Dietary cinnamaldehyde activation of TRPA1 antagonizes high-salt induced hypertension through restoring renal tubular mitochondrial dysfunction.

BACKGROUND: Renal proximal tubule plays a pivotal role in regulating sodium reabsorption and thus blood pressure. Transient receptor potential ankyrin 1 (TRPA1) has been reported to protect against renal injury by modulating mitochondrial function. We hypothesize that the activation of TRPA1 by its agonist cinnamaldehyde may mitigates high salt intake induced hypertension by inhibiting urinary sodium reabsorption through restoration of renal tubular epithelial mitochondrial function. METHODS: Trpa1-deficient (Trpa1-/-) mice and wild-type (WT) mice were fed standard laboratory chow [normal diet (ND) group, 0.4% salt], standard laboratory chow with 8% salt [high-salt diet (HS) group] or standard laboratory chow with 8% salt plus 0.015% cinnamaldehyde [high-salt plus cinnamaldehyde diet (HSC) group] for six months. Urinary sodium excretion, ROS production, mitochondrial function and the expression of NHE3 and Na+/K+-ATPase of renal proximal tubules were determined. RESULTS: Chronic dietary cinnamaldehyde supplementation reduced tail systolic blood pressure and 24-hour ambulatory arterial pressure in HS-fed WT mice. Compared with the mice fed HS, cinnamaldehyde supplementation significantly increased urinary sodium excretion, inhibited excess ROS production and alleviated mitochondrial dysfunction of renal proximal tubules in WT mice. However, these effects of cinnamaldehyde were absent in Trpa1-/- mice. Furthermore, chronic dietary cinnamaldehyde supplementation blunted HS-induced upregulation of NHE3 and Na+/K+-ATPase in WT mice but not Trpa1-/- mice. CONCLUSION: The present study demonstrated that chronic activation of Trpa1 attenuates HS-induced hypertension by inhibiting urinary sodium reabsorption through restoring renal tubular epithelial mitochondrial function. Renal TRPA1 may be a potential target for the management of excessive dietary salt intake-associated hypertension.

Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants.

The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike (S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the wild-type, B.1.1.7, B.1.351, B.1.617.2, and the newly emerging Omicron variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. Furthermore, our safety assessment of RQ3013 in NHP showed no observable adverse effects. These results provide strong support for the evaluation of RQ3013 in clinical trials and suggest that it may be a promising candidate for broad protection against COVID-19 and its variants.

A high-salt diet promotes hypertrophic scarring through TRPC3-mediated mitochondrial Ca2+ homeostasis dysfunction.

Diet High in salt content have been associated with cardiovascular disease and chronic inflammation. We recently demonstrated that transient receptor potential canonical 3 (TRPC3) channels regulate myofibroblast transdifferentiation in hypertrophic scars. Here, we examined how high salt activation of TRPC3 participates in hypertrophic scarring during wound healing. In vitro, we confirmed that high salt increased the TRPC3 protein expression and the marker of myofibroblast alpha smooth muscle actin (α-SMA) in wild-type mice (WT) primary cultured dermal fibroblasts but not Trpc3-/- mice. Activation of TRPC3 by high salt elevated cytosolic Ca2+ influx and mitochondrial Ca2+ uptake in dermal fibroblasts in a TRPC3-dependent manner. High salt activation of TRPC3 enhanced mitochondrial respiratory dysfunction and excessive ROS production by inhibiting pyruvate dehydrogenase action, that activated ROS-triggered Ca2+ influx and the Rho kinase/MLC pathway in WT mice but not Trpc3-/- mice. In vivo, a persistent high-salt diet promoted myofibroblast transdifferentiation and collagen deposition in a TRPC3-dependent manner. Therefore, this study demonstrates that high salt enhances myofibroblast transdifferentiation and promotes hypertrophic scar formation through enhanced mitochondrial Ca2+ homeostasis, which activates the ROS-mediated pMLC/pMYPT1 pathway. TRPC3 deficiency antagonizes high salt diet-induced hypertrophic scarring. TRPC3 may be a novel target for hypertrophic scarring during wound healing.

Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo.

Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 â€‹ng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 â€‹ng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 â€‹mg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 â€‹mg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 â€‹mg/kg. These results highlight the potential for future modifications of the mAbs for practical use.

Dual roles and evolutionary implications of P26/poxin in antagonizing intracellular cGAS-STING and extracellular melanization immunity.

