Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

D-LMBmap: a fully automated deep-learning pipeline for whole-brain profiling of neural circuitry.

Recent proliferation and integration of tissue-clearing methods and light-sheet fluorescence microscopy has created new opportunities to achieve mesoscale three-dimensional whole-brain connectivity mapping with exceptionally high throughput. With the rapid generation of large, high-quality imaging datasets, downstream analysis is becoming the major technical bottleneck for mesoscale connectomics. Current computational solutions are labor intensive with limited applications because of the exhaustive manual annotation and heavily customized training. Meanwhile, whole-brain data analysis always requires combining multiple packages and secondary development by users. To address these challenges, we developed D-LMBmap, an end-to-end package providing an integrated workflow containing three modules based on deep-learning algorithms for whole-brain connectivity mapping: axon segmentation, brain region segmentation and whole-brain registration. D-LMBmap does not require manual annotation for axon segmentation and achieves quantitative analysis of whole-brain projectome in a single workflow with superior accuracy for multiple cell types in all of the modalities tested.

Uncovering the complexity of childhood undernutrition through strain-level analysis of the gut microbiome.

BACKGROUND: Undernutrition (UN) is a critical public health issue that threatens the lives of children under five in developing countries. While evidence indicates the crucial role of the gut microbiome (GM) in UN pathogenesis, the strain-level inspection and bacterial co-occurrence network investigation in the GM of UN children are lacking. RESULTS: This study examines the strain compositions of the GM in 61 undernutrition patients (UN group) and 36 healthy children (HC group) and explores the topological features of GM co-occurrence networks using a complex network strategy. The strain-level annotation reveals that the differentially enriched species between the UN and HC groups are due to discriminated strain compositions. For example, Prevotella copri is mainly composed of P. copri ASM1680343v1 and P. copri ASM345920v1 in the HC group, but it is composed of P. copri ASM346549v1 and P. copri ASM347465v1 in the UN group. In addition, the UN-risk model constructed at the strain level demonstrates higher accuracy (AUC = 0.810) than that at the species level (AUC = 0.743). With complex network analysis, we further discovered that the UN group had a more complex GM co-occurrence network, with more hub bacteria and a higher clustering coefficient but lower information transfer efficiencies. Moreover, the results at the strain level suggested the inaccurate and even false conclusions obtained from species level analysis. CONCLUSIONS: Overall, this study highlights the importance of examining the GM at the strain level and investigating bacterial co-occurrence networks to advance our knowledge of UN pathogenesis.

Effect of interpregnancy interval on the risk of gestational diabetes mellitus during a second pregnancy.

BACKGROUND: Interpregnancy interval (IPI) is associated with the risk of GDM in a second pregnancy. However, an optimal IPI is still need to be determined based on the characteristics of the population. This study aimed to analyze the effect of interpregnancy interval (IPI) on the risk of gestational diabetes mellitus (GDM) in the Chinese population. METHODS: We conducted a retrospective cohort study on female participants who had consecutive deliveries at Peking University Shenzhen Hospital from 2013 to 2021. The IPI was categorized into 7 groups and included into the multivariate logistic regression model with other confound factors. Analysis was also stratified based on age of first pregnancy, BMI, and history of GDM. Adjusted OR values (aOR) and 95% confidence intervals (CI) calculated. The regression coefficient of IPI months on GDM prediction risk was analyzed using a linear regression model. RESULTS: A total of 2,392 participants were enrolled. The IPI of the GDM group was significantly greater than that of the non-GDM group (P < 0.05). Compared with the 18-24 months IPI category, participants with longer IPIs (24-36 months, 36-48 months, 48-60 months, and ≥ 60 months) had a higher risk of GDM (aOR:1.585, 2.381, 2.488, and 2.565; 95% CI: 1.021-2.462, 1.489-3.809, 1.441-4.298, and 1.294-5.087, respectively). For participants aged < 30 years or ≥ 30 years or without GDM history, all longer IPIs (≥ 36 months) were all significantly associated with the GDM risk in the second pregnancy (P < 0.05), while any shorter IPIs (< 18 months) was not significantly associated with GDM risk (P > 0.05). For participants with GDM history, IPI 12-18 months, 24-36 months, 36-48 months, and ≥ 60 months were all significantly associated with the GDM risk (aOR: 2.619, 3.747, 4.356, and 5.373; 95% CI: 1.074-6.386, 1.652-8.499, 1.724-11.005, and 1.078-26.793, respectively), and the slope value of linear regression (0.5161) was significantly higher compared to participants without a history of GDM (0.1891) (F = 284.168, P < 0.001). CONCLUSIONS: Long IPI increases the risk of GDM in a second pregnancy, but this risk is independent of maternal age. The risk of developing GDM in a second pregnancy for women with GDM history is more significantly affected by IPI.

