Effect of three NiTi files on transportation of the apical foramen.

AIM: To compare landed and nonlanded rotary file overinstrumentation on transportation of the apical foramen in the curved canals of extracted teeth. METHODOLOGY: Severely curved molar root canals (n = 45) were distributed into three equal groups (n = 15) according to angle (mean 54°) and radius of curvature (mean 5 mm). Canals were overinstrumented 0.5 mm beyond the foramen to a size 35 master apical file using landed (ProFile ISO), nonlanded (ProFile Vortex) or nonlanded, reduced shape memory (Vortex Blue) files. Post-instrumentation images of the apical foramen were compared with pre-instrumentation control images for differences in area, circularity and ratio of Feret's diameters. Groups were compared using anova or Kruskal-Wallis tests with significance of P < 0.05. RESULTS: There were no differences between pre-treatment groups in the parameters tested. All groups demonstrated alterations in the geometry of the apical foramen. There were no significant differences between ProFile ISO, ProFile Vortex or Vortex Blue in area, circularity and ratio of Feret's diameters. CONCLUSIONS: Landed, nonlanded and nonlanded reduced shape memory files all produced transportation of the apical foramen when overinstrumented by 0.5 mm in severely curved canals. There was no difference between these file systems with regard to the degree of this effect.

Differentiating dental pulp cells via RGD-dendrimer conjugates.

Traumatic dental injuries are often irreversible, underscoring the need for therapies that protect dental pulp cells and enhance their regeneration. We hypothesized that generation 5 poly amido amine (PAMAM) dendrimers (G5), functionalized with fluorescein isothiocyanate (FL) and αVß3-specific, cyclic arginine-glycine-aspartic acid (RGD) peptides, will bind to dental pulp cells (DPCs) and modulate their differentiation. Dental pulp cells and mouse odontoblast-like cells (MDPC-23) (±) treated with G5-FL-RGD were analyzed via Western blot, RT-PCR, and quantitative PCR. Transcription of dental differentiation markers was as follows: Dentin matrix protein (DMP-1), dentin sialoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) as well as vascular endothelial growth factor (VEGF) all increased via the JNK pathway. Long-term G5-RGD treatment of dental pulp cells resulted in enhanced mineralization as examined via Von Kossa assay, suggesting that PAMAM dendrimers conjugated to cyclic RGD peptides can increase the odontogenic potential of these cells.

A genetic determinant in Streptococcus gordonii Challis encodes a peptide with activity similar to that of enterococcal sex pheromone cAM373, which facilitates intergeneric DNA transfer.

Enterococcus faecalis strains secrete multiple peptides representing different sex pheromones that induce mating responses by bacteria carrying specific conjugative plasmids. The pheromone cAM373, which induces a response by the enterococcal plasmid pAM373, has been of interest because a similar activity is also secreted by Streptococcus gordonii and Staphylococcus aureus. The potential to facilitate intergeneric DNA transfer from E. faecalis is of concern because of extensive multiple antibiotic resistance, including vancomycin resistance, that has emerged among enterococci in recent years. Here, we characterize the related pheromone determinant in S. gordonii and show that the peptide it encodes, gordonii-cAM373, does indeed induce transfer of plasmid DNA from E. faecalis into S. gordonii. The streptococcal determinant camG encodes a lipoprotein with a leader sequence, the last 7 residues of which represent the gordonii-cAM373 heptapeptide SVFILAA. Synthetic forms of the peptide had activity similar to that of the enterococcal cAM373 AIFILAS. The lipoprotein moiety bore no resemblance to the lipoprotein encoded by E. faecalis. We also identified determinants in S. gordonii encoding a signal peptidase and an Eep-like zinc metalloprotease (lspA and eep, respectively) similar to those involved in processing certain pheromone precursors in E. faecalis. Mutations generated in camG, lspA, and eep each resulted in the ablation of gordonii-cAM373 activity in culture supernatants. This is the first genetic analysis of a potential sex pheromone system in a commensal oral streptococcal species, which may have implications for intergeneric gene acquisition in oral biofilms.

