The MRE11-RAD50-NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair.

Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11-RAD50-NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and enabling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.

PELI2 is a negative regulator of STING signaling that is dynamically repressed during viral infection.

The innate immune cGAS-STING pathway is activated by cytosolic double-stranded DNA (dsDNA), a ubiquitous danger signal, to produce interferon, a potent anti-viral and anti-cancer cytokine. However, STING activation must be tightly controlled because aberrant interferon production leads to debilitating interferonopathies. Here, we discover PELI2 as a crucial negative regulator of STING. Mechanistically, PELI2 inhibits the transcription factor IRF3 by binding to phosphorylated Thr354 and Thr356 on the C-terminal tail of STING, leading to ubiquitination and inhibition of the kinase TBK1. PELI2 sets a threshold for STING activation that tolerates low levels of cytosolic dsDNA, such as that caused by silenced TREX1, RNASEH2B, BRCA1, or SETX. When this threshold is reached, such as during viral infection, STING-induced interferon production temporarily downregulates PELI2, creating a positive feedback loop allowing a robust immune response. Lupus patients have insufficient PELI2 levels and high basal interferon production, suggesting that PELI2 dysregulation may drive the onset of lupus and other interferonopathies.

PELI1 Selectively Targets Kinase-Active RIP3 for Ubiquitylation-Dependent Proteasomal Degradation.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.

Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

BECC438b TLR4 agonist supports unique immune response profiles from nasal and muscular DTaP pertussis vaccines in murine challenge models.

The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.

The role of forkhead-associated (FHA)-domain proteins in plant biology.

The forkhead-associated (FHA) domain, a well-characterized small protein module that mediates protein-protein interactions by targeting motifs containing phosphothreonine, is present in many regulatory molecules like protein kinase, phosphatases, transcription factors, and other functional proteins. FHA-domain containing proteins in yeast and human are involved in a large variety of cellular processes such as DNA repair, cell cycle arrest, or pre-mRNA processing. Since the first FHA-domain protein, kinase-associated protein phosphatase (KAPP) was found in plants, the interest in plant FHA-containing proteins has increased dramatically, mainly due to the important role of FHA domain-containing proteins in plant growth and development. In this review, we provide a comprehensive overview of the fundamental properties of FHA domain-containing proteins in plants, and systematically summarized and analyzed the research progress of proteins containing the FHA domain in plants. We also emphasized that AT5G47790 and its homologs may play an important role as the regulatory subunit of protein phosphatase 1 (PP1) in plants.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Use of pulsatile gonadotropin-releasing hormone (GnRH) in patients with functional hypothalamic amenorrhea (FHA) results in monofollicular ovulation and high cumulative live birth rates: a 25-year cohort.

PURPOSE: To analyze outcomes of pulsatile administration of gonadotropin-releasing hormone (GnRH) in infertile women diagnosed with functional hypothalamic amenorrhea (FHA). METHODS: A single-center retrospective cohort study was conducted from 1996 to 2020. Sixty-six patients with the diagnosis FHA that underwent therapy using the pulsatile GnRH pump for conception were included and analyzed. The primary outcome was the live birth rate (LBR). Secondary outcomes were the number of dominant follicles, ovulation rate, biochemical pregnancy rate (BPR), clinical pregnancy rate (CPR), miscarriage rate, and multiple pregnancy rate. A matched control group was selected to compare the birth weight of newborn children. RESULTS: During the study period, 66 patients with FHA underwent 82 treatments (14 of 66 patients had more than one treatment) and a total of 212 cycles (ovulation induction attempts) using pulsatile GnRH. The LBR per treatment was 65.9%. The ovulation rate per cycle was 96%, and monofollicular ovulation was observed in 75% of cycles. The BPR per treatment was 80.5%, and the cumulative CPR per treatment was 74.4%. The miscarriage rate was 11.5%. One dizygotic twin pregnancy was observed (1.6%). Average newborn birth weight (NBW) from patients with FHA was comparable to the control group. CONCLUSION(S): In patients with FHA, excellent pregnancy rates were achieved using the subcutaneous GnRH pump. The high cumulative LBR with normal NBW as well as low rates of multiple gestation indicate that the pulsatile GnRH pump represents a safer and more physiologic alternative to ovulation induction with injectable gonadotropins. TRIAL REGISTRATION: Ethics Committee Northwest and Central Switzerland (Ethikkommission Nordwest- und Zentralschweiz - EKNZ) - Project-ID 2020-01612.

