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Optimization of Culture Media Parameters of Saccharomyces Cerevisiae PTCC 5209 for Maximized Invertase Enzyme Production
Full text linkBackground and Objective: Invertase enzyme or D-fructofuranosidfructohydrolase EC (3.2.1.26) is a member of the hydrolase family and responsible for the decomposition of sucrose into fructose and glucose. In recent years, extensive research has been carried out to increase the industrial production of invertase enzyme.
Material and Methods: This study focused on maximizing invertase production in Saccharomyces cerevisiae by optimizing culture media conditions. Elements of the culture media were investigated using monofunctional optimization method. Moreover, basic salt culture media, containing compounds such as Na₂HPO₄, K₂HPO₄, MgSO₄ and CaCl₂, were used. Then, growth curve of the yeast was plotted and results showed that the highest growth rate occurred within 38 h and the strongest enzyme activity occurred within 18 h. Optimizing the culture conditions showed that yeast provided the most activity with 1% sucrose as a carbon source, urea and 0.5% meat peptone as nitrogen sources, pH 5, 30 °C and shaking speed of 150 rpm. In this research, 3-l fermentor was used to assess yeast growth and enzyme activity at a larger scale.
Results and Conclusion: Results of this study showed that the highest OD value was included at 48 h and the highest enzyme activity was recorded at 28 and 96 h. The difference between the time of maximum growth and peak enzyme activity indicated the need of careful control of fermentation time to prevent unnecessary biomass accumulation. Therefore, further research in the field of advanced fermentation and optimization of yeast strains can help researchers achieve the highest secretion and enzyme activity.
Keywords: Invertase, Optimization, Saccharomyces cerevisiae PTCC 5209, Yeast fermentation
Introduction
Enzymes are macromolecules that play a critical role in enabling the chemical transformations needed for sustaining biological processes. Enzymes are classified based on the types of reactions they catalyze, reflecting their diverse catalytic activities [1]. Enzymes are used in several industrial processes, including baking, brewing, detergents, fermented products, pharmaceuticals, textiles and leather processing and include a crucial role in the pharmaceutical and diagnostic industries [2]. One of the enzymes that has been most discussed in recent years is the invertase enzyme. Invertase (D-fructofuranosid fructohydrolase, EC 3.2.1.26) catalyzes the hydrolysis of the α-1,4-glycosidic bonds between D-glucose and D-fructose in sucrose and transfers the αβ-D-O-fructofuranoside residue to an acceptor substrate [3]. Thus, invertase functions under high sucrose concentrations showing transferase activity. This dual characteristic classifies it within the group of transferases, referred to as fructosyltransferases (EC 2.4.1.9) [4]. In addition, invertase can hydrolyze other oligosaccharides, including kestose, raffinose and stachyose [5]. Nowadays, invertase is widely used for commercial purposes in various industries such as foods, beverages, pharmaceuticals and biosensors. It facilitates the conversion of sucrose and linked glycosides into simple commercial carbohydrates. Saccharomyces sp. invertase is the most common commercial source, compared to others. Yeast invertase is a β-fructosidase, whereas the fungus produces an α-glucosidase type of invertase. These two types of invertase include various catalytic mechanisms. The β-fructosidase hydrolyzes the sucrose from the fructose end, while the α-glucosidase hydrolyzes sucrose from the glucose end. The two reactions yield a mixture named invert syrup, which consists of glucose and fructose. Due to the high sweetness of fructose, the invert syrup is much sweeter than sucrose. Fructose is more appropriate than glucose for diabetic patients and enhances iron absorption in children [6]. The generally recognized as safe (GRAS) S. cerevisiae is a preferred protein-production host due to its well-understood genetics, collection of molecular biology tools that enable precise strain engineering and significant tolerance to industrial and chemical stresses [7]. First, invert sugar was produced using chemical method by the hydrolysis of sucrose with acid. Before identification of the invertase enzyme this method was highly used; however, acid hydrolysis of sucrose includes several disadvantages such as byproduct generation and low efficiency, limiting its industrial uses [8]. Invertase is a glycoprotein rich in mannose residues that belongs to the glycoside hydrolase (GH) family and consists of 370 enzymes [9]. Various isoforms with distinct characteristics of invertase are located in various parts of the cell and produced in intracellular and extracellular forms [10]. The major strain for the production of the invertase enzyme for the industries is S. cerevisiae [11]. In addition to its ability to catalyze and hydrolyze several sugars, invertase is capable of degrading numerous chemical compounds such as rhamnose and stachyose. As the first known protein in the role of biological catalysts, this enzyme has formed one of the most fundamental principles in enzymology. This characteristic has led to suggest invertase as a basis for the development of various models used in the study of enzyme reaction kinetics [12]. Previous studies' major focus was on conventional yeast strains and standard fermentation conditions, focusing primarily on basic production and biochemical characterization [3, 4]. Optimization of culture conditions, particularly nitrogen sources, has been verified as effective in enhancing enzyme yield [13]. The goal of this research was to enhance invertase activity. Invertase production and activity highly depend on the microbial strain, culture media and environmental conditions. However, systematic assessment of S. cerevisiae PTCC 5209 with optimized nitrogen sources is limited. This study demonstrated that combining this strain with two nitrogen sources enhanced the enzyme yield, while Amicon ultrafiltration efficiently concentrated the enzyme. The novelty of this study was linked to the combined approach of strain selection and nutritional optimization, including use of a combination of nitrogen sources, to maximize invertase production under controlled culture conditions.
Materials and Methods
2.1. Materials
Chemicals and mineral salts in this study included carbon sources of molasses (Brix 80, Jahan Alcohol, Iran) and sucrose (Merck, Germany); nitrogen sources of yeast extract (Leiber, Germany), meat peptone (Sigma-Aldrich, Germany), urea (pharmaceutical grade; Behansar, Iran) and diammonium phosphate (Merck, Germany); mineral salts of calcium chloride (Merck, Germany), magnesium sulfate (Merck, Germany), disodium hydrogen phosphate (Merck-DNA Biotech, Germany), dipotassium hydrogen phosphate (Merck, Germany), sodium potassium tartrate (Merck, Germany) and sodium acetate trihydrate (Merck, Germany); agar (Ibresco, Germany); sodium hydroxide (Merck, Germany); and glucose ( Merck, Germany).
2.2. Microorganism
The yeast strain of S. cerevisiae PTCC 5209 was provided by the Persian Type Culture Collection (PTCC, Iran). The strain was cultivated in yeast peptone dextrose adenine (YPDA) media (pH 5). For further experiments, glycerol stocks and slants were prepared.
