Meta-QTLs, ortho-meta QTLs and related candidate genes for yield and its component traits under water stress in wheat (Triticum aestivum L.).

Meta-QTLs (MQTLs), ortho-MQTLs, and related candidate genes (CGs) for yield and its seven component traits evaluated under water deficit conditions were identified in wheat. For this purpose, a high density consensus map and 318 known QTLs were used for identification of 56 MQTLs. Confidence intervals (CIs) of the MQTLs were narrower (0.7-21 cM; mean = 5.95 cM) than the CIs of the known QTLs (0.4-66.6 cM; mean = 12.72 cM). Forty-seven MQTLs were co-located with marker trait associations reported in previous genome-wide association studies. Nine selected MQTLs were declared as 'breeders MQTLs' for use in marker-assisted breeding (MAB). Utilizing known MQTLs and synteny/collinearity among wheat, rice and maize, 12 ortho-MQTLs were also identified. A total of 1497 CGs underlying MQTLs were also identified, which were subjected to in-silico expression analysis, leading to identification of 64 differentially expressed CGs (DECGs) under normal and water deficit conditions. These DECGs encoded a variety of proteins, including the following: zinc finger, cytochrome P450, AP2/ERF domain-containing proteins, plant peroxidase, glycosyl transferase, glycoside hydrolase. The expression of 12 CGs at seedling stage (3 h stress) was validated using qRT-PCR in two wheat genotypes, namely Excalibur (drought tolerant) and PBW343 (drought sensitive). Nine of the 12 CGs were up-regulated and three down-regulated in Excalibur. The results of the present study should prove useful for MAB, for fine mapping of promising MQTLs and for cloning of genes across the three cereals studied. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01301-z.

Meta-QTLs for multiple disease resistance involving three rusts in common wheat (Triticum aestivum L.).

KEY MESSAGE: In wheat, multiple disease resistance meta-QTLs (MDR-MQTLs) and underlying candidate genes for the three rusts were identified which may prove useful for development of resistant cultivars. Rust diseases in wheat are a major threat to global food security. Therefore, development of multiple disease-resistant cultivars (resistant to all three rusts) is a major goal in all wheat breeding programs worldwide. In the present study, meta-QTLs and candidate genes for multiple disease resistance (MDR) involving all three rusts were identified using 152 individual QTL mapping studies for resistance to leaf rust (LR), stem rust (SR), and yellow rust (YR). From these 152 studies, a total of 1,146 QTLs for resistance to three rusts were retrieved, which included 368 QTLs for LR, 291 QTLs for SR, and 487 QTLs for YR. Of these 1,146 QTLs, only 718 QTLs could be projected onto the consensus map saturated with 2, 34,619 markers. Meta-analysis of the projected QTLs resulted in the identification of 86 MQTLs, which included 71 MDR-MQTLs. Ten of these MDR-MQTLs were referred to as the 'Breeders' MQTLs'. Seventy-eight of the 86 MQTLs could also be anchored to the physical map of the wheat genome, and 54 MQTLs were validated by marker-trait associations identified during earlier genome-wide association studies. Twenty MQTLs (including 17 MDR-MQTLs) identified in the present study were co-localized with 44 known R genes. In silico expression analysis allowed identification of several differentially expressed candidate genes (DECGs) encoding proteins carrying different domains including the following: NBS-LRR, WRKY domains, F-box domains, sugar transporters, transferases, etc. The introgression of these MDR loci into high-yielding cultivars should prove useful for developing high yielding cultivars with resistance to all the three rusts.

Clinical Profile and Prevalence of H Pylori, Iga-Ttg, and Spectrum of Duodenal Abnormalities in Non Ulcer Dyspepsia.

