treatment effectively reduces cell stemness,induces apoptosis,and re-sensitizes breast cancer cells to fulvestrant.

Objective:To establish a GC method for the determination of related substances in myristyl alcohol.Methods:Use a column packed with polyethylene glycol as the stationary phase(HP-INNOWAX,0.25 mm×30 m,0.25 μm).Maintain the column temperature at 90℃,raise the temperature to 180℃by 5℃per minutes,maintain for 25 minutes.The temperatures of injection port is 270℃.The temperatures of detector is 280℃.Cal-culate the content by normalization method.Result:Lauryl alcohol,myristyl alcohol,pentadecanol,cetyl alcohol,stearyl alcohol and oleyl alcohol showed good linear relationships within the ranges of 6.2-123.8,4.2-134.2,5.2-104.2,4.5-90.3,4.8-96.0,4.8-95.2 μg·mL-1,respectively,with the correlation coefficient above 0.999 8.The limit of detection of aforementioned fatty alcohols were 0.2,0.2,0.2,0.3,0.5 and 0.5 μg·mL-1,which were more sensitive than the method in USP monotherapy Myristyl Alcohol.The content of lauryl alcohol was 0.2%-0.6%and that of cetyl alcohol was 0.1%-0.9%in 5 batches of samples.Sum of re-lated fatty alcohols and unspecified impurities was 0.7%-2.5%.Conclusion:The method established in this study has good separation effect,high specificity and high sensitivity,and solved the problem of difficult identifica-tion of related fatty alcohols caused by high noise at the baseline of the method in USP monotherapy myristyl alco-hol.It can be used for the the determination of related substances in myristyl alcohol.It also provides reference and guidance for the quality management and control of other fatty alcohols.

Objective·To determine hyaluronan(HA)expression in the endocrine-resistant microenvironment of estrogen receptor-positive(ER+)breast cancer and elucidate its impact on the acquired resistance.Methods·Chemiluminescent immunoassay was used to quantify HA levels in the culture supernatants of fulvestrant-resistant breast cancer cells.An immunofluorescence(IF)assay was performed to visualize the colocalization of CD44 and HA in MCF7/FulR cells.Using an established adaptive endocrine-resistant breast cancer mouse model,HA expression in resistant breast cancer tissues was assessed by immunohistochemistry(IHC)assay.Single-cell RNA sequencing(scRNA-seq)and RNA sequencing(RNA-seq)were conducted to examine transcriptomic profiles and alterations in HA-related genes in resistant breast cancer cells.Flow cytometry(FCM)was utilized to measure the proportion of CD44+CD24-cells in MCF7/FulR.The correlation between HA synthesis genes and cell stemness was investigated in clinical ER+breast cancers from GEO data sets.Hyaluronidase(HAase)treatment was applied to remove the"HA coat",and RT-qPCR and Western blotting analysis were carried out to monitor changes in stemness-related molecules.CCK-8 assays,flow cytometry(FCM),and Hoechst 33258 staining were performed to determine changes in apoptosis and fulvestrant efficiency after HAase treatment.Results·IF results revealed that compared with MCF7 cells,the"HA coat"on the surface of MCF7/FulR cells was significantly thickened.IHC demonstrated markedly increased HA retention in fulvestrant-resistant mouse breast cancer tissues.ScRNA-seq and RNA-seq analyses indicated elevated expression of stemness-related genes and HA synthesis-associated genes in fulvestrant-resistant breast cancer cells.Correlation analysis revealed a positive association between HA synthesis and cancer stemness in ER+breast cancer.IF and RT-qPCR results demonstrated that removing the HA coating from the surface of MCF7/FulR cells led to a significant reduction in the expression of stemness-related molecules;concurrently,CCK-8 assays,FCM analysis,and Hoechst 33258 staining revealed that"HA coat"clearance reduced MCF7/FulR'tolerance to fulvestrant and increased apoptosis.Conclusion·Endocrine-resistant breast cancer cells develop an enriched"HA coat",which promotes stemness in fulvestrant-resistant tumors.Disruption of this HA coat through HAase treatment effectively reduces cell stemness,induces apoptosis,and re-sensitizes breast cancer cells to fulvestrant.

Objective To observe prenatal ultrasonic findings of fetal malformation of cortical development(MCD).Methods Totally 37 singleton fetuses with MCD diagnosed by fetal cranial MRI and/or genetic examination were retrospectively enrolled,and prenatal ultrasonic manifestations were observed.Results Among 37 fetuses,29(29/37,78.38％)were found with abnormal Sylvian fissure,26(26/37,70.27％)with widened/asymmetrical lateral ventricles,22(22/37,59.46％)with abnormal surface sulci,14(14/37,37.84％)with abnormal cavity of septum pellucidum(CSP)/corpus callosum(CC),11(11/37,29.73％)with midline deviation/curvature,11(11/37,29.73％)with widened subarachnoid space,10(10/37,27.03％)with head circumference＜2 standard deviation below mean value,9(9/37,24.32％)with abnormalities in the cerebellar hemisphere/vermis,7(7/37,18.92％)with abnormal echoes in the brain parenchyma,7(7/37,18.92％)with midline cysts and 2(2/37,5.41％)with enlarged ganglion eminence.Conclusion Fetal MCD mainly presented as abnormal Sylvian fissure,widened/asymmetrical lateral ventricles and abnormal surface sulci,which might accompanied by abnormal CSP/CC,midline deviation/curvature,widened subarachnoid space and abnormal head circumference in prenatal ultrasound.

