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HIV-1 Genotypes Related to Failure of Nelfinavir as the First Protease Inhibitor Treatment

HIV-1 Genotypes Related to Failure of Nelfinavir as the First Protease Inhibitor Treatment

With the establishment of the National Net of HIV Genotyping (RENAGENO) by the National Program of STD/AIDS of the Ministry of Health, the profile of the mutations occurring after failure of nelfinavir as the first protease inhibitor in our setting became available. This net has been operating since February 2002. Within this context, the Project “GERAIS” – Grupo de Estudos em Resistência aos anti-retrovirais [Study Group on Resistance to Antiretrovirals] was created, with the main purpose of contributing to the evaluation of the efficacy of this new technology. Our study was proposed in this context, with the primary objective of evaluating the characteristics of the HIV-1 genotyping test after therapeutic failure. Its secondary objectives include: phylogenetic analysis of HIV-1 and determination the of protease gene mutations, detection of mutations related to nucleosides (NAM) and the time of use of ARV, and the number of secondary mutations and their relation to duration of treatment.
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A Treatment with a Protease Inhibitor Recombinant from the Cattle Tick (Rhipicephalus Boophilus microplus) Ameliorates Emphysema in Mice

A Treatment with a Protease Inhibitor Recombinant from the Cattle Tick (Rhipicephalus Boophilus microplus) Ameliorates Emphysema in Mice

The Kunitz type serine protease inhibitor is a canonical inhibitor of serine proteases which domain has molecular mass around 7000 Da with three disulfide bridges, characterizing a protein with a stabilized structure [11]. Rhipicephalus (B.) microplus is a very important bovine ectoparasite with an extensive geographic distribution in tropical and subtropical regions of the world, especially in Brazil [12]. In both the larvae and eggs of these ticks, a serine proteinase-inhibiting activity has been described that protects the host from infection by pathogens or parasites, inhibits fungal or bacterial proteinases, and which likely regulates proteinases involved in coagulation or cytokine activation [13].
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The effect of an HIV-1 viral protease inhibitor on staurosporine-induced apoptosis in immortalized mesangial cells

The effect of an HIV-1 viral protease inhibitor on staurosporine-induced apoptosis in immortalized mesangial cells

The effect of the HIV-1 protease inhibitor Ac-Leu-Val-phenylalanine (PI; Calbiochem, CA, USA) on the rate of staurosporine-induced apoptosis was then examined. Cells were cultivated in 24-well plates as described above. After reaching a confluence of around 80-90%, the cells were treated for 24 hours with one of the following: i) vehicle (methanol, 20 µ l); ii) staurosporine (10 nM); iii) protease inhibitor (900 nM – this concentration is the pCI 50 for this drug); iv) staurosporine (10 nM) plus protease inhibitor (900 nM). Incubation of immortalized mesangial cells with PI alone for 2 hours had no acute effect on cell viability or the rate of apoptosis, when compared with vehicle controls (Figure 2). Increasing the time of incubation to 24 hours (to coincide with the incubation time required for staurosporine- induced apoptosis) had no significant effect on either the viability or rate of apoptosis (Figure 2). Incubation of the cells with 10 nM stauros- porine, which was the concentration shown to induce a significant amount of apoptosis in preliminary experiments, again caused a significant rise in the rate of apoptosis observed, with a slight decrease in the viability. However,
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Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.
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The first serine protease inhibitor from Lasiodora sp (Araneae: Theraphosidae) hemocytes

The first serine protease inhibitor from Lasiodora sp (Araneae: Theraphosidae) hemocytes

The final purification of EILaH was obtained by a reversed phase chromatography and the homogeneity of preparation was demon- strated by detection of a single peptide on SDS-PAGE and a single molecular mass by MALDI-TOF, revealing the absence of contam- inant molecules. These results indicate that this chromatographic step removed trypsin inhibitors other than EILaH that were present in the material from affinity column. The removal of a great num- ber of other inhibitors may be the reason for the low activity yield after this purification stage, as shown in Table 1. SDS-PAGE sep- aration efficiency of proteins with a molecular mass of less than 10 kDa is usually low but this method was able to detect EILaH pep- tide (8 kDa) as well as the serine protease inhibitor isolated from B. microplus (8 kDa) [41].
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In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

The findings assembled herein lead us to suggest that E-64 can interfere in some crucial process in relation to the parasite survival, but until today little is known about the efficacy and safety of this protease inhibitor in vivo. So, it is suitable that the concentrations assessed in vitro must be tested in vivo before a definitive statement can be made on its antiparasitic potential. Even so, our results provide insights on protease inhibitors targeting parasite cysteine proteases open perspectives for future investigations in order to confirm the real antigiardial potential of E-64 and other potent inhibitors of cysteine proteases.
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Frequência de tabagismo e das mutações N34S e P55S do gene Serine Protease Inhibitor...

