Classical Pathway Research Articles

Aromatic Amino Acids Promote Lipid Metabolism Disorders by Increasing Hepatic Bile Acid Synthesis

BackgroundObesity is a progressive metabolic disease that begins with lipid metabolism disorders. Aromatic amino acids (AAAs), including tryptophan, phenylalanine, and tyrosine, have diverse biological activities as nutrients. However, the underlying mechanisms by which AAAs affect lipid metabolism are unclear. ObjectivesThis study was designed to investigate the possible roles and underlying molecular mechanisms of AAA in the pathogenesis of lipid metabolism disorders. MethodsWe added an AAA mixture to the high-fat diet (HFD) of mice. Glucose tolerance test was recorded. Protein expression of hepatic bile acid (BA) synthase and mRNA expression of BA metabolism-related genes were determined. Hepatic BA profiles and gut microbial were also determined in mice. ResultsThe results showed that AAA significantly increased body weight and white adipose tissue, aggravated liver injury, impaired glucose tolerance and intestinal integrity, and significantly increased hepatic BA synthesis by inhibiting intestinal farnesoid X receptor (FXR). Moreover, AAA increased the content of total BA in the liver and altered the hepatic BA profile, with elevated levels of lithocholic acid, glycochenodeoxycholic acid, and glycoursodeoxycholic acid. AAA markedly increased the levels of proteins involved in BA synthesis (cholesterol 7α-hydroxylase and oxysterol 7α-hydroxylase) and inhibited the intestinal FXR. Gut microbial composition also changed, reducing the abundance of some beneficial bacteria, such as Parvibacter and Lactobacillus. ConclusionsUnder HFD conditions, AAAs stimulate BA synthesis in both the classical and alternative pathways, leading to aggravation of liver injury and fat deposition. Excessive intake of AAA disrupts BA metabolism and contributes to the development of lipid metabolism disorders, suggesting that AAA may be a causative agent of lipid metabolism disorders.

High Ki67 Gene Expression Is Associated With Aggressive Phenotype in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) with high Ki67 protein expression, the most commonly used cell proliferation marker, is associated with an aggressive biologic phenotype; however, conventional immunostaining is hampered by variability in institutional protocol, specific antibody probe, and by assessor subjectivity. To this end, we hypothesized that Ki67 gene (MKi67) expression would identify highly proliferative HCC, and clarify its association with oncologic outcome, tumor progression, and immune cell population in the tumor microenvironment (TME). Furthermore, we sought to identify the cell-cycle gene expression profile that confers this aggressive phenotype. A total of 473 HCC patients with clinicopathological data associated with transcriptome were selected for this study: 358 patients from The Cancer Genome Atlas (TCGA) as the testing cohort, and 115 from GSE76427 as the validation cohort. Each cohort was divided into a highly proliferative group (MKi67-high) and the low MKi67 group (MKi67-low) by the median of Ki67 gene (MKi67) expression levels. MKi67-high HCC patients had worse disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS) independent of histological grade in the TCGA cohort. MKi67 expression correlated with histological grade and tumor size. MKi67 expression increased throughout the HCC carcinomatous sequence from normal liver, cirrhotic liver, early HCC, and advanced HCC. MKi67-high HCC was associated with higher intratumor heterogeneity, homologous recombination deficiency, and altered fraction as well as intratumoral infiltration of T helper type 1 (Th1) and Th2 cells, but lower interferon-gamma response and M2 macrophage infiltration. Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, Myc target v1 and mitotic spindle), MTORC1 signaling, DNA repair, PI3K MTOR signaling, and unfolded protein response were all enriched in the MKi67-high HCC (false discovery rate (FDR) < 0.25). High MKi67 gene expression identified highly proliferative HCC with aggressive biology involving classical pathways in cell cycle regulation and DNA repair, as well as poor overall oncologic outcomes. This suggests potential for personalized treatment strategies, but validation and refinement of these observations require further research to elucidate the underlying mechanisms and validate therapeutic targeting of these pathways in MKi67-high HCC tumors.