2.3. Media
For enzyme production, the optimized culture media contained Na₂HPO₄ (2.5 g l-1), K₂HPO₄ (2.5 g l-1), meat peptone (10 g l-1), MgSO₄ (0.05 M) and CaCl₂ (0.01 M). The primary pH of the media was adjusted to 5.0 before sterilization.
2.4. Invertase assay
The culture media were centrifuged at 9,000 rpm for 20 min at 4 °C and the supernatant was collected as the crude enzyme source for the invertase assay. Invertase activity was investigated by measuring the quantity of reducing sugars released from sucrose using DNS method according to Miller [14]. The reaction mixture contained 0.4 ml of 1% (w/v) sucrose as substrate, 1.2 ml of 0.1 M acetate buffer (pH 5.0) and 0.4 ml of the crude enzyme supernatant. The mixture was incubated at room temperature (RM) for 30 min. After incubation, 0.25 ml of the reaction mixture was added to 1 ml of DNS reagent to terminate the reaction and the tubes were boiled for 10 min using water bath. After cooling to RM, the absorbance was measured at 540 nm using UV-vis spectrophotometer. One unit of invertase activity was reported as the quantity of enzyme needed to release 1 µmol of glucose per minute under the assay conditions.
2.5. Invertase activity calculation
Enzyme activity (mol min-1 ml-1) or (U ml-1) =
2.6. Carbon source optimization
Two carbon sources were assessed to investigate the optimal substrate for invertase production, including molasses with a Brix of 80 and sucrose. The culture media were supplemented with either 1% (v/v) molasses or 1% (w/v) sucrose and the pH was adjusted to 5.0. Each 250-ml flask containing 100 ml of the media was inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator.
2.7. Sucrose concentration optimization
The culture media were prepared with various concentrations of sucrose ranging from 1 to 4% (w/v) to optimize invertase production [15]. With pH 5 in 250-ml flasks, culture media were inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator.
2.8. Nitrogen source optimization
Four nitrogen sources were selected to investigate the best nitrogen source for invertase activity. These nitrogen sources were urea, yeast extract, meat peptone [16] and diammonium phosphate (DAP). The culture media were supplemented with 1% (w/v) of each nitrogen source and pH was adjusted to 5.0. Each 250-ml flask containing 100 ml of the media was inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator.
2.9. Combination nitrogen source optimization
To enhance invertase production, various combinations of nitrogen sources were assessed, including 0.5% yeast extract and 0.5% meat peptone, 0.5% urea and 0.5% meat peptone, 0.5% urea and 0.5% yeast extract, and 0.5% yeast extract and 0.5% diammonium phosphate. Each combination was prepared in 100 ml of the culture media (pH adjusted to 5.0) using 250-ml flasks, inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker.
2.10. Optimization pH
Briefly, 100 ml of the production media were distributed into each 250-ml flask and pH was adjusted to 3, 4, 5, 6, 7 and 8 [17]. These were inoculated with S. cerevisiae and incubated at 30 °C for 24 h at 150 rpm using shaker incubator. Then, the supernatant was used for enzyme assay and investigation of invertase activity.
2.11. Acetate buffer pH optimization
To investigate that at what pH the enzyme was most active, acetate buffer was prepared at various pH levels between 4 and 7 and enzyme activity was assessed using enzyme assay with the acetate buffer at various pH levels.
2.12. Shaker incubator rpm optimization
To investigate the effect of the rpm of the shaker incubator on enzyme activity, the culture media containing microorganisms were incubated at 30 °C for 24 h at 150 and 160 rpm after inoculation, and enzyme activity was assessed, as described in the previous steps.
2.13. Shaker incubator temperature optimization
To investigate the Optimal shaker temperature to achieve higher enzyme activity, the culture media containing microorganisms were transferred into shakers at 25, 30 and 32 °C and 150 rpm after inoculation. After 24 h, enzyme assay was used as previously described.
2.14. Growth curve and enzyme activity
Yeast growth and invertase activity were monitored over 48 h. Cell growth was assessed spectrophotometrically at 600 nm every 2 h and the growth curve was plotted. Enzyme activity was investigated approximately every 4 h using standard invertase assay with each measurement carried out in duplicate (n = 2). Results were reported as mean ±SD (standard deviation).
2.15. Effects of temperature on enzyme activity
The crude enzyme from the supernatant was incubated at 30 to 90 °C for 30 min using water bath [18]. Enzyme activity was then assessed using standard invertase assay and absorbance of the samples was read at 540 nm.
2.16. Effects of substrate concentration and kinetic parameters (Michaelis-Menten equation)
The effect of substrate concentration on invertase activity was assessed using sucrose at final concentrations ranging from 0 to 300 mM under standard assay conditions. The reactions were carried out at constant temperatures of 30 and 50 °C. Kinetic parameters, including the Michaelis-Menten constant (Km) and the maximum reaction rate (Vmax), were calculated using non-linear regression analysis and Michaelis-Menten model.
2.17. The SMF fermentor
To assess enzyme production at a larger scale, fermentation was carried out using 3-l laboratory-scale bioreactor (working volume of 2.0 l; Zist Fan Sanat Iranian, Iran). The bioreactor was equipped with a Rushton-type impeller, an air sparger and automatic control systems for temperature, pH and dissolved oxygen (DO). Fermentation was carried out at 30 °C with pH 5.0. The culture was agitated at 150 rpm and continuously aerated with sterile air, while DO was set at approximately 5 mg l-1 throughout the process to ensure adequate oxygen transfer and homogeneous mixing. To minimize the risk of contamination, no sampling was carried out during the first 24 h after inoculation. Then, samples were collected at regular intervals to investigate yeast growth (OD₆₀₀) and invertase activity and growth and enzyme activity profiles were recorded. For each sample, OD₆₀₀ was measured in duplicate (technical replicates) to monitor cell growth and invertase activity was assessed in duplicate. Data were present as mean ±SD and error bars in figures represented SD.
2.18. Amicon (ultra centrifugal filter of 10 kDa)
Briefly, 10-kDa Amicon ultrafiltration was used to concentrate the crude invertase enzyme and simultaneously remove low-molecular-weight (LMW) salts and other solutes in the supernatant. The enzyme solution was processed according to the manufacturer’s instructions and the concentrated enzyme was collected for activity assays.
2.19. Statistical analysis
All experiments, except fermentation studies, were carried out in duplicate as independent biological replicates (n = 2). Enzyme activity for each replicate was calculated individually and results were present as mean ±SD. For fermentation samples, only one fermentor was used; each sample was assessed in duplicate (technical replicates) and OD₆₀₀ and enzyme activity were reported as mean ±SD. Michaelis-Menten plots were generated using GraphPad Prism 8 (GraphPad, USA) and the mean values of duplicate measurements. Standard deviations were not included in the fitting analysis since averaged values were used for each substrate concentration.