The Rome IV criterion for a diagnosis of NUD is chronic or recurrent epigastric pain within the last 3 months and an onset of symptoms at least 6 months prior to presentation. The term functional Dyspepsia and idiopathic dyspepsia are often used as well. Symptoms include ulcer-like dyspepsia; gastroparetic-like (nausea, early satiety, and post-prandial pain), and undifferentiated. Pathogenesis of NUD is not completely known yet. Several mechanisms have been proposed to be responsible for these symptoms. Although there is strong evidence of an association between H. pylori infection and NUD, Celiac Disease and NUD. Being a tropical country, the prevalence of infections is parasitic cause. Dyspepsia is likely to be more in India. However, the present data from India as scares in literature. Hence the present study was planned to decipher the clinical profile, prevalence of H. pylori, IgA tTG, spectrum of duodenal biopsy abnormalities in NUD patients. MATERIAL: This Descriptive Observational study was carried out in the Gastro Enterology center in GOI research institute from August 2020 to March 2021. Initially, 200 dyspepsia patients were selected. 50 patients were excluded due to various reasons. Finally, 150 patients who met the Rome 4 criteria for NUD/Functional Dyspepsia were recruited. The inclusion criteria were patients above 18 years of age, dyspepsia for >/- 6 months, and no evidence of underlying malignancy, pan gastritis, previous gastric ulcers, and pancreatitis. The patients underwent routine blood investigations like haemogram and biochemistry, Rapid Urease Test (RUT), Upper Gastro-Intestinal Endoscopy, Duodenal Biopsy, and Serum IgA-tTG antibody. OBSERVATION: The mean age was 46.3 yrs. +/- 14.12 yrs, of which 49.3% were females and 50.70% were males. The prevalence of Epigastric Pain Syndrome (EPS) was found in 37.3%, Post Prandial Distress Syndrome (PDS) in 30.7%, and 32% had both EPS+PDS. 38% of the NUD patients were positive on Rapid Urease Test (RUT) suggesting H. pylori infection. 88.7% of NUD patients were IgA-tTG antibody negative and 11.3% serologically positive. The Duodenal biopsy was normal in 48% of patients, 21.3% had mild inflammation/duodenitis, 8% chronic duodenitis and 22.7% had various grades of Celiac Disease (as per Marsh Grading). These 22.7% showing evidence of Celiac Disease on histopathological examination showed Marsh Grade 1 in 12.7%, Grade-2 in 2%, Grade 3A in 6.7%, and Grade 3B in 1.3%. Only 17.6% of biopsy positive had IgA-tTG antibody positivity but only 4% of total cases were positive for both biopsy and IgA-tTG antibody (p-value 0.05). Eosinophilic infiltration in duodenum common in NUD patients. It was observed that 17.33% (26/150) NUD patients had duodenal eosinophilia. Further, look for the association of duodenal eosinophilia with various diseases. 33.33% (19/57) H. pylori patients had duodenal eosinophilia with p-value < 0.001. It was also observed that 7.52% (7/93) others like normal individual, Chronic duodenitis, mild inflammation/ duodenitis had Duodenal eosinophilia. CONCLUSION: The prevalence of H. pylori and IgA-tTG antibodies in non-ulcer dyspepsia patients was 38% and 11.3% respectively. The spectrum of Duodenum biopsy abnormalities in NUD patients included mild inflammation/ duodenitis, Chronic duodenitis, and Celiac Disease. 22.7% of NUD patients had various degrees of celiac disease morphology on D2 biopsy and only 17.6% of these biopsy positive patients were positive for IgA-tTG. Only 4% of total NUD patients were positive for both biopsy and IgA-tTG antibody labeled as Celiac Disease (CeD). There is a significant association between H. pylori and duodenal eosinophilia.

Helicobacter pylori infection in non-ulcer dyspepsia: A cross-sectional study.

Background: Helicobacter pylori infection has been known to be associated with dyspepsia for more than two decades; however, studies on this topic in India are limited. This study was carried out to estimate the Helicobacter pylori infection rates in non-ulcer dyspepsia. Methods: Helicobacter pylori infection rates detected by rapid urease test (RUT) positivity were analyzed in 235 patients presenting to a tertiary care center with dyspepsia having no evidence of peptic ulcer disease on esophagogastroduodenoscopy. Results: In this study, the prevalence of Helicobacter pylori infection diagnosed by the RUT was found to be 40.85%. Gender-based prevalence was found to be 40.14% and 41.93% for men and women, respectively. The highest prevalence was found in the age group of 30-39 years. The most common area of involvement was the isolated antrum of the stomach as seen in 93 patients. Conclusion: This study shows a modest RUT positivity rate for Helicobacter pylori infection with the commonest site of involvement being the antrum of the stomach. Further studies will be needed to assess the prevalence of Helicobacter pylori in the community to analyze the extent of infection.

Chemical and mechanical influence of root canal irrigation on biofilm removal from lateral morphological features of simulated root canals, dentine discs and dentinal tubules.

