An efficient system for in vitro propagation of Bouchea fluminensis (Vell.) Mold. (Verbenaceae)

This study aimed to establish and propagate in vitro plants of Bouchea fluminensis, a medicinal species known in Brazil as gervão-falso ("false verbena"), evaluating the influences of different growth regulators on in vitro multiplication and rooting stages, as well as examining ex vitro acclimatization of rooted plants. Explants were established on Murashige and Skoog medium at half strength of salts and vitamins without growth regulators. For multiplication, the explants were subjected to combinations of 6-benzyladenine (BA; 0, 2.5, 5.0 and 7.5 µM) and α-naphthalene-acetic acid (NAA; 0, 0.2, 0.4 and 0.6 µM). The medium found to induce the greatest number of shoot was that containing 5 µM of BA (NAA-free). For rooting, we evaluated three auxins (NAA, indole-3-acetic acid and indole-3-butyric acid; 0.1, 0.2, 0.3 and 0.4 µM), as well as a control. No differences were observed between the control and the other treatments. The auxin-free medium was deemed the most suitable, because it ensures the lowest cost in the micropropagation procedures. We obtained 100% survival of the acclimatized seedlings, and the plants showed normal vegetative and reproductive development, suggesting that the micropropagation did not alter the biological cycle of this species. The results show the importance and potential of micropropagation for biodiversity conservation of Bouchea fluminensis

Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment

Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single- Domain Antibody Fragments.

Submitted by EMERSON LEAL ([email protected]) on 2018-07-12T14:10:06Z No. of bitstreams: 1 Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.PDF: 2085939 bytes, checksum: ee0b2f63f746338bb86bb2d5e895816d (MD5)Approved for entry into archive by EMERSON LEAL ([email protected]) on 2018-07-12T20:22:29Z (GMT) No. of bitstreams: 1 Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.PDF: 2085939 bytes, checksum: ee0b2f63f746338bb86bb2d5e895816d (MD5)Made available in DSpace on 2018-07-12T20:22:29Z (GMT). No. of bitstreams: 1 Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.PDF: 2085939 bytes, checksum: ee0b2f63f746338bb86bb2d5e895816d (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Universidade Federal Fluminense. Rio de Janeiro, RJ, Brasil.Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Empresa Brasileira de Pesquisa Agropecuária. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Universidade Federal de Rondônia. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil. / Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenomtherapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I byWestern blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) inmice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem

Amino acid sequence alignment of anti-BthTX-I and BthTX-II VHHs.

<p>The clones were clustered in four groups based on sequence homologies. The framework regions (FR) as well as the complementarity determining regions (CDR) are indicated with arrows; two conserved cysteines are shaded; VHH hallmark substitutions in FR2 and FR4 are bolded and underlined. Cluster I (KF498607, KF498608, KF498609, and KC329718), Cluster II (KC329709, KC329714), Cluster III (KC329713), and Cluster IV (KC329710, KC329711, KC329712, KC329715, KC329716, KC329717). The colon (:) represents highly conserved amino acids; the asterisk (*) represents identical amino acid residues; the period (.) means somewhat similar but different amino acids and blank represents dissimilar amino acids or gaps.</p

<i>In vitro</i> inhibition of phospholipase activity.

<p>The quantification of phospholipase activity inhibition by selected VHHs was assayed using synthetic fluorescent phospholipid. To verify the ability of VHHs to inhibit the phospholipase activity of BthTX-II, the toxin was pre-incubated with selected VHHs for 30 minutes at 37°C in different proportions (1:5; 1:10 and 1:40 w/w). Inhibition of phospholipase activity by (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718. BthTX-II activity on the phospholipid, in the absence of VHH, was used as a positive control, and considered as having 100% activity. The negative controls were carried out using medium reaction without BthTX-II. All measurements were performed in duplicate. Error bars represent standard deviation.</p

VHHs affinity to BthTX-I and BthTX-II.

