Comparative Biochemical, Structural, and Functional Analysis of Recombinant Phospholipases D from Three Loxosceles Spider Venoms.

Spiders of Loxosceles genus are widely distributed and their venoms contain phospholipases D (PLDs), which degrade phospholipids and trigger inflammatory responses, dermonecrosis, hematological changes, and renal injuries. Biochemical, functional, and structural properties of three recombinant PLDs from L. intermedia, L. laeta, and L. gaucho, the principal species clinically relevant in South America, were analyzed. Sera against L. gaucho and L. laeta PLDs strongly cross-reacted with other PLDs, but sera against L. intermedia PLD mostly reacted with homologous molecules, suggesting underlying structural and functional differences. PLDs presented a similar secondary structure profile but distinct melting temperatures. Different methods demonstrated that all PLDs cleave sphingomyelin and lysophosphatidylcholine, but L. gaucho and L. laeta PLDs excelled. L. gaucho PLD showed greater "in vitro" hemolytic activity. L. gaucho and L. laeta PLDs were more lethal in assays with mice and crickets. Molecular dynamics simulations correlated their biochemical activities with differences in sequences and conformations of specific surface loops, which play roles in protein stability and in modulating interactions with the membrane. Despite the high similarity, PLDs from L. gaucho and L. laeta venoms are more active than L. intermedia PLD, requiring special attention from physicians when these two species prevail in endemic regions.

Venomous Loxosceles Species (Araneae, Haplogynae, Sicariidae) from Brazil: 2n♂ = 23 and X1X2Y Sex Chromosome System as Shared Characteristics.

The family Sicariidae comprises the genera Hexophthalma, Sicarius and Loxosceles. This latter is subdivided in eight monophyletic groups based on genitalia morphology and molecular analyses: amazonica, gaucho, laeta, and spadicea (South America); reclusa (North America); rufescens (Mediterranean); spinulosa and vonwredei (Africa). In Brazil, the genus Loxosceles is represented by 50 species. The mitotic and meiotic characteristics of eight Loxosceles species were analyzed in order to discuss the chromosome evolution, as well as the correspondence between cytogenetic data and morphological/molecular data for the delimitation of the South American groups of species belonging to this genus. All species studied in this work showed 2n♂ = 23, including a X1X2Y sex chromosome system (SCS). Despite the similarity of diploid number and SCS, the species studied here differed regarding the chromosome morphology of some autosomal pairs, presence of secondary constrictions, size of X chromosomes and localization of Ag-NOR/rDNA sites. Based on all these chromosomal data, we verified a close relationship between Loxosceles species belonging to the amazonica and gaucho groups. Transmission electron microscopy (TEM) analysis of spread pachytene cells of L. gaucho showed regular synapsis between homologous autosomal chromosomes, but asynaptic behavior of the sex chromosomes. The axial elements of the sex chromosomes undergo conspicuous morphological modifications resulting in shortening of their length.

Clinical aspects, diagnosis and management of Loxosceles spider envenomation: literature and case review.

The genus Loxosceles comprises 140 species widely distributed around the world. These spiders are nocturnal, sedentary and remarkably nonaggressive, although they cause accidents in humans with wide degrees of severity, generating signs and symptoms that define the clinical condition known as loxoscelism. Its local signs and symptoms were first reported in 1872, and over the years, a large medical literature has been accumulated; unfortunately, it is not always trustworthy. Assessing the reliability of such information, we reviewed 120 case reports of loxoscelism published in 84 articles over the past 20 years. This search allowed us to gather information on the clinical aspects, diagnosis and treatment of loxoscelism, showing that the severity of these accidents has multiple degrees and that it is influenced by many factors. Thus, coupled with epidemiological and species occurrence information, this study can be a useful tool for the clinical practice of loxoscelism. It may support and provide a multidisciplinary view that should be taken into consideration when establishing the therapeutic approach in cases of Loxosceles envenomation.

Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.

Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D.

Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.

Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus.

Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

Novel insights into the application of recombinant mutated phospholipases D as antigens for developing new strategies against Loxoscelism.

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: identification and functional characterization.