AIM: To investigate the anti-biofilm efficacy of irrigation using a simulated root canal model, the chemical effect of irrigants against biofilms grown on dentine discs and their impact on biofilm viscoelasticity, the efficacy of the irrigants in decontaminating infected dentinal tubules and the capacity of bacteria to regrow. METHODOLOGY: Biofilm removal, viscoelastic analysis of remaining biofilms and bacterial viability were evaluated using a simulated root canal model with lateral morphological features, dentine discs and a dentinal tubule model, respectively. Experiments were conducted using a two-phase irrigation protocol. Phase 1: a modified salt solution (RISA) and sodium hypochlorite (NaOCl) were used at a low flow rate to evaluate the chemical action of the irrigants. Ultrasonic activation (US) of a chemically inert solution (buffer) was used to evaluate the mechanical efficacy of irrigation. Phase 2: a final irrigation with buffer at a high flow rate was performed for all groups. Optical coherence tomography (OCT), low load compression testing (LLCT) and confocal scanning laser microscopy analysis were used in the different models. One-way analysis of variance (anova) was performed for the OCT and LLCT analysis, whilst Kruskal-Wallis and Wilcoxon ranked tests for the dentinal tubule model. RESULTS: US and high flow rate removed significantly more biofilm from the artificial lateral canal. For biofilm removal from the artificial isthmus, no significant differences were found between the groups. Within-group analysis revealed significant differences between the steps of the experiment, with the exception of NaOCl. For the dentine discs, no significant differences regarding biofilm removal and viscoelasticity were detected. In the dentinal tubule model, NaOCl exhibited the greatest anti-biofilm efficacy. CONCLUSIONS: The mechanical effect of irrigation is important for biofilm removal. An extra high flow irrigation rate resulted in greater biofilm removal than US in the artificial isthmus. The mechanical effect of US seemed to be more effective when the surface contact biofilm-irrigant was small. After the irrigation procedures, the remaining biofilm could survive after a 5-day period. RISA and NaOCl seemed to alter post-treatment remaining biofilms.

Biofilm removal from a simulated isthmus and lateral canal during syringe irrigation at various flow rates: a combined experimental and Computational Fluid Dynamics approach.

AIM: (i) To quantify biofilm removal from a simulated isthmus and a lateral canal in an artificial root canal system during syringe irrigation with NaOCl at different concentrations and delivered at various flow rates (ii) to examine whether biofilm removal is further improved by a final high-flow-rate rinse with an inert irrigant following irrigation with NaOCl. (iii) to simulate the irrigant flow in these areas using a computer model (iv) to examine whether the irrigant velocity calculated by the computer model is correlated to biofilm removal. METHODOLOGY: Ninety-six artificial root canals with either a simulated isthmus or lateral canal were used. A dual-species in vitro biofilm was formed in these areas using a Constant Depth Film Fermenter. NaOCl at various concentrations (2, 5 and 10%) or adhesion buffer (control) was delivered for 30 s by a syringe and an open-ended needle at 0.033, 0.083, or 0.166 mL s-1 or passively deposited in the main root canal (phase 1). All specimens were subsequently rinsed for 30 s with adhesion buffer at 0.166 mL s-1 (phase 2). The biofilm was scanned by Optical Coherence Tomography to determine the percentage of the remaining biofilm. Results were analysed by two 3-way mixed-design ANOVAs (α = 0.05). A Computational Fluid Dynamics model was used to simulate the irrigant flow inside the artificial root canal system. RESULTS: The flow rate during phase 1 and additional irrigation during phase 2 had a significant effect on the percentage of the remaining biofilm in the isthmus (P = 0.004 and P < 0.001). Additional irrigation during phase 2 also affected the remaining biofilm in the lateral canal significantly (P ≤ 0.007) but only when preceded by irrigation at medium or high flow rate during phase 1. The effect of NaOCl concentration was not significant (P > 0.05). Irrigant velocity in the isthmus and lateral canal increased with increasing flow rate and it was substantially correlated to biofilm removal from those areas. CONCLUSIONS: The irrigant flow rate affected biofilm removal in vitro more than NaOCl concentration. Irrigant velocity predicted by the computer model corresponded with the pattern of biofilm removal from the simulated isthmus and lateral canal.

Genome-wide analysis of H3K4me3 and H3K27me3 modifications due to Lr28 for leaf rust resistance in bread wheat (Triticum aestivum).