<p>Representative sensorgrams of the interaction were measured in a Biacore T200 system (GE Healtcare). Purified VHHs (KF498607, KF498608 and KC329718) at concentrations of 6.25 to 0.049 μg/mL were flowed over a BthTX-I-coated CM5 chip and 25 to 0.006 μg/mL were used for immobilized BthTX-II. (A) KF498607 x BthTX-I; (B) KF498607 x BthTX-II; (C) KF498608 x BthTX-I; (D) KF498608 x BthTX-II; (E) KC329718 x BthTX-I; (F) KC329718 x BthTX-II. Assays were injected in a flow rate of 30 μl/min at 37°C. RU indicates resonance units.</p

Interaction analysis by SPR.

<p>Interaction analysis by SPR.</p

<i>In vivo</i> neutralization of <i>B</i>. <i>jararacussu</i> venom and PLA₂-induced myotoxicity by VHHs.

<p>Plasma creatine kinase (CK) levels were measured at 340 nm to determine VHH ability to inhibit the myotoxicity caused by <i>B</i>. <i>jararacussu</i> venom or PLA₂s, or by a related toxin, MTX-I, a PLA₂ isolated from <i>B</i>. <i>brazili</i>. For this, groups of 5 animals were injected with PBS, VHH KF498607, VHH KC329718, BthTX-I, BthTX-I + VHH KF498607, BthTX-I + VHH KC329718, BthTX-II, BthTX-II + VHH KF498607, BthTX-II + VHH KC329718, <i>B</i>. <i>jararacussu</i> venom, <i>B</i>. <i>jararacussu</i> venom + VHH KF498607, <i>B</i>. <i>jararacussu</i> venom + VHH KC329718, MTX-I, MTX-I + VHH KC329718, MTX-I + VHH KC329718. Before administration to mice, venom or PLA₂s and antibody preparations were pre-incubated at 37°C for 1 h, in a proportion 1:5 or 1:10 (w/w). The negative control was performed with PBS or VHHs, and as a positive control, animals were injected with <i>B</i>. <i>jararacussu</i> venom, BthTX-I, BthTX-II or MTX-I without the addition of VHH. Bonferroni’s test was used for significance analysis. (*) P <0.05. Error bars represent standard deviation.</p

Docking results showing binding sites of VHHs on surfaces of PLA₂s.

<p>(A) KF498607 (blue ribbon) and BthTX-I (red surface); (B) KC329718 (blue ribbon) and BthTX-I (red surface); (C) KF498607 (blue ribbon) and BthTX-II (orange surface); (D) KC329718 (blue ribbon) and BthTX-II (orange surface). The interaction sites were enlarged to show hydrogen bonds formed between the amino acid residues. Both VHHs covered the enzymatic groove surface areas of the modeled BthTX-II and inserted the CDR3 into the catalytic cleft, as well as being able to interact with amino acid residues of the PLA₂ C-termini.</p

Selection of anti-BthTX-I and anti-BthTX-II VHHs.

<p>VHHs from the recombinant phage displayed library were submitted separately against immobilized BthTX-I or BthTX-II. After one round of panning, 82 and 28 clones selected for BthTX-I and BthTX-II, respectively, presented VHHs. ELISA assays were performed to verify clone reactivity. Twenty-eight (28) and six (06) clones recognized BthTX-I and BthTX-II. (A) Clonal reactivity of VHHs (1–40) selected against BthTX-I, NC–negative control; (B) Clonal reactivity of VHHs (41–83) selected against BthTX-I, Cross-reactivity of VHH 82 with BthTX-II, NC–negative control; (C) Clonal reactivity of VHHs (1–37) selected against BthTX-II, Cross-reactivity of 02, 09, 20, 21, 28 and 30 VHHs with BthTX-I, NC–negative control. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. All measurements were performed in triplicate. The negative control was performed using the llama pre-immune serum. Error bars represent standard deviation.</p