UNLABELLED: Brown spider (Loxosceles genus) bites have been reported worldwide. The venom contains a complex composition of several toxins, including phospholipases-D. Native or recombinant phospholipase-D toxins induce cutaneous and systemic loxoscelism, particularly necrotic lesions, inflammatory response, renal failure, and hematological disturbances. Herein, we describe the cloning, heterologous expression and purification of a novel phospholipase-D toxin, LiRecDT7 in reference to six other previously described in phospholipase-D toxin family. The complete cDNA sequence of this novel brown spider phospholipase-D isoform was obtained and the calculated molecular mass of the predicted mature protein is 34.4 kDa. Similarity analyses revealed that LiRecDT7 is homologous to the other dermonecrotic toxin family members particularly to LiRecDT6, sharing 71% sequence identity. LiRecDT7 possesses the conserved amino acid residues involved in catalysis except for a conservative mutation (D233E) in the catalytic site. Purified LiRecDT7 was detected as a soluble 36 kDa protein using anti-whole venom and anti-LiRecDT1 sera, indicating immunological cross-reactivity and evidencing sequence-epitopes identities similar to those of other phospholipase-D family members. Also, LiRecDT7 exhibits sphingomyelinase activity in a concentration dependent-manner and induces experimental skin lesions with swelling, erythema and dermonecrosis. In addition, LiRecDT7 induced a massive inflammatory response in rabbit skin dermis, which is a hallmark of brown spider venom phospholipase-D toxins. Moreover, LiRecDT7 induced in vitro hemolysis in human erythrocytes and increased blood vessel permeability. These features suggest that this novel member of the brown spider venom phospholipase-D family, which naturally contains a mutation (D233E) in the catalytic site, could be useful for future structural and functional studies concerning loxoscelism and lipid biochemistry. HIGHLIGHTS: 1- Novel brown spider phospholipase-D recombinant toxin contains a conservative mutation (D233E) on the catalytic site. 2-LiRecDT7 shares high identity level with isoforms of Loxosceles genus. 3-LiRecDT7 is a recombinant protein immunodetected by specific antibodies to native and recombinant phospholipase-D toxins. 4-LiRecDT7 shows sphingomyelinase-D activity in a concentration-dependent manner, but less intense than other isoforms. 5-LiRecDT7 induces dermonecrosis and inflammatory response in rabbit skin. 6-LiRecDT7 increases vascular permeability in mice. 7-LiRecDT7 triggers direct complement-independent hemolysis in erythrocytes.

Partial characterization of Loxosceles anomala (Mello-Leitão, 1917) venom: A brown spider of potential medical concern.

The spider's genus Loxosceles (also known as "brown spiders") is one of the few ones of medical importance in Brazil, being Loxosceles anomala a species of common occurrence in the Southeast region. This species is usually smaller in size than the other members of the Loxosceles group. A single human accident involving L. anomala was reported to date and the clinical picture shared similar characteristics with accidents caused by other Loxosceles species. Despite the potential relevance of L. anomalafor loxocelism in Minas Gerais state, its venom activity has never been characterized. In this work, we provide a preliminary characterization of L. anomala venom, considering its most relevant enzymatic activities and its venom immunorecognition by current therapeutic antivenoms. The results showed that L. anomala venom is immunorecognised by therapeutic antivenoms and by anti-phospholipase D antibodies. Its venom also shows enzymatic activities (sphingomyelinase activity, fibrinogenolytic) described for other Loxosceles venoms. This work contributes to a better knowledge on the venom content and activities of synanthropic Loxosceles species that have the potential of causing relevant human accidents.

The Health Status of Horses Used for at Least Six Complete Cycles of Loxoscelic Antivenom Production.

Antivenom production against Loxosceles venom relies on horses being immunized and bled for plasma harvest. One horse can partake in several cycles of antivenom production, which will require years of constant venom and adjuvant inoculation and bleeding. The actual impact on the health of horses that participate in several antivenom-producing cycles is unknown. Therefore, this study aimed to evaluate the general health status of horses that underwent at least six cycles of loxoscelic antivenom production. Seven crossbred horses that had partaken in six to eight complete antivenom-producing cycles were used and established as the immunized group (IG). Under the same handling and general management, eleven horses were established as the control group (CG). The horses were evaluated regarding their general clinical status and had their blood sampled, and an ECG recorded. The IG presented lower RBC and PCV, despite keeping values within inferior limits for the species. Renal function was not impaired, and liver-related enzymes were higher than those in the CG, probably due to liver exertion from immunoglobulin synthesis. ECG showed some abnormalities in the IG, such as atrioventricular block and a wandering atrial pacemaker, corroborated by an increase in CK-MB. The cardiovascular abnormalities were mainly found in the horses that participated in several antivenom-producing cycles. The overall results indicate that these horses had some impairment of their general health status. Once available, some alternative, less toxic antigens should replace the venom for immunization of horses used for antivenom production.

Brown Spider Venom Phospholipase-D Activity upon Different Lipid Substrates.