Prenatal diagnosis of fetuses with Emanuel syndrome: Results of ultrasound examination and invasive genetic testing.

OBJECTIVE: To investigate prenatal manifestations of Emanuel syndrome (ES) by retrospectively analyzing the results of prenatal diagnosis. METHODS: Thirteen fetuses were collected from five hospitals, of which six were confirmed with 47,der(22)t(11;22; ES) by karyotype and chromosomal microarray analysis (CMA). Seven were diagnosed with 46,t(11;22) balanced translocations by karyotype, including one de novo mosaic 46,XX,t(11;22). In 3/7, CMA was performed but did not identify chromosomal imbalances. The results of prenatal diagnoses were reviewed, including ultrasound examinations and genetic testing. RESULTS: In ES fetuses, the derivative 22 was consistently inherited from the mother, while in the balanced translocation group, the t(11;22) chromosome was of paternal origin in 3/6 cases, All ES fetuses presented with multiple abnormalities by ultrasound examinations. Diaphragm hernia (3/6), Dandy-Walker complex (3/6), and kidney aplasia (3/6), were the most common ultrasound findings. Sonographic soft markers such as increased nuchal translucency, increased nuchal fold thickness appeared in 3 cases and all of these were associated with other anomalies. However, none of the ultrasound findings differentiated ES from other genetic syndromes during fetal period. CONCLUSIONS: In this series, in fetuses with a der(22), the derivative chromosome was consistently of maternal origin. In contrast, 46,t(11;22) balanced translocations were of maternal or paternal origin. The results contribute to the literature regarding the fetal phenotype of ES. Due to the absence of specific features distinguishing ES from other genetic syndromes, confirming the diagnosis through invasive genetic testing is necessary.

Differential effects of different delivery methods on progression to severe postpartum hemorrhage between Chinese nulliparous and multiparous women: a retrospective cohort study.

BACKGROUND: Delivery methods are associated with postpartum hemorrhage (PPH) both in nulliparous and multiparous women. However, few studies have examined the difference in this association between nulliparous and multiparous women. This study aimed to explore the difference of maternal and neonatal characteristics and delivery methods between Chinese nulliparous and multiparous women, and then examine the differential effects of different delivery methods on PPH between these two-type women. METHODS: Totally 151,333 medical records of women who gave birth between April 2013 to May 2016 were obtained from the electronic health records (EHR) in a northern province, China. The severity of PPH was estimated and classified into blood loss at the level of < 900 ml, 900-1500 ml, 1500-2100 ml, and > 2100 ml. Neonatal and maternal characteristics related to PPH were derived from the same database. Multiple ordinal logistic regression was used to estimate associations. RESULTS: Medical comorbidities, placenta previa and accreta were higher in the nulliparous group and the episiotomy rate was higher in the multiparous group. Compared with spontaneous vaginal delivery (SVD), the adjusted odds (aOR) for progression to severe PPH due to the forceps-assisted delivery was much higher in multiparous women (aOR: 9.32; 95% CI: 3.66-23.71) than in nulliparous women (aOR: 1.70; 95% CI: 0.91-3.18). The (aOR) for progression to severe PPH due to cesarean section (CS) compared to SVD was twice as high in the multiparous women (aOR: 4.32; 95% CI: 3.03-6.14) as in the nulliparous women (aOR: 2.04; 95% CI: 1.40-2.97). However, the (aOR) for progression to severe PPH due to episiotomy compared to SVD between multiparous (aOR: 1.24; 95% CI: 0.96-1.62) and nulliparous women (aOR: 1.55; 95% CI: 0.92-2.60) was not significantly different. The (aOR) for progression to severe PPH due to vacuum-assisted delivery compared to SVD in multiparous women (aOR: 2.41; 95% CI: 0.36-16.29) was not significantly different from the nulliparous women (aOR: 1.05; 95% CI: 0.40-2.73). CONCLUSIONS: Forceps-assisted delivery and CS methods were found to increase the risk of severity of the PPH. The adverse effects were even greater for multiparous women. Episiotomy and the vacuum-assisted delivery, and SVD were similar to the risk of progression to severe PPH in either nulliparous or multiparous women. Our findings have implications for the obstetric decision on the choice of delivery methods, maternal and neonatal health care, and obstetric quality control.