Results and Discussion
3.1. Carbon source optimization
After 24 h of yeast incubation in culture media containing 1% molasses or 1% sucrose as carbon sources, enzyme activity was assessed to investigate the most suitable substrate. Sucrose was selected as the optimal carbon source since molasses interfered with the DNS assay due to its strong reaction with the reagent, making accurate quantification of enzyme activity unreliable.
3.2. Sucrose concentration optimization
After selecting sucrose as the preferred carbon source for enzyme production, various concentrations of sucrose (1–4% w/v) were assessed to investigate the optimal level for maximum enzyme activity. The results showed that the highest enzyme activity was achieved at 1% sucrose.
3.3. Nitrogen source optimization
Culture media containing yeast extract, meat peptone, urea and diammonium phosphate (DAP) were assessed as nitrogen sources. After 24 h of incubation, the enzyme assay results showed that the media containing pharmaceutical-grade urea as the nitrogen source included the highest invertase activity.
3.4. Combination of nitrogen sources
To enhance enzyme production, various combinations of nitrogen sources were assessed. After 24 h of incubation, the results indicated that the media containing a combination of meat peptone and pharmaceutical-grade urea included the highest invertase activity.
3.5. Media pH optimization
Media of various pH levels between 3 and 8 were prepared. After 24 h of yeast inoculation and enzyme assay, the culture media with pH of 5 included the highest enzyme activity. At pH 7 and pH 8, enzyme activity could not be assessed due to the precipitation in the culture media.
3.6. The pH acetate buffer
The buffer used in the assay included acetate buffer. To achieve and ensure the appropriate pH for the invertase enzyme, buffers with various pH levels between 3 and 7 were prepared. Then, the enzyme assay was carried out. The invertase enzyme showed the highest activity at pH 5.
3.7. The rpm optimization
To assess the effects of agitation speed on enzyme activity, shaking rates of 150 and 160 rpm were assessed. The results showed that the culture agitated at 150 rpm included the highest invertase activity.
3.8. Shaker incubator temperature optimization
After inoculation, the culture flasks were incubated at 25, 30 and 35 °C for 24 h. Enzyme assay results showed that the highest invertase activity was achieved at 30 °C.
3.9. Growth curve and enzyme activity curve
The growth and enzyme activity curves were monitored over 48 h by measuring cell growth (OD₆₀₀) every 2 h and invertase activity approximately every 4 h, except during the early stage of inoculation, when enzyme secretion did not begin. The growth curve showed two distinct logarithmic phases, likely due to the presence of dual nitrogen sources in the media. The highest enzyme activity was observed at 18 and 48 h, while the maximum optical density (OD₆₀₀) occurred at 38 h. These results indicate that the peak enzyme secretion did not coincide with the time of maximum yeast growth (biomass accumulation).
3.10. Fermentor
During the first 24 h of fermentation, no sampling was carried out to minimize the risk of contamination. After this time, samples were collected at regular intervals to monitor cell growth and enzyme activity. The highest optical density (OD₆₀₀) was recorded at 48 h, while the maximum invertase activity occurred at 28 and 96 h. Similar to the shake-flask experiments, the peak of enzyme activity did not match with the maximum cell growth, indicating that invertase secretion was not directly correlated with the biomass accumulation.
3.11. Effects of temperature
The crude enzyme was incubated at 30, 40, 60, 70 and 90 °C for 30 min, followed by activity measurement using standard assay. The highest invertase activity was observed within the temperature range of 40–60 °C, with similar activity levels at 40 and 60 °C, indicating that the enzyme preserved its substantial stability and catalytic efficiency across this range.
3.12. Michaelis-Menten equation
The Michaelis–Menten parameters of invertase were investigated through non-linear regression at 30 and 50 °C, as summarized in Table 1. At 30 °C, the Km and Vmax values were 63.35 mM and 0.8557 µmol min-1, respectively. At 50 °C, Km increased to 124.2 mM and Vmax to 2.283 µmol min-1. The higher Km at 50 °C indicated decreased affinity of invertase for sucrose at increased temperatures, suggesting that structural changes or increased flexibility of the enzyme at higher temperatures might affect substrate binding. The increase in Vmax at 50 °C showed that the catalytic rate could increase at higher temperatures; however, the overall efficiency was moderated by the decreased substrate affinity.
3.13. Amicon ultra centrifugal filter
In this study, Amicon 10-kDa filter was used to concentrate the enzyme. Before centrifugation, the volume in the tube was 3.5 ml and after centrifugation, the rest of volume in the Amicon filter was 1.4 ml; hence, this was 2.5 times further concentrated.
The results of this study showed that the yeast reached its highest growth rate within 38 h, while the best enzyme activity was seen within 18 h, highlighting the fact that enzyme secretion was not strictly coupled to growth, a phenomenon reported for S. cerevisiae MK, where the maximum invertase production occurred at 48 h while biomass increased up to 96 h [19]. These observations highlighted the importance of optimizing harvest time to achieve maximal enzyme yield. Such differences highlighted the importance of selecting the optimal harvest time to maximize enzyme activity. The present results, including that the invertase enzyme pH optimization was 5, were similar to those of Al-Saady’s study [16] but differed from Shankar's study [18], where maximum activity was observed at pH 6 for invertase from S. cerevisiae MTCC 170, suggesting that strain-specific variations in invertase isoforms or differences in post-translational modifications and culture conditions such as media composition, temperature and aeration might further affect the pH profile. In 2024, Dokuzparmak investigated the effect of pulp on the activity of the invertase enzyme in S. cerevisiae and the results showed that the invertase enzyme activity increased by 2.5 times. In the current study, a combination of 1% sucrose, 0.5% urea and meat peptone at pH 5 and 30 °C and 150 rpm yielded the highest enzyme activity, aligning with Dokuzparmak’s findings under comparable conditions [20]. However, it is noteworthy that the optimization was carried out using one-factor-at-a-time (OFAT) approach, which did not account for potential interactions between the factors. For example, the optimal sucrose concentration investigated by one nitrogen source may shift, when a various nitrogen source is used. Future studies can use factorial design or response surface methodology (RSM) to investigate such interactions and achieve f
Development of Specific Primers for Detection of Buffalo (Bubalus bubalis) Cytochrome b Gene in Processed Food Products
Full text linkBackground and Objective: Buffalo plays a role in providing animal protein in Indonesia. Their meat and skins are widely used as raw materials for processed foods. However, the high demand for buffalo products is not proportionate with the current supply; thus, creating chances for food fabricating. This practice is not only illegal but also causes serious risks to food safety and halal compliance. The detection of adulteration in food product can be carried out using polymerase chain reaction technique and specific primers that target buffalo DNA. This research aimed to develop primers that could accurately identify buffalo DNA.
