Salivary proteins and microbiota as biomarkers for early childhood caries risk assessment

Early childhood caries （ECC） is a term used to describe dental caries in children aged 6 years or younger. Oral streptococci, such as Streptococcus mutans and Streptococcus sorbrinus, are considered to be the main etiological agents of tooth decay in children. Other bacteria, such as Prevotella spp. and Lactobacillus spp., and fungus, that is, Candida albicans, are related to the development and progression of ECC. Biomolecules in saliva, mainly proteins, affect the survival of oral microorganisms by multiple innate defensive mechanisms, thus modulating the oral microflora. Therefore, the protein composition of saliva can be a sensitive indicator for dental health. Resistance or susceptibility to caries may be significantly correlated with alterations in salivary protein components. Some oral microorganisms and saliva proteins may serve as useful biomarkers in predicting the risk and prognosis of caries. Current research has generated abundant information that contributes to a better understanding of the roles of microorganisms and salivary proteins in ECC occurrence and prevention. This review summarizes the microorganisms that cause caries and tooth-protective salivary proteins with their potential as functional biomarkers for ECC risk assessment. The identification of biomarkers for children at high risk of ECC is not only critical for early diagnosis but also important for preventing and treating the disease.

Cloning and RNA interference analysis of the salivary protein C002 gene in Schizaphis graminum

The full-length c DNA of functionally-unknown salivary protein C002 in Schizaphis graminum was cloned using rapid amplification of c DNA ends（RACE） and designated as Sg C002（Gen Bank accession no. KC977563）. It is 767 bp long and encodes a protein of 190 amino acid residues with a predicted mass of 21.5 k Da and a predicted cleavage site of N-terminal signal peptide between the 24 th and the 25 th residues. Sg C002 is specifically expressed in salivary gland with the highest level at the 2nd instar. Introducing Sg C002-specific 476-si RNA, but not 546-si RNA to aphids through artificial diet significantly suppressed Sg C002 expression. Silencing Sg C002 gene led to lethality of the aphid on wheat plants, but not on pure artificial diet. Our study demonstrated that artificial diet-mediated RNAi can be a useful tool for research on the roles of genes in aphid salivary gland, and also provided new insights into the characteristics of C002 in wheat aphids.

Pilot study on binding of bovine salivary proteins to grit silicates and plant phytoliths

Mostly fed with grass in fresh or conserved form,cattle and other livestock have to cope with silicate defence bodies from plants(phytoliths)and environmental silicates(grit),which abrade tooth enamel and could additionally interact with various salivary proteins.To detect potential candidates for silicate-binding proteins,bovine whole saliva was incubated with grass-derived phytoliths and silicates.Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed.After intense washing,the powder fractions were loaded onto 1D-polyacrylamide gels,most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates.All materials were mainly bound by bovine odorant-binding protein,bovine salivary protein 30×10^(3) and carbonic anhydrase VI.The phytolith/silicate fraction showed additional stronger interaction with haemoglobinβand lactoperoxidase.Conceivably,the binding of these proteins to the surfaces may contribute to biological processes occurring on them.

Pilot study on binding of bovine salivary proteins to grit silicates and plant phytoliths

Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants （phytoliths） and environmental silicates （grit）, which abrade tooth enamel and could additionally interact with various salivary proteins. To detect potential candidates for silicate-binding proteins, bovine whole saliva was incubated with grass-derived phytoliths and silicates. Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed. After intense washing, the powder fractions were loaded onto 1D-polyacrylamide gels, most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates. All materials were mainly botmd by bovine odorant-binding protein, bovine salivary protein 30× 10^3 and carbonic anhydrase VI. The phytolith/silicate fraction showed additional stronger interaction with haemoglobin β and lactoperoxidase. Conceivably, the binding of these proteins to the surfaces may contribute to biological processes occurring on them.

Research progress toward the influence of mosquito salivary proteins on the transmission of mosquito-borne viruses

Mosquito-borne viruses(MBVs)are a large class of viruses transmitted mainly through mosquito bites,including dengue virus,Zika virus,Japanese encephalitis virus,West Nile virus,and chikungunya virus,which pose a major threat to the health of people around the world.With global warming and extended human activities,the incidence of many MBVs has increased significantly.Mosquito saliva contains a variety of bioactive protein components.These not only enable blood feeding but also play a crucial role in regulating local infection at the bite site and the remote dissemination of MBVs as well as in remodeling the innate and adaptive immune responses of host vertebrates.Here,we review the physiological functions of mosquito salivary proteins(MSPs)in detail,the influence and the underlying mechanism of MSPs on the transmission of MBVs,and the current progress and issues that urgently need to be addressed in the research and development of MSP-based MBV transmission blocking vaccines.