Effects of VEGF and FGF2 on the revascularization of severed human dental pulps.

The long-term outcome of replanted avulsed permanent teeth is frequently compromised by lack of revascularization, resulting in pulp necrosis. The purpose of this study was to evaluate the effects of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) on the revascularization of severed human dental pulps. Tooth slices were prepared from non-carious human molars and treated with 0-50 ng/mL rhVEGF(165) or rhFGF-2 for 7 days in vitro. Both angiogenic factors enhanced pulp microvessel density compared with untreated controls (p < 0.05). Tooth slices were also treated with 0 or 50 ng/mL rhVEGF(165) for one hour prior to implantation into the subcutaneous space of immunodeficient mice. Treatment with rhVEGF(165) increased pulp microvessel density in vivo (p < 0.05). These results demonstrate that rhVEGF(165) enhanced neovascularization of severed human dental pulps and suggest that topical application of an angiogenic factor prior to replantation might be beneficial for the treatment of avulsed teeth.

Effects of prolonged exposure to alkaline pH on Enterococcus faecalis survival and specific gene transcripts.

INTRODUCTION: The persistence of Enterococcus faecalis in treated root canals has been attributed to its resistance to the high pH of antimicrobial agents used during treatment, but the specific mechanisms are not clear. We investigated the survival and gene expression of E. faecalis maintained in alkaline media. METHODS: E. faecalis JH2-2 was maintained in media at pH 7, 10, 11 and 12 at either 25 degrees C or 37 degrees C for 1 week (168 h). At 24, 48, 72, 120 and 168 h, cell viability was determined in parallel with real-time quantitative polymerase chain reaction analyses of stress response genes (dnaK, fba, ftsZ, GroEL, napA, pbp5, tsf and tuf). RESULTS: After 1 week the E. faecalis showed survival levels of 100% in pH 7, 1% in pH 10, 0.001% in pH 11 and 0.00001% in pH 12 media. At 37 degrees C increased levels of gene transcripts occurred between 72 and 120 h in pH 7 media for ftsZ and dnaK, and in pH 10 media for ftsZ, pbp5, dnaK, napA, tsf, fba and GroEL. No increase in transcripts was observed at 37 degrees C in media at pH 11 or pH 12, nor at 25 degrees C in any media. CONCLUSION: Transcripts of ftsZ, a gene involved in cell division, increased by 37-fold after 120 h at pH 10 at 37 degrees C. Overall, the greatest increase in levels of gene transcripts occurred in cultures maintained in pH 10 media at 37 degrees C. These data may assist in understanding the survival strategies of E. faecalis following prolonged exposure to alkaline pH levels.

Survival of Enterococcus faecalis in root canals ex vivo.

AIM: The hypotheses tested in this study were that: (i) Enterococcus faecalis can survive long-term entombment in root filled teeth without additional nutrients, (ii) initial cell density influences the survival of E. faecalis in instrumented root canals and (iii) gelatinase-production capacity influences the survival of E. faecalis in root canals. METHODOLOGY: The root canals of 150 extracted single canal teeth were instrumented to apical size 60 and divided into six groups of 25. Within each group 10 canals were inoculated with either gelatinase-producing E. faecalis OG1-S and the other 10 with its gelatinase-defective mutant E. faecalis OG1-X. Five canals per group were kept as uninoculated controls. The root canals in groups 1 and 2 were inoculated with 10(6) bacteria, incubated for 48 h at 37 degrees C then filled with gutta-percha and zinc-oxide eugenol sealer. Root canals were inoculated with 10(6), 10(5), 10(4) and 10(3) bacteria in groups 3-6, respectively, and left unfilled. All teeth were sealed coronally with glass-ionomer cement. After 6- (groups 1, 3-6) and 12-month (group 2) incubation at 37 degrees C in 100% humidity, root fragments were analysed for presence of E. faecalis, using culture, polymerase chain reaction and histological methods. RESULTS: Viable E. faecalis was recovered from all root filled teeth and from 95-100% of unfilled inoculated teeth. Initial cell density and gelatinase production did not influence the recovery of viable E. faecalis (P > 0.05; chi-square test). Enterococcus faecalis 16S rRNA gene products were present in all inoculated teeth and absent in all noninoculated controls. Dentinal tubule infection was evident under light microscopy in sections from inoculated teeth after 48-h, 6- and 12-month incubation. CONCLUSIONS: Enterococcus faecalis inoculated into root canals maintained viability for 12-months ex vivo. The clinical implications are that viable E. faecalis entombed at the time of root filling could provide a long-term nidus for subsequent infection.