P26, a homolog of the viral-encoded nuclease poxin that neutralizes the cGAS-STING innate immunity, is widely distributed in various invertebrate viruses, lepidopteran insects, and parasitoid wasps. P26/poxin from certain insect viruses also retains protease activity, though its biological role remains unknown. Given that many P26s contain a signal peptide, it is surmised that P26 may possess certain extracellular functions. Here, we report that a secretory baculoviral P26 suppresses melanization, a prominent insect innate immunity against pathogen invasion. P26 targets the cofactor of a prophenoloxidase-activating protease, and its inhibitory function is independent of nuclease activity. The analysis of P26/poxin homologs from different origins suggests that the ability to inhibit the extracellular melanization pathway is limited to P26s with a signal peptide and not shared by the homologs without it. These findings highlight the independent evolution of a single viral suppressor to perform dual roles in modulating immunity during virus-host adaptation.

The Micrococcus luteus infection activates a novel melanization pathway of cSP10, cSP4, and cSP8 in Helicoverpa armigera.

Melanization is a key immune response mediated by serine protease (SP) cascade in insects. Multiple SP pathways exist in different species and it is unclear how conserved these cascades are. The cotton bollworm Helicoverpa armigera is a major worldwide agricultural pest. We reported a conserved melanization pathway in this species, which consists of SP41, cSP1, and cSP6. In this study, we attempted to identify an insect pathogen that elicits the cascade and test whether or not there are other SP cascades in H. armigera. After Micrococcus luteus, Enterobacter cloacae, Beauveria bassiana, or Helicoverpa armigera nucleopolyhedrovirus were injected into larvae, pathogen-induced hemolymph samples were collected for in vitro biochemical assays, which failed to detect proSP41 or procSP1 activation. In contrast, we found that procSP4, a protein proposed to participate in H. armigera melanization, was activated in M. luteus infected hemolymph. We further revealed that cSP8 was a prophenoloxidase (PPO) activating protease downstream of cSP4, and cSP4 was activated by cSP10. The pathway of cSP10-cSP4-cSP8 activated PPO in vitro. Efficiently cleaved procSPH11 and procSPH50 by cSP8 substantially enhanced phenoloxidase activity, suggesting they work together as a cofactor for cSP8 mediated PPO activation. Hemolymph from larvae challenged with M. luteus or its peptidoglycan effectively activated procSP10. Collectively, these results revealed a new PPO activation cascade specifically triggered by the bacterium. In addition, we found that the PPO activation cascades in H. armigera and Manduca sexta are conserved.

In vitro and in vivo efficacy of a novel nucleoside analog H44 against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus that causes fever, hemorrhage, and multi-organ failure, with an average fatality rate of ∼40% in humans. Currently, there are no available vaccines or drugs for the treatment of Crimean-Congo hemorrhagic fever (CCHF). Favipiravir (T-705), a nucleoside analog, protects against CCHFV infection in animal models. Here, we evaluated the anti-CCHFV efficacy of several nucleoside analogs, including some well-known compounds such as remdesivir (GS-5734), EIDD-1931 and its prodrug molnupiravir (EIDD-2801), as well as a novel T-705-derived compound H44. T-705, H44, and EIDD-1931 inhibited CCHFV infection in vitro while GS-5734 had no inhibitory effect. All three nucleoside analogs functioned at the "post-entry" stage of virus infection. However, EIDD-2801 failed to protect type I interferon receptor knockout (IFNAR)-/- mice from CCHFV infection. H44, similar to T-705, conferred 100% protection to IFNAR-/- mice against lethal CCHFV challenge, even with delayed administration. This study provided in vitro and in vivo data regarding the anti-CCHFV efficacy of different nucleosides and identified a novel compound, H44, as a promising drug candidate for the treatment of CCHFV infection in vivo.

Covalently Engineered Protein Minibinders with Enhanced Neutralization Efficacy against Escaping SARS-CoV-2 Variants.