Salivary C-reactive protein and mean platelet volume as possible diagnostic markers for late-onset neonatal pneumonia

BACKGROUND Neonatal sepsis,a formidable threat to newborns,is a leading cause of neonatal mortality,with late-onset sepsis manifesting after 72 hours post-birth being particularly concerning.Pneumonia,a prevalent sepsis presentation,poses a significant risk,especially during the neonatal phase when lung defenses are compromised.Accurate diagnosis of pneumonia is imperative for timely and effective interventions.Saliva,a minimally invasive diagnostic medium,holds great promise for evaluating infections,especially in infants.AIM To investigate the potential of serum C-reactive protein(CRP),salivary CRP(sCRP),and mean platelet volume(MPV)as diagnostic markers for late-onset neonatal pneumonia(LONP).METHODS Eighty full-term neonates were systematically examined,considering anthropometric measurements,clinical manifestations,radiology findings,and essential biomarkers,including serum CRP,sCRP,and MPV.RESULTS The study reveals noteworthy distinctions in serum CRP levels,MPV,and the serum CRP/MPV ratio between neonates with LONP and healthy controls.MPV exhibited a robust discriminatory ability[area under the curve(AUC)=0.87]with high sensitivity and specificity at a cutoff value of>8.8.Correlations between serum CRP,sCRP,and MPV were also identified.Notably,sCRP demonstrated excellent predictive value for serum CRP levels(AUC=0.89),underscoring its potential as a diagnostic tool.CONCLUSION This study underscores the diagnostic promise of salivary and serum biomarkers,specifically MPV and CRP,in identifying and predicting LONP among neonates.These findings advocate for further research to validate their clinical utility in larger neonatal cohorts.

Identification of salivary proteins in the whitefly Bemisia tabaci by transcriptomic and LC-MS/MS analyses

The whitefly Bemisia tabaci is a notorious agricultural pest of many crops worldwide.Although it is thought that B.tabaci secretes saliva into the host plant to counter plant defenses,knowledge on the whitefly salivary proteome is limited.Here,we characterized the gene/protein repertoires of B.tabaci salivary glands and secreted saliva by transcriptomic and liquid chromatography tandem mass spectroscopy analyses.A total of 698 salivary gland-enriched unigenes and 171 salivary proteins were identified.Comparative analysis between the B.tabaci salivary proteins and those of different arthropod species revealed numerous similarities in proteins associated with binding,hydrolysis,and oxidation-reduction,which demonstrates a degree of conservation across herbivorous saliva.There were 74 proteins only identified in B.tabaci saliva,of which 34 were B.tabaci-specific.In addition,13 salivary proteins,of which I1 were B.tabaci-specific,were differentially regulated when B.tabaci fed on different hosts.Our results provide a good resource for future functional studies of whitefly salivary effectors,and might be useful in pest management.

Enamel matrix proteins in promoting saliva lubrication

Anti-wear performance of human enamel in the mouth is closely related to the lubrication of salivary pellicle.It is well known that the inorganic hydroxyapatite(HA)of the enamel plays an important role in the adsorption and pellicle-forming of salivary proteins on the enamel,but the role of enamel matrix proteins remains unclear.In this study,the adsorption and lubrication behavior of salivary proteins on original,heated,and deproteinated enamel surfaces was comparatively investigated using an atomic force microscopy and nano-indentation/scratch techniques.Compared with that on the original enamel surface,the adsorption and lubrication behavior of salivary proteins remains almost unchanged on the heated enamel surface(where the enamel matrix proteins are denatured but the size of HA crystalline nanoparticles keeps constant)but exhibits an obvious compromise on the deproteinated enamel surface(where the enamel matrix proteins are removed and agglomeration of HA crystallites occurs).The HA agglomeration weakens the electrostatic interaction of enamel surfaces with salivary proteins to cause a distinct negative influence on the adsorption and pellicle-forming of salivary proteins.Further,the negative effect is confirmed with a quartz crystal microbalance with dissipation.In summary,by regulating enamel nanostructure for appropriate electrostatic interactions between salivary proteins and enamel surfaces,the enamel matrix proteins play an essential role in the adsorption and pellicle-forming of salivary proteins on human enamel,and then contribute to saliva lubrication,which provides the enamel with an anti-wear mechanism.The findings will promote and assist the design of enamel-inspired anti-wear materials.

