Wnt/beta-catenin signaling controls development of the blood–brain barrier

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown

Wnt/β-catenin signaling controls development of the blood–brain barrier

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Safety, immunogenicity, and reactogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines given as fourth-dose boosters following two doses of ChAdOx1 nCoV-19 or BNT162b2 and a third dose of BNT162b2 (COV-BOOST): a multicentre, blinded, phase 2, randomised trial

Background Some high-income countries have deployed fourth doses of COVID-19 vaccines, but the clinical need, effectiveness, timing, and dose of a fourth dose remain uncertain. We aimed to investigate the safety, reactogenicity, and immunogenicity of fourth-dose boosters against COVID-19.Methods The COV-BOOST trial is a multicentre, blinded, phase 2, randomised controlled trial of seven COVID-19 vaccines given as third-dose boosters at 18 sites in the UK. This sub-study enrolled participants who had received BNT162b2 (Pfizer-BioNTech) as their third dose in COV-BOOST and randomly assigned them (1:1) to receive a fourth dose of either BNT162b2 (30 µg in 0·30 mL; full dose) or mRNA-1273 (Moderna; 50 µg in 0·25 mL; half dose) via intramuscular injection into the upper arm. The computer-generated randomisation list was created by the study statisticians with random block sizes of two or four. Participants and all study staff not delivering the vaccines were masked to treatment allocation. The coprimary outcomes were safety and reactogenicity, and immunogenicity (antispike protein IgG titres by ELISA and cellular immune response by ELISpot). We compared immunogenicity at 28 days after the third dose versus 14 days after the fourth dose and at day 0 versus day 14 relative to the fourth dose. Safety and reactogenicity were assessed in the per-protocol population, which comprised all participants who received a fourth-dose booster regardless of their SARS-CoV-2 serostatus. Immunogenicity was primarily analysed in a modified intention-to-treat population comprising seronegative participants who had received a fourth-dose booster and had available endpoint data. This trial is registered with ISRCTN, 73765130, and is ongoing.Findings Between Jan 11 and Jan 25, 2022, 166 participants were screened, randomly assigned, and received either full-dose BNT162b2 (n=83) or half-dose mRNA-1273 (n=83) as a fourth dose. The median age of these participants was 70·1 years (IQR 51·6–77·5) and 86 (52%) of 166 participants were female and 80 (48%) were male. The median interval between the third and fourth doses was 208·5 days (IQR 203·3–214·8). Pain was the most common local solicited adverse event and fatigue was the most common systemic solicited adverse event after BNT162b2 or mRNA-1273 booster doses. None of three serious adverse events reported after a fourth dose with BNT162b2 were related to the study vaccine. In the BNT162b2 group, geometric mean anti-spike protein IgG concentration at day 28 after the third dose was 23 325 ELISA laboratory units (ELU)/mL (95% CI 20 030–27 162), which increased to 37 460 ELU/mL (31 996–43 857) at day 14 after the fourth dose, representing a significant fold change (geometric mean 1·59, 95% CI 1·41–1·78). There was a significant increase in geometric mean anti-spike protein IgG concentration from 28 days after the third dose (25 317 ELU/mL, 95% CI 20 996–30 528) to 14 days after a fourth dose of mRNA-1273 (54 936 ELU/mL, 46 826–64 452), with a geometric mean fold change of 2·19 (1·90–2·52). The fold changes in anti-spike protein IgG titres from before (day 0) to after (day 14) the fourth dose were 12·19 (95% CI 10·37–14·32) and 15·90 (12·92–19·58) in the BNT162b2 and mRNA-1273 groups, respectively. T-cell responses were also boosted after the fourth dose (eg, the fold changes for the wild-type variant from before to after the fourth dose were 7·32 [95% CI 3·24–16·54] in the BNT162b2 group and 6·22 [3·90–9·92] in the mRNA-1273 group).Interpretation Fourth-dose COVID-19 mRNA booster vaccines are well tolerated and boost cellular and humoral immunity. Peak responses after the fourth dose were similar to, and possibly better than, peak responses after the third dose

Early postoperative delayed hearing loss: Patterns of behavioural and electrophysiological auditory responses following vestibular schwannoma surgery