Material and Methods: The primers were designed based on the sequence of the buffalo cytochrome b gene. The effectiveness of these primers in recognizing and amplifying buffalo DNA was assessed using polymerase chain reaction technique. Specificity assessments were carried out to assess the primer capability to detect buffalo DNA, with comparative controls including cattle, goat, chicken, pig, dog and mouse DNA. Sensitivity assessments were carried out to assess the minimum DNA concentration detectable by polymerase chain reaction. Then, the ability of these primers in identifying buffalo skin crackers was assessed against controls of cow skin crackers, goat skin crackers, and crispy chicken skin and pork skin crackers.
Results and Conclusion: The polymerase chain reaction results indicated that the Buffalo_5.1 primer pair (forward 5’-TTAGTACTATTCGCACCCGACCTC-3’ and reverse 5’-TCGTTGTTTGGATGTATGTAGCAG-3’) successfully amplified buffalo DNA specifically, with a detection limit of up to 10-3 ng μl-1 (assuming that the solution included a density of 1 g ml-1 equal to 10-⁷ % w/w), which could potentially increase to 10-5 ng μl-1 (10-⁷ % w/w) under optimal conditions. Furthermore, it was able to detect buffalo DNA in buffalo-skin cracker products even at low DNA purity levels. These results suggest that the Buffalo_5.1 primer includes the potential to serve as a reliable molecular marker in polymerase chain reaction-based food authentication studies.
Keywords: Buffalo, Food adulteration, Forward and reverse primers, PCR
Introduction
Indonesia, the fourth most populous country in the world, faces a high demand for animal protein, including buffalo meat. However, domestic production is insufficient to meet national needs. In 2023, buffalo meat production reached only 22,110 tons, while the total national demand for beef and buffalo meat was 680,019 tons and is projected to increase to 724,188 tons in 2024 (1,2). To overcome this problem, in 2023, Indonesia imported large quantities of buffalo products, nearly 100,000 tons, majorly from India (3). Other than serving as a protein source, buffalo commodities support the tourism sector through the production of local delicacies such as rambak crackers made from buffalo (Bubalus bubalis) skin. The increasing demand and limited supply, however, create opportunities for food fraud that threaten food safety and halal compliance. Reliable authentication is therefore essential. Molecular methods based on nucleic acid analysis, particularly polymerase chain reaction (PCR), are widely used for species identification because of their high sensitivity and the inherent stability of DNA molecules. In various research, the PCR technique targeting mitochondrial DNA has commonly been used for species identification in food products (4–7); however, specific molecular biomarkers for detecting adulteration in buffalo-derived and processed products are underdeveloped.
Mitochondrial DNA offers greater effectiveness than nuclear DNA because it exists in thousands of copies per cell, enabling reliable detection even from limited or degraded samples. It yields higher amplification success in extensively processed food products where DNA fragmentation is severe (5). The mitochondrial cytochrome b (cytb) gene has long been used as a molecular marker for species identification in meat authentication (8). The cytb gene shows a lower mutation rate compared to other genes within mitochondrial DNA loci (9). In previous research, the cytb gene sequence has been used as a basis for designing species-specific primers for dogs, which have proven effective in detecting dog meat contamination in meatball products (10), verifying that the leather garment material is from cowhide (11), as well as for halal authentication (4,12–17).
Although previous studies have developed cytb-based primers for buffalo DNA detection, most studies were designed for raw or minimally processed meats, with reported amplicon sizes ranging from approximately 655 to 106 bp (18, 19). Amplicon size critically affects detection performance in processed foods, where DNA degradation is extensive—larger fragments often fail to amplify, while overly short targets may decrease specificity. Therefore, current primers are inappropriate for highly processed non-meat buffalo derivatives such as buffalo skin crackers (rambak), which include severe thermal and chemical treatments.
Advanced molecular approaches such as real-time PCR and loop-mediated isothermal amplification (LAMP) have improved the sensitivity of buffalo DNA detection, achieving detection limits nearly 1% (20, 21). However, these techniques need costly reagents and specialized instrumentation, limiting their practicality for routine food inspection or field assessing. Despite these developments, a significant research gap persists that the current cytb-based primers are not optimized for detecting buffalo DNA in highly processed non-meat derivatives such as buffalo skin crackers (rambak) that include intense thermal and chemical treatments leading to severe DNA degradation. To address this limitation, the present study aimed to design and assess species-specific cytb primers with moderate amplicon lengths optimized for degraded DNA. This study provided a foundation for reliable authentication of processed buffalo products and contributed to the broader goal of establishing standardized molecular protocols for food authenticity verification and halal certification.
Materials and Methods
2.1. Primer Design Specific for Buffalo
The primer was designed based on the cytb coding sequence from the National Center for Biotechnology Information (NCBI) database
(https://www.ncbi.nlm.nih.gov/). The sequences were aligned with cytb nucleotide sequences from other species using BIOEDIT software to identify unique polymorphic sequences that are specific for the buffalo species. The selected polymorphic sequences were then used as references in the design of specific primers. The primer design process was facilitated by the Primer3Plus software (https://primer3plus.com/). Once appropriate primer candidates were identified, an in-silico assessment was carried out using basic local alignment search tool (BLAST), available on the NCBI website
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify that the primers specifically amplify buffalo DNA. Then, in-silico analysis was carried out using NetPrimer, available on the Premier Biosoft website
(https://www.premierbiosoft.com/netprimer/) to assess the melting temperature (Tm), GC percentage, GC clamp, potential secondary structure formation (hairpin, self-dimer, cross-dimer), repeats and runs. The primers were synthesized by Integrated DNA Technologies, Singapore.
2.2. Sample Preparation
The sample included buffalo meat, beef, goat, chicken, pork, dog, rat, buffalo skin crackers, beef skin crackers, goat skin crackers, crispy chicken skin and pork skin crackers from various stores. Each sample was packaged in plastic containers and stored in a freezer at -20 °C to preserve its freshness and prevent cross contaminations.
