Characterization of the chemosensory protein EforCSP3 and its potential involvement in host location by Encarsia formosa

Chemosensory proteins(CSPs) perform several functions in insects.This study performed the gene expression,ligand-binding,and molecular docking assays on the EforCSP3 identified in the parasitoid wasp Encarsia formosa,to determine whether EforCSP3 functions in olfaction,especially in host location and host preference.The results showed that EforCSP3 was highly expressed in the female head,and its relative expression was much higher in adults than in other developmental stages.The fluorescence binding assays suggested that the EforCSP3 exhibited high binding affinities to a wide range of host-related volatiles,among which dibutyl phthalate,1-octene,β-elemene,and tridecane had the strongest binding affinity with EforCSP3,besides α-humulene and β-myrcene,and should be assessed as potential attractants.Protein structure modeling and molecular docking predicted the amino acid residues of EforCSP3possibly involved in volatile binding.α-Humulene and β-myrcene attracted E.formosa in a previous study and exhibited strong binding affinities with EforCSP3 in the current study.In conclusion,EforCSP3 may be involved in semiochemical reception by E.formosa.

Post-COVID-19 persistent olfactory,gustatory,and trigeminal chemosensory disorders:Definitions,mechanisms,and potential treatments

The nose and the oral cavities are the main sites for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)entry into the body.Smell and taste deficits are the most common acute viral manifestations.Persistent smell disorders are the most common and bothersome complications after SARS-CoV-2 infection,lasting for months to years.The mechanisms and treatment of persistent post-coronavirus disease 2019(COVID-19)smell and taste disorders are still challenges.Information sources for the review are PubMed,Centers for Disease Control and Prevention,Ovid Medline,Embase,Scopus,Web of Science,International Prospective Register of Systematic Reviews,Cumulative Index to Nursing and Allied Health Literature,Elton Bryson Stephens Company,Cochrane Effective Practice and Organization of Care,Cooperation in Science and Technology,International Clinical Trials Registry Platform,World Health Organization,Randomized Controlled Trial Number Registry,and MediFind.This review summarizes the up-to-date information about the prevalence,patterns at onset,and prognoses of post-COVID-19 smell and taste disorders,evidence for the neurotropism of SARS-CoV-2 and the overlap between SARSCoV-1,Middle East respiratory syndrome coronavirus,and SARS-CoV-2 in structure,molecular biology,mode of replication,and host pathogenicity,the suggested cellular and molecular mechanisms for these post-COVID19 chemosensory disorders,and the applied pharmacotherapies and interventions as trials to treat these disorders,and the recommendations for future research to improve understanding of predictors and mechanisms of these disorders.These are crucial for hopeful proper treatment strategies.

Functional Characteristics of a Novel Chemosensory Protein in the Cotton Bollworm Helicoverpa armigera (Hübner)

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera （Hvbner） was obtained from antennal eDNA libraries and expressed in Escherichia coll. The real time quantitative PCR （RT-qPCR） results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isobomeol, nerolidol, 2- nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33-38 （αl）, 40-48 （α2）, 62-72 （α3）, 80-96 （α4）, 98-108 （α5）, and 116-119 （α6）, two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trpl01, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.

Roles of the main olfactory and vomeronasal systems in the detection of androstenone in inbred strains of mice

We investigated the role of the main olfactory and accessory olfactory systems （MOS and AOS respectively） in the detection of androstenone. We used the following experimental approaches： behavioral, surgical removal of the vomeronasal organ （VNX） followed by histochemical verification and Fos immunohistochemistry. Using a Y-maze paradigm we estimated sensitivity of NZB/B1NJ and CBA/J mice to androstenone. CBA mice were 2,000-fold more sensitive to androstenone than NZB mice. VNX caused a 4-tol6-fold decrease in sensitivity to androstenone in highly-sensitive CBA mice, but did not affect thresholds in NZB mice. Results indicate the involvement of the MOS and AOS in the detection of androstenone. We observed a specific pattern of Fos-positive cells in the main olfactory bulb of CBA mice but not in NZB mice subsequent to exposure of mice to androstenone; the compound activated cells in the accessory olfactory bulb in both strains of mice, indicating the involvement of the vomeronasal organ. Patterns of Fos-positive cells in the vomeronasal organ were recorded subsequent to exposure to androstenone. Fos-positive receptor cells in the vomeronasal organ of CBA and NZB mice were different, in CBA mice Fos-positive cells were noted in both the basal and apical zones, however, in NZB mice activation was observed only in the apical zone [Current Zoology 56 （6）： 813-818, 2010].