Імунологічні особливості у вагітних із великими акушерськими синдромами

Aim of the research – to evaluate the adaptive capabilities of the immunological system in pregnant women who had complications from the group of major obstetric syndromes (МОS) for the further development of an algorithm for the prevention of this complication. Materials and methods. The main study group (MG) - 99 pregnant women who had a complication from the group of MOS, which was clinically manifested by placental insufficiency (PI); control group (CG) - 50 practically healthy pregnant women with a healthy reproductive history and an uncomplicated course of this pregnancy. The number (x109/l) and relative (%) of lymphocyte subpopulations CD3+ (T-lymphocytes), CD4+ (helper-inducers), CD8+ (cytotoxic suppressors), CD56+ (natural killers), CD19+ (B-lymphocytes) were determined; the presence of antiphospholipid antibodies (APA); total hemolytic activity of the classical way of complement activation (CH50); levels of IgG, IgM, IgA. Statistical processing of research results was carried out using standard Microsoft Excel 5.0 and Statistical 6.0 programs. Results. In the I and II trimesters of pregnancy in MG and CG, a tendency to decrease was observed, СM4+ (51.8±2.5% and 48.9±1.6; 58.4±2.3% and 55.6±2.6%), and СD8+ (24.6±2.4% and 21.2±1.2%; 31.3±2.5% and 30.9±2.7%), which led to an increase in the immunoregulatory index (СD4+/СD8+) in MG (р&lt;0.05). The level of СD19+, during gestation there was an increase in their relative number in MG (13.6±0.98%; 16.4±2.03% and 21.1±2.01%) (p&lt;0.05); a predominance of the percentage of CD56+ lymphocytes, which have the properties of natural killers, was observed in pregnant MG (12.3±1.7%; 15.1±1.7%; 13.9±1.73% vs. 8.6±1.4%; 8.1±1.18%; 7.2±0.98%; p&lt;0.05). Hemolytic activity of the classical pathway of complement activation (CH50) in MG pregnant women was 1.5 times higher compared to CG pregnant women (p&lt;0.05). APA, represented by IgG to cardiolipin, were detected in 47 (47.5%) pregnant women MG and 4 (8.0%) women pregnant CG (p&lt;0.05). Conclusions. Іn pregnant women with МОS, there is an immune maladaptation that progressively increases in the dynamics of gestation and is manifested by excessive activation of natural killers (CD56+), an increase in the immunoregulatory coefficient (CD4+/CD8+), an increase in the percentage of B-lymphocytes (CD19+), dysimmunoglobulinemia, which is accompanied by high indicators of the general hemolytic activity of the classical pathway of complement activation (CH50), the presence of APA. The research was conducted according to principles of Declaration of Helsinki. Protocol of research was proved by local ethical committee, mentioned in institution’s work. A informed sonsennt was collected in order to carry out the research. The author is stating no conflict of interests is declared.

Insect Antimicrobial Peptides as Guardians of Immunity and Beyond: A Review.

Antimicrobial peptides (AMPs), as immune effectors synthesized by a variety of organisms, not only constitute a robust defense mechanism against a broad spectrum of pathogens in the host but also show promising applications as effective antimicrobial agents. Notably, insects are significant reservoirs of natural AMPs. However, the complex array of variations in types, quantities, antimicrobial activities, and production pathways of AMPs, as well as evolution of AMPs across insect species, presents a significant challenge for immunity system understanding and AMP applications. This review covers insect AMP discoveries, classification, common properties, and mechanisms of action. Additionally, the types, quantities, and activities of immune-related AMPs in each model insect are also summarized. We conducted the first comprehensive investigation into the diversity, distribution, and evolution of 20 types of AMPs in model insects, employing phylogenetic analysis to describe their evolutionary relationships and shed light on conserved and distinctive AMP families. Furthermore, we summarize the regulatory pathways of AMP production through classical signaling pathways and additional pathways associated with Nitric Oxide, insulin-like signaling, and hormones. This review advances our understanding of AMPs as guardians in insect immunity systems and unlocks a gateway to insect AMP resources, facilitating the use of AMPs to address food safety concerns.