2.3. DNA Isolation using Chloroform-Isoamyl Alcohol (24:1) Method
Briefly, DNA isolation was carried out under aseptic condition using 20 mg of the sample. Then, 500 μl of salt-tris-EDTA (STE) buffer was added as the lysis buffer, with 40 µl of 10% SDS and 20 µl of proteinase-K at a concentration of 20 mg ml-1. The mixture was vortexed and incubated at 55 °C and 800 rpm overnight until the cell membranes were lysed using thermo shaker (Biosan TS-100, Latvia). Then, mixture was centrifuged (TOMY MDX-310, Japan) at 12,000 rpm for 10 min at 29 °C. The supernatant, which contained the crude DNA components, was collected at 500 μl and transferred to a fresh microtube. Then, 500 μl of chloroform-isoamyl alcohol solution (24:1) and 40 μl of 5 M NaCl were added to the supertnatant. The microtube was shaked until the solution was homogenized and then centrifuged at 12,000 rpm for 10 min at 29 °C. The pellet was discarded and 400 μl of the supernatant were transferred to a fresh microtube. The supernatant was mixed with 400 μl of chloroform-isoamyl alcohol (24:1) and shaked until the mixrure was homogenized. The mixture was centrifuged at 12,000 rpm for 10 min at 29 °C. The pellet was discarded and the supernatant was transferred to a fresh microtube. Then, 40 μl of 5 M NaCl and 800 μl of cold absolute ethanol were added to the supernatant, followed by incubation at -20 °C for 2.5 h to precipitate the DNA. The mixture was centrifuged at 12,000 rpm for 10 min at 4 °C to allow the DNA to settle into a pellet. The pellet was treated with 500 μl of 70% ethanol and recentrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was discarded and the pellet was dried at 55 °C for 30 min until the ethanol evaporated using thermomixer. Pellet was dissolved in 50 μl of TBE buffer at pH 7.6. This solution contained the DNA isolate from the sample, which could be analyzed for purity and concentration using nanodrop spectrophotometer. The concentration and purity of the DNA isolate were verifyed using Implen NanoPhotometer (Model NP 80, Germany) at 260/280 nm.
2.4. Reaction Components and PCR Program Settings
The PCR cocktails included 0.5 µl of forward and reverse primers (10 μM), 1 µl of 50 ng µl-1 DNA sample isolate, 5 µl of MyTaq HS red mix (Bioline BIO-25048, Germany) and 3 µl of nuclease-free water (Invitrogen AM9932, USA) (22). The amplification steps consisted of initial denaturation (95 °C for 60 s); followed by denaturation (95 °C for 15 s), annealing (63 °C for 15 s), elongation (72 °C for 10 s) and final elongation (72 °C for 60 s); as well as cooling (4 °C) (Bioline, 2014). The PCR instrument was PCR gradient thermocycler (SensoQuest, Germany). The PCR results were analyzed using gel electrophoresis system (Mupid-exU, Japan) with agarose gel concentration of 1.5% at 50 V for 55 min.
2.5. Primer Specificity Assay
The specificity assay was carried out using target DNA (buffalo) and non-target DNA (cattle, goats, chickens, pigs and rats) at a concentration of 50 ng µl-1. The assay was carried out in two repetitions. The specificity of the primers could be verifyed after analyzing the amplicon bands resulting from electrophoresis. A primer was considered specific if only the amplicon band from the target DNA was present; which in this research, context referred to the cytb gene fragment from the buffalo's mitochondria.
Primer Sensitivity Assay
The primer sensitivity assay was carried out involving a gradient of buffalo DNA concentrations, which included 100, 10, 1, 10-1, 10-2, 10-3, 10-4 and 10-5 ng μl-1. The sensitivity of the primers was assessed based on the lowest concentration of buffalo mitochondrial DNA that could be amplified. The primer sensitivity assay was carried out in three repetitions.
Primer Sampling Assay
The primer sampling assay was carried out using processed food products in the form of crackers made from animal skin. The positive sample consisted of buffalo skin crackers, while the negative samples included cattle skin crackers, goat skin crackers, crispy chicken skin and pig skin crackers. The positive control consisted of pig DNA with a concentration of 100 ng μl-1 and negative control consisted of the nuclease free water (Invitrogen AM9932, USA) were used in this step to assess the validity of the results. The primer sampling assay was carried out in two repetitions.
Results and Discussion
3.1. Cytochrome b (cytb) Primer Development
The primer was designed using in-silico analysis. The designed primer candidates have met the ideal criteria for all parameters, with the exception of self-dimer formation. The Buffalo_5.1 reverse primer included a ∆G value of -8.76 kcal mol-1. Primers with a ∆G value less than -6 kcal/mol were still used, despite the potential for secondary structure formation. This shortcoming was addressed through the optimization of the PCR procedure, as is commonly practiced in studies involving PCR techniques (23). Primer development based on in-silico analysis provides a predictive result but may differ from actual PCR conditions. In-silico analysis of primer specificity often cannot be used as a reference for identifying good primer specificity (24). Therefore, primer validation using the PCR process is essential to identify primers that have been developed to include good quality.
3.2. Primer Specificity Validation
Primer specificity was assessed via its ability to hybridize specifically with target DNA without producing DNA amplicon products from non-target species using PCR method. The PCR amplification product visualization showed that the Buffalo_5.1 primer pair specifically amplified buffalo DNA and did not amplify DNA from other samples (cattle, goats and mice). This demonstrated that the designed primer was specific and complementary to the buffalo DNA. The results showed consistency upon repetition. In the first and the second trials, the visible DNA amplicon band was a single band that corresponded to the target size of 216 bp (Figure 1).
The specificity assay results for the Buffalo_5.1 primer pair verify that the 5.1 primer pair show good performance for specificity and amplification pattern consistency. The specificity of this primer is critical as it ensures reliability when used for assessment the presence of adulteration or the authentication of a material. Primer specificity is key in PCR-based adulteration assessment as it assesss the validity, accuracy and credibility of detection results. Specific primers accurately detect the target DNA because they only bind to the DNA sequence of the target species/product. This prevents the amplification of unwanted DNA from other organisms. Furthermore, the presence of specific primers helps avoid the generation of false positive data, as non-specific primers may bind to similar DNA from other species, leading to results that indicate adulteration when it does not actually exist. The level of specificity of Buffalo_5.1 primer pair was similar to that of cytb-based assay using duplex PCR by (18) that differentiated buffalo against cattle, which produced amplicons of 249 bp (buffalo) and 655 bp (cow) (18). In degraded or processed samples, universal cytb primers that produce shorter fragments (~148 bp) have been shown further effective in amplifying damaged or fragmented DNA (30). Similarly, primers developed from the cytb gene sequence successfully identified unprocessed ruminant skin (31). and could clearly separate four species (buffalo, cattle, sheep and goat) with various cytb fragment sizes (106–308 bp) for each species using blood samples (19). Compared with these primers, the primers developed in this study (single target 216 bp, buffalo specific) will complement the detection capabilities of the current primers.