Molecular and in vitro biochemical assessment of chemosensory protein 10 from brown planthopper Nilaparvata lugens at acidic pH

Chemosensory proteins(CSPs)are important molecular components of the insect olfactory system,which are involved in capturing,binding,and transporting hydrophobic odour molecules across the sensillum in sensillar lymph in regulating insect behavior.This protein family(CSPs)is also involved in many other systems that are not linked to olfactory receptors in olfactory sensilla.The brown planthopper(BPH)is a monophagous pest of rice that causes damage by sucking phloem sap and transmitting a number of diseases caused by viruses.In this study,fluorescence competitive binding assay and fluorescence quenching assay at acidic p H were performed as well as homology modelling to describe the binding affinity of Nlug CSP10.Fluorescence competitive binding assay(FCBA)demonstrated that Nlug CSP10 bound strongly to nonadecane,farnesene,and 2-tridecanone at acidic p H.The results of FCBA indicated that Nlug CSP10 bound different ligands at the physiological p H(5.0)of the bulk sensillum lymph.Fluorescence quenching assay demonstrated that Nlug CSP10 generated a stable complex with 2-tridecanone,while two ligands nonadecane and farnesene collided due to molecular collisions.The interaction of selected ligands with the modelled structure of Nlug CSP10 was also analyzed,which found the key amino acids(Gln23,Gln24,Gln25,Asn27,Met33,Ser34,Ile35,Tyr36,Asn42,Met43,Val45,Asn46,Asn93,Arg96,Ala97,Lys99,and Ala100)in Nlug CSP10 that were involved in binding of volatile compounds.The present study contributes to the binding profile of Nlug CSP10 that promotes the development of behaviorally active ligands based on BPH olfactory system.

Chromosome-level genome assembly of Cylas formicarius provides insights into its adaptation and invasion mechanisms

Cylasformicarius is one of the most important pests of sweet potato worldwide, causing considerable ecological and economic damage.This study improved the effect of comprehensive management and understanding of genetic mechanisms by examining the functional genomics of C. formicarius.Using Illumina and PacBio sequencing, this study obtained a chromosome-level genome assembly of adult weevils from lines inbred for 15 generations.The high-quality assembly obtained was 338.84 Mb, with contig and scaffold N50 values of 14.97 and 34.23 Mb, respectively.In total, 157.51 Mb of repeat sequences and 11 907 protein-coding genes were predicted.A total of 337.06 Mb of genomic sequences was located on the 11 chromosomes, accounting for 99.03%of the total length of the associated chromosome.Comparative genomic analysis showed that C. formicarius was sister to Dendroctonus ponderosae, and C. formicarius diverged from D. ponderosae approximately 138.89 million years ago (Mya).Many important gene families expanded in the C. formicarius genome were involved in the detoxification of pesticides, tolerance to cold stress and chemosensory system.To further study the role of odorant-binding proteins (OBPs) in olfactory recognition of C. formicarius, the binding assay results indicated that Cfor OBP4–6 had strong binding affinities for sex pheromones and other ligands.The high-quality C. formicarius genome provides a valuable resource to reveal the molecular ecological basis, genetic mechanism, and evolutionary process of major agricultural pests;it also offers new ideas and new technologies for ecologically sustainable pest control.

Effect of drug therapies on self-reported chemosensory outcomes after COVID-19

Objective:The aim of this study was to assess the relative efficacy of medications used following severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)infection on self-reported alterations in taste and/or smell function.Methods:Seven hundred and fourteen persons with self-reported postcoronavirus disease 2019(post-COVID-19)chemosensory disorders were personally interviewed regarding specific medications they were administered following the acute phase of the disease.The dependent measure—self-reported total recovery of chemosensory symptoms—was subjected to stepwise logistic regression.Independent predictors included demographic and clinical variables,in addition to specific medications used to mitigate disease symptoms(i.e.,systemic corticosteroids,oseltamivir,vitamin C,ibuprofen,hydroxychloroquine,azithromycin,ivermectin,nitazoxanide,anticoagulants,and zinc).Results:The median time between COVID-19 symptom onset and the interviews was 81 days(interquartile range:60-104).Of the 714 subjects,249(34.9%)reported total recovery of their chemosensory function;437(61.2%)had at least one treatment since the beginning of the disease.Women and those with more comorbidities had undergone more treatments.The recovery rates of the treated and nontreated groups did not differ significantly.Nonetheless,respondents who had used nitazoxanide tended to have a higher rate of self-reported taste or smell recovery.Those who took oral zinc were less likely to improve.Conclusions:No medication employed during the first months after SARS-CoV-2 infection had a clear positive effect on returning self-reported smell or taste function to normal,although nitrazoxide trended in a positive direction.Oral zinc had a negative effect on the reported recovery of these senses.