KEY MESSAGE: Present study revealed a complex relationship among histone H3 methylation (examined using H3K4/K27me3 marks), cytosine DNA methylation and differential gene expression during Lr28 mediated leaf rust resistance in wheat. During the present study, genome-wide histone modifications were examined in a pair of near isogenic lines (NILs) (with and without Lr28 in the background of cv. HD2329). The two histone marks used included H3K4me3 (an activation mark) and H3K27me3 (a repression mark). The results were compared with levels of expression (using RNA-seq) and DNA methylation (MeDIP) data obtained using the same pair of NILs. Some of the salient features of the present study include the following: (i) large scale differential binding sites (DBS) were available for only H3K4me3 in the susceptible cultivar, but for both H3K4me3 and H3K27me3 in its resistant NIL; (ii) DBSs for H3K27me3 mark were more abundant (> 80%) in intergenic regions, whereas DBSs for H3K4me3 were distributed in all genomic regions including exons, introns, intergenic, TTS (transcription termination sites) and promoters; (iii) fourteen (14) genes associated with DBSs showed co-localization for both the marks; (iv) only a small fraction (7% for H3K4me3 and 12% for H3K27me3) of genes associated with DBSs matched with the levels of gene expression inferred from RNA-seq data; (v) validation studies using qRT-PCR were conducted on 26 selected representative genes; results for only 11 genes could be validated. The proteins encoded by important genes involved in promoting infection included domains generally carried by R gene proteins such as Mlo like protein, protein kinases and purple acid phosphatase. Similarly, proteins encoded by genes involved in resistance included those carrying domains for lectin kinase, R gene, aspartyl protease, etc. Overall, the results suggest a very complex network of downstream genes that are expressed during compatible and incompatible interactions; some of the genes identified during the present study may be used in future validation studies involving RNAi/overexpression approaches.

Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue.

Chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are associated with changes in extracellular matrix (ECM) composition and abundance affecting the mechanical properties of the lung. This study aimed to generate ECM hydrogels from control, severe COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV], and fibrotic human lung tissue and evaluate whether their stiffness and viscoelastic properties were reflective of native tissue. For hydrogel generation, control, COPD GOLD IV, and fibrotic human lung tissues were decellularized, lyophilized, ground into powder, porcine pepsin solubilized, buffered with PBS, and gelled at 37°C. Rheological properties from tissues and hydrogels were assessed with a low-load compression tester measuring the stiffness and viscoelastic properties in terms of a generalized Maxwell model representing phases of viscoelastic relaxation. The ECM hydrogels had a greater stress relaxation than tissues. ECM hydrogels required three Maxwell elements with slightly faster relaxation times (τ) than that of native tissue, which required four elements. The relative importance (Ri) of the first Maxwell element contributed the most in ECM hydrogels, whereas for tissue the contribution was spread over all four elements. IPF tissue had a longer-lasting fourth element with a higher Ri than the other tissues, and IPF ECM hydrogels did require a fourth Maxwell element, in contrast to all other ECM hydrogels. This study shows that hydrogels composed of native human lung ECM can be generated. Stiffness of ECM hydrogels resembled that of whole tissue, while viscoelasticity differed.

Complex relationship between DNA methylation and gene expression due to Lr28 in wheat-leaf rust pathosystem.

Differential DNA methylation due to Lr28 was examined in susceptible (S) wheat cv. HD2329 and its resistant (R) near isogenic line (NIL) (HD2329+Lr28) using two approaches: methylation sensitive amplified polymorphism (MSAP) and methylated DNA immunoprecipitation (MeDIP). S/R lines each had a large number of hypomethylated genes and relatively fewer hypermethylated genes at 96 hai (hours after inoculation) relative to 0 hbi (hours before inoculation), suggesting activation of many genes during the passage of time (96 hai), although identity of genes may differ in S and R lines. When R NIL was compared with S cultivar, there were many hypermethylated and fewer hypomethylated genes in R NIL relative to S cultivar, suggesting that many genes that are active in S cultivar are silenced in R NIL, both at 0 hbi and at 96 hai. Level of methylation was generally abundant in intergenic regions followed by that in promoters, transcription termination sites (TTSs) and exons/introns. Hypermethylation in promoter and gene body regions was not always associated with inhibition of gene expression and vice-versa, indicating that more than one regulatory mechanisms may control the expression of genes due to pathogen attack in presence and absence of Lr28. MSAP analysis also showed abundance of mCG methylation in S cultivar and that of mCCG methylation in R NIL (at 96 hai), suggesting differences in methylation context in NILs with and without Lr28. The results of the present study improved our understanding of the epigenetic control of leaf rust resistance in wheat.

The influence of time and irrigant refreshment on biofilm removal from lateral morphological features of simulated root canals.