Fragment-wise design of inhibitors to 3C proteinase from enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD), which can spread its infection to central nervous and other systems with severe consequence. A key factor in the replication of EV71 is its 3C proteinase (3C(pro)), a significant drug target. Peptidomimetics were employed as inhibitors of this enzyme for developing antivirals. However, the peptide bonds in these peptidomimetics are a source of low bioavailability due to their susceptibility to protease digestion. To produce non-peptidomimetic inhibitors by replacing these peptide bonds, it would be important to gain better understanding on the contribution of each component to the interaction and potency. METHODS: A series of compounds of different lengths targeting 3C(pro) and having an α,ß-unsaturated ester as the warhead were synthesized and their interactions with the enzyme were evaluated by complex structure analyses and potency assays for a better understanding on the relationship between potency and evolution of interaction. RESULTS: The P2 moiety of the compound would need to be oriented to interact in the S2 site in the substrate binding cleft and the P3-P4 moieties were required to generate sufficient potency. A hydrophobic terminal group will benefit the cellular uptake and improve the activity in vivo. CONCLUSIONS AND GENERAL SIGNIFICANCE: The data presented here provide a basis for designing a new generation of non-peptidomimetics to target EV71 3C(pro).

Prenatal Identification and Confirmation of Jacobsen Syndrome: A Series of Four Cases.

Jacobsen syndrome (JBS) is a rare contiguous gene disorder caused by partial deletion of the distal part of the long arm of chromosome 11. Only a few prenatal cases of JBS have been reported, and data on prenatal ultrasonographic findings are relatively scarce. We analysed four cases of JBS diagnosed prenatally in our centre. All four cases received ultrasound examination in the second trimester. Cardiac defects and intrauterine growth retardation (IUGR) were present in three cases. Ventriculomegaly, shortened femur length and pyelectasis were found in two cases. According to the literature, IUGR, pyelectasis and ventriculomegaly are common prenatal phenotypes of JBS. In addition, cardiac defects, trigonocephaly and shortened femur are also found. Our presentation of these cases provides more ultrasonic information for the prenatal diagnosis of this rare disease. Key Words: Ultrasound, Prenatal diagnosis, Jacobsen syndrome, Chromosomal abnormalities, Fetal malformation.

Phenotypic and functional alteration of CD45+ immune cells in the decidua of preeclampsia patients analyzed by mass cytometry (CyTOF).