Following vestibular schwannoma excision, a subset of cases has been reported in which hearing is present immediately after surgery, but is lost in the early postoperative period. Such cases have rarely been reported, and the postoperative audiological data collected from patients in these cases lacks the time resolution necessary to determine the pathophysiological mechanism responsible for the pattern of hearing loss. The present study aimed to more clearly define delayed hearing loss by collecting detailed data documenting changes in behavioural and electrophysiological auditory responses following vestibular schwannoma surgery. In particular, we aimed to use this data to determine the time course of changes in auditory function and to identify whether the site of impairment was cochlear or neural. Preoperative and daily postoperative monitoring of auditory function was performed in 19 patients undergoing vestibular schwannoma excision via the retrosigmoid approach at Christchurch Public Hospital. The pre- and postoperative assessment battery included pure-tone and speech audiometry, tympanometry, tone decay, distortion product otoacoustic emissions (DPOAEs), and auditory brainstem response (ABR) measurement. Intraoperative ABR was performed in four cases in which clear preoperative waveforms were present. Transtympanic electrocochleography (ECochG) was carried out if wave I was lost in the early postoperative period. Thirteen of the 19 patients suffered immediate anacusis following surgery and six had measurable hearing postoperatively. The behavioural and electrophysiological data collected in each case is discussed with regard to the likely pathophysiology of pre- and postoperative hearing loss. No patients demonstrated behavioural evidence of delayed hearing loss, however a gradual deterioration of ABR in the early postoperative period was observed in Case 16. ECochG and DPOAEs in this case indicated the presence of cochlear function although the patient presented with immediate postoperative anacusis in the ipsilateral ear. These results are consistent with postoperative retrograde degeneration of the cochlear nerve

Investigations into the Effects of Middle Ear Surgery on Inner Ear Function

Middle ear surgical procedures are typically associated with a high rate of improvement in air-conduction thresholds and a low rate of sensorineural hearing loss in the conventionally assessed frequency range (0.25 – 8 kHz). Hearing loss in the extended high-frequency (EHF) range (8 – 16 kHz), however, may be common, although its characteristics are not well understood. To elucidate the effects of middle ear surgery on auditory function, prospective investigations were performed to provide data that allowed transient and permanent changes in EHF hearing to be distinguished, and to establish the nature of EHF hearing loss. Changes in hearing at 0.25 to 16 kHz were documented in 88 patients following stapedectomy, ossiculoplasty, and tympanoplasty. Hearing was measured preoperatively, and 1 week, 1, 3, 6, and 12 months postoperatively. Results showed that elevation of EHF air-conduction thresholds occurred frequently following all three surgeries and was most severe one week postoperatively. Although significant recovery of hearing was recorded by three months, 12 months after surgery, 50% of patients who underwent stapedectomy, 42% who had a tympanoplasty and 20% who underwent ossiculoplasty retained a reduction in their highest audible frequency. A TEAC HP-F100 bone-conduction transducer was modified for use in EHF audiometry and used in a small pilot study to demonstrate that EHF hearing loss following stapedectomy may be composed of both conductive and sensorineural elements. It was hypothesised that changes to utricular responses reflective of trauma to the vestibular portion of the inner ear may also occur following middle ear surgery. Measurements of tap-evoked ocular vestibular evoked myogenic potentials (oVEMPs) were performed in the same group of patients in which audiometric data was collected. Overall, the oVEMP data provided no evidence of a postoperative change in utricular responses. To assess clinical importance of EHF hearing loss, its role in one aspect of auditory function, localisation ability, was investigated in 46 participants; 23 with EHF hearing loss and 23 with normal EHF hearing. Overall, the results agreed with previous studies that localisation accuracy decreased when was EHF spectral content was removed by filtering, however the difference between hearing groups was significant only when speakers were positioned in the lateral vertical orientation. Regardless of the clinical consequences, the increased vulnerability of EHF hearing acuity to the effects of middle ear surgery provides a useful model which could be used to efficiently assess the effect of technical factors of surgery or the efficacy of ototherapeutic treatments on hearing outcomes

Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F

Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, nuclear targets for PI3K are not known. Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5 and Raf/MEK pathways from E2F regulation. Protein kinase B (PKB) is activated by IL-2 via PI3K. The expression of an active PKB is sufficient to induce E2F activity. Inhibition of PI3K inhibits phosphorylation of Rb, induction of cyclin D3, and degradation of p27 kip1. These results establish a crucial PI3K/PKB–mediated link between the IL-2 receptor and the cell cycle machinery