A conserved odorant receptor identified from antennal transcriptome of Megoura crassicauda that specifically responds to cis-jasmone

Herbivore-induced plant volatiles(HIPVs)play a key role in the interactions between plants and herbivorous insects,as HIPVs can promote or deter herbivorous insects’behavior.While aphids are common and serious phloem-feeding pests in farmland ecosystems,little is known about how aphids use their sensitive olfactory system to detect HIPVs.In this study,the antennal transcriptomes of the aphid species Megoura crassicauda were sequenced,and expression level analyses of M.crassicauda odorant receptors(ORs)were carried out.To investigate the chemoreception mechanisms that M.crassicauda uses to detect HIPVs,we performed in vitro functional studies of the ORs using 11 HIPVs reported to be released by aphid-infested plants.In total,54 candidate chemosensory genes were identified,among which 20 genes were ORs.McraOR20 and McraOR43 were selected for further functional characterization because their homologs in aphids were quite conserved and their expression levels in antennae of M.crassicauda were relatively high.The results showed that McraOR20 specifically detected cis-jasmone,as did its ortholog ApisOR20 from the pea aphid Acyrthosiphon pisum,while McraOR43 did not respond to any of the HIPV chemicals that were tested.This study characterized the ability of the homologous OR20 receptors in the two aphid species to detect HIPV cis-jasmone,and provides a candidate olfactory target for mediating aphid behaviors.

Differential expression of pancreatic protein and chemosensing receptor m RNAs in NKCC1-null intestine

AIM: To investigate the intestinal functions of the NKCC1 Na^+-K^+-2Cl cotransporter(SLC12a2 gene), differential m RNA expression changes in NKCC1-null intestine were analyzed.METHODS: Microarray analysis of m RNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice(n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed.RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas m RNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

Disruption of Zfh3 abolishes mulberry-specific monophagy in silkworm larvae

Feeding behavior is critical for insect survival and fitness.Most researchers have explored the molecular basis of feeding behaviors by identifying and elucidating the function of olfactory receptors(ORs)and gustatory receptors(GRs).Other types of genes,such as transcription factors,have rarely been investigated,and little is known about their potential roles.The silkworm(Bombyx mori)is a well-studied monophagic insect which primarily feeds on mulberry leaves,but the genetic basis of its monophagy is still not un-derstood.In this report,we focused on a transcription factor encoded by the Zfh3 gene,which is highly expressed in the silkworm central and peripheral nervous systems,in-cluding brain,antenna,and maxilla.To investigate its function,Zfh3 was abrogated using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9(Cas9)mutagenesis.Since Zfh3 knockout homozygotes are not viable,we stud-ied feeding behavior in heterozygotes,and found that disruption of Zfh3 affects both gus-tation and olfaction.Mutant larvae lose preference for mulberry leaves,acquire the ability to consume an expanded range of diets,and exhibit improved adaptation to the Mo artifi-cial diet,which contains no mulberry leaves.These results provide the first demonstration that a transcription factor modulates feeding behaviors in an insect.

Comparison of trigeminal lateralization with differing stimulants

Trigeminal-specific stimulants have been shown to activate different receptors preferentially and this likely accounts for variation in sensory perception.It is unclear whether trigeminal sensitivity is similar across different transient receptor potential(TRP)receptors or if dysfunction of different receptors results in differing patient symptoms.Therefore,a prospective cohort study was conducted,consisting of trigeminal lateralization testing with three different stimulants(eucalyptol,isothiocyanate,acetic acid),olfaction testing with Sniffin'Sticks,and measurement of various patient‐reported outcome measures(PROMs).A total of 50 participants were enrolled across the olfactory spectrum.Mean TDI score was 27.1±8.3(range 7.0–39.5)with 38%normosmic and 62%dysosmic.Mean trigeminal lateralization scores out of 20 in the overall cohort were 16.18(2.78)for eucalyptol,14.94(3.49)for mustard oil,and 15.28(3.68)for vinegar.Eucalyptol showed a significant correlation with threshold scores of Sniffin'Sticks.A significant correlation was found between acetic acid and various PROMs.None of the lateralization scores of the trigeminal stimulants correlated to each other significantly and there was no correlation to age.The lack of correlation suggests that the measured sensitivity of one type of TRP receptor may not translate to similar sensitivity of the other receptors.Additional investigations with TRPV1 and TRPA1 agonists are needed to corroborate our findings.