The high primer specificity enables the identification of complex mixed materials. Processed meat products typically undergo various processes that cause DNA fragmentation such as grinding, cooking and drying. If adulteration assessment uses primers with great specificity, even small targeted DNA fragments are still detected. The cytb gene has widely been used as a marker for identifying specific species across mammals, birds and insects. The authentication of cattle leather has successfully been achieved using cytb gene sequence (12). Halal authentication using this gene has been reported by several researchers (12–17,32,33).
3.3. Primer Sensitivity Assay
Sensitivity assessment was carried out to assess the lowest concentration of buffalo DNA that could be detected by Buffalo_5.1 primer during the PCR reaction. Based on the analysis of the DNA amplicon bands (Figure 2), variation in sensitivity levels between the two repetitions was detected.
From the three trials, two trials showed that the primer was able to detect buffalo DNA down to the lowest concentration of 10-3 ng µl-1. One trial, however, the sensitivity reached to 10-5 ng µl-1. The distinct results between the trials might be attributed to the quality of the DNA (34). The DNA degradation is one of the factors leading to the failure of DNA analysis using PCR (35). This issue can increase when the primer fails to bind to the severely degraded target DNA sequence. The DNA concentration and volume may vary between PCR reagent manufacturers. Referring to the manufacturer's recommendation, the recommended DNA template included 200 ng per reaction with a total volume of 50 µl. Furthermore, suspected EDTA residue from the extraction might cause primer inconsistency in amplifying DNA at concentrations of 10-3 to 10⁻⁵ ng µl-1 (36). Nevertheless, the Buffalo_5.1 primer designed in this research was quite reliable in detecting DNA at least until a concentration of 10-3 ng µl-1. Assuming that the solution included a density of 1 g ml-1, 10-3 ng µl-1 equal to 10-7 % w/w and the potential to reach 10-5 ng µl-1 equal to 10-9 % w/w under optimal conditions.
The sensitivity level of Buffalo_5.1 primer was still considered good when viewed from the perspective of its use for food fraud detection. In comparison, detection of adulteration using primers based on mitochondrial D-loop capable in detecting the contamination down to 1% and very faint/inconsistent results at 1% in autoclaved meat emulsion (37). Discrimation of beef and buffalo in Malaysian meat curry and burger products using primers developed based on mitochondrial genes showed sensitivity down to 1% meat in mixtures of 0/0.01 ng DNA (38). Other research which developed a detection method for meat fraud using multiplex PCR and 12S rRNA showed sensitivity of 10-1 ng µl-1 for pig species and 10-2 ng µl-1 for cattle, chicken and donkey species (39). Another study that developed a detection method for pig DNA using real-time PCR indicated that the smallest concentration of pig DNA that could be detected is 10-3 ng µl-1 (40). The article also explained that the sensitivity of real-time PCR was higher than that of conventional PCR. Identification of buffalo skin using universal cytb amplification and RFLP (RsaI enzyme) distinguished buffalo aganst cattle in skins down to ~10% buffalo skin in mixed hide samples (31). This finding strengthened that the Buffalo_5.1 primer included a good sensitivity result.
3.4. The Efectivity Assay of The Primer in Processed Food Products
Assessments were carried out to assess the reliability of the Buffalo_5.1 primer pair using PCR technique in detecting buffalo DNA in processed food products. The potential challenge during the DNA preparation from processed food products is the possibility of DNA degradation during the food processing. In this study, DNA was successfully isolated from several animal skin crackers which are commonly consumed in the community. Several samples were identified having protein contamination, which could be resolved by adding proteinase-K enzyme into the DNA sample.
From the agarose gel electrophoresis visualization result (Figure 3), it was evident that the PCR amplicon band was only present in the positive control and the buffalo skin cracker sample.
Samples of cow skin crackers, goat skin crackers, crispy chicken skin and pork crackers did not show detectable amplicon bands. The positive control included DNA isolated from fresh meat of buffallo, while the negative control included nuclease-free water. Positive amplification indicated that Buffalo_5.1 primer was still capable of recognizing buffalo DNA sequences in the buffalo skin cracker sample, despite the potential for DNA degradation during food preparation process.
The similar results from the repetitions verified that Buffalo_5.1 primer pair was not only specific but also demonstrated good performance in detecting the presence of buffalo DNA in processed food products derived from skin. The skin crackers have been boiled, dried and fried. Study of the effectivity of primers targeting the cytb was used to amplify DNA isolated from the skins of cattle, buffalo, goats and pigs (41) after putrefaction, heating or processing of mixed meats (42), beef curries, burger products under boiling, autoclaving, microwave cooking (37), minced meat, frozen rolls, boiled meat, meat balls, jerky which processed in various ways (43), but none of them involved drying followed by frying The succes of Buffalo_5.1 primers to amplify DNA from skin crackers demonstrated that this primers were capable in identifying bufallo DNA sequences even from products that have undergone drying and frying processes.
The Buffalo_5.1 primer developed in this study combined moderate amplicon size and high species specificity, enabling reliable amplification of severely degraded DNA from processed buffalo products. Compared with previously reported Cyt b assays, this achieved a superior detection limit (10⁻³ ng μl⁻¹) while maintaining simplicity and cost-effectiveness for routine authentication and halal verification. The skin cracker products in the market generally include similar shapes and physical appearances, whether they made from cow, buffalo or pig skin. This morphological similarity mak
The Effectiveness of Dialectical Temperament-Based Therapy on Marital Satisfaction, Intimacy, Commitment, and Reduction of Sexual Dysfunctions among Couples in District 12 of Tehran
Full text linkBackground and Aim: Recognizing the pivotal role of marital relationship quality in overall family health, this study aimed to assess the effectiveness of dialectical temperament-based therapy on marital satisfaction, intimacy, commitment, and reduction of sexual dysfunction among couples residing in District 12 of Tehran.
Materials and Methods: A quasi-experimental design was employed, consisting of pretest, posttest, and longitudinal follow-up stages. The sample consisted of ten couples who were selected through convenience sampling from Valiasr Health Center during the 2024–2025 period. Standardized questionnaires were administered at various stages to comprehensively evaluate the outcomes, including the ENRICH Marital Satisfaction Questionnaire, the Walker and Thompson Intimacy Scale, the Adams and Jones Commitment Scale, the Sexual Dysfunction Questionnaire, and the Mojahedi Temperament Questionnaire. The therapeutic intervention consisted of six structured sessions, combining elements of dialectical behavior therapy with traditional temperament theory, with content tailored to the unique temperament profiles of each couple.