Frequência de tabagismo e das mutações N34S e P55S do gene Serine Protease Inhibitor...

Em 2000, a análise de pacientes com pancreatite, nos quais não foram identificadas mutações no gene PRSS1, permitiu a identificação da mutação N34S no exon 3 do gene SPINK1 (serine protease inhibitor Kazal type 1) (56). Após novos trabalhos, ficou clara a associação desta mutação com a pancreatite, e em metanalise realizada por Aoun et al. (57), foram incluídos 24 estudos caso-controle que investigaram a frequência desta mutação em suas populações, concluindo-se que a mutação N34S está fortemente associada à pancreatite crônica, tanto de etiologia idiopática e tropical, como alcoólica, porém com menor impacto nesta última.
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Virologic failure of protease inhibitor-based second-line antiretroviral therapy without resistance in a large HIV treatment program in South Africa.

Virologic failure of protease inhibitor-based second-line antiretroviral therapy without resistance in a large HIV treatment program in South Africa.

Methodology/ Principal Findings: HIV-infected patients $15 years of age who had failed protease inhibitor (PI)-based second-line ART (2 consecutive HIV RNA tests .1000 copies/ml on lopinavir/ritonavir, didanosine, and zidovudine) were identified retrospectively. Patients with virologic failure were continued on second-line ART. Genotypic testing for drug resistance was performed on frozen plasma samples obtained closest to and after the date of laboratory confirmed second- line ART failure. Of 322 HIV-infected patients on second-line ART, 43 were adults with confirmed virologic failure, and 33 had available plasma for viral sequencing. HIV-1 RNA subtype C predominated (n = 32, 97%). Mean duration on ART (SD) prior to initiation of second-line ART was 23 (17) months, and time from second-line ART initiation to failure was 10 (9) months. Plasma samples were obtained 7(9) months from confirmed failure. At second-line failure, 22 patients (67%) had wild-type virus. There was no major resistance to PIs found. Eleven of 33 patients had a second plasma sample taken 8 (5.5) months after the first. Median HIV-1 RNA and the genotypic resistance profile were unchanged.
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Quantitative proteomic analysis of oral brush biopsies identifies secretory leukocyte protease inhibitor as a promising, mechanism-based oral cancer biomarker.

Quantitative proteomic analysis of oral brush biopsies identifies secretory leukocyte protease inhibitor as a promising, mechanism-based oral cancer biomarker.

A decrease in the almost fifty percent mortality rate from oral cancer is needed urgently. Improvements in early diagnosis and more effective preventive treatments could affect such a decrease. Towards this end, we undertook for the first time an in-depth mass spectrometry-based quantitative shotgun proteomics study of non-invasively collected oral brush biopsies. Proteins isolated from brush biopsies from healthy normal tissue, oral premalignant lesion tissue (OPMLs), oral squamous cell carcinoma (OSCC) and matched control tissue were compared. In replicated proteomic datasets, the secretory leukocyte protease inhibitor (SLPI) protein stood out based on its decrease in abundance in both OPML and OSCC lesion tissues compared to healthy normal tissue. Western blotting in additional brushed biopsy samples confirmed a trend of gradual decreasing SLPI abundance between healthy normal and OPML tissue, with a larger decrease in OSCC lesion tissue. A similar SLPI decrease was observed in-vitro comparing model OPML and OSCC cell lines. In addition, exfoliated oral cells in patients’ whole saliva showed a loss of SLPI correlated with oral cancer progression. These results, combined with proteomics data indicating a decrease in SLPI in matched healthy control tissue from OSCC patients compared to tissue from healthy normal tissue, suggested a systemic decrease of SLPI in oral cells correlated with oral cancer development. Finally, in-vitro experiments showed that treatment with SLPI significantly decreased NF-kB activity in an OPML cell line. The findings indicate anti-inflammatory activity in OPML, supporting a mechanistic role of SLPI in OSCC progression and suggesting its potential for preventative treatment of at-risk oral lesions. Collectively, our results show for the first time the potential for SLPI as a mechanism-based, non-invasive biomarker of oral cancer progression with potential in preventive treatment.
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Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