Quantitative real-time PCR detection of oral Enterococcus faecalis in humans.

OBJECTIVE: Enterococcus faecalis is consistently associated with recurrent root canal infections. Only low concentrations of E. faecalis in the human mouth have been demonstrated by culture techniques. Quantitative detection strategies more sensitive than culturing, such as quantitative PCR (qPCR), could provide more illuminating data. DESIGN: Thirty outpatients attending the University of Michigan Graduate Endodontic Clinic for endodontic treatment provided oral rinse samples that were analysed for E. faecalis using qPCR and microbiological culturing. A SYBR Green I qPCR protocol was developed for the quantifiable detection of E. faecalis and total bacteria in oral rinse samples using primers designed to target the 16S rRNA gene. Annealing temperature and primer, magnesium ion, and dimethyl sulfoxide concentrations were investigated for optimisation of the protocol; a minimum sensitivity limit of 23 rRNA copies (an estimated six E. faecalis cells) was established for E. faecalis in pure culture, and 104 rRNA copies (an estimated 26 E. faecalis cells) in mixed culture. RESULTS: In qPCR assays, based on extrapolation from estimated rRNA gene copy numbers, E. faecalis comprised 0.0006-0.0047% of a total bacteria load that ranged from 5.92 x 10(5) to 5.69 x 10(7) cells/ml of oral rinse. E. faecalis was detected in five (17%) samples in concentrations from 114 to 490 cells/ml. In parallel culture assays E. faecalis were detected in only two samples (7%) of the five identified by qPCR and in concentrations 30 and 240 CFU/ml. CONCLUSIONS: qPCR reported a higher incidence of E. faecalis in oral rinse samples than culture techniques and afforded greater sensitivity.

Influence of irrigant needle depth in removing bioluminescent bacteria inoculated into instrumented root canals using real-time imaging in vitro.

AIM: To test the hypothesis that the mechanical efficacy of irrigation in reducing bacteria in the root canal is dependent on depth of placement of the irrigation needle. METHODOLOGY: The root canals of 30 permanent cuspids were instrumented to apical size 60 using a crown-down technique. A suspension of the bioluminescent reporter strain Pseudomonas fluorescens 5RL was inoculated into each canal of sterilized teeth. Emission of bioluminescence (photons s(-1)) from each tooth was quantified on four sequential occasions using luminometry and bioluminescence imaging: (i) background, (ii) after inoculation, (iii) after irrigating the inoculated teeth with 3 mL of a nonantimicrobial irrigant delivered either 1 mm (group 1, n = 15) or 5 mm (group 2, n = 15) from working length (WL) using a 28G safety-ended irrigating needle, (iv) after an additional 3 mL irrigation (total 6 mL). Intragroup and intergroup comparisons were made using Wilcoxon matched pairs and Mann-Whitney tests, respectively. RESULTS: In group 1, there was a mean log10 decrease in bacteria of 0.68 +/- 0.26 after 3 mL of irrigant compared with 1.19 +/- 0.48 after 6 mL (P < 0.001); in group 2 the mean log10 decrease was 0.58 +/- 0.28 after 3 mL of irrigant compared with 0.69 +/- 0.35 after 6 mL (P < 0.02) (Wilcoxon matched pairs). Using 3 mL of irrigant, needle depth did not have a significant effect on reduction of intracanal bacteria (P = 0.407), but the effect became significant when 6 mL of irrigant was used (P < 0.002) (Mann-Whitney tests). CONCLUSIONS: The mechanical efficacy of 6 mL of irrigant in reducing intracanal bacteria was significantly greater when delivered 1 mm compared with 5 mm from WL.

Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp.

BACKGROUND/AIMS: Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. METHODS: Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. RESULTS: Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n=23), and response to pheromones in E. faecalis culture filtrate (n=16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n=31) but not in Enterococcus faecium (n=2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. CONCLUSIONS: Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones.

Prevalence, phenotype and genotype of oral enterococci.

This study investigated the prevalence, phenotype and genotype of oral enterococci. Enterococci were detected in oral rinse samples from 11% of 100 patients receiving endodontic treatment and 1% of 100 dental students with no history of endodontic treatment (P=0.0027). All enterococcal isolates were identified as Enterococcus faecalis. Viable counts ranged from 1 x 10 to 6 x 103 colony forming units per mL of oral rinse sample. Potential virulence traits expressed by oral E. faecalis strains included production of hemolysin (n=4) and gelatinase (n=4), and response to pheromones in E. faecalis culture filtrate (n=1). Six strains produced bacteriocin. All strains were susceptible to ampicillin, benzylpenicillin, gentamicin and vancomycin. There was no evidence of metal-ion resistance. One isolate produced hemolysin, gelatinase and bacteriocin, was resistant to several antibiotics, and responded to the pheromone cPD1. Pulsed-field gel electrophoresis and plasmid analysis showed that oral E. faecalis exhibited widespread genetic polymorphism, with plasmids detected in seven strains.

Orthograde retreatment and apexification after unsuccessful endodontic treatment, retreatment and apicectomy.

AIM: To describe a case where a second orthograde retreatment was successful in the management of an infected mandibular right first molar that previously had received both orthograde and retrograde treatments. SUMMARY: Periapical surgery is unlikely to be successful unless the root canal system has been adequately debrided and sealed. A case is described where orthograde endodontic treatment, retreatment and apicectomy were unsuccessful in the management of and infected mandibular right first molar. The periapical radiolucency eventually disappeared following a second orthograde retreatment. Teh second retreatment included 12 months of intracanal calcium hydroxide placement to promote apexification, thus allowing subsequent controlled obturation with gutta percha and AH26. At a 5-year review following completion of treatment, the tooth remained asymptomatic and was in normal function. KEY LEARNING POINTS: Orthograde retreatment is a treatment option to manage refractory lesions in teeth that have previously received endodontic treatment, retreatment and apicectomy. Orthograde retreatment using long-term intracanal calcium hydroxide can help promote root-end closure of a resected apex.

The antimicrobial efficacy of 'MGP' gutta-percha in vitro.

AIM: To determine whether 'MGP' gutta-percha (Westport, CT, USA), a commercially available gutta-percha containing iodoform, inhibits the growth of potential endodontic pathogens. METHODOLOGY: Inocula of Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus sanguis, Fusobacterium nucleatum and Actinomyces odontolyticus were spread onto the surface of agar plates. 'MGP' gutta-percha cones presoaked in sterile water were transferred to the inoculated agar and incubated at 37 degrees C aerobically or anaerobically as required for optimal growth. Identical studies were performed using iodoform-free gutta-percha and sterile paper disks saturated with 10% povidone-iodine. Following incubation, zones of inhibition around the 'MGP' gutta-percha, iodoform-free gutta-percha and disks were evaluated. RESULTS: Povidone-iodine inhibited all the strains. Iodoform-free gutta-percha inhibited S. sanguis and A. odontolyticus. 'MGP' gutta-percha inhibited S. aureus, S. sanguis, A. odontolyticus and F. nucleatum. Neither iodoform-free gutta-percha nor 'MGP' gutta-percha inhibited growth of E. faecalis, E. coli or P. aeruginosa. CONCLUSIONS: Compared to iodoform-free gutta-percha, iodoform-containing 'MGP' gutta-percha had an inhibitory effect in vitro on S. aureus and F. nucleatum, but not on E. faecalis, E. coli or P. aeruginosa.