The conserved GOLD domain in the Plasmodiophora brassicae effector Pb257 is required for triggering cell death and root swelling

Plasmodiophora brassicae is one of the biggest threats to cruciferous plants and one of the most economically significant diseases worldwide. However, our current understanding of its pathogenic mechanisms remains limited. In this study, we have identified an effector, Pb257, which plays a crucial role in the virulence of P. brassicae. The expression pattern analysis revealed continuous induction of Pb257 during both primary and secondary infections. Ectopic expression of Pb257 strongly triggered cell death in Nicotiana benthamiana and several other plants, which was dependent on SOBIR1 and the salicylic acid pathway but not BAK1, a central molecular regulator, to mediate classical defense pathways. Overexpression of Pb257 increased susceptibility of Brassica rapa to P. brassicae. Silencing of the Pb257-encoding gene led to reduce root swelling. Further investigation showed that the conserved GOLD domain within Pb257 was essential for inducing root enlargement, which is similar to the mild symptoms of clubroot, indicating that it is an important effector for promoting root cell enlargement. GOLD domain-containing family proteins were widely present in the kingdoms of protozoa, fungi, and animalia, and Pb257 was clustered into a clade of protozoa, suggesting the encoding gene as a potential marker gene for classification of P. brassicae. The GOLD domain-containing proteins are known regulators involved in human cell proliferation and immune system disruption, however, no previous reports have described a pathogen-derived GOLD domain protein with elicitor activity. Our findings demonstrate that Pb257 functions as a crucial effector contributing to the virulence of P. brassicae and could be used as a potential molecular target for controlling clubroot disease.

The molecular determinants of classical pathway complement inhibition by OspEF-related proteins of Borrelia burgdorferi

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp/protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.

Maintenance of persistent transmission of a plant arbovirus in its insect vector mediated by the Toll-Dorsal immune pathway

Throughout evolution, arboviruses have developed various strategies to counteract the host's innate immune defenses to maintain persistent transmission. Recent studies have shown that, in addition to bacteria and fungi, the innate Toll-Dorsal immune system also plays an essential role in preventing viral infections in invertebrates. However, whether the classical Toll immune pathway is involved in maintaining the homeostatic process to ensure the persistent and propagative transmission of arboviruses in insect vectors remain unclear. In this study, we revealed that the transcription factor Dorsal is actively involved in the antiviral defense of an insect vector (Laodelphax striatellus) by regulating the target gene, zinc finger protein 708 (LsZN708), which mediates downstream immune-related effectors against infection with the plant virus (Rice stripe virus, RSV). In contrast, an antidefense strategy involving the use of the nonstructural-protein (NS4) to antagonize host antiviral defense through competitive binding to Dorsal from the MSK2 kinase was employed by RSV; this competitive binding inhibited Dorsal phosphorylation and reduced the antiviral response of the host insect. Our study revealed the molecular mechanism through which Toll-Dorsal-ZN708 mediates the maintenance of an arbovirus homeostasis in insect vectors. Specifically, ZN708 is a newly documented zinc finger protein targeted by Dorsal that mediates the downstream antiviral response. This study will contribute to our understanding of the successful transmission and spread of arboviruses in plant or invertebrate hosts.

The engineered exosomes targeting ferroptosis: A novel approach to reverse immune checkpoint inhibitors resistance.

