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Simple Summary: In this study, we present the characterization of Aedes aegypti mosquito salivary protein, LTRIN (lymphotoxin β receptor inhibitor), a key facilitator of ZIKV transmission. Injected into the human host during blood-feeding, LTRIN interacts with the human receptor LTβR. We found that LTRIN exists as a thermostable protein with homodimers consisting of an alpha helix-dominant secondary structure with two EF-hand motifs, crucial for withstanding temperature fluctuations during blood-feeding. Despite being an EF-hand protein, its secondary structure remains unaffected by Ca2+ binding. ELISA assays and HDX-MS experiments identified the binding region of LTRIN with LTβR. Disrupting the calcium-binding ability in the second EF-hand motif significantly impacted the interaction between LTRIN, particularly the 15 kDa C-terminal protein, ΔLTRIN, and LTβR. Moreover, the decrease in binding affinity of full-length LTRIN suggests the presence of only the truncated form, ΔLTRIN, in the mosquito's saliva, as the N-terminal region likely covers the interaction site. This insight provides a basis for developing inhibitors against LTRIN as a potential treatment or vaccine target for ZIKV. Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTβR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTβR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTβR using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTβR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV. [ABSTRACT FROM AUTHOR]

Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTβR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTβR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTβR using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTβR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV.

Malaria is initiated as Plasmodium sporozoites are injected into the dermis when an infected mosquito probes on a vertebrate host for a blood meal. Factors in the mosquito saliva, such as AgTRIO, can alter the ability of Anopheles gambiae to transmit Plasmodium. We therefore used CRISPR-Cas9-mediated genome editing to generate AgTRIO knockout (KO) A. gambiae and examined the ability of these mosquitoes to probe on a vertebrate host. AgTRIO KO mosquitoes showed a diminished host probing capacity and required repetitive probing to locate a blood resource to complete a blood meal. This increased probing resulted in enhanced Plasmodium transmission to the vertebrate host. Our data demonstrate the importance of the A. gambiae saliva protein AgTRIO in probing and its influence on the ability of mosquitoes to transmit malaria. [Display omitted] • AgTRIO knockout mosquitoes show a diminished host probing capacity • AgTRIO knockout mosquitoes require repetitive probing to complete a blood meal • Increased probing results in enhanced Plasmodium transmission to the vertebrate host Chuang et al. use CRISPR-Cas9 to generate AgTRIO knockout mosquitoes and demonstrate that AgTRIO is required for mosquitoes to probe efficiently during a blood meal. Lacking AgTRIO causes mosquitoes to take longer when probing to take a blood meal. This increased probing results in enhanced Plasmodium transmission to the vertebrate host. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the mineral-promoting effects of in-office bleaching agent on enamel subsurface lesions. Enamel subsurface lesions were divided into following groups; D: demineralized samples without any further treatment, DS: samples were further immersed in fresh saliva, DSR: samples were immersed in saliva followed by remineralization buffer, and DSBR: samples were immersed in saliva, subjected to in-office bleaching, and then immersed in remineralization buffer. The control group (CONT) consisted of untreated enamel specimens. Transverse microradiography showed that integrated mineral loss was significantly lower in the DSBR group than in the DSR group. Confocal laser Raman analysis revealed that v1 phosphate peak height of 959 cm-1 and mineral to matrix ratio of peak heights 959 cm-1 to 1,610 cm-1 in the DSBR group were similar to those in the CONT. In-office bleaching can promote enamel remineralization by altering or removing proteins infiltrated to enamel subsurface lesions. [ABSTRACT FROM AUTHOR]