Leafhopper salivary carboxylesterase suppresses JA-Ile synthesis to facilitate initial arbovirus transmission in rice phloem

Plant jasmonoyl-L-isoleucine(JA-Ile)is a major defense signal against insect feeding,but whether or how insect salivary effectors suppress JA-Ile synthesis and thus facilitate viral transmission in the plant phloem remains elusive.Insect carboxylesterases(CarEs)are the third major family of detoxification enzymes.Here,we identify a new leafhopper CarE,CarE10,that is specifically expressed in salivary glands and is secreted into the rice phloem as a saliva component.Leafhopper CarE10 directly binds to rice jasmonate resistant 1(JAR1)and promotes its degradation by the proteasome system.Moreover,the direct association of CarE10 with JAR1 clearly impairs JAR1 enzyme activity for conversion of JA to JA-Ile in an in vitro JAIle synthesis system.A devastating rice reovirus activates and promotes the co-secretion of virions and CarE10 via virus-induced vesicles into the saliva-storing salivary cavities of the leafhopper vector and ultimately into the rice phloem to establish initial infection.Furthermore,a virus-mediated increase in CarE10 secretion or overexpression of CarE10 in transgenic rice plants causes reduced levels of JAR1 and thus suppresses JA-Ile synthesis,promoting host attractiveness to insect vectors and facilitating initial viral transmission.Our findings provide insight into how the insect salivary protein CarE10 suppresses host JA-Ile synthesis to promote initial virus transmission in the rice phloem.

A secreted salivary effector from Riptortus pedestris impairs soybean defense through modulating phytohormone signaling pathways

Riptortus pedestris(Fabricius),one of the major piercing-sucking insects in soybeans,causes delayed plant senescence and abnormal pods,known as staygreen syndrome.Recent research has shown that direct feeding of this insect is the major cause of soybean staygreen syndrome.However,it remains unclear whether R.pedestris salivary proteins play vital roles in insect infestation.Here,we found that 4 secretory salivary proteins can induce cell death in Nicotiana benthamiana by transient heterologous expression.The cell death induced by Rp2155 relies on the nucleotide-binding leucine-rich repeat helper,HSP90.Tissue-specificity assays indicated that Rp2155 is specifically expressed in the salivary gland of R.pedestris and is significantly induced during insect feeding.The expression of salicylic acid(SA)-,jasmonic acid(JA)-related genes was increased in soybean when fed by Rp2155-silenced R.pedestris.More importantly,soybean staygreen symptoms caused by R.pedestris were significantly alleviated when Rp2155 was silenced.Together,these results suggest that the salivary effector Rp2155 is involved in promoting insect infestation by suppressing the JA and SA pathways,and it can be considered as a potential RNA interference target for insect control.

Insect-specific RNA virus affects the stylet penetration activity of brown citrus aphid(Aphis citricidus)to facilitate its transmission

Sap-sucking insects often transmit plant viruses but also carry insect viruses,which infect insects but not plants.The impact of such insect viruses on insect host biology and ecology is largely unknown.Here,we identified a novel insect-specific virus carried by brown citrus aphid(Aphis citricidus),which we tentatively named Aphis citricidus picornavirus(AcPV).Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses,suggesting that these viruses represent a new family in order Picornavirales.Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference,resulting in asymptomatic tolerance.Importantly,we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants.AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration,thus promoting its transmission among aphids with plants as an intermediate site.The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling.Together,our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors,thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

Effect of Salivary Antimicrobial Factors on Microbial Composition of Tongue Coating in Patients with Coronary Heart Disease with Phlegm-Stasis Syndrome

Objective: Phlegm-stasis syndrome is one of the most common traditional Chinese medicine(TCM) syndromes and found in 59% of patients with coronary heart disease(CHD) in routine TCM clinical practice in China. One of the diagnostic criteria of phlegm-stasis syndrome is its characteristic white-greasy and thin tongue coating. We have previously reported that different types of tongue coating have different microbiome characteristics that can be used as diagnostic markers. However, the microbial characteristics of tongue coating of CHD patients with phlegm-stasis syndrome, including coating feature and underlying reason of formation, have rarely been reported. Herein, we examined the characteristic microbiome of tongue coating, and discussed the cause of tongue coating formation via salivary proteins in patients with phlegm-stasis syndrome. Methods: We examined white-greasy and thin tongue coatings obtained from 10 patients with CHD having phlegm-stasis syndrome(n = 10), and compared with those of patients with almost no coating – patients with Qi-Yin deficiency syndrome(n = 10) – and healthy controls(n = 10). 16S r RNA sequencing of tongue coating microbiome and isobaric tags for relative and absolute quantitation(i TRAQ)-based quantitative analysis of salivary proteins were used to detect tongue coating and salivary protein separately. Salivary levels of s Ig A, lysozyme, and amylase were detected by ELISA. Results: We identified Candidatus_Saccharimonas and Candidate_division_TM7_norank as the prominent members of tongue coating in patients with CHD having phlegm-stasis syndrome. Salivary proteins involved in biological processes, pentose phosphate pathway, and complement and coagulation cascades were among the differentially expressed proteins identified in patients with CHD having phlegm-stasis syndrome on i TRAQ analysis. Moreover, the formation of microbiota in tongue coating was associated with salivary s Ig A, lysozyme, and saliva flow rate. Conclusions: We explored the characteristics of microbial composition of tongue coating patients with CHD having phlegm-stasis syndrome and identified correlations between salivary proteins and microbiome formation, providing a theoretical and mechanistic basis for tongue coating formation.