Brown spider envenomation results in dermonecrosis, characterized by an intense inflammatory reaction. The principal toxins of brown spider venoms are phospholipase-D isoforms, which interact with different cellular membrane components, degrade phospholipids, and generate bioactive mediators leading to harmful effects. The Loxosceles intermedia phospholipase D, LiRecDT1, possesses a loop that modulates the accessibility to the active site and plays a crucial role in substrate. In vitro and in silico analyses were performed to determine aspects of this enzyme's substrate preference. Sphingomyelin d18:1/6:0 was the preferred substrate of LiRecDT1 compared to other Sphingomyelins. Lysophosphatidylcholine 16:0/0:0 was preferred among other lysophosphatidylcholines, but much less than Sphingomyelin d18:1/6:0. In contrast, phosphatidylcholine d18:1/16:0 was not cleaved. Thus, the number of carbon atoms in the substrate plays a vital role in determining the optimal activity of this phospholipase-D. The presence of an amide group at C2 plays a key role in recognition and activity. In silico analyses indicated that a subsite containing the aromatic residues Y228 and W230 appears essential for choline recognition by cation-π interactions. These findings may help to explain why different cells, with different phospholipid fatty acid compositions exhibit distinct susceptibilities to brown spider venoms.

The C-terminal mutation beyond the catalytic site of brown spider phospholipase D significantly impacts its biological activities.

Loxosceles spider envenomation results in dermonecrosis, principally due to phospholipases D (PLDs) present in the venom. These enzymes have a strongly conserved sequence, 273ATXXDNPW280, in the C-terminal region (SMD-tail) that make contact with ß-sheets of the TIM barrel, in which the amino acids Asp277 and Trp280 establish the energetically strongest contacts. The SMD-tail is conserved in PLDs from different species but absent in the non-toxic PLD ancestral glycerophosphodiester phosphodiesterases (GDPDs). This work aims to understand the role of the C-terminal region in the structural stability and/or function of phospholipases D. Through site-directed mutagenesis of the rLiD1 protein (recombinant Loxosceles intermedia dermonecrotic protein 1), we produced two mutants: rLiD1D277A and rLiD1W280A (both with sphingomyelinase activity), in which Asp277 and Trp280 were replaced by alanine. rLiD1D277A showed similar sphingomyelinase activity but at least 2 times more dermonecrotic activity than rLiD1 (wild-type protein). Conversely, while the rLiD1W280A displayed a slight increase in sphingomyelinase activity, its biological activity was similar or lower compared to rLiD1, potentially due to its decreased thermostability and formation of amyloid aggregates. In conclusion, these new findings provide evidence that SMD-tail mutants impact the structure and function of these proteins and point out that residues outside the active site can even increase the function of these enzymes.

Systemic Loxoscelism, Less Frequent but More Deadly: The Involvement of Phospholipases D in the Pathophysiology of Envenomation.

Bites of Loxosceles spiders can lead to a set of clinical manifestations called loxoscelism, and are considered a public health problem in many regions. The signs and symptoms of loxoscelism are divided into cutaneous and systemic forms. The former is more frequent and includes signs of envenoming at the bite site or neighboring regions. Systemic loxoscelism, although much less frequent, is associated with complications, and can even lead to death. It may include intravascular hemolysis, acute renal failure, and thrombocytopenia. Loxosceles venoms are enriched with phospholipases D (PLDs), which are a family of isoforms found at intra-species and inter-species levels. Under experimental conditions, these enzymes reproduce the main clinical signs of loxoscelism, including an exacerbated inflammatory response at the bite site and dermonecrosis, as well as thrombocytopenia, intravascular hemolysis, and acute renal failure. The role of PLDs in cutaneous loxoscelism was described over forty years ago, when studies identified and purified toxins featured as sphingomyelinase D. More recently, the production of recombinant PLDs and discoveries about their structure and mechanism has enabled a deeper characterization of these enzymes. In this review, we describe these biochemical and functional features of Loxosceles PLDs that determine their involvement in systemic loxoscelism.

Production and Functional Evaluation of Anti-Loxosceles Sera Raised by Immunizations of Rabbits with Mutated Recombinant Phospholipases-D.

Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles. The main species involved in accidents in South America are L. intermedia, L. laeta, and L. gaucho. The only specific treatment is the anti-Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A (L. gaucho), W230A (L. intermedia), and H12A-H47A (L. laeta). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti-Loxosceles sera for treatments of Loxoscelism.

Brown spider (Loxosceles sp.) bite and COVID-19: A case report.

We present the case of a 32-year-old male patient hospitalized during the COVID-19 pandemic because of a Brown spider bite on his lower lip. The Brown spider accident occurred in southern Brazil; at hospital admission, the patient presented on his lip: edema, pustules, necrotic regions, and ulcerations. The patient complained of lower back pain, fever and dyspnea. Laboratory tests showed monocytosis, leukocytosis, neutrophilia, increased D-dimer levels, C-reactive protein, glutamate-pyruvate transaminase, delta bilirubin, creatine phosphokinase, procalcitonin, and fibrinogen. The patient was hospitalized and a multi-professional team carried out the treatment. The medical team diagnosed loxoscelism with moderate changes. The dentist treated the oral cavity. The patient began to develop nausea, vomiting, and desaturation episodes during hospitalization. A computed tomography of the chest was performed, which showed signs of viral infection. The RT-PCR test for COVID-19 was positive. The systemic conditions worsened (renal dysfunction, systemic inflammatory response, pulmonary complications). This condition may have resulted from the association of the two diseases (loxoscelism and COVID-19), leading to the patient's death. This case illustrates the difficulties and risks in treating patients with venomous animal accidents during the pandemic, and the importance of a multi-professional team in treating such cases.