Objective:To investigate whether short chain fatty acid(SCFAs) intervention has an antidepressant effect by improving gut microbiota dysregulation and regulating the TLR4/MyD88/NF-κB inflammatory pathway in depression model mice.Methods:Totally 60 SPF grade male C57BL/6 J mice aged 6-8 weeks were randomly divided into three groups: control group, depression model group, and SCFAs group, with 20 mice in each group.The mice in depression model group and SCFAs group were given the chronic unpredictable mild stress (CUMS) stimulations for 8 weeks to establish the depression model.From the 6th week, SCFAs group mice were given a mixed solution of short chain fatty acid salts for drinking, until modeling was completed, meanwhile mice in the model group were given 0.78% NaCl solution for drinking.The depression-like behavior was assessed using the sucrose preference test (SPT) and forced swimming test (FST) following modeling, and the open field test (OFT) was employed to evaluate the anxiety-like behavior of mice.16S rRNA gene sequence was used to analyze the gut microbiota of mice.The activation of astrocytes and TLR4/MyD88/NF-κB inflammatory pathway in hippocampus was determined by immunofluorescence staining and Western blot.SPSS 26.0 was used for statistical analysis, one-way ANOVA was used for comparison among the three groups, and LSD- t test was used for further pairwise comparisons. Results:There were statistically significant differences in the sugar water preference rate, the immobility time in FST, and the percentage of activity time in OFT among the three groups ( F=10.554, 10.912, 12.599, all P<0.05).The the sugar water preference rate and the percentage of activity time in OFT of the depression model group were both lower than those of the control group (both P<0.05), and the immobility time in FST was higher than that of the control group ( P<0.05).The sugar water preference rate in SCFAs group((84.7±3.5)%, (75.3±6.0)%)and the percentage of activity time in OFT((7.4±1.4)%, (3.2±0.9)%) were both higher than those in the depression model group(both P<0.05 ), while the immobility time in FST was shorter than that in the depression model group((110.5±21.5) s, (148.0±20.1) s, P<0.05).There was a statistical difference in the β diversity of gut microbiota among three groups ( P=0.001).At the family level, compared with the depression model group, the relative abundance of Rikenellaceaee and Bacteroidaceae increased in the SCFAs group, while the relative abundance of Clostridia_UCG-014 decreased.At the genus level, the relative abundance of Clostridia_UCG-014 and Prevotella decreased, while the relative abundance of Alistipes increased (all P<0.05).The immunofluorescence results showed that there was a statistically significant difference in GFAP expression levels among the three groups of mice ( F=16.565, P=0.004).The GFAP expression in the depression model group was higher than that in the control group and SCFAs group (both P<0.05).The Western blot results showed that there were statistically significant differences in the expression levels of TLR4, MYD88, and NF-κB ptoteins in the hippocampal tissue of the three groups ( F=70.59, 174.39, 14.40, all P<0.05).The protein levels of TLR4, MyD88, and NF-κB in the depression model group were all higher than those in the control group and SCFAs group (all P<0.05). Conclusion:SCFAs can ameliorate the depressive-like behavior in depression model mice and reduce the activation of astrocytes in the hippocampus, which may be associated with the improvement of dysregulated gut microbiota and down-regulation of the TLR4/MyD88/NF-κB pathway protein.

Objective:To explore the diagnostic value of ultrasound in posterior fossa anomalies (PFA) at 11-13 + 6 gestational weeks by measuring brainstem (BS), brainstem-to-occipital bone (BSOB) diameter and BS/BSOB ratio. Methods:A total of 209 normal fetuses (control group) were randomly selected from Beijing Obstetrics and Gynecology Hospital, Capital Medical University, between March 2018 and November 2021. Reference ranges for BS, BSOB diameter and BS/BSOB ratio were obtained on the mid-sagittal view of the fetal profile and the relationship of three parameters and crown-rump length (CRL) was investigated. The intra- and inter-observer reliabilities were determined by intraclass correlation coefficient (ICC) in 30 normal fetuses. Fourteen fetuses diagnosed with PFA in the same period including 10 cases of cystic posterior fossa malformations (cPFM) and 4 cases of open spine bifida (OSB) were retrospectively selected to compare BS, BSOB diameter and the BS/BSOB ratio with control group.Results:BS and BSOB diameters were successfully obtained in all control fetuses (100%), and the intra- and inter-observer reliabilities for BS and BSOB diameters were good (ICC=0.877, 0.846 and 0.939, 0.895). In the control group, BS and BSOB diameter linearly correlated with CRL ( r=0.867, 0.794; all P<0.001), while the BS/BSOB ratio was 0.75 (0.71, 0.79). There were significant differences of BSOB diameter and BS/BSOB ratio between control group and PFA group (all P<0.05). Except for one isolated vermian hypoplasia (VH), the BSOB diameters in 9 (90%) cases of cPFM were above the 95th percentile of the calculated normal range and were below the 5th percentile in 4(100%) cases of OSB.Except for one isolated VH, the BS/BSOB ratio in 9 (90%) cases of cPFM was below the 5th percentile of the calculated normal range. The BS/BSOB ratio in 4 (100%) cases of OSB was above the 95th percentile of the calculated normal range. Conclusions:The measurements of BS and BSOB diameter are feasible with good repeatability. Abnormal BSOB diameter and BS/BSOB ratio are suggestive for PFA. The posterior fossa of isolated VH can be normal in the first trimester.