Malaria begins when Plasmodium-infected Anopheles mosquitoes take a blood meal on a vertebrate. During the initial probing process, mosquitoes inject saliva and sporozoites into the host skin. Components of mosquito saliva have the potential to influence sporozoite functionality. Sporozoite-associated mosquito saliva protein 1 (SAMSP1; AGAP013726) was among several proteins identified when sporozoites were isolated from saliva, suggesting it may have an effect on Plasmodium. Recombinant SAMSP1 enhanced sporozoite gliding and cell traversal activity in vitro. Moreover, SAMSP1 decreased neutrophil chemotaxis in vivo and in vitro, thereby also exerting an influence on the host environment in which the sporozoites reside. Active or passive immunization of mice with SAMSP1 or SAMSP1 antiserum diminished the initial Plasmodium burden after infection. Passive immunization of mice with SAMSP1 antiserum also added to the protective effect of a circumsporozoite protein monoclonal antibody. SAMSP1 is, therefore, a mosquito saliva protein that can influence sporozoite infectivity in the vertebrate host. [ABSTRACT FROM AUTHOR]

Surface treatments are designed to promote modified implant surfaces with positive interactions with the surrounding living tissues. However, the inadvertent early contact of these surfaces with oral fluids during surgery may lead to undesired conditions affecting osseointegration. This study aimed to investigate the possible alterations in the physico-chemical properties of modified-surfaces caused by early saliva exposure. Titanium (Ti) surfaces were exposed to three different samples of human saliva and later analyzed for protein adhesion, physico-chemical surface alterations, and osteogenic cell-viability. The results indicated that surface roughness was the most significant factor influencing saliva protein adsorption; moreover, hydrophilic surfaces had critically lost their characteristics after contact with saliva. Decreased cell viability was observed in cultures after contact with saliva. Early contact with saliva might negatively influence modified surface properties and local cell viability. Careful surgical insertion of implants with hydrophilic surfaces is recommended, particularly in sites where saliva interaction is prone to occur. [ABSTRACT FROM AUTHOR]

The present study evaluated the capability of saliva analysis combining membrane protein purification with surface-enhanced Raman spectroscopy (SERS) for noninvasive detection of nasopharyngeal carcinoma (NPC). A rapid and convenient protein purification method based on cellulose acetate membrane was developed. A total of 659 high-quality SERS spectra were acquired from purified proteins extracted from the saliva samples of 170 patients with pathologically confirmed NPC and 71 healthy volunteers. Spectral analysis of those saliva protein SERS spectra revealed specific changes in some biochemical compositions, which were possibly associated with NPC transformation. Furthermore, principal component analysis combined with linear discriminant analysis (PCA-LDA) was utilized to analyze and classify the saliva protein SERS spectra from NPC and healthy subjects. Diagnostic sensitivity of 70.7%, specificity of 70.3%, and diagnostic accuracy of 70.5% could be achieved by PCA-LDA for NPC identification. These results show that this assay based on saliva protein SERS analysis holds promising potential for developing a rapid, noninvasive, and convenient clinical tool for NPC screening. [ABSTRACT FROM AUTHOR]

Due to the recorded spreading of ticks in past years, a higher incidence of tick-borne diseases (TBDs) can be expected in the future in endemic areas, but can also pose an emerging public health concern in areas where they have not yet been recognized. Assessment of the exposure of vulnerable hosts to ticks would be a very helpful tool for TBD epidemiological studies, as well as for their proper managing. To confirm previous tick bites, the method of choice is detection of antibodies in host serum as markers developed against injected tick saliva proteins during feeding. We recently showed that the recombinant form of Ixodes ricinus AV422 saliva protein (r IrAV422) can serve for detection of markers in experimentally infested rats. Here we examine whether it can be used in the same manner in naturally exposed hosts. We chose hunting dogs as good sentinel animals. The study group consisted of 15 dogs that varied in breed, age, sex, previous tick infestation history and repellent treatment. Western blot analysis with r IrAV422 as an antigen confirmed the presence of tick bite markers in all analysed dogs. For some of the dogs, their previous tick infestation history was unclear, which emphasizes the usefulness of r IrAV422 for revealing it. Since hunting dogs are naturally infested with different ticks, the potential of r IrAV422 in assessment of general exposure to ticks is highlighted. Use of r IrAV422 can also be helpful in veterinary practice and research as a tool for validation of the efficiency of tick repellent products. [ABSTRACT FROM AUTHOR]

Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen responsive genes. The data suggest that Armet is an effector protein mediating aphid--plant interactions [ABSTRACT FROM AUTHOR]