Clinical studies in COVID-related olfactory disorders:Review of an institutional experience

Objective:To share a single institutional experience with clinical research on COVID-related olfactory dysfunction(OD).Data Source/Method:Narrative review of published original data and ongoing clinical trials on COVID-related OD at Washington University from 2020 to 2023.Results:There were three new diagnostic-/patient-reported outcome measures developed and tested.We report five clinical trials of interventions for COVID-related olfactory disorders:combined Visual-Olfactory Training(VOLT)with patient-preferred scents versus standard olfactory training(VOLT trial),oral gabapentin versus placebo(Gabapentin for the Relief of Acquired Chemosensory Experience trial),nasal theophylline irrigations versus placebo(Smell Changes and Efficacy of Nasal Theophylline trial),stellate ganglion block(single-arm),and mindfulness-based stress reduction(MBSR)versus lifestyle intervention(MBSR trial).Conclusions:Initial intervention trials for COVID-related OD have shown potential for improving subjective and objective olfactory outcomes.However,there remains no gold standard treatment that definitively outperforms placebo in controlled trials.Therefore,continued investigation of novel therapeutic strategies for COVID-related OD is necessary to maximize olfactory outcomes for affected patients.

The role of chemosensory protein 10 in the detection of behaviorally active compounds in brown planthopper, Nilaparvata lugens

Chemosensory proteins(CSPs)play important roles in insects’chemoreception,although their specific functional roles have not been fully elucidated.In this study,we conducted the developmental expression patterns and competitive binding assay as well as knock‐down assay by RNA interference both in vitro and in vivo to reveal the function of NlugCSP10 from the brown planthopper(BPH),Nilaparvata lugens(Stål),a major pest in rice plants.The results showed that NlugCSP10 messenger RNA was significantly higher in males than in females and correlated to gender,development and wing forms.The fluorescence binding assays revealed that NlugCSP10 exhibited the highest binding affinity with cis‐3‐hexenyl acetate,eicosane,and(+)‐β‐pinene.Behavioral assay revealed that eicosane displayed attractant activity,while cis‐3‐hexenyl acetate,similar to(+)‐β‐pinene significantly repelled N.lugens adults.Silencing of NlugCSP10,which is responsible for cis‐3‐hexenyl acetate binding,significantly disrupted cis‐3‐hexenyl acetate communication.Overall,findings of the present study showed that NlugCSP10 could selectively interrelate with numerous volatiles emitted from host plants and these ligands could be designated to develop slow‐release mediators that attract/repel N.lugens and subsequently improve the exploration of plans to control this insect pest.

Bioinformatics-Based Identification of Chemosensory Proteins in African Malaria Mosquito, Anopheles gambiae

Chemosensory proteins (CSPs) are identifiable by four spatially conserved Cys-teine residues in their primary structure or by two disulfide bridges in their tertiary structure according to the previously identified olfactory specific-D related proteins. A genomics- and bioinformatics-based approach is taken in the present study to identify the putative CSPs in the malaria-carrying mosquito, Anopheles gambiae. The results show that five out of the nine annotated candidates are the most possible Anopheles CSPs of A. gambiae. This study lays the foundation for further functional identification of Anopheles CSPs, though all of these candidates need additional experimental verification.

Molecular cloning and comparative analysis of transcripts encoding chemosensory proteins from two plant bugs, Lygus lineolaris and Lygus hesperus

Chemosensory proteins(CSPs)are soluble carrier proteins typically characterized by a six‐helix bundle structure joined by two disulfide bridges and a conserved Cys spacing pattern(C1‐X6‐8‐C2‐X16‐21‐C3‐X2‐C4).CSPs are functionally diverse with reported roles in chemosensation,immunity,development,and resistance.To expand our molecular understanding of CSP function in plant bugs,we used recently developed transcriptomic resources for Lygus lineolaris and Lygus hesperus to identify 17 and 14 CSP‐like sequences,respectively.The Lygus CSPs are orthologous and share significant sequence identity with previously annotated CSPs.Three of the CSPs are predicted to deviate from the typical CSP structure with either five or seven helical segments rather than six.The seven helix CSP is further differentiated by an atypical C3‐X3‐C4 Cys spacing motif.Reverse transcriptase PCR‐based profiling of CSP transcript abundance in adult L.lineolaris tissues revealed broad expression for most of the CSPs with antenna specific expression limited to a subset of the CSPs.Comparative sequence analyses and homology modeling suggest that variations in the amino acids that comprise the Lygus CSP binding pockets affect the size and nature of the ligands accommodated.

