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.

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.

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).