Antimicrobial susceptibility of oral isolates of Enterobacter cloacae and Klebsiella pneumoniae from a southern Chinese population.

The antibiotic susceptibilities of 59 Enterobacter cloacae and 39 Klebsiella pneumoniae human oral isolates collected from a southern Chinese population in Hong Kong were investigated for their susceptibility to eight antibiotics: ampicillin, cephalothin, cefuroxime, ceftazidime, ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole using the E-Test method for direct quantification of minimum inhibitory concentrations. Most strains were sensitive to all antibiotics except ampicillin and cephalothin. Ampicillin resistance was exhibited by 82% of K. pneumoniae and 69% of E. cloacae isolates. Eighty-eight percent of E. cloacae isolates were resistant to cephalothin. Several strains fell within the intermediate category of sensitivity for ampicillin (E. cloacae and K. pneumoniae), cefuroxime (E. cloacae) and tetracycline (K. pneumoniae). Comparison with other Hong Kong data suggests that resistance rates to cefuroxime, ceftazidime, ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole exhibited by the oral isolates are generally lower than in enterobacters and Klebsiella spp. isolated from urine, skin and soft tissues in Hong Kong populations.

A 4-year longitudinal study of the oral prevalence of enteric gram-negative rods and yeasts in Chinese children.

A 4-year longitudinal study of the oral prevalence of enteric gram-negative rods and yeasts in 116 Chinese primary school children in Hong Kong was conducted. The oral prevalence of enteric gram-negative rods for each consecutive year was 25.3%, 37.0%, 24.0% and 25.8% respectively, with a weighted mean of 27.9%. Enterobacteriaceae, which comprised 57% of all enteric gram-negative rods, were more common in children with no caries experience. The oral prevalence of yeasts for each consecutive year was 7.7%, 12.0%, 14.4% and 15.5% respectively, with a weighted mean of 12.5%. Candida albicans comprised 84% of all yeasts isolated. Oral yeast carriage was significantly associated with caries prevalence. While the oral prevalence of enteric gram-negative rods in primary school children in Hong Kong may be higher than in other parts of the world, repeated isolation of either enteric gram-negative rods or Candida spp. from individual children over the 4-year study period was rare, suggesting that carriage of these organisms is transient.

The influence of incubation conditions on the adherence of oral Enterobacteriaceae to HeLa cells.

The adherence of 12 oral isolates and 4 type strains of Enterobacteriaceae (equally representing Enterobacter cloacae, Klebsiella pneumoniae, Escherichia coli and Citrobacter freundii) to HeLa cell monolayers following five different incubation conditions (sucrose, D-mannose, serum, MEM and Candida albicans GDH 1957) was investigated. Incubation with sucrose and D-mannose resulted in the greatest and least adherence, respectively. The presence of preadherent C. albicans GDH 1957 on the HeLa cells tended to enhance the adherence of certain strains of E. cloacae and C. freundii, but had no overall impact on Enterobacteriaceae adherence. While heterogeneity of behaviour existed between strains within species, E. cloacae was the most, and K. pneumoniae the least, adherent species irrespective of incubation conditions. Haemagglutination assays indicated the presence of mannose-resistant type 1 fimbriae associated with all Enterobacteriaceae. In clinical terms, the variations in adherence properties observed in vitro may contribute to an understanding of the different prevalence rates of oral Enterobacteriaceae reported in the literature.

The oral prevalence of aerobic and facultatively anaerobic gram-negative rods and yeasts in semi-recluse human vegetarians.