PURPOSE: To evaluate the relationship between salivary protein's change and dental caries in patients after oral administration of glucocorticoid. METHODS: Sixty patients were divided equally into the experimental group and the control group. The mixed saliva samples of the experimental group before (T1) and after (T2) taking GC orally, as well as the baseline (C1) and after 12 months (C2) of the control group were collected. IgA, LDH, IZM were examined in both groups. The decayed-missing-filled surface (DMFS), decayed-missing-filled tooth (DMFT), caries severity index (CSI) in both groups were recorded. All statistical analyses were performed using SPSS17.0 software package. RESULTS: After taking GC orally, the concentration of IgA and IZM were significantly lower than the control group (P<0.01), while the concentration of LDH was significantly higher than the control group (P<0.01). Before and after taking GC orally, the salivary protein's change was significantly correlated with DMFF, DMFS and CSI. CONCLUSIONS: After taking glucocorticoid orally, the concentration of LZM, IgA, LDH in saliva changes. LZM, IgA, LDH take part in the occurrence and development of dental caries as the important part of oral immunization. Supported by Research Fund of Science and Technology Commission of Shanghai Municipality(081411967000). [ABSTRACT FROM AUTHOR]

Grape seed and skin proanthocyanidins undergo significant changes during grape maturation. Those changes have been associated with a decreased astringency of grapes collected at later maturation stages. We have now compared the ability of proanthocyanidin-rich extracts from Carménère grape seeds collected at two stages of berry development (veraison vs. harvest) both to interact with salivary proteins and to produce astringency. Interaction of proanthocyanidins with the salivary protein was assessed by analysing the concentration-dependent effect of seed extracts both on inducing salivary protein precipitation and on restricting diffusion of the salivary protein on cellulose membranes. Seed extracts from grapes collected at veraison compared with those of grapes collected at harvest displayed a higher ability both to interact with the salivary protein fraction and to produce astringency. As a whole, our observations show that astringency is closely dependent on the magnitude of the interactions between proanthocyanidins and the salivary protein fraction. [ABSTRACT FROM AUTHOR]

Adsorption of protein from saliva on hydroxyapatite was compared with adsorption of several typical proteins with different electric charges, i.e. lysozyme, human serum albumin, β-lactoglobulin and ovalbumin. Adsorbed amounts of these proteins were determined and electrophoretic mobilities of protein-covered hydroxyapatite particles were measured, at different values for the adsorbed mass and, therefore, at various degrees of surface coverage. Also, adsorption kinetics were investigated by streaming potential measurements of a hydroxyapatite surface in contact with a protein solution, allowing monitoring of changes in the zeta-potential of the protein-covered hydroxyapatite surface in real time. The adsorbed amounts show that, as compared to most of the other proteins, the saliva proteins have remarkably low adsorption affinity. The measured values for the electrophoretic mobilities indicate that the positively charged proteins in the saliva mixture preferentially adsorb onto the negatively charged hydroxyapatite surface; this is most pronounced at low protein concentration in solution (i.e. at low coverage of the surface by the protein). Preferential uptake of the positively charged saliva proteins during the initial stages of the adsorption process is also concluded from the results of the kinetics experiments. Preferential adsorption of positive proteins is somewhat suppressed by the presence of Ca2+ ions in the medium. The results suggest that an acquired pellicle on a tooth in an oral environment contains a significant fraction of positively charged proteins. The positively charged proteins in the pellicle reduce the zeta-potential at the tooth surface to low values; consequently, electrostatic forces are expected to play only a minor role in the interaction with other components (e.g. bacterial cells). [Copyright &y& Elsevier]