The rapid emergence and spread of escaping mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly challenged our efforts in fighting against the COVID-19 pandemic. A broadly neutralizing reagent against these concerning variants is thus highly desirable for the prophylactic and therapeutic treatments of SARS-CoV-2 infection. We herein report a covalent engineering strategy on protein minibinders for potent neutralization of the escaping variants such as B.1.617.2 (Delta), B.1.617.1 (Kappa), and B.1.1.529 (Omicron) through in situ cross-linking with the spike receptor binding domain (RBD). The resulting covalent minibinder (GlueBinder) exhibited enhanced blockage of RBD-human angiotensin-converting enzyme 2 (huACE2) interaction and more potent neutralization effect against the Delta variant than its noncovalent counterpart as demonstrated on authentic virus. By leveraging the covalent chemistry against escaping mutations, our strategy may be generally applicable for restoring and enhancing the potency of neutralizing antibodies to SARS-CoV-2 and other rapidly evolving viral targets.

Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera.

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

Transient Receptor Potential Channel Canonical Type 3 Deficiency Antagonizes Myofibroblast Transdifferentiation In Vivo.

OBJECTIVE: Myofibroblast transformation has been shown to be associated with the reactive oxygen species- (ROS-) producing enzyme NADPH oxidase (Nox4). Inhibition of transient receptor potential channel canonical type 3 (TRPC3) attenuates mitochondrial calcium handling and ROS production in the vasculature of hypertensive rats. However, it remains elusive whether TRPC3 regulates mitochondrial calcium and ROS production and participates in myofibroblast transdifferentiation during wound healing. METHODS AND RESULTS: In this study, we demonstrated that activation of TRPC3 by transforming growth factor ß (TGFß (TGFαSMA). Inhibition of TRPC3 with its specific inhibitor, Pyr3, significantly decreased TGFß (TGFαSMA). Inhibition of TRPC3 with its specific inhibitor, Pyr3, significantly decreased TGFß (TGFß (TGFTrpc3-/- mice exhibited significantly attenuated myofibroblast transdifferentiation, as demonstrated by decreased αSMA). Inhibition of TRPC3 with its specific inhibitor, Pyr3, significantly decreased TGFß (TGFß (TGFTrpc3-/- mice exhibited significantly attenuated myofibroblast transdifferentiation, as demonstrated by decreased Trpc3+/+ mice. In addition, Trpc3-/- mice exhibited significantly attenuated myofibroblast transdifferentiation, as demonstrated by decreased. CONCLUSIONS: Our data indicate that TGFß1-mediated activation of TRPC3 enhances mitochondrial calcium and ROS production, which promotes myofibroblast transdifferentiation and HTS formation. Inhibition of the TRPC3-mediated Nox4/pSmad2/3 pathway may be a useful strategy to limit HTS formation after injury.ß (TGF.

High-salt intake increases TRPC3 expression and enhances TRPC3-mediated calcium influx and systolic blood pressure in hypertensive patients.

Enhanced transient receptor potential canonical subtype 3 (TRPC3) expression and TRPC3-mediated calcium influx in monocytes from hypertensive rats and patients are associated with increased blood pressure. Daily salt intake is closely related to hypertension, but the relationship between TRPC3 expression and salt intake has not yet been evaluated in hypertensive patients. Using reverse transcription-polymerase chain reaction, we studied the expression of TRPC3 and TRPC3-related store-operated calcium entry (SOCE) in peripheral blood mononuclear cells (PBMCs) from hypertensive and normotensive control subjects. Measurement of SOCE was performed using the fluorescent dye Fura-2 AM. Participants were divided into a low-salt group (<9 g) and a high-salt group (≥9 g) based on 24-h urinary sodium excretion. Increased TRPC3 mRNA expression levels and SOCE were observed in THP-1 cells after high-NaCl treatment. However, administration of the TRPC3-specific inhibitor Pyr3 significantly decreased the effect. Furthermore, the TRPC3 mRNA expression levels in PBMCs from high-salt intake patients with essential hypertension were significantly higher than those in low-salt intake patients compared with those in normotensive control subjects. We also observed significantly increased TRPC3-mediated SOCE in PBMCs from hypertensive subjects (but not from normotensive control subjects), with calcium concentration correlating with salt intake. More importantly, TRPC3 mRNA levels showed a significant correlation with salt intake and systolic blood pressure in patients with essential hypertension. This study demonstrated, for the first time, that increased TRPC3 mRNA levels are associated with elevated salt intake and systolic blood pressure in hypertensive patients.

Genome Analysis of a Novel Clade II.b Alphabaculovirus Obtained from Artaxa digramma.