Limited data exist on the oral ecology of vegetarians. Hence the dental and periodontal status, and the oral prevalence of aerobic and facultatively anaerobic Gram-negative rods (AGNR) and yeasts, were studied in 36 semi-recluse, vegetarian, Buddhist monks and nuns in Hong Kong. The oral prevalence of AGNR and yeasts was 61.1% and 33.3%. There was no correlation between the prevalence of AGNR and/or yeasts and the incidence of carious or filled teeth and the health status of the periodontium. Rather, the results of this study combined with those of previous studies suggest that increasing age and the consumption of food prepared in communal kitchens might be more important contributory factors in the oral prevalence of AGNR than the nature of the diet itself or the health of the dentition and periodontium.

The adherence of oral isolates of Enterobacteriaceae to HeLa cells. An in vitro method using image analysis.

An in vitro model and an image analysis were designed to improve on existing quantification methods in the assessment of the adherence of Enterobacteriaceae to human epithelial cell monolayers. Adherence to HeLa cell monolayers of three oral isolates and one type strain from each of four species of Enterobacteriaceae over two incubation time periods was examined. Correction for actual cell area and a cube root transformation of the data to stabilize variance were applied. While behaviour varied between strains within species, E. cloacae was the most, and K. pneumoniae the least, adherent species irrespective of the incubation period. Increasing the incubation period from 30 min to 60 min resulted in greater adherence for E. cloacae, E. coli, and C. freundii, but not K. pneumoniae strains. The method permits the reliable measurement and valid analysis of the adherence of Enterobacteriaceae to cultured epithelial cell monolayers.

The oral prevalence of aerobic and facultatively anaerobic gram-negative rods and yeasts in Hong Kong Chinese.

Saline oral rinse samples were obtained from 300 community-dwelling Hong Kong Chinese attending an outpatient dental clinic to determine the oral prevalence of aerobic and facultatively anaerobic Gram-negative rods (AGNR) and yeasts. The oral prevalence of AGNR was 41.7%. Enterobacteriaceae species comprised 73% of all AGNR isolated, with an overall prevalence of 32%. There was no difference in prevalence between females (n = 190) and males (n = 110). Morning samples (n = 154) yielded a significantly higher prevalence of AGNR (54.5%) and Enterobacteriaceae (42.2%) than afternoon samples (n = 146) (28.1 and 21.2%, respectively; p < 0.01, p < 0.01). Subjects over 50 yr had a significantly higher prevalence of AGNR than those aged 30-49 yr (p < 0.01). The most commonly isolated AGNR species were Enterobacter cloacae and Klebsiella pneumoniae pneumoniae. The oral prevalence of yeasts was 24%, with Candida albicans forming 77% of all yeasts isolated. Subjects taking medication (n = 38) or wearing dentures (n = 38) had a significantly higher oral yeast prevalence of 36.8 (p < 0.05) and 44.7% (p < 0.01), respectively. Yeast prevalence was significantly higher in subjects over 50 yr than those aged 30-49 yr (p < 0.05) and 15-29 yr (p < 0.05). Comparisons with previous studies suggest that the oral prevalence of AGNR in Chinese may be higher in Hong Kong than in other parts of the world.

Oral and oropharyngeal prevalence of Enterobacteriaceae in humans: a review.

Members of the Enterobacteriaceae family are widely distributed in nature and exhibit substantial diversity in ecology, host range and pathogenic potential for man. While wide discrepancies in methodology exist between epidemiological studies, the available data indicate an increased prevalence of oral and/or oropharyngeal Enterobacteriaceae carriage in patients with illnesses of varying severity compared with healthy subjects. This paper reviews the prevalence of Enterobacteriaceae in the oral and oropharyngeal region of healthy human subjects and those affected by different disease entities, and discusses the complexities associated with collating and interpreting such data. The effect of antimicrobials and antiseptics on oral and oropharyngeal Enterobacteriaceae has also been reviewed, while highlighting the gaps in knowledge and future research directions.