Results: Following the intervention, marital satisfaction increased by 19.5%, intimacy by 23%, and commitment by 19%. In addition, a 22.9% decrease in sexual dysfunction was observed. These improvements remained stable across 3-, 6-, and 12-month follow-up assessments, evidencing the durability of therapeutic effects.
Conclusion: The findings deliver empirical support for the effectiveness of dialectical temperament-based therapy as an innovative, culturally attuned method for enhancing marital quality and addressing sexual health issues. The results highlight the importance of integrated, locally adapted psychological interventions in promoting family well-being
Structural Equation Modeling of the Relationship Between Dark Personality Traits and Subjective Well-Being in Women on the Verge of Divorce, with an Emphasis on the Mediating Role of Emotion Regulation
Full text linkBackground and Aim: The present study aimed to investigate the structural model of dark personality traits (Machiavellianism, narcissism, and psychopathy), interpersonal emotion regulation, and psychological well-being in married women experiencing thoughts of separation.
Materials and Methods: This descriptive-correlational study used a structural equation modeling (SEM) approach. The statistical population consisted of married women in Tehran who had thoughts of separation in 2024, from whom 188 individuals were selected using convenience sampling. Participants completed the Short Dark Triad (SD3), Interpersonal Emotion Regulation (IRI) questionnaire, and the Ryff Psychological Well-Being questionnaire. Data were analyzed using Pearson correlation coefficients and structural equation modeling with AMOS software.
Results: The results indicated that dimensions of dark personality traits had a significant adverse effect on the ability of interpersonal emotion regulation; in particular, Machiavellianism and narcissism directly weakened interpersonal emotion regulation and reduced psychological well-being. Although psychopathy did not have a direct effect on interpersonal emotion regulation, it exerted the most substantial negative impact on psychological well-being. The mediating role of interpersonal emotion regulation in the relationship between narcissism and psychological well-being was confirmed, whereas such mediation was not observed for Machiavellianism and psychopathy.
Conclusion: Dark personality traits—especially narcissism—lead to reduced psychological well-being in women on the verge of divorce, with interpersonal emotion regulation serving as a key mechanism in this relationship. It is recommended that couples therapy interventions focus on training interpersonal emotion regulation strategies and enhancing components of psychological well-being
Effects of Gradual Reintroduction of Visual Architectural Distractors on Sensory Profiles and Visual Attention in Children with Autism: an In-novation in Sensory Integration Therapy, a pilot study
Full text linkBackground: Objective measures such as eye tracking offer promise to quantify attention modulation. We report a controlled intervention in which visual distractors in the therapy room were removed and then systematically reintroduced, examining changes in sensory profiles and eye-tracking metrics. Methods: Five children with ADHD were enrolled in intervention group and 5 as controls. At baseline, all participants completed the Short Sensory Profile-2 (SSP-2) and an eye-tracking session to detect primary visual distractors. During a first 3-month, all distractors were removed in both groups, and standard therapy proceeded. After 3 months, all assessments were repeated. In the intervention group, over the next 3 months, architectural distractors were gradually reintroduced (one every 3 weeks) while the control group remained in the distraction-free environment. At the end of 6 months, SSP2and eye tracking were reassessed and compared. Results: In the intervention group, mean total SSP2score improved) from 130 ± 8 at baseline to 145 ± 10 after the first 3 months (p = 0.02), then declined modestly to 138 ± 9 after the second 3 months (p = 0.04). Eye-tracking metrics showed significant reductions in distractor engagement in the second period relative to the midpoint (p<0.03). The control group showed continued gradual improvement in SSP2 (128 ± 7 → 135 ± 9 → 140 ± 10) but no major change in distractor zone metrics. The net SSP2change in intervention group attenuated compared to control (p = 0.04). Conclusions: Findings support the use of eye tracking as an objective outcome measure in sensory-based interventions and highlight the importance of environmental control in autism therapy. Larger controlled trials are needed
Reduction of plasma levels of E-selectin and P-selectin induced-intensity training in obese boys
No full textBackground : Obesity is an epidemic problem that cause of atherosclerosis development and increase risk of cardiovascular diseases. Increase of adhesion molecule such as the P-selectin and E-selectin in obese people have key role in atherosclerosis phenomena. The purpose of this study was to investigate of high-intensity and moderate- intensity aerobic training on plasma levels of P-selectin and E-selectin in obese boys. Methods: In the semi-experimental study 36 obese student boys )age 13.4 ± 0.69 years old, height 163.06 ± 7.88 cm and weight 85.72 ± 8.42 kg) were randomly assigned to three groups: high-intensity aerobic training (n= 12), moderate-intensity aerobic training (n= 12) and control (n=12). The training was performed at 50-65% maximal heart rate (moderate-intensity) and 70-85% maximal heart rate (high-intensity), 3 day per week for 10- weeks. The blood sample was collected 48-h before and 48-h after last sessions of exercise training. The P-selectin and E-selectin were measured by ELISA. Data were analyzed using two-way repeated ANOVA with SPPS version 18.Results: The results of tow-way ANOVA analysis indicated that body percent fat (P = 0.028) and body weight (P = 0.042) were significantly decreased after 10- weeks training intervention. There is a significantly decrese of body fat percent and body weight in high-intensity training compared with moderate-intensity training (P= 0.027). The results of tow-way ANOVA analysis indicated that VO2max value significantly increased (P = 0.018). The plasma levels of P-selectin (P=0.001) and E-selectin (P=0.016) were significantly decreased after training intervention. Additionally, there is a significantly decrease of P-selectin in high-intensity training compared with moderate-intensity training (P= 0.034), but there is no significantly different between high-intensity training and moderate-intensity training in plasma levels of E-selectin in obese boys (P> 0.05). Conclusion: It seems that high-intensity aerobic training compared with moderate-intensity training has greater reduction in plasma levels of E-selectin, P-selectin, body weight and BMI, and increase of VO2max
Metaverse (Virtual World)
Full text linkThe concept of parallel digital realities offering experiences that mirror or transcend the limitations of the physical world has a long history, predating the internet itself. However, advancements in recent decades, such as near-ubiquitous mobile phone adoption andhigh-speed internet proliferation, have brought the notion of a blended physical and digital reality closer to realization. The Metaverse refers to this convergence, facilitated by computing devices and immersive technologies like virtual reality (VR), augmented reality (AR) and mixed reality (MR). While this vision of a fully realized virtual world remains in its early stages, with its components still under development, the potential of the Metaverse to offer significant opportunities for humanity is clear. However, these opportunities are accompanied by ethical and legal challenges, prompting the critical question of who is responsible for regulating the Metaverse to ensure compliance with ethical and legal frameworks. Undoubtedly, the coming years will necessitate the development of some form of regulation and rule-making to govern human interactions within these digitally connected worlds
شناسایی ژن های پیش آگهی و تشخیص سارکوما: مبتنی بر بیوانفورماتیک و یادگیری عمیق: شناسایی ژنهای پیشآگهی سارکوما
No full textزمینه و هدف: سارکوم بافت نرم (STS) یک سرطان نادر با منشا مزانشیمی است که در کودکان 15 درصد و در بزرگسالان 1 درصد مرگومیر ناشی از سرطان را ایجاد میکند. این سرطان به دلیل چالشهای تشخیصی معمولاً دیر شناسایی میشود و با تشخیص دیرهنگام به پیشرفت بیماری منجر میشود. نرخ بقاء پنجساله STS برابر با 65 درصد است که برای سارکومهای با درجه بالا این نرخ به 10 درصد کاهش مییابد. هدف این مطالعه شناسایی ژنهای پیشآگهی و تشخیصی در STS با استفاده از روشهای بیوانفورماتیک و یادگیری عمیق است.