AIM: To evaluate the effect of irrigant refreshment and exposure time of a 2% sodium hypochlorite solution (NaOCl) on biofilm removal from simulated lateral root canal spaces using two different flow rates. METHODOLOGY: A dual-species biofilm was formed by a Constant Depth Film Fermenter (CDFF) for 96 h in plug inserts with anatomical features resembling an isthmus or lateral canal-like structures. The inserts were placed in a root canal model facing the main canal. NaOCl 2% and demineralized water (control group) were used as irrigant solutions. Both substances were applied at a flow rate of 0.05 and 0.1 mL s-1 . The samples were divided into three groups with zero, one or two refreshments in a total exposure time of 15 min. A three-way analysis of variance (anova) was performed to investigate the interaction amongst the independent variables and the effect of consecutive irrigant refreshment on percentage of biofilm removal. A Tukey post hoc test was used to evaluate the effect of each independent variable on percentage biofilm removal in the absence of statistically significant interactions. RESULTS: For the lateral canal, NaOCl removed significantly more biofilm irrespective of the number of refreshments and exposure time (P = 0.005). There was no significant effect in biofilm removal between the consecutive irrigant refreshments measured in the same biofilm. For the isthmus, NaOCl removed significantly more biofilm irrespective of the number of refreshments and exposure time; both NaOCl and a high flow rate removed significantly more biofilm when the exposure time was analysed (P = 0.018 and P = 0.029, respectively). Evaluating the effect of consecutive irrigant refreshment on the same biofilm, 2% NaOCl, 0.1 mL s-1 flow rate and one or two refreshments removed significant more biofilm (P = 0.04, 0.034 and 0.003, <0.001, respectively). CONCLUSIONS: In this model, refreshment did not improve biofilm removal from simulated lateral root canal spaces. NaOCl removed more biofilm from the lateral canal- and isthmus-like structure. A higher flow rate removed significantly more biofilm from the isthmus-like structure. There was always remaining biofilm left after the irrigation procedures.

Brucellosis in India: results of a collaborative workshop to define One Health priorities.

Brucellosis is an important zoonosis worldwide. In livestock, it frequently causes chronic disease with reproductive failures that contribute to production losses, and in humans, it causes an often-chronic febrile illness that is frequently underdiagnosed in many low- and middle-income countries, including India. India has one of the largest ruminant populations in the world, and brucellosis is endemic in the country in both humans and animals. In November 2017, the International Livestock Research Institute invited experts from government, national research institutes, universities, and different international organizations to a one-day meeting to set priorities towards a "One Health" control strategy for brucellosis in India. Using a risk prioritization exercise followed by discussions, the meeting agreed on the following priorities: collaboration (transboundary and transdisciplinary); collection of more epidemiological evidence in humans, cattle, and in small ruminants (which have been neglected in past research); Economic impact studies, including cost effectiveness of control programmes; livestock vaccination, including national facilities for securing vaccines for the cattle population; management of infected animals (with the ban on bovine slaughter, alternatives such as sanctuaries must be explored); laboratory capacities and diagnostics (quality must be assured and better rapid tests developed); and increased awareness, making farmers, health workers, and the general public more aware of risks of brucellosis and zoonoses in general. Overall, the meeting participants agreed that brucellosis control will be challenging in India, but with collaboration to address the priority areas listed here, it could be possible.

Meta-QTL analysis and identification of candidate genes for drought tolerance in bread wheat (Triticum aestivum L.).

Meta-QTL (MQTL) analysis for drought tolerance was undertaken in bread wheat to identify consensus and robust MQTLs using 340 known QTLs from 11 earlier studies; 13 MQTLs located on 6 chromosomes (1D, 3B, 5A, 6D, 7A and 7D) were identified, with maximum of 4 MQTLs on chromosome 5A. Mean confidence intervals for MQTLs were much narrower (mean, 6.01 cM; range 2.07-19.46 cM), relative to those in original QTLs (mean, 13.6 cM; range, 1.0-119.1 cM). Two MQTLs, namely MQTL4 and MQTL12, were major MQTLs with potential for use in marker-assisting breeding. As many as 228 candidate genes (CGs) were also identified using 6 of the 13 MQTLs. In-silico expression analysis of these 228 CGs allowed identification of 14 important CGs, with + 3 to - 8 fold change in expression under drought (relative to normal conditions) in a tolerant cv. named TAM107. These CGs encoded proteins belonging to the following families: NAD-dependent epimerase/dehydratase, protein kinase, NAD(P)-binding domain protein, heat shock protein 70 (Hsp70), glycosyltransferase 2-like, etc. Important MQTLs and CGs identified in the present study should prove useful for future molecular breeding and for the study of molecular basis of drought tolerance in cereals in general and wheat in particular.