AGGF1 inhibits the expression of inflammatory mediators and promotes angiogenesis in dental pulp cells.

OBJECTIVES: To determine the role of angiogenic factor with G-patch and FHA domain 1 (AGGF1) in inflammatory response of human dental pulp cells (DPCs) and the underneath mechanism and to explore its role in angiogenesis. MATERIALS AND METHODS: The expression of AGGF-1 in human healthy and inflammatory pulp tissues was detected by immunohistochemistry. RT-qPCR and Western blot were used to evaluate the expression of AGGF1 in DPCs stimulated by lipopolysaccharide (LPS). After AGGF1 was knocked down, the expression of LPS-induced inflammatory cytokines in DPCs was quantified by RT-qPCR and ELISA. Immunofluorescence and Western blot were used to assess the activation of NF-κB signaling. Inflammatory cytokines were detected by RT-qPCR and ELISA in DPCs pretreated with NF-κB pathway inhibitors before LPS stimulation, and then the effect of AGGF1 on angiogenesis was also evaluated. RESULTS: AGGF1 expression increased in inflammatory dental pulp tissues. In DPCs stimulated by LPS, AGGF1 was upregulated in a dose-dependent manner (P < 0.05). In AGGF1 knockdown cells, the expression of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1/CCL-2) increased by LPS stimulation (P < 0.001). Nuclear translocation of p65 was promoted, and the addition of NF-κB inhibitors inhibited the expression of inflammatory factors. Meanwhile, knockdown of AGGF1 inhibited vascularization. CONCLUSIONS: AGGF1 inhibited the synthesis of inflammatory cytokines through NF-κB signaling pathway and promoted the angiogenesis of DPCs. CLINICAL RELEVANCE: This study might shed light in the treatment of pulpitis and regeneration of dental pulp tissues; however, more clinical trials are required to validate these findings.

Neuroendocrine Effects of Carnitines on Reproductive Impairments.

Carnitines are quaternary amines involved in various cellular processes such as fatty acid uptake, ß-oxidation and glucose metabolism regulation. Due to their neurotrophic activities, their integrative use has been studied in several different physio-pathological conditions such as anorexia nervosa, chronic fatigue, vascular diseases, Alzheimer's disease and male infertility. Being metabolically active, carnitines have also been proposed to treat reproductive impairment such as functional hypothalamic amenorrhea (FHA) and polycystic ovary syndrome (PCOS) since they improve both hormonal and metabolic parameters modulating the neuroendocrine impairments of FHA. Moreover, they are capable of improving the lipid profile and the insulin sensitivity in patients with PCOS.

DNA repair functional analyses of NBN hypomorphic variants associated with NBN-related infertility.

Nijmegen breakage syndrome caused by biallelic pathogenic variants of the DNA-damage response gene NBN, is characterized by severe microcephaly, cancer proneness, infertility, and karyotype abnormalities. We previously reported NBN variants in siblings suffering from fertility defects. Here, we identify a new founder NBN variant (c.442A>G, p.(Thr148Ala)) in Lebanese patients associated with isolated infertility. Functional analyses explored preserved or altered functions correlated with their remarkably mild phenotype. Transcript and protein analyses supported the use of an alternative transcript with in-frame skipping of exons 4-5, leading to p84-NBN protein with a preserved forkhead-associated (FHA) domain. The level of NBN was dramatically reduced and the MRN complex delocalized to the cytoplasm. Interestingly, ataxia-elangiectasia mutated (ATM) also shifted from the nucleus to the cytoplasm, suggesting some interaction between ATM and the MRN complex at a steady state. The ATM pathway activation, attenuated in typical patients with NBS, appeared normal under camptothecin treatment in these new NBN-related infertile patients. Cell cycle checkpoint defect was present in these atypical patients, although to a lesser extent than in typical patients with NBS. In conclusion, we report three new NBN-related infertile patients and we suggest that preserved FHA domain could be responsible for the mild phenotype and intermediate DNA-damage response defects.

Overexpression of PP1-NIPP1 limits the capacity of cells to repair DNA double-strand breaks.