Artaxa digramma is a lepidopteran pest distributed throughout southern China, Myanmar, Indonesia, and India. Artaxa digramma nucleopolyhedrovirus (ArdiNPV) is a specific viral pathogen of A. digramma and deemed as a promising biocontrol agent against the pest. In this study, the complete genome sequence of ArdiNPV was determined by deep sequencing. The genome of ArdiNPV contains a double-stranded DNA (dsDNA) of 161,734 bp in length and 39.1% G+C content. Further, 149 hypothetical open reading frames (ORFs) were predicted to encode proteins >50 amino acids in length, covering 83% of the whole genome. Among these ORFs, 38 were baculovirus core genes, 22 were lepidopteran baculovirus conserved genes, and seven were unique to ArdiNPV, respectively. No typical baculoviral homologous regions (hrs) were identified in the genome. ArdiNPV had five multi-copy genes including baculovirus repeated ORFs (bros), calcium/sodium antiporter B (chaB), DNA binding protein (dbp), inhibitor of apoptosis protein (iap), and p26. Interestingly, phylogenetic analyses showed that ArdiNPV belonged to Clade II.b of Group II Alphabaculoviruses, which all contain a second copy of dbp. The genome of ArdiNPV was the closest to Euproctis pseudoconspersa nucleopolyhedrovirus, with 57.4% whole-genome similarity. Therefore, these results suggest that ArdiNPV is a novel baculovirus belonging to a newly identified cluster of Clade II.b Alphabaculoviruses.

TRPC3 deficiency attenuates high salt-induced cardiac hypertrophy by alleviating cardiac mitochondrial dysfunction.

Long-term high salt intake leads to cardiac hypertrophy, but the mechanism remains elusive. Transient receptor potential channel, canonical 3(TRPC3), located in mitochondria, regulates mitochondrial calcium and reactive oxygen species(ROS) production. Herein, we investigated whether TRPC3 participates in high salt-induced cardiac hypertrophy by impairing cardiac mitochondrial function. High salt treatment increased the expression of mitochondrial TRPC3 in cardiomyocytes, accompanied by enhanced mitochondrial calcium uptake and elevated ROS production. Inhibition of TRPC3 significantly reduced high salt-induced ROS generation, promoted ATP production by stimulating oxidative phosphorylation, and increased enzyme activity in mitochondria in cardiomyocytes. Additionally, TRPC3 deficiency inhibited high salt-induced cardiac hypertrophy in vivo. A long-term high salt diet increased cardiac mitochondrial TRPC3 expression, elevated expression of cardiac hypertrophic markers atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC) and decreased ATP production and mitochondrial complex I and II enzyme activity in a TRPC3-dependent manner. TRPC3 deficiency antagonises high salt diet-mediated cardiac hypertrophy by ameliorating TRPC3-mediated cardiac mitochondrial dysfunction. TRPC3 may therefore represent a novel target for preventing high salt-induced cardiac damage.

Genome Characteristics of the Cyclophragma Undans Nucleopolyhedrovirus: A Distinct Species in Group I of Alphabaculovirus.

The Cyclophragma undans nucleopolyhedrovirus (CyunNPV), a potential pest control agent, was isolated from Cyclophragma undans (Lepidoptera: Lasiocampidae), an important forest pest. In the present study, we performed detailed genome analysis of CyunNPV and compared its genome to those of other Group I alphabaculoviruses. Sequencing of the CyunNPV genome using the Roche 454 sequencing system generated 142,900 bp with a G + C content of 45%. Genome analysis predicted a total of 147 hypothetical open reading frames comprising 38 baculoviral core genes, 24 lepidopteran baculovirus conserved genes, nine Group I Alphabaculovirus conserved genes, 71 common genes, and five genes that are unique to CyunNPV. In addition, the genome contains 13 homologous repeated sequences (hrs). Phylogenetic analysis groups CyunNPV under a distinct branch within clade "a" of Group I in the genus Alphabaculovirus. Unlike other members of Group I, CyunNPV harbors only nine of the 11 genes previously determined to be specific to Group I viruses. Furthermore, the CyunNPV lacks the tyrosine phosphatase gene and the ac30 gene. The CyunNPV F-like protein contains two insertions of continuous polar amino acids, one at the conventional fusion peptide and a second insertion at the pre-transmembrane domain. The insertions are likely to affect the fusion function and suggest an evolutionary process that led to inactivation of the F-like protein. The above findings imply that CyunNPV is a distinct species under Group I Alphabaculovirus.