H3K4/K9 acetylation and Lr28-mediated expression of six leaf rust responsive genes in wheat (Triticum aestivum).

Development of leaf rust-resistant cultivars is a priority during wheat breeding, since leaf rust causes major losses in yield. Resistance against leaf rust due to Lr genes is partly controlled by epigenetic modifications including histone acetylation that is known to respond to biotic/abiotic stresses. In the present study, enrichment of H3K4ac and H3K9ac in promoters of six defense responsive genes (N-acetyltransferase, WRKY 40, WRKY 70, ASR1, Peroxidase 12 and Sarcosine oxidase) was compared with their expression in a pair of near-isogenic lines (NILs) for the gene Lr28 following inoculation with leaf rust pathotype '77-5'; ChIP-qPCR was used for this purpose. The proximal and distal promoters of these genes contained a number of motifs that are known to respond to biotic stresses. The enrichment of two acetylation marks changed with passage of time; changes in expression of two of the six genes (N-acetyltransferase and peroxidase12), largely matched with changes in H3K4/H3K9 acetylation patterns of the two promoter regions. For example, enrichment of both the marks matched with higher expression of N-acetyltransferase gene in susceptible NIL and the deacetylation (H3K4ac) largely matched with reduced gene expression in resistant NIL. In peroxidase12, enrichment of H3K4ac and H3K9ac largely matched with higher expression in both the NILs. In the remaining four genes, changes in H3 acetylation did not always match with gene expression levels. This indicated complexity in the regulation of the expression of these remaining four genes, which may be controlled by other epigenetic/genetic regulatory mechanisms that need further analysis.

Further studies on sugar transporter (SWEET) genes in wheat (Triticum aestivum L.).

SWEET proteins represent one of the largest sugar transporter family in the plant kingdom and play crucial roles in plant development and stress responses. In the present study, a total of 108 TaSWEET genes distributed on all the 21 wheat chromosomes were identified using the latest whole genome sequence (as against 59 genes reported in an earlier report). These 108 genes included 14 of the 17 types reported in Arabidopsis and also included three novel types. Tandem duplications (22) and segmental duplications (5) played a significant role in the expansion of TaSWEET family. A number of cis-elements were also identified in the promoter regions of TaSWEET genes, indicating response of TaSWEET genes during development and also during biotic/abiotic stresses. The TaSWEET proteins carried 4-7 trans-membrane helices (TMHs) showing diversity in structure. Phylogenetic analysis using SWEET proteins of wheat and 8 other species gave four well-known clusters. Expression analysis involving both in silico and in planta indicated relatively higher expression of TaSWEET genes in water/heat sensitive and leaf rust resistant genotypes. The results provided insights into the functional role of TaSWEETs in biotic and abiotic stresses, which may further help in planning strategies to develop high yielding wheat varieties tolerant to environmental stresses.

Chemical efficacy of several NaOCl concentrations on biofilms of different architecture: new insights on NaOCl working mechanisms.

AIM: To investigate the anti-biofilm efficacy and working mechanism of several NaOCl concentrations on dual-species biofilms of different architecture as well as the changes induced on the architecture of the remaining biofilms. METHODOLOGY: Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were co-cultured under different growth conditions on saliva-coated hydroxyapatite discs. A constant-depth film fermenter (CDFF) was used to grow steady-state, four-day mature biofilms (dense architecture). Biofilms were grown under static conditions for 4 days within a confined space (less dense architecture). Twenty microlitres of buffer, 2-, 5-, and 10% NaOCl were applied statically on the biofilms for 60 s. Biofilm disruption and dissolution, as well as bubble formation, were evaluated with optical coherence tomography (OCT). The viscoelastic profile of the biofilms post-treatment was assessed with low load compression testing (LLCT). The bacteria/extracellular polysaccharide (EPS) content of the biofilms was examined through confocal laser scanning microscopy (CLSM). OCT, LLCT and CLSM data were analysed through one-way analysis of variance (ANOVA) and Tukey's HSD post-hoc test. Linear regression analysis was performed to test the correlation between bubble formation and NaOCl concentration. The level of significance was set at a < 0.05. RESULTS: The experimental hypothesis according to which enhanced biofilm disruption, dissolution and bubble formation were anticipated with increasing NaOCl concentration was generally confirmed in both biofilm types. Distinct differences between the two biofilm types were noted with regard to NaOCl anti-biofilm efficiency as well as the effect that the several NaOCl concentrations had on the viscoelasticity profile and the bacteria/EPS content. Along with the bubble generation patterns observed, these led to the formulation of a concentration and biofilm structure-dependent theory of biofilm removal. CONCLUSIONS: Biofilm architecture seems to be an additional determining factor of the penetration capacity of NaOCl, and consequently of its anti-biofilm efficiency.