Immune checkpoint inhibitors (ICIs) have been extensively used in immunological therapy primarily due to their ability to prolong patient survival. Although ICIs have achieved success in cancer treatment, the resistance of ICIs should not be overlooked. Ferroptosis is a newly found cell death mode characterized by the accumulation of reactive oxygen species (ROS), glutathione (GSH) depletion, and glutathione peroxidase 4 (GPX4) inactivation, which has been demonstrated to be beneficial to immunotherapy and combining ferroptosis and ICIs to exploit new immunotherapies may reverse ICIs resistance. Exosomes act as mediators in cell-to-cell communication that may regulate ferroptosis to influence immunotherapy through the secretion of biological molecules. Thus, utilizing exosomes to target ferroptosis has opened up exciting possibilities for reversing ICIs resistance. In this review, we summarize the mechanisms of ferroptosis improving ICIs therapy and how exosomes regulate ferroptosis through adjusting iron metabolism, blocking the ROS accumulation, controlling ferroptosis defense systems, and influencing classic signaling pathways and how engineered exosomes target ferroptosis and improve ICIs efficiency.

Indole-3-acetic acid alleviates DSS-induced colitis by promoting the production of R-equol from Bifidobacterium pseudolongum

ABSTRACT Background Inflammatory bowel disease (IBD) is characterized by immune-mediated, chronic inflammation of the intestinal tract. The occurrence of IBD is driven by the complex interactions of multiple factors. The objective of this study was to evaluate the therapeutic effects of IAA in colitis. Method C57/BL6 mice were administered 2.5% DSS in drinking water to induce colitis. IAA, Bifidobacterium pseudolongum, and R-equol were administered by oral gavage and fed a regular diet. The Disease Activity Index was used to evaluate disease activity. The degree of colitis was evaluated using histological morphology, RNA, and inflammation marker proteins. CD45+ CD4+ FOXP3+ Treg and CD45+ CD4+ IL17A+ Th17 cells were detected by flow cytometry. Analysis of the gut microbiome in fecal content was performed using 16S rRNA gene sequencing. Gut microbiome metabolites were analyzed using Untargeted Metabolomics. Result In our study, we found IAA alleviates DSS-induced colitis in mice by altering the gut microbiome. The abundance of Bifidobacterium pseudolongum significantly increased in the IAA treatment group. Bifidobacterium pseudolongum ATCC25526 alleviates DSS-induced colitis by increasing the ratio of Foxp3+T cells in colon tissue. R-equol alleviates DSS-induced colitis by increasing Foxp3+T cells, which may be the mechanism by which ATCC25526 alleviates DSS-induced colitis in mice. Conclusion Our study demonstrates that IAA, an indole derivative, alleviates DSS-induced colitis by promoting the production of Equol from Bifidobacterium pseudolongum, which provides new insights into gut homeostasis regulated by indole metabolites other than the classic AHR pathway.

Characterization of individual bile acids in vivo utilizing a novel low bile acid mouse model.

Bile acids (BAs) are signaling molecules synthesized in the liver initially by CYP7A1 and CYP27A1 in the classical and alternative pathways, respectively. BAs are essential for cholesterol clearance, intestinal absorption of lipids, and endogenous modulators of farnesoid x receptor (FXR). FXR is critical in maintaining BA homeostasis and gut-liver crosstalk. Complex reactions in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs. In this study, we characterized the in vivo effects of three-day feeding of cholic acid (CA), deoxycholic acid (DCA), or ursodeoxycholic acid (UDCA) at physiological/non-hepatotoxic concentrations in a novel low-BA mouse model (Cyp7a1-/-/Cyp27a1-/-, DKO). Liver injury, BA levels and composition and BA signaling by the FXR-fibroblast growth factor 15 (FGF15) axis were determined. Overall, higher basal inflammation and altered lipid metabolism in DKO mice might be associated with low BAs. CA, DCA, and UDCA feeding activated FXR signals with tissue specificity. Dietary CA and DCA similarly altered tissue BA profiles to be less hydrophobic, while UDCA promoted a more hydrophobic tissue BA pool with the profiles shifted toward non-12α-OH BAs and secondary BAs. However, UDCA did not offer any overt protective effects as expected. These findings allow us to determine the precise effects of individual BAs in vivo on BA-FXR signaling and overall BA homeostasis in liver physiology and pathologies.

Mechanisms of YAP1-mediated trophoblast ferroptosis in recurrent pregnancy loss.