We report two cases of Joubert syndrome initially tentatively diagnosed by prenatal ultrasound in the first or second trimester which were thereafter confirmed by whole exome sequencing (WES). Case 1 was one of the twins who presented with increased intracranial transparency (IT) and thinner brainstem at 12 +1 gestational weeks. Ultrasound at 18 +2 weeks found multiple intracranial malformations, "molar tooth sign (MTS)" at the midbrain-hindbrain junction level in the cerebral cross section, and bilateral ventriculomegaly. Enlarged and echogenic kidneys and oligohydramnios were also detected. In case 2, ultrasound image at 17 +5 weeks of gestation indicated multiple intra-and extra cranial and extracranial malformations, MTS in the midbrain-hindbrain junction plane, bilateral ventriculomegaly, unclear cavum septum pellucidum. Extracranial anomalies were bilateral multicystic enlarged kidneys, invisible bladder, and oligohydramnios. Both fetuses underwent amniocentesis, which showed normal karyotype and no copy number variation was detected. However, variation of the TMEM67 gene (c.312+5G>A at introns 2 and c.1175C>G at exon12) was detected in both fetuses by WES, supporting the diagnosis of Joubert syndrome. Selective reduction and termination of pregnancy were performed on case 1 and case 2 at 18 +5 and 19 weeks of gestation, respectively.

OBJECTIVE：To optimi ze the ratio of four comp onents of Compound renshen jianti formulation （Panax ginseng ， Dioscorea oppositifolia ，Lycium barbarum fruit，Alpinia oxyphylla ），and to investigate its anti-fatigue activity and acute toxicity. METHODS：The water extract of Compound renshen jianti formulation was prepared by water extraction ，concentration and decompression drying. By single factor tests ，using weight-bearing swimming time as index ，the effects of four factors were investigated，such as the amount of P. ginseng ，D. oppositifolia ，L. barbarum fruit，A. oxyphylla . On the basis of single factor tests，using comprehensive score of weight-bearing swimming time ，serum urea nitrogen content ，liver glycogen content and AUC of blood lactate after exercise as index ，the formulation was optimized by Box-Behnken response surface method. The mice was divided into blank control group （water），positive control group （Renshen hongjingtian capsules ，0.135 g/kg）and compound low-dose，medium-dose and high-dose groups [the optimal ratio of Compound renshen jianti formulation extract （called“optimal compound formulation ”for short ）4.08，8.16，12.24 g/kg，by crude drug] ，intragastric administration of drug or distilled water 20 mL/kg，once a day ，for consecutive 30 d. The weight-bearing swimming time ，the contents of serum urea nitrogen ，liver glycogen and blood lactate AUC after exercise were used to optimize its anti-fatigue activity of optimal compound formulation. The comprehensive score was calculated based on the measured data of mice in the compound formulation middle-dose group ， and the difference between it and the theoretical prediction value was compared. The mice were given optimal compound formulation intragastrically （total dose 16.00 g/kg， by extract）. The general state ， body mass change ， toxic characteristics and death of mice were observed and recorded for 14 days. Median lethal dose （LD50）and maximum tolerated dose （MTD）were measured. RESULTS ：The optimal formulation ratio of Compound renshen jianti formulation included that P. ginseng 1.5 g，D. oppositifolia 10 g，L. barbarum fruit 10 g，A. oxyphylla 3 g. Results of anti-fatigue activity validation test showed that the optimal compound formulation could significantly prolonged weight-bearing swimming time ，reduced serum content of urea nitrogen ，blood lactate content and its AUC （except for low-dose group ），while significantly increased the content of liver glycogen （P＜0.05 or P＜0.01）. Average comprehensive score of medium-dose group was 96.95，which was only 0.06％ different from the theoretical prediction value of 97.01. The results of acute toxicity test showed that there was no death in mice. The oral MTD of the optimal compound formulation was more than 15 g/kg，which was non-toxic. CONCLUSIONS ：The optimal Compound renshen jianti formulation has effective anti-fatigue activity of mice ，and has no significant toxic effect.