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e . 10 - 15 nm 2 , 5.5 nm 2 and 4 nm 2 per polymer chain, respectively. The adsorption of different proteins on the PEO grafted surfaces was measured in real time by reflectometry. Furthermore, the change of the zeta-potential of such surfaces resulting from adsorption of the proteins was determined using the streaming potential method. Both the protein adsorption and the zeta-potential were monitored for 1 h after exposure of the protein solution to the surface. The adsorption pattern for a mixture of saliva proteins was compared to those observed for a number of well-defined model-proteins (lysozyme, human serum albumin, β-lactoglobulin and ovalbumin). The results of the adsorption kinetics and streaming potential measurements indicate that the effect of the PEO layer on protein adsorption primarily depends on the size and the charge of the protein molecules. The saliva proteins are strongly blocked for adsorption, whereas the change in the zeta-potential is larger than for the other proteins (except lysozyme). It is concluded that positively charged protein molecules, having dimensions larger than those of lysozyme, are involved in the initial stage of adsorption from saliva onto a negatively charged surface. [ABSTRACT FROM AUTHOR]

SUMMARY The gene encoding a 36-kDa (p36) immunomodulatory protein present in saliva of Dermacentor andersoni was cloned in prokaryotic and eukaryotic expression vectors. A polymerase chain reaction (PCR)-generated cDNA lacking signal peptide was cloned into the Escherichia coli expression vector pET28 and a similar sequence was cloned into pIB/V5-His-TOPO expression vector for stable transfection of insect cells, High 5 ™. The 26-kDa molecular mass of p36 expressed by bacteria is in agreement with that predicted from the translated full-length cDNA sequence. Eukaryotic-cell-expressed p36 consisted of multiple forms with molecular masses between 34 and 36 kDa. These multiple forms were attributed to differences in post-translational modifications. N-linked mannose was detected on insect-cell-expressed and tick-derived p36. Multiple bands remained after endoglycosidase removal of N-linked sugars, indicating the presence of other modifications. Both bacterial- and insect-cell-expressed p36 reacted on immunoblots with polyclonal antibodies raised against tick-derived p36. Insect-cell-expressed p36 suppressed T-lymphocyte-mitogen-driven in vitro proliferation of splenocytes from tick-naïve mice in a dose-dependent manner. Bacterial-cell-expressed p36 lacked immunomodulatory activity. [ABSTRACT FROM AUTHOR]

The present study evaluated the capability of saliva analysis combining membrane protein purification with surface-enhanced Raman spectroscopy (SERS) for noninvasive detection of nasopharyngeal carcinoma (NPC). A rapid and convenient protein purification method based on cellulose acetate membrane was developed. A total of 659 high-quality SERS spectra were acquired from purified proteins extracted from the saliva samples of 170 patients with pathologically confirmed NPC and 71 healthy volunteers. Spectral analysis of those saliva protein SERS spectra revealed specific changes in some biochemical compositions, which were possibly associated with NPC transformation. Furthermore, principal component analysis combined with linear discriminant analysis (PCA-LDA) was utilized to analyze and classify the saliva protein SERS spectra from NPC and healthy subjects. Diagnostic sensitivity of 70.7%, specificity of 70.3%, and diagnostic accuracy of 70.5% could be achieved by PCA-LDA for NPC identification. These results show that this assay based on saliva protein SERS analysis holds promising potential for developing a rapid, noninvasive, and convenient clinical tool for NPC screening., ((2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen-responsive genes. The data suggest that Armet is an effector protein mediating aphid-plant interactions., (© FASEB.)

Many arboviral diseases are uncontrolled, and the viruses that cause them are globally emerging or reemerging pathogens that produce significant disease throughout the world. The increased spread and prevalence of disease are occurring during a period of substantial scientific growth in the vector-borne disease research community. This growth has been supported by advances in genomics and proteomics, and by the ability to genetically alter disease vectors. For the first time, researchers are elucidating the molecular details of vector host-seeking behavior, the susceptibility of disease vectors to arboviruses, the immunological control of infection in disease vectors, and the determinants that facilitate transmission of arboviruses from a vector to a host. These discoveries are facilitating the development of novel strategies to combat arboviral disease, including the release of transgenic mosquitoes harboring dominant lethal genes, the introduction of arbovirus-blocking microbes into mosquito populations, and the development of acquisition- and transmission-blocking therapeutics. Understanding the role of the vector in arbovirus transmission has provided critical practical and theoretical tools to control arboviral disease.