The purpose of our study is to investigate the function of YAP1 in the trophoblast ferroptosis and maternal-fetal interface communication of RPL. We collected 25 villous tissues and detected the expression of YAP1. Cell counting kit-8 assay, scratch wound-healing assay, and Matrigel invasion assay were performed to observe the proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Subsequently, measured the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), SLC7A11, SOD2, and GPX4. Ultimately, the use of ferroptosis activator (erastin) and inhibitor (Ferrostatin-1, fer-1) further confirmed the regulation by YAP1. In addition, established an in vitro-induced cell model to study the effect of YAP1 on the decidualization process. Finally, animal models were implemented for further confirmation. We found that YAP1 was downregulated in RPL patients. Overexpression of YAP1 could significantly enhance the proliferation, migration, and invasion of trophoblasts, and inhibit ferroptosis. Knocking down YAP1 exhibited the opposite effect. Rescue experiments have shown that YAP1 could upregulate the expression of SLC7A11 and GPX4, which are key molecules in the classic pathway of ferroptosis. In addition, the decidualization was impaired when hESCs were treated with conditioned medium of YAP1 knockdown trophoblasts. Moreover, we found that Yap1, Slc7a11, and Gpx4 were downregulated in the RPL mice, along with increased MDA and decreased GSH. Downregulation of YAP1 induces ferroptosis, thereby damaging the trophoblast invasion processes, which also disturbs the communication at the maternal-fetal interface. Our study identified YAP1 as a potential key molecule in the pathogenesis of RPL.

High rates of nitrogen removal in aerated VFCWs treating sewage through C-N-S cycle

The efficiency of deep aerated vertical flow constructed wetlands (DA-VFCWs) being operated in Hyderabad, India, was evaluated herein using physicochemical analysis and 16S rRNA amplicon sequencing. The results showed 2–4-fold higher removal rate coefficients for Biochemical oxygen demand (1.32–––3.53 m/d) and nitrogen (0.88––1.36 m/d) in DA-VFCWs than those of passive VFCWs. Elevated sulfate concentration in the DA-VFCWs effluent (84–113 mg/L) indicated possibility of sulfur-driven autotrophic denitrification (SDAD) as a major pathway operating in these wetlands besides the classical nitrogen removal pathways. The presence of nitrifiers (3.09–10.02 %), heterotrophic and aerobic denitrifiers (0.79–0.83 %), anammox bacteria (1.31–2.22 %) and SDAD bacteria (0.08–0.73 %) in the biofilm samples collected from the DA-VFCWs exemplify an interplay of Carbon-Nitrogen-Sulfur cycles in these systems. If proven, the presence of an operational SDAD pathway in DA-VFCWs can help reduce surface area requirement in VFCWs substantially besides alleviating biological clogging of the wetland substrate.

Interleukin 3 Inhibits Glutamate-Cytotoxicity in Neuroblastoma Cell Line.

Interleukin 3 (IL-3) is a well-known pleiotropic cytokine that regulates the proliferation and differentiation of hematopoietic progenitor cells, triggering classical signaling pathways such as JAK/STAT, Ras/MAPK, and PI3K/Akt to carry out its functions. Interestingly, the IL-3 receptor is also expressed in non-hematopoietic cells, playing a crucial role in cell survival. Our previous research demonstrated the expression of the IL-3 receptor in neuron cells and its protective role in neurodegeneration. Glutamate, a principal neurotransmitter in the central nervous system, can induce cellular stress and lead to neurotoxicity when its extracellular concentrations surpass normal levels. This excessive glutamate presence is frequently observed in various neurological diseases. In this study, we uncover the protective role of IL-3 as an inhibitor of glutamate-induced cell death, analyzing the cytokine's signaling pathways during its protective effect. Specifically, we examined the relevance of JAK/STAT, Ras/MAPK, and PI3 K signaling pathways in the molecular mechanism triggered by IL-3. Our results show that the inhibition of JAK, ERK, and PI3 K signaling pathways, using pharmacological inhibitors, effectively blocked IL-3's protective role against glutamate-induced cell death. Additionally, our findings suggest that Bcl-2 and Bax proteins may be involved in the molecular mechanism triggered by IL-3. Our investigation into IL-3's ability to protect neuronal cells from glutamate-induced damage offers a promising therapeutic avenue with potential clinical implications for several neurological diseases characterized by glutamate neurotoxicity.