In Figure 4A, clade IV presents tryptogalinin together with TdPI, another potent human b-tryptase inhibitor from the hard tick Rhipicephalus appendiculatus [5]. Both peptides possess the same Cys-Lys-Ala (C-K-A) motif that form the enzyme-inhibitor interactive site (P1) and a slightly shifted Cys framework compared to the other Kunitz peptides from the phylogram (see Figure 4B and Figure S2). TdPI has an overall altered Kunitz domain structure due to a lack of an alpha-helix, shortening of a loop region, differences in disulfide-bridges (four as opposed to three found in classical Kunitz), and a relocation of the N-terminus [5]. Structural differences, compared with classical Kunitz peptides, in the loop regions of TdPI generate an ‘‘arrow-like’’ structure that increases TdPI association with the compact binding site of trypsin and b-tryptase. We used the web-server DiANNA [72] to predict the disulfide bridges – also verified by our homology modeling (see below) – demonstrating that both TdPI and tryptogalinin share similar disulfide bridges (except for the additional Ia disulfide bridge in TdPI, Figure 4B). As most Kunitz protease inhibitors, but unlike TdPI, tryptogalinin possesses six Cys residues forming three disulfide bridges. The orders of the disulfide bridges, however, differ from that of classical Kunitz proteins since they form a pattern similar to TdPI – the first disulfide bridge is in the same conformation as the Ib disulfide bridge of TdPI (Figure 4B). Figure 2. A–B. Titration of the targeted serine proteases with the recombinant protein. An inhibition screening (A) using 14 vertebrate serine proteases – the asterisk denotes with p-values # 0.05 and #50% inhibition. Inhibition curves (B) were produced for the targeted enzymes by plotting the concentration of the inhibitor and the estimated percentage of the enzymatic activity. Since the amount of enzyme used in the assay varies for each protease, we represent the molar excess of the I. scapularis Kunitz peptide to achieve 50% inhibition of the respective enzyme, when compared to the amount of enzyme used in each assay (also see Materials and Methods).
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A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development.

PbICP-C deletion attenuates blood stage parasites The few PbICP-negative merosomes successfully isolated in vitro were injected into naı¨ve mice and were capable of establishing a blood stage infection confirming a recent study in which the entire pbicp gene was deleted [31]. In contrast to our data, no effect on blood stage development was observed in the previous study. This was surprising because attempts to knockout the icp gene in the closely related parasite P. yoelii [30] and the human parasite P. falciparum [22] have failed, suggesting an important role for this inhibitor during the blood stage. Indeed, we did not succeed to delete this gene in P. berghei using straight knockout approaches suggesting an important role for this inhibitor during the blood stage as well. Since gene deletion in the previous study occurred in the blood stage [31], selective pressure to compensate for the loss of the inhibitor was high and the obtained knockout clones may have carried modifications in addition to the pbicp knockout. In our study, we obtained blood stage parasites by injecting mice with hepatocyte-derived mero- zoites and, thus, the parasites did not have the chance to adapt to the loss of PbICP by compensatory mutations or epigenetic modifications. Furthermore, we showed a complete reversion of the pbicp knockout phenotype by add-back transfection of PbICP KO parasites, strongly suggesting that the observed effects
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Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response

Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response

The Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. In order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki ¼ 2.5 nM), BmCl1 (Ki ¼ 1.8 nM) and cathepsin B (Ki ¼ 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. The mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. In order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity.
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Liver safety of two nucleoside analogs plus efavirenz, nevirapine or a ritonavir-boosted protease inhibitor in HIV/HCV-coinfected drug-naïve patients

Liver safety of two nucleoside analogs plus efavirenz, nevirapine or a ritonavir-boosted protease inhibitor in HIV/HCV-coinfected drug-naïve patients