Preeclampsia (PE) is a severe placenta-related pregnancy disease that has been associated with maternal systemic inflammation and immune system disorders. However, the distribution and functional changes in immune cells of the maternal-placental interface have not been well characterized. Herein, cytometry by time-of-flight mass spectrometry (CyTOF) was used to investigate the immune atlas at the decidua, which was obtained from four PE patients and four healthy controls. Six superclusters were identified, namely, T cells, B cells, natural killer (NK) cells, monocytes, granulocytes, and others. B cells were significantly decreased in the PE group, among which the reduction in CD27+CD38+ regulatory B cell (Breg)-like cells may stimulate immune activation in PE. The significantly increased migration of B cells could be linked to the significantly overexpressed chemokine C-X-C receptor 5 (CXCR5) in the PE group, which may result in the production of excessive autoantibodies and the pathogenesis of PE. A subset of T cells, CD11c+CD8+ T cells, was significantly decreased in PE and might lead to sustained immune activation in PE patients. NK cells were ultimately separated into four subsets. The significant reduction in a novel subset of NK cells (CD56-CD49a-CD38+) in PE might have led to the failure to suppress inflammation at the maternal-fetal interface during PE progression. Moreover, the expression levels of functional markers were significantly altered in the PE group, which also inferred that shifts in the decidual immune state contributed to the development of PE and might serve as potential treatment targets. This is a worthy attempt to elaborate the differences in the phenotype and function of CD45+ immune cells in the decidua between PE and healthy pregnancies by CyTOF, which contributes to understand the pathogenesis of PE, and the altered cell subsets and markers may inspire the immune modulatory therapy for PE.

[Clinical features of abnormal chromosome karyotypes in twin pregnancies complicated with structural abnormalities].

OBJECTIVE: To investigate the clinical features of the abnormal chromosome karyotypes in twin pregnancies complicated with fetal malformations. METHODS: Totally 181 twin pregnancies (362 fetuses) in which one or two fetuses had abnormalities diagnosed by ultrasound were referred to the First Affiliated Hospital of Sun Yat-Sen University from January, 2000 to September, 2010. They were divided into different groups according to (1) maternal age: the cases with maternal age ≥ 35 were divided into advanced pregnancy group (105 fetuses), and those with maternal age < 35 were divided into young pregnancy group (203 fetuses); (2) conceived method: those conceived by assisted reproductive technology were divided into assisted reproductive group (81 fetuses), and the natural conception pregnancies were divided into natural conception group (227 fetuses); (3) chorionicity: the monochorionic twin (MCT) pregnancies were divided into MCT group (123 fetuses), and the dichorionic twin (DCT) pregnancies were divided into DCT group (185 fetuses); (4) structural abnormalities: 205 fetuses with structural abnormalities were divided into the abnormal fetal group, and 103 fetuses without structural abnormalities were divided into the normal fetal group. All fetuses were examined by the ultrasound and chromosomes were examined in 308 fetuses. RESULTS: (1) The karyotype of fetuses: among 181 twin pregnancies, 23 cases had chromosomal abnormalities in 1 or 2 fetuses (12.7%, 23/181), and chromosomes were examined in both fetuses in 20 of 23 cases. Twenty-six of 308 fetuses were found with abnormal chromosomes (8.4%, 26/308), and the aneuploid was the most common type of abnormal karyotypes (53.8%, 14/26). Twenty-one of 205 fetuses with malformations were found with abnormal karyotypes (10.2%, 21/205). (2) Seven of 123 fetuses in MCT group were with abnormal karyotypes (5.7%, 7/123), and 19 of 185 fetuses in DCT group were with abnormal karyotypes (10.3%, 19/185). There was no statistical difference of abnormal chromosome incidence between the two groups. There were 14 fetuses with aneuploid in DCT group (7.6%, 14/185); but there was no fetus with aneuploid in MCT group. There was statistical difference between these two groups. In two cases of DCT group, only one fetus with malformation received chromosome examination because another fetus was dead, and the karyotypes were trisomy 21 and trisomy 18 respectively. Both fetuses of the rest 17 cases received chromosome examination, and the chromosomes of both fetuses in each pregnancy were different. Fifteen of 19 fetuses with abnormal chromosomes in DCT group were complicated with structural abnormalities, and 7 fetuses of 4 twin pregnancies in MCT group were with chromosomal abnormalities. (3) The comparison of the abnormal karyotype incidence between the advanced pregnancy group and young pregnancy group: the abnormal karyotype incidence of the advanced pregnancy group was 7.6% (8/105), and that was 8.9% (18/203) in young pregnancy group. There was no statistical difference between the two groups (P > 0.05). Six of 105 fetuses in advanced pregnancy group were aneuploids (5.7%, 6/105), and 8 of 203 fetuses in young pregnancy group were aneuploids (3.9%, 8/203). The aneuploid incidence in advanced pregnancy group was significantly higher than that in young pregnancy group (P < 0.05).(4) The comparison of the abnormal karyotype incidence between the assisted reproductive group and the natural conception group: 11 of 81 fetuses were with the abnormal karyotypes in assisted reproductive group (13.6%, 11/81), and 15 of 227 fetuses were with the abnormal karyotypes in assisted reproductive group (6.6%, 15/227). There was statistical difference between the two groups (P < 0.05). There were 7 fetuses with the aneuploid in assisted reproductive group (8.6%, 7/81) and 7 fetuses with the aneuploid in natural conception group (3.1%, 7/227), which showed no statistical difference (P > 0.05). (5) The comparison of the abnormal karyotype incidence between the abnormal fetal group and normal fetal group: 21 of 205 fetuses in abnormal fetal group were with abnormal karyotypes (10.2%, 21/205), and 5 of 103 fetuses in normal fetal group were with abnormal karyotypes (4.9%, 5/103). There was no statistical difference (P > 0.05). 13 fetuses in abnormal fetal group were with the aneuploid (6.3%, 13/205), and only one fetus in normal fetal group was aneuploid (1.0%, 1/103). There was statistical difference between the two groups (P < 0.05). CONCLUSIONS: Aneuploid is the most common abnormal karyotype in twin pregnancy complicated with fetal abnormalities, especially trisomy 21. Aneuploid mainly occurs in only one fetus of DCT, and chromosomal discordance is usually found in DCT. While in MCT, the twin fetuses with the same abnormal karyotype may have different phenotypes. The results suggest that it is necessary to analyze both karyotypes of twins even if only one fetus is complicated with structural abnormalities.