Comparative analysis of basal and etoposide-induced alterations in gene expression by DNA-PKcs kinase activity.

Background: Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical nonhomologous end-joining pathway, but it also has roles in modulating gene expression and thus, the overall cellular response to DNA damage. Methods: Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Results: Downregulated genes were mostly involved in protein, sugar, and nucleic acid biosynthesis pathways in both genotypes, but enriched biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/β-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Finally, we have used qPCR to validate selected genes among the differentially regulated pathways to validate RNA sequence data. Conclusion: Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trials as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.

O232: Enzymatic blood group conversion prevents complement activation in an ABO-incompatible model of human kidney transplantation

Abstract Introduction Recently, enzymatic blood group conversion has been used to convert human kidneys from blood group A to universal blood group O using bacterial enzymes from Flavonifractor plautii. However, little is known about the functional consequences of antigen removal when a treated kidney is transplanted in an ABO-incompatible (ABOi) recipient. Here, we investigated classical complement activation during ABOi conditions in enzyme treated (ABOe) vs control human type A kidneys. Method Three biological pairs of type A human kidneys rejected for transplantation and offered for research were used in this study. One kidney per pair was treated with 1mg/L of FpGalNAc deacetylase and FpGalactosaminidase during 6hrs of hypothermic machine perfusion while the contralateral kidney was perfused without enzymes. Both kidneys were subsequently perfused for 4hrs in ABOi conditions using normothermic machine perfusion (10% AB serum; mouse anti-A IgM titre 1:128; type O red blood cells). Tissue-bound components of classical complement activation were assessed in pre- and post-perfusion biopsies. Results Following ABOi perfusion, control kidneys showed peritubular deposition of the classical complement component C1qA and further complement activation components C4d and C5b-9. The staining directly co-localised with regions of anti-A staining. In ABOe kidneys, no anti-A staining was observed, with no C1qA, C4d, or C5b-9 staining found in post-perfusion biopsies. Conclusions We have shown for the first time that ABOe kidneys do not activate the classical complement pathway in ABOi conditions. This work shows the potential of the enzymatic blood group conversion to evade hyperacute antibody-mediated rejection during renal transplantation.

Complement System Response to Adeno-Associated Virus Vector Gene Therapy.

Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.

A single C1q A chain promotes leukocytes phagocytosis/chemotaxis and mediates bacterial clearance in Japanese flounder Paralichthys olivaceus

Complement component 1q (C1q) is the first component involved in the activation of complement classical pathway and acts as a linker of the innate and adaptive immune system. The C1q is a macromolecular containing three chains i.e. C1q A, C1q B, and C1q C. In teleost, the biological functions of C1q A chain is still unclear. In this study, we investigated the immunological properties of a C1q A chain from Japanese flounder (Paralichthys olivaceus) (PoC1qA). PoC1qA containing the conserved CLR and C1q domain, shares 45.4%–78.6% identities with that of other teleost and mammals. PoC1qA expression pattern was constitutive in tissues, with higher expression in spleen and liver. Besides, the PoC1qA expression was notably regulated by pathogenic invasion. In vitro the recombinant PoC1qA (rPoC1qA) could directly bind a variety of bacteria, and affect bacterial growth and survival. Moreover, PoC1qA could influence the hemolysis and bactericidal activity of fish serum. Additionally, rPoC1qA could interact with the membrane of peripheral blood leucocytes (PBLs), promoting the PBLs phagocytosis and chemotaxis, and enhancing the activation of PBLs. Consistently, in vivo rPoC1qA could restrain the bacterial replication in immune tissues. To sum up, our observations added the first evidence that an A chain of teleost C1q plays vital roles in mediating PBLs activity and antibacterial immunity.