Factors affecting the chemical efficacy of 2% sodium hypochlorite against oral steady-state dual-species biofilms: Exposure time and volume application.

AIM: To study the influence of time and volume of 2% sodium hypochlorite (NaOCl) on biofilm removal and to investigate the changes induced on the biofilm architecture. Steady-state, dual-species biofilms of standardized thickness and a realistic contact surface area between biofilms and NaOCl were used. METHODOLOGY: Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 biofilms were grown on saliva-coated hydroxyapatite discs within sample holders in the Constant Depth Film Fermenter (CDFF) for 96 h. Two per cent NaOCl was statically applied for three different time intervals (60, 120 and 300 s) and in two different volumes (20 and 40 µL) over the biofilm samples. The diffusion-driven effects of time and volume on biofilm disruption and dissolution were assessed with Optical Coherence Tomography (OCT). Structural changes of the biofilms treated with 2% NaOCl were studied with Confocal Laser Scanning Microscopy (CLSM) and Low Load Compression Testing (LLCT). A two-way analysis of variance (2-way anova) was performed, enabling the effect of each independent variable as well as their interaction on the outcome measures. RESULTS: Optical coherence tomography revealed that by increasing the exposure time and volume of 2% NaOCl, both biofilm disruption and dissolution significantly increased. Analysis of the interaction between the two independent variables revealed that by increasing the volume of 2% NaOCl, significant biofilm dissolution could be achieved in less time. Examination of the architecture of the remaining biofilms corroborated the EPS-lytic action of 2% NaOCl, especially when greater volumes were applied. The viscoelastic analysis of the 2% NaOCl-treated biofilms revealed that the preceding application of higher volumes could impact their subsequent removal. CONCLUSIONS: Time and volume of 2% NaOCl application should be taken into account for maximizing the anti-biofilm efficiency of the irrigant and devising targeted disinfecting regimes against remaining biofilms.

Chemical biofilm removal capacity of endodontic irrigants as a function of biofilm structure: optical coherence tomography, confocal microscopy and viscoelasticity determination as integrated assessment tools.

AIM: To investigate the influence of biofilm structure on the biofilm removal capacity of endodontic irrigants and to study changes in the architecture of the remaining biofilms. METHODOLOGY: Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were cocultured under different growth conditions on saliva-coated hydroxyapatite discs. A constant depth film fermenter (CDFF) was used to grow steady-state 4-day biofilms. Biofilms were grown under static conditions for 4 and 10 days within a confined space. Twenty microlitres of 2% NaOCl, 2% Chlorhexidine (CHX), 17% Ethylene-diamine-tetra-acetic acid (EDTA) and buffer were applied statically on the biofilms for 60 s. Biofilm removal was evaluated with optical coherence tomography (OCT). Post-treated biofilms were assessed via low load compression testing (LLCT) and Confocal laser scanning microscopy (CLSM). Optical coherence tomography data were analysed through a two-way analysis of variance (ANOVA). Low load compression testing and CLSM data were analysed through one-way ANOVA and Dunnett's post hoc test. The level of significance was set at a < 0.05. RESULTS: The initial biofilm structure affected the biofilm removal capacity of the irrigants. NaOCl demonstrated the greatest chemical efficacy against the biofilms and was significantly more effective on the static than the CDFF biofilms (P < 0.001). CHX was ineffective and caused a rearrangement of the biofilm structure. Ethylene-diamine-tetra-acetic acid exhibited a distinct removal effect only on the CDFF biofilms. Biofilm age influenced the structure of the remaining biofilms. The 4-day grown remaining biofilms had a significantly different viscoelastic pattern compared to the respective 10-day grown biofilms (P ≤ 0.01), especially in the NaOCl-treated group. Confocal laser scanning microscopy analysis confirmed the CHX-induced biofilm structural rearrangement. CONCLUSIONS: Biofilm structure is an influential factor on the chemical efficacy of endodontic irrigants. Optical coherence tomography allows biofilm removal characteristics to be studied. NaOCl should remain the primary irrigant. Ethylene-diamine-tetra-acetic acid was effective against cell-rich/EPS-poor biofilms. Chlorhexidine did not remove biofilm, but rather rearranged its structure.