Introduction of Intracapsular Rotary-cut Procedures (IRCP): A Modified Hysteromyomectomy Procedures Facilitating Fertility Preservation.

Uterine fibroids are common benign tumors in the female reproductive system. A hysterectomy is the most effective treatment for symptomatic fibroids. For patients desiring pregnancy, laparoscopic intracapsular myomectomy (LM) is an alternative surgery option. Although LM is widely accepted to treat fibroids, it is technically demanding with risk of excessive bleeding and difficult suturing, especially in cases with large fibroids or tumors in unusual locations. Therefore, we developed an intracapsular rotary-cut procedure (IRCP) as a modification of laparoscopic intracapsular myomectomy, with the intention to minimize risks of LM and help uterine healing. A summary of the improvements to the IRCP is described: 1) making an incision at the site of the fibroid with a length of one-third to one-half of the fibroid's diameter at a depth reaching the fibroid's surface; 2) holding the fibroid stably and making rotary cuts on the fibroid at a depth of 0.5-1 cm within its pseudo-capsule while pulling it outward slightly, making sure not to cut off any pieces of the fibroid; and 3) repeating the cutting-and-pulling until the longest dimension of the fibroid is outside the incision. The multiple cuts are to minimize the diameter and extend the length of the fibroid. When the multiple cuts cause half of the fibroid body to "shrink", the fibroid is squeezed out by contraction of the surrounding myometrium. Evaluation of the outcomes of IRCP showed that the time of enucleation and suturing, intraoperative bleeding, and decline of hemoglobin were significantly lower in the IRCP group than the LM group. As for reproductive outcomes, the full-term live birth rate of the IRCP group was significantly higher than that of LM group. However, there was no difference in delivery modes between the two groups. In conclusion, IRCP significantly benefits fertility preservation by minimizing damage to the uterus, protecting myofibers of the pseudo-capsule, and resulting in a shallower residual cavity, which eases stitching and causes less bleeding. It is worthwhile to adopt IRCP in younger patients who desire preservation of their fertility.