A novel teleost C1q C chain (PoC1qC) participates in complement and leukocytes antibacterial immunity

Complement C1q composed of C1q A, B, C chains is the initial vital protein of classical complement pathway, playing an essential role in innate immune response. Whereas in teleost, the immune roles of C1q C chain are largely scarce. In this research, we studied the complement/peripheral blood leukocytes (PBLs) activation and antibacterial potentials mediated by Japanese flounder (Paralichthys olivaceus) C1q C chain (PoC1qC). The PoC1qC shares 56.7%–87.6% identities with that of teleost and mammals. The expression of PoC1qC can be detected in various organs, notably up-regulated by V. harveyi stimulation, and down-regulated by E. tarda infection. The recombinant PoC1qC (rPoC1qC) could directly bind to different bacteria and exhibit antibacterial potential. Consistently, rPoC1qC and anti-rPoC1qC antibody could remarkably enhance or suppress the complement hemolytic and bactericidal activity, respectively. Furthermore, rPoC1qC could directly interact with PBLs, resulting in the activation of proliferation, chemotaxis, and phagocytosis of PBLs, and the generation of ROS/acid phosphatase activity of PBLs. Lastly, we found in vivo rPoC1qC could promote the host to resistant microbial invasion. Taken together, our findings provide the first evidence that PoC1qC played pivotal roles in complement and PBLs activation and antibacterial activity.

Transient Binding Dynamics of Complement System Pattern Recognition Molecules on Pathogens.

Previous studies of pattern recognition molecules (PRMs) of the complement system have revealed difficulties in observing binding on pathogens such as Aspergillus fumigatus and Escherichia coli, despite complement deposition indicative of classical and lectin pathway activation. Thus, we investigated the binding dynamics of PRMs of the complement system, specifically C1q of the classical pathway and mannose-binding lectin (MBL) of the lectin pathway. We observed consistently increasing deposition of essential complement components such as C4b, C3b, and the terminal complement complex on A. fumigatus and E. coli. However, C1q and MBL binding to the surface rapidly declined during incubation after just 2-4 min in 10% plasma. The detachment of C1q and MBL can be linked to complement cascade activation, as the PRMs remain bound in the absence of plasma. The dissociation and the fate of C1q and MBL seem to have different mechanistic functions. Notably, C1q dynamics were associated with local C1 complex activation. When C1s was inhibited in plasma, C1q binding not only remained high but further increased over time. In contrast, MBL binding was inversely correlated with total and early complement activation due to MBL binding being partially retained by complement inhibition. Results indicate that detached MBL might be able to functionally rebind to A. fumigatus. In conclusion, these results reveal a (to our knowledge) novel "hit-and-run" complement-dependent PRM dynamic mechanism on pathogens. These dynamics may have profound implications for host defense and may help increase the functionality and longevity of complement-dependent PRMs in circulation.

The Current Understanding of Mechanistic Pathways in Zeolite Crystallization.

Zeolite catalysts and adsorbents have been an integral part of many commercial processes and are projected to play a significant role in emerging technologies to address the changing energy and environmental landscapes. The ability to rationally design zeolites with tailored properties relies on a fundamental understanding of crystallization pathways to strategically manipulate processes of nucleation and growth. The complexity of zeolite growth media engenders a diversity of crystallization mechanisms that can manifest at different synthesis stages. In this review, we discuss the current understanding of classical and nonclassical pathways associated with the formation of (alumino)silicate zeolites. We begin with a brief overview of zeolite history and seminal advancements, followed by a comprehensive discussion of different classes of zeolite precursors with respect to their methods of assembly and physicochemical properties. The following two sections provide detailed discussions of nucleation and growth pathways wherein we emphasize general trends and highlight specific observations for select zeolite framework types. We then close with conclusions and future outlook to summarize key hypotheses, current knowledge gaps, and potential opportunities to guide zeolite synthesis toward a more exact science.