Increased expression of CSP and CYP genes in adult silkworm females exposed to avermectins

We analyzed 20 chemosensory protein （CSP） genes of the silkworm Bombyx mori. We found a high number of retrotransposons inserted in introns. We then analyzed expression of the 20 BrnorCSP genes across tissues using quantitative real-time polymerase chain reaction （PCR）. Relatively low expression levels of BmorCSPs were found in the gut and fat body tissues. We thus tested the effects of endectocyte insecticide abamectin （B 1 a and Blb avermectins） on BmorCSP gene expression. Quantitative real-time PCR experi- ments showed that a single brief exposure to insecticide abamectin increased dramatically CSP expression not only in the antennae but in most tissues, including gut and fat body. Furthermore, our study showed coordinate expression of CSPs and metabolic cytochrome P450 enzymes in a tissue-dependent manner in response to the insecticide. The function of CSPs remains unknown. Based on our results, we suggest a role in detecting xenobiotics that are then detoxified by cytochrome P450 anti-xenobiotic enzymes.

Disturbance cues in freshwater prey fishes： Does urea function as an ＇early warning cue＇ in juvenile convict cichlids and rainbow trout？

Freshwater vertebrate and invertebrate prey species commonly rely on chemosensory information, including non-injury released disturbance cues, to assess local predation threats. We conducted laboratory studies to （1） determine if urea can function as a disturbance cue in juvenile convict cichlids and rainbow trout and （2） determine if the background level of urea influences the behavioral response to a subsequent pulse of urea （＇background noise＇ hypothesis）. In the first series of trials, juve- nile cichlids and trout were exposed to urea at varying concentrations （0 to 0.5 mg L-1 for cichlids and 0 to 1.0 mg L1 for trout）. Our results suggest that both cichilds and trout exhibited functionally similar responses to urea and conspecific disturbance cues and that increasing the concentration of urea results in an increase intensity of antipredator behaviour. In the second series of trials, we pre-exposed cichlids or trout to intermediate or high concentrations of urea （or a distilled water control） and then tested for the response to a second pulse of urea at at intermediate or high concentrations （versus a distilled water control）. Our results demon- strate that pre-exposure to urea reduces or eliminates the response to a second pulse of urea, supporting the background noise hy- pothesis. Together, our results suggest that pulses of urea, released by disturbed or stressed individuals, may function as an early warning signal in freshwater prey species

Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemo-sensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolution-ary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among pri-mates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

Influence of medications on taste and smell

Medications frequently have chemosensory side effects that can adversely affect compliance with medical treatment regimens. Hundreds of drugs have been reported to induce unpleasant tastes and/or odors as well as altered chemosensations when administered alone or in combination with other medications. Some chemosensory complaints are due to the sensory properties of the drug itself such as aversive bitter and metallic tastes. However, most chemo-sensory side effects of drugs are due to alterations in the transduction pathways, biochemical targets, enzymes, and transporters by the offending medications. Studies of chemosensory perception in medicated older individuals have found that taste and smell loss is greatest for those consuming the largest number of prescription drugs. There are no standard treat-ments for drug-induced chemosensory disorders because each drug has unique biological ef-fects. However, there are a few treatment options to ameliorate chemosensory alterations including addition of simulated flavors to food to compensate for losses and to override offend-ing tastes and smells.

The role of bitter and sweet taste receptors in upper airway innate immunity: Recent advances and future directions

Bitter(T2R)and sweet(T1R)taste receptors have been implicated in sinonasal innate immunity and in the pathophysiology of chronic rhinosinusitis(CRS).Taste receptors are expressed on several sinonasal cell types including ciliated epithelial cells and solitary chemosensory cells.Bitter agonists released by pathogenic microbes elicit a T2R dependent signaling cascade which induces the release of bactericidal nitric oxide,increases mucociliary clearance,and promotes secretion of antimicrobial peptides.Genetic variation conferred by polymorphisms in T2R related genes is associated with differential CRS susceptibility,symptomatology and post-treatment outcomes.More recently,based on our understanding of T1R and T2R function,investigators have discovered novel potential therapeutics in T2R agonists and T1R antagonists.This review will discuss bitter and sweet taste receptor function in sinonasal immunity,explore the emerging diagnostic and therapeutic implications stemming from the most recent findings,and suggest directions for future research.