Development and evaluation of an improved laparoscopic myomectomy adopting intracapsular rotary-cut procedures.

OBJECTIVES: To develop and evaluate the efficacy and feasibility of Intracapsular Rotary-cut Procedure (IRCP) in laparoscopic myomectomy (LM). STUDY DESIGN: It is a retrospective study conducted in Peking University Shenzhen Hospital (PUSH) in Shenzhen, China. A total of 96 patients who were treated with LM because of big and moderate size of myomas in PUSH from January 2013 and December 2015 were enrolled in this study. Among them 35 were managed with IRCP -integrated LM as the study group, while the other 61 were treated with normal LM as the control group. The core steps of IRCP include minimized uterine incision, rotary cuttings in the fibroid inside the pseudocapsule, and preserving the fibers and vessels of the fibroid pseudocapsule. The operation data and outcome variables of each patient in the two groups were collected and compared to evaluate the efficacy and feasibility of this new improved surgery procedure. RESULTS: The ages, body mass indexes, main indication for myomectomy, and the diameters, numbers and types of the fibroids of patients in the two groups were similar. There is no significant difference in enucleation time and days of postoperative in-hospital staying between the two groups. In comparison with normal LM, application of IRCP significantly reduced the length of uterine incisions (P=0.000), the overall time of operations (P=0.001), and the time for uterine cavity suturing (P=0.000), and application of IRCP was also associated with less operation bleeding (P=0.003) and postoperative hemoglobin dropping (P=0.001). Five cases (8.2%) in the control group were referred to laparotomy, while no case was done so in the study group. No patient in the study group needed blood transfusion but six patients (9.8%) in the control group did. Follow-up to all the patients showed no uterine malignant tumor. CONCLUSIONS: Our study provided strong supportive evidences showing that Intracapsular Rotary-cut Procedures is safe and practicable. The less uterine incision length, less surgery time, and less bleeding than the normal LM procedures all suggests that IRCP should be adopted in LM for facilitation to the operation procedures, minimization of operative invasiveness, and protection to patients' infertility.

Human chorionic gonadotropin, fetal sex and risk of hypertensive disorders of pregnancy: A nested case-control study.

OBJECTIVES: To assess whether human chorionic gonadotropin (HCG) and fetal sex are two independent risk factors for hypertensive pregnancy in the early second-trimester of pregnancy. METHODS: This was a retrospective nested case-control study based on a cohort of 2521 singleton pregnancies, among whom we recruited 98 hypertensive pregnancies (subdivided into severe preeclampsia, n=34; mild preeclampsia, n=29 and gestational hypertension, n=35) and 196 normotensive pregnancies. Maternal serum HCG levels were measured at 15-20 weeks of gestation and fetal sex was determined from the neonatal record. Mann-Whitney U and chi-square tests were performed to assess differences of HCG levels and fetal sex between groups. Logistic regressions were performed to evaluate the effect of HCG and fetal sex on hypertensive pregnancy. RESULTS: There were 35 male and 63 female fetuses in the hypertensive group, and 102 male and 94 female fetuses in the normotensive group (p=0.008). HCG (MoM) levels were significantly higher in only severe preeclamptic pregnancies (n=34) (p=0.013). There were no significant differences of the HCG (MoM) levels between male and female fetuses in each sub-group. aOR for increased maternal HCG levels and female fetus were 2.4 (95% CI: 1.434-3.954) and 2.9 (95% CI: 1.227-6.661) respectively in severe preeclamptic pregnancies compared with normotensive pregnancies. CONCLUSIONS: There is a female preponderance in hypertensive pregnancies. Increased HCG levels and female fetus are two independent risk factors for severe preeclampsia in the early second-trimester of pregnancy.