Correlation of In Vitro Susceptibility Based on MICs and Squalene Epoxidase Mutations with Clinical Response to Terbinafine in Patients with Tinea Corporis/Cruris.

Recalcitrant dermatophytoses are on the rise in India. High MICs of terbinafine (TRB) and squalene epoxidase (SQLE) gene mutations conferring resistance in Trichophyton spp. have been recently documented. However, studies correlating laboratory data with clinical response to TRB in tinea corporis/cruris are lacking. For this study, we investigated the clinicomycological profile of 85 tinea corporis/cruris patients and performed antifungal susceptibility testing by CLSI microbroth dilution and SQLE mutation analysis of the isolates obtained and correlated these with the responses to TRB. Patients confirmed by potassium hydroxide (KOH) mounting of skin scrapings were started on TRB at 250 mg once a day (OD). If >50% clinical clearance was achieved by 3 weeks, the same dose was continued (group 1). If response was <50%, the dose was increased to 250 mg twice a day (BD) (group 2). If the response still remained below 50% after 3 weeks of BD, the patients were treated with itraconazole (ITR; group 3). Overall, skin scrapings from 64 (75.3%) patients yielded growth on culture. Strikingly, all isolates were confirmed to be Trichophyton interdigitale isolates by internal transcribed spacer (ITS) sequencing. Thirty-nine (61%) of the isolates had TRB MICs of ≥1 µg/ml. Complete follow-up data were available for 30 culture-positive patients. A highly significant difference in modal MICs to TRB among the three treatment response groups was noted (P = 0.009). Interestingly, 8 of the 9 patients in group 3 harbored isolates exhibiting elevated TRB MICs (8 to 32 µg/ml) and SQLE mutations. The odds of achieving cure with TRB MIC < 1 µg/ml strains were 2.5 times the odds of achieving cure with the strain exhibiting MIC ≥1 µg/ml.

Complete cure of Fusarium solani sp. complex onychomycosis with Qs NdYAG treatment.

The incidence of non dermatophytic mould (NDM) onychomycosis (OM) has been steadily increasing Fusarium spp is the most common cause of NDM OM in most geographical locations. Fusarium spp and other NDMs are largely resistant to commonly used anti-fungals. The successful use of laser and light based devices has been demonstrated in dermatophytic OM, but there is no previous report of their successful use in any NDM OM. We describe a patient with OM caused by Fusarium solani spp, who was clinically (with a normal appearing nail) and mycologically (with negative microscopy and culture on repeated samples) cured of her infection following treatment with 2 sessions of Qs NdYAG (532nm and 1064nm) given 1 month apart.

A prospective cohort study assessing the appearance of retrieved aesthetic orthodontic archwires.

OBJECTIVES: To investigate the appearance of three esthetic nickel titanium (NiTi) wires after 6 weeks of intra-oral cycling and to determine the association between objective and subjective measures of esthetics. SETTING AND SAMPLE POPULATION: A prospective cohort study was undertaken involving participants undergoing upper fixed orthodontic appliance treatment with ceramic brackets. MATERIALS AND METHODS: Fifty participants were assigned to one of three groups of NiTi esthetic wires (American Orthodontics Ever White™, Forestadent Biocosmetic™ and GAC High Aesthetic™), with wires retrieved after 6 weeks in situ. Participants completed a bespoke questionnaire exploring perceptions of wire esthetics. Objective measurement of coating loss was undertaken using a custom arch wire jig. RESULTS: American Orthodontics Ever White™ had the greatest mean coating loss (50.7%) followed by Forestadent Biocosmetic™ (6%), with GAC High Aesthetic TM undergoing minimal loss (0.07%) (P < .001). The majority of coating loss with the American Orthodontics Ever White™ wires arose in the anterior region while Forestadent Biocosmetic™ wires and GAC High Aesthetic™ wires exhibited coating loss posteriorly (P < .001). These findings were reflected in the subjective assessment with a negative correlation found between coating loss and final Visual Analogue Scale scores (P < .001). CONCLUSIONS: Considerable esthetic variation between arch wires following 6 weeks of intraoral cycling was identified in this prospective cohort study. Intraoral cycling has a negative impact on participant perception of arch wire esthetics, and objective and subjective assessment of wire esthetics appears to be consistent.