روشها: در این مطالعه دادههای RNA-Seq از 261 نمونه سارکوم بافت نرم (STS) از پایگاه داده TCGA شامل 125 نمونه توموری و 128 نمونه نرمال به همراه اطلاعات بالینی بیماران تحلیل گردید. دادهها پس از پیشپردازش با نرمافزار R و بستههای Limma و edgeR نرمالسازی شدند. ژنهای بیان افتراقی (DEGs) با معیارهای |logFC|≥1.5 و P<0.05 شناسایی شدند. برای شناسایی ژنهای کلیدی از روشهای یادگیری ماشین (ML) و یادگیری عمیق (DL) استفاده گردید. عملکرد مدلها با شاخصهای AUC، دقت، F1-Score، R² و ماتریس سردرگمی ارزیابی شد. همچنین، تحلیلهای غنیسازی مسیر (GO و KEGG) و شبکههای برهمکنش پروتئین-پروتئین (PPI) انجام شد.
یافتهها: در مجموع 5204 ژن بیان افتراقی بین بافتهای توموری و نرمال شناسایی شد. از میان این ژنها، 16 ژن با بیان افزایشی شامل HIST1H1E، CARTPT، MAGEA8، HIST1H4E، RPA4 و ANGPTL3 با کاهش بقاء کلی بیماران ارتباط داشتند. در مقابل، 11 ژن دیگر مانند LECT2، ADAM21P1، GDF7، APOM و PIPOX با افزایش بقاء کلی بیماران مرتبط بودند. تحلیل ROC نشان داد که ژن A1CF به عنوان یک نشانگر زیستی مستقل با AUC=0.70 قدرت تمایز بالایی دارد. همچنین ترکیبهای A1CF-ATP6V0D2 (AUC=0.743) و A1CF-LECT2 (AUC=0.796) قدرت تشخیصی بهتری نسبت به تکژن ارائه دادند.
نتیجهگیری: مطالعه حاضر نشان داد که ژن A1CF و ترکیبات A1CF-ATP6V0D2 و A1CF-LECT2 به عنوان بیومارکرهای بالقوه برای تشخیص و پیشآگهی سارکوم بافت نرم (STS) شناسایی شدند. این یافتهها نشاندهنده پتانسیل استفاده از بیوانفورماتیک و یادگیری ماشین در تشخیص زودهنگام و درمان شخصیشده بیماران است
Management of Cracked and Weakened Endodontically Treated Teeth Using Fiber-reinforced Composites: A Case Series
Full text linkRestoring endodontically treated teeth (ETT) that exhibit cracks, enlarged roots, or weakened root walls is a frequent challenge in dental practice. The present study describes three cases in which contemporary restorative techniques were employed and suggests that applying Ribbond tape (RT) to ETT can improve fracture resistance and better prevent the propagation of cracks compared with traditional methods. Although extensive in vitro research has been conducted on fiber-reinforced composites, studies evaluating the clinical use and durability of fiber-reinforced composites to restore ETT are limited. This report strictly adhered to the case report (CARE) guidelines, and the treatments were initiated only after signed informed consents were obtained from the patients. Therefore, the old restorations were removed from the teeth that required intervention and composite resin core build-up was created, followed by endodontic treatments or retreatment. Subsequently, the endodontic accesses were reinforced with RT. The protective restorations were performed and bonded. The two-year follow-ups showed that the patients had complete remission of signs and symptoms, and they remain under monitoring. The study emphasizes the importance of internal reinforcement of ETT and strengthening weakened walls with a resin core build-up reinforced with fibers like RT. This approach enhances mechanical retention, inhibit fracture propagation, and establish a strong chemical bond between RT and resin. It is suggested to be a promising strategy for increasing the longevity and strength of the teeth, providing a conservative and effective alternative to traditional methods
Vital Pulp Therapy: Evidence-Based Techniques and Outcomes
Full text linkVital pulp therapy (VPT) comprises a range of conservative treatment modalities aimed at preserving the vitality of the compromised dental pulp in primary and permanent teeth. This PubMed-based review comprehensively evaluates seven distinct VPT techniques; including their historical development, broad definitions, clinical protocols and treatment outcomes as evidenced by systematic reviews and meta-analyses. The VPT modalities covered in this review include stepwise excavation, indirect pulp capping, direct pulp capping, miniature pulpotomy, partial pulpotomy, full pulpotomy and partial pulpectomy. Stepwise excavation, as a minimally-invasive option, has demonstrated effectiveness in reducing the risk of pulp exposure, particularly in permanent teeth. Clinical outcomes of indirect and direct pulp capping are promising; specifically with the application of advanced calcium silicate cements. Miniature and partial pulpotomies emphasize the importance of precise definitions and standardised protocols due to their subtle differences. Full pulpotomy has emerged as a viable alternative to root canal treatment, achieving comparable success rates in managing irreversible pulpitis. While partial pulpectomy remains the most invasive approach, it has shown potential in managing complex cases, such as root resorption, through selective tissue preservation. Despite advancements in biomaterials and technique standardisation, challenges remain, including variability in clinical protocols, limited high-quality evidence, and the need for long-term studies to better evaluate anticipated outcomes. Nevertheless, emerging biotechnologies hold promise for enhancing the precision and predictability of VPT procedures in the future