Driven by the European AIDS Treatment Group (EATG) and Community recommendations on universal access to HIV services for migrants and ethnic minorities in Europe, this session will add[r]

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Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides

Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides

The huge interest in protease inhibitors has focused on natural inhibitors from different sources, particularly leguminous plants that are capable of regulating a number of relevant biological processes. These inhibitors belong to the well-characterized Bowman-Birk Inhibitor (BBI) and Kunitz-type inhibitor families [14]. In particular, BBI have been the most widely investigated molecules from the physicochemical, structural and functional points of view. BBIs play an important role in plant defense mechanisms against pathogens [15–17] and in various biological processes and therapeutic applications. They are involved in the inhibition of intracellular protein hydrolysis, in transcription and cell cycle, and cell invasion [18,19] . In addition, these inhibitors have also been described as anticarcinogenic agents acting on the prevention and suppression of cancer in several organs and tissues in vitro and in vivo [20–25].
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Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

of T. infestans T. cruzi – infected (Fig. 4B). These results suggested more than one role of this molecule in T. infestans. Since triatomine insects use cathepsins for protein digestion in posterior midgut [27], Tigutcystatin could be also involved in regulation of endoge- nous cysteine proteases in stomach, thus controlling undesired proteolysis in this compartment. On the other hand, cystatins are related to innate immunity in insects [4]. Tigutcystatin might also have a defensive role when T. infestans is infected with T. cruzi by inhibiting cruzipain, a parasite cysteine protease used to invade host cells [28].
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Effect of Natural Polymorphisms in the HIV-1 CRF02_AG Protease on Protease Inhibitor Hypersusceptibility

Effect of Natural Polymorphisms in the HIV-1 CRF02_AG Protease on Protease Inhibitor Hypersusceptibility

Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibi- tor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molec- ular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did in- crease susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M poly- morphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These find- ings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mu- tations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.
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Randomized trial of time-limited interruptions of protease inhibitor-based antiretroviral therapy (ART) vs. continuous therapy for HIV-1 infection.

Randomized trial of time-limited interruptions of protease inhibitor-based antiretroviral therapy (ART) vs. continuous therapy for HIV-1 infection.

Genotypic Resistance Testing. Viral RNA was extracted from plasma using the QIAamp Viral RNA Mini kit (QIAGEN Group, Germany). RNA extractions were incubated at 37 uC for 2 hours with 1–5 U Heparinase (Sigma Aldrich) and 40 U RNase Inhibitor in a 10 mM Tris pH 7.5, 1 mM CaCl 2 solution, to a total volume of 140 m l, and then re-extracted to remove the enzymes. Sequencing of the pol gene was done using an in-house assay [20] and BigDye Terminators v3.1 on an ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Consensus sequences were aligned and manually edited using the Sequencher v4.5 software (GeneCodes, Ann Arbor, MI). Genotypic resistance was defined using the Stanford Genotypic Resistance Interpretation Algorithm (http://hivdb.stanford.edu/) and the December 2009 International AIDS Society drug resistance mutation list.
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Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

Rapid mutations of the HIV-1 genome due to erroneous HIV Reverse Transcriptase (RT) [9,10] and host RNA processing elements [11], cause immunity escape mutations to accumulate in the HIV-1 genome. CTL escape mutations may interfere with the processing and presentation of the CTL epitope or attenuate CTL T-Cell receptor interaction with the peptide-MHC complex rendering the epitope ineffective [12,13]. In the majority of HIV-1 infected individuals, effective immune responses are mostly transient mainly due to the acquisition of immunity escape mutations by HIV-1. The development of antiretrovirals have provided additional protection against HIV infection. Antiretro- viral drugs, such as HIV protease inhibitors (PI) and HIV reverse transcriptase inhibitors (RTI) inhibit steps in the HIV-1 viral replication cycle and have a large negative impact on viral load [14]. Still, ARV resistance can also accumulate, the mechanism involving interference with the binding of an ARV to the target site and rendering the drug ineffective [15].
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Clonagem, expressão e caracterização de um inibidor de tripsina presente no mosquito Aedes aegypti

Clonagem, expressão e caracterização de um inibidor de tripsina presente no mosquito Aedes aegypti

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K i ) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito’s development.
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