Direct Amination of Alcohols Catalyzed by Aluminum Triflate: An Experimental and Computational Study.

Among the best-performing homogeneous catalysts for the direct amination of activated secondary alcohols with electron-poor amine derivatives, metal triflates, such as aluminum triflate, Al(OTf)3 , stand out. Herein we report the extension of this reaction to electron-rich amines and activated primary alcohols. We provide detailed insight into the structure and reactivity of the catalyst under working conditions in both nitromethane and toluene solvent, through experiment (cyclic voltammetry, conductimetry, NMR spectroscopy), and density functional theory (DFT) simulations. Competition between aniline and benzyl alcohol for Al in the two solvents explains the different reactivities. The catalyst structures predicted from the DFT calculations were validated by the experiments. Whereas a SN 1-type mechanism was found to be active in nitromethane, we propose a SN 2 mechanism in toluene to rationalize the much higher selectivity observed when using this solvent. Also, unlike what is commonly assumed in homogeneous catalysis, we show that different active species may be active instead of only one.

Efficacy of a Hearing Aid Noise Reduction Function.

Noise reduction systems have been implemented in hearing aids to improve signal-to-noise ratio and listening comfort. The aim of this study was to evaluate the efficacy of hearing aid noise reduction for Mandarin speakers. The results showed a significant improvement in acceptable noise levels and speech reception thresholds with noise reduction turned on. Sound quality ratings also suggested that most listeners preferred having noise reduction turned on for listening effort, listening comfort, speech clarity, and overall sound quality. These results suggest that the noise reduction system used in this study might improve sentence perception in steady-state noise, noise tolerance, and sound quality, although not all listeners preferred aggressive noise reduction. However, due to large interindividual variation, clinical application of the results should be on an individual basis.

Genome analysis of a novel Group I alphabaculovirus obtained from Oxyplax ochracea.

Oxyplax ochracea (Moore) is a pest that causes severe damage to a wide range of crops, forests and fruit trees. The complete genome sequence of Oxyplax ochracea nucleopolyhedrovirus (OxocNPV) was determined using a Roche 454 pyrosequencing system. OxocNPV has a double-stranded DNA (dsDNA) genome of 113,971 bp with a G+C content of 31.1%. One hundred and twenty-four putative open reading frames (ORFs) encoding proteins of >50 amino acids in length and with minimal overlapping were predicted, which covered 92% of the whole genome. Six baculoviral typical homologous regions (hrs) were identified. Phylogenetic analysis and gene parity plot analysis showed that OxocNPV belongs to clade "a" of Group I alphabaculoviruses, and it seems to be close to the most recent common ancestor of Group I alphabaculoviruses. Three unique ORFs (with no homologs in the National Center for Biotechnology Information database) were identified. Interestingly, OxocNPV lacks three auxiliary genes (lef7, ie-2 and pcna) related to viral DNA replication and RNA transcription. In addition, OxocNPV has significantly different sequences for several genes (including ie1 and odv-e66) in comparison with those of other baculoviruses. However, three dimensional structure prediction showed that OxocNPV ODV-E66 contain the conserved catalytic residues, implying that it might possess polysaccharide lyase activity as AcMNPV ODV-E66. All these unique features suggest that OxocNPV represents a novel species of the Group I alphabaculovirus lineage.

Inhibition of melanization by serpin-5 and serpin-9 promotes baculovirus infection in cotton bollworm Helicoverpa armigera.

Melanization, an important insect defense mechanism, is mediated by clip-domain serine protease (cSP) cascades and is regulated by serpins. Here we show that proteolytic activation of prophenoloxidase (PPO) and PO-catalyzed melanization kill the baculovirus in vitro. Our quantitative proteomics and biochemical experiments revealed that baculovirus infection of the cotton bollworm, Helicoverpa armigera, reduced levels of most cascade members in the host hemolymph and PO activity. By contrast, serpin-9 and serpin-5 were sequentially upregulated after the viral infection. The H. armigera serpin-5 and serpin-9 regulate melanization by directly inhibiting their target proteases cSP4 and cSP6, respectively and cSP6 activates PPO purified from hemolymph. Furthermore, serpin-5/9-depleted insects exhibited high PO activities and showed resistance to baculovirus infection. Together, our results characterize a part of the melanization cascade in H. armigera, and suggest that natural insect virus baculovirus has evolved a distinct strategy to suppress the host immune system.