Clinical Effects of the Immunization Protocol Using Loxosceles Venom in Naïve Horses.

Bites of brown spiders (Loxosceles spp.) are responsible for dermonecrotic lesions and potentially systemic envenoming that can lead to death. The only effective therapy is the use of the antivenom, usually produced in horses. However, little is known about the consequences of the systematic use of the Loxosceles venom and adjuvants and of the bleedings on antivenom-producing horses. Thus, the aim of this study was to evaluate the clinical changes in horses in their first immunization protocol for Loxosceles antivenom production. Eleven healthy horses, never immunized, were evaluated in three different periods: T0 (before immunization); T1 (after their first venom immunization); and T2 (after their first bleeding). Horses were clinically evaluated, sampled for blood, and underwent electrocardiographic (ECG) recordings. Several suppurated subcutaneous abscesses occurred due to the use of Freund's adjuvants and thrombophlebitis due to systematic venipunctures for the bleeding procedures. ECG showed arrhythmias in few horses in T2, such as an increase in T and R waves. In summary, the immunization protocol impacted on horses' health, especially after bleeding for antivenom procurement.

Protective Effectiveness of an Immunization Protocol Against the Toxic Effects of Loxosceles intermedia Venom in Rabbits.

Loxosceles spp. (brown spiders) bites are responsible for the development of a syndrome consisting mainly of dermonecrotic lesions, and also systemic effects. Rabbits are one of the main experimental models used for better understanding the systemic and local effects of Loxosceles venom. The aim of this study is to evaluate the toxic and protective effects of rabbits immunized with Loxosceles spp. venom. Male New Zealand rabbits were allocated as a control group (CG; n = 5) that received adjuvant (Montanide) and phosphate-buffer saline (PBS), or as venom group (VG; n = 5) that received 21 µg of Loxosceles venom using Montanide as adjuvant. After five immunization cycles, a trial with 7 µg of Loxosceles intermedia (L. intermedia) venom was performed, and dermonecrotic lesions were measured. The rabbits were then euthanized, and their organs were collected for histopathology analysis. Rabbits that had undergone Loxosceles venom immunization protocol showed minor clinical disturbances during the experimental period. The used immunization protocol protected the rabbits against the toxic effect of the Loxosceles venom because they showed minor clinical disturbances during the experimental period.

A protective vaccine against the toxic activities following Brown spider accidents based on recombinant mutated phospholipases D as antigens.

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.

Biotechnological potential of Phospholipase D for Loxosceles antivenom development.

Loxoscelism is one of the most important forms of araneism in South America. The Health Authorities from countries with the highest incidence and longer history in registering loxoscelism cases indicate that specific antivenom should be administered during the first hours after the accident, especially in the presence or at risk of the most severe clinical outcome. Current antivenoms are based on immunoglobulins or their fragments, obtained from plasma of hyperimmunized horses. Antivenom has been produced using the same traditional techniques for more than 120 years. Although the whole composition of the spider venom remains unknown, the discovery and biotechnological production of the phospholipase D enzymes represented a milestone for the knowledge of the physiopathology of envenomation and for the introduction of new innovative tools in antivenom production. The fact that this protein is a principal toxin of the venom opens the possibility of replacing the use of whole venom as an immunogen, an attractive alternative considering the laborious techniques and low yields associated with venom extraction. This challenge warrants technological innovation to facilitate production and obtain more effective antidotes. In this review, we compile the reported studies, examining the advances in the expression and application of phospholipase D as a new immunogen and how the new biotechnological tools have introduced some degree of innovation in this field.

From taxonomy to molecular characterization of brown spider venom: An overview focused on Loxosceles similis.

Loxosceles spp. (Araneae, Sicariidae), known as brown spiders, are distributed in temperate and tropical regions worldwide. Accidents caused by these spiders are known as loxoscelism and constitute a public health problem, especially in Brazil. The present review describes the taxonomy, distribution, and ecological profile of brown spiders, as well as the molecular and biochemical aspects of Loxosceles venom. Additionally, it presents an overview on L. similis, a species found in the Southeastern region of Brazil. In this region, the number of Loxosceles accidents has been increasing in the past few years, thus calling attention to its raising importance as a medically relevant spider species in Brazil.