The ubiquitously expressed nuclear protein NIPP1 (also known as PPP1R8) recruits phosphoproteins for regulated dephosphorylation by the associated protein phosphatase PP1. To bypass the PP1 titration artifacts seen upon NIPP1 overexpression, we have engineered covalently linked fusions of PP1 and NIPP1, and demonstrate their potential to selectively explore the function of the PP1:NIPP1 holoenzyme. By using inducible stable cell lines, we show that PP1-NIPP1 fusions cause replication stress in a manner that requires both PP1 activity and substrate recruitment via the ForkHead Associated domain of NIPP1. More specifically, PP1-NIPP1 expression resulted in the build up of RNA-DNA hybrids (R-loops), enhanced chromatin compaction and a diminished repair of DNA double-strand breaks (DSBs), culminating in the accumulation of DSBs. These effects were associated with a reduced expression of DNA damage signaling and repair proteins. Our data disclose a key role for dephosphorylation of PP1:NIPP1 substrates in setting the threshold for DNA repair, and indicate that activators of this phosphatase hold therapeutic potential as sensitizers for DNA-damaging agents.

FHA2 is a plant-specific ISWI subunit responsible for stamen development and plant fertility.

Imitation Switch (ISWI) chromatin remodelers are known to function in diverse multi-subunit complexes in yeast and animals. However, the constitution and function of ISWI complexes in Arabidopsis thaliana remain unclear. In this study, we identified forkhead-associated domain 2 (FHA2) as a plant-specific subunit of an ISWI chromatin-remodeling complex in Arabidopsis. By in vivo and in vitro analyses, we demonstrated that FHA2 directly binds to RLT1 and RLT2, two redundant subunits of the ISWI complex in Arabidopsis. The stamen filament is shorter in the fha2 and rlt1/2 mutants than in the wild type, whereas their pistil lengths are comparable. The shorter filament, which is due to reduced cell size, results in insufficient pollination and reduced fertility. The rlt1/2 mutant shows an early-flowering phenotype, whereas the phenotype is not shared by the fha2 mutant. Consistent with the functional specificity of FHA2, our RNA-seq analysis indicated that the fha2 mutant affects a subset of RLT1/2-regulated genes that does not include genes involved in the regulation of flowering time. This study demonstrates that FHA2 functions as a previously uncharacterized subunit of the Arabidopsis ISWI complex and is exclusively involved in regulating stamen development and plant fertility.

TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha.

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control.

Pertactin-Negative and Filamentous Hemagglutinin-Negative Bordetella pertussis, Australia, 2013-2017.

During the 2008-2012 pertussis epidemic in Australia, pertactin (Prn)-negative Bordetella pertussis emerged. We analyzed 78 isolates from the 2013-2017 epidemic and documented continued expansion of Prn-negative ptxP3 B. pertussis strains. We also detected a filamentous hemagglutinin-negative and Prn-negative B. pertussis isolate.

Pellino1 specifically binds to phospho-Thr18 of p53 and is recruited to sites of DNA damage.

Pellino1 is an E3 ubiquitin ligase that plays a key role in positive regulation of innate immunity signaling, specifically required for the production of interferon when induced by viral double-stranded RNA. We report the identification of the tumor suppressor protein, p53, as a binding partner of Pellino1. Their interaction has a Kd of 42 ±â€¯2 µM and requires phosphorylation of Thr18 within p53 and association with the forkhead-associated (FHA) domain of Pellino1. We employed laser micro-irradiation and live cell microscopy to show that Pellino1 is recruited to newly occurring DNA damage sites, via its FHA domain. Mutation of a hitherto unidentified nuclear localization signal within the N-terminus of Pellino1 led to its exclusion from the nucleus. This study provides evidence that Pellino1 translocates to damaged DNA in the nucleus and has a functional role in p53 signaling and the DNA damage response.

Identification of common antigens of three pathogenic Nocardia species and development of DNA vaccine against fish nocardiosis.

Fish nocardiosis is a chronic granulomatous bacterial disease and three pathogens have been reported so far, including Nocardia asteroids, N. seriolae and N. salmonicida. However, the absence of antigen markers is a bottleneck for developing effective vaccines against fish nocardiosis. In this study, the antigenicity of whole-cell protein of these three pathogenic Nocardia species were profiled by immunoproteomic analysis and 7 common immunogenic proteins were identified as follows: molecular chaperone DnaK (DnaK), molecular chaperone GroEL (GroEL), 30 S ribosomal protein S1 (RpsA), TerD family protein (TerD), FHA domain-containing protein (FHA), 50 S ribosomal protein L7/L12 (RplL) and PspA/IM30 family protein (PspA). Furthermore, the DNA vaccine encoding FHA gene against fish nocardiosis was developed and its efficacy was investigated in hybrid snakehead. The results suggested that it needed at least 7 d to transport pcDNA-FHA DNA vaccine from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection. In addition, non-specific immunity paraments (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were used to evaluate the immune response induced in pcDNA-FHA vaccinated hybrid snakehead, it proved that all these mentioned immune activities were significantly enhanced after immunization. The results also showed hybrid snakehead vaccinated with pcDNA-FHA had higher survival rate (79.33%) compared with the controls after challenge with N. seriolae, indicating that the pcDNA-FHA DNA vaccine can supply immune protection against N. seriolae infection. Taken together, this study may warrant further development of these common immunogenic proteins as the antigens for vaccine or diagnosis and facilitate the prevention and treatment of fish nocardiosis.

Identification of the TIFAB Gene as a Susceptibility Locus for Coronary Artery Aneurysm in Patients with Kawasaki Disease.

Kawasaki disease (KD) is a self-limiting systemic vasculitis of unknown etiology. KD is often complicated by coronary artery aneurysms (CAAs), which develop in about 20-25% of untreated children and 3-5% of children treated with intravenous immunoglobulin therapy. To identify the risk loci for CAA susceptibility in patients with KD, we performed a genome-wide association study (GWAS) using our previous Illumina HumanOmni1-Quad BeadChip data (296 KD patients) and a new replication study in an independent sample set (713 KD patients) by grouping KD patients without CAA (control) versus KD patients with extremely large aneurysms (diameter ≥ 5 mm) (case). Among 44 candidate single -nucleotide polymorphisms (SNPs) selected from the initial GWAS data (33 cases vs. 215 controls), a SNP (rs899162) located 7 kb upstream of the TIFAB gene on chromosome five was replicated in an independent sample (12 cases vs. 532 controls). In the combined analysis (45 cases vs. 747 controls), the SNP (rs899162) showed a highly significant association with CAA formation (diameter ≥ 5 mm) in patients with KD (odds ratio = 3.20, 95% confidence interval = 2.02-5.05, Pcombined = 1.95 × 10-7). These results indicate that the TIFAB gene may act as a CAA susceptibility locus in patients with KD.

Screening and Genomic Characterization of Filamentous Hemagglutinin-Deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure.

Aggf1 attenuates neuroinflammation and BBB disruption via PI3K/Akt/NF-κB pathway after subarachnoid hemorrhage in rats.

BACKGROUND: Neuroinflammation and blood-brain barrier (BBB) disruption are two critical mechanisms of subarachnoid hemorrhage (SAH)-induced brain injury, which are closely related to patient prognosis. Recently, angiogenic factor with G-patch and FHA domain 1 (Aggf1) was shown to inhibit inflammatory effect and preserve vascular integrity in non-nervous system diseases. This study aimed to determine whether Aggf1 could attenuate neuroinflammation and preserve BBB integrity after experimental SAH, as well as the underlying mechanisms of its protective roles. METHODS: Two hundred forty-nine male Sprague-Dawley rats were subjected to the endovascular perforation model of SAH. Recombinant human Aggf1 (rh-Aggf1) was administered intravenously via tail vein injection at 1 h after SAH induction. To investigate the underlying neuroprotection mechanism, Aggf1 small interfering RNA (Aggf1 siRNA) and PI3K-specific inhibitor LY294002 were administered through intracerebroventricular (i.c.v.) before SAH induction. SAH grade, neurological score, brain water content, BBB permeability, Western blot, and immunohistochemistry were performed. RESULTS: Expression of endogenous Aggf1 was markedly increased after SAH. Aggf1 was primarily expressed in endothelial cells and astrocytes, as well as microglia after SAH. Administration of rh-Aggf1 significantly reduced brain water content and BBB permeability, decreased the numbers of infiltrating neutrophils, and activated microglia in the ipsilateral cerebral cortex following SAH. Furthermore, rh-Aggf1 treatment improved both short- and long-term neurological functions after SAH. Meanwhile, exogenous rh-Aggf1 significantly increased the expression of PI3K, p-Akt, VE-cadherin, Occludin, and Claudin-5, as well as decreased the expression of p-NF-κB p65, albumin, myeloperoxidase (MPO), TNF-α, and IL-1ß. Conversely, knockdown of endogenous Aggf1 aggravated BBB breakdown, inflammatory response and neurological impairments at 24 h after SAH. Additionally, the protective roles of rh-Aggf1 were abolished by LY294002. CONCLUSIONS: Taken together, exogenous Aggf1 treatment attenuated neuroinflammation and BBB disruption, improved neurological deficits after SAH in rats, at least in part through the PI3K/Akt/NF-κB pathway.