Pancreatic Digestion Research Articles

Comparison of two methods used to culture and purify rat retinal Müller cells.

To study two methods for culturing and purifying Sprague-Dawley (SD) rat retinal Müller cells and determine which one is better. The passage culture method of Müller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method. After culturing retinal cells for one month through these two methods, fluorescence-activated cell sorter (FACS), RT-PCR, and immunohistochemistry technology were performed to examine the enrichment and purity of Müller glial cells, and carried out two-sample approximate t test using SSPS 13.0 to further compare the Müller cell positive rate in both methods. The statistical results showed that the purity of Müller cells was 83.2%±5.16% in group A, and the purity was 98.5%±1.08% in group B. The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant (t=-9.178, P<0.005). The results clearly exhibited a difference between the purity of Müller cells cultured by the complete pancreatic enzyme digestion method (group A) and the repeated incomplete pancreatic enzyme digestion method (group B). Compared with the complete pancreatic enzyme digestion method, this novel method was more efficient and a higher purity of Müller cells could be obtained using this approach.

Detection on effect of different processed Cibotium barometz on osteoblasts by CCK-8

The pancreatic enzyme-II type collagenase digestion method was adopted for primary culture of osteoblasts, inoculation and passage. They were identified by alkaline phosphatase dye-liquor. N-butanol extract fractions from different processed products of Cibotium barometz were prepared. The above osteoblasts were jointly cultured with protocatechuic acid, protocatechuic aldehyde, kojic acid and the mixed control liquid of the above three substances, and their proliferation was detected by CCK-8. Various n-butanol extract fractions from different processed products of C. barometz showed a significant proliferative effect on osteoblasts in the order of the wined > the heated > the salted > the sand-heated and wined system > the alcohol-processed > the steamed > the crude. The q test showed no significant difference among sand-heated, alcohol-processed and steamed C. barometz, no significant difference between heated and salted C. barometz. Various control substances also showed a certain proliferative effect on osteoblasts in the order of the mixed control > protocatechuic aldehyde > protocatechuic acid > kojic acid. The q test showed no significant difference between protocatechuic aldehyde and protocatechuic acid. All of n-butanol extract fractions from different processed products of C. barometz showed a significant effect on osteoblast proliferation, of which wined C. barometz showed the best effect. All of phenolic compounds such as protocatechuic aldehyde, protocatechuic acid and kojic acid showed a significant proliferative effect on osteoblasts.

Immunological behavior of in vitro digested egg‐white lysozyme

Besides its antimicrobial properties, lysozyme (LYS) is one of the major allergens from hen egg. This paper addresses the identification of the peptides produced upon in vitro gastrointestinal digestion of LYS, together with their IgE-binding and biological activity as a contribution to the understanding of what makes it a relevant allergen. Simulated in vitro gastrointestinal digestion together with IgE binding, basophil degranulation, and peripheral blood mononuclear cells stimulation experiments were carried out. Identification of the fragments released was performed by HPLC-MS/MS and the immunoreactive products were analyzed by MALDI-TOF/TOF. Results showed that in vitro gastric and gastroduodenal digests of LYS maintained IgE binding, basophil activation capacity, and preserved T-cell immunogenicity. These biological activities could be attributed to either the persistence of intact LYS, due to incomplete gastric degradation and subsequent duodenal precipitation, the formation of fragment f(24-129) by chymotrypsin action on the soluble intact protein, or the release, upon combined gastric and pancreatic digestion, of immunoreactive peptides linked by disulphide bonds containing the epitopes f(57-83) and f(108-122). The pH of gastric hydrolysis greatly determined the extent of subsequent duodenal digestion of LYS and the disclosure of relevant epitopes that could increase its allergenic potential.

Comparison of New Preservation Solutions, HN-1 and University of Wisconsin Solution, in Pancreas Preservation for Porcine Islet Isolation.

For pancreatic islet transplantation, maintaining organ viability after pancreas procurement is critical and a major determinant for better graft function and survival. University of Wisconsin (UW) solution is currently the gold standard for abdominal organ preservation and the pancreas in particular. However, in the use of UW preservation solution for islet transplantation, there are disadvantages to be overcome, such as the inhibition of collagenase activity during pancreatic digestion. In this study, we compared UW solution with HN-1 solution in pancreas preservation for islet isolation. Islet yield was significantly greater in the HN-1 group than the UW group both before and after purification. In the in vitro assay, the adenosine triphosphate content in cultured islets was significantly higher in the HN-1 group than in the UW group. Furthermore, in streptozotocin-induced diabetic nude mice, the islet graft function of the HN-1 group was superior to that of the UW group. We concluded that the use of HN-1 solution is a promising approach for optimal pancreas preservation in islet transplantation.

Histidine-Tryptophan-Ketoglutarate and University of Wisconsin Solution Demonstrate Equal Effectiveness in the Preservation of Human Pancreata Intended for Islet Isolation: A Large-Scale, Single-Center Experience

We previously reported a small-scale study on the efficacy of histidine-tryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution on pancreas preservation for islet isolation. In this large-scale, retrospective analysis (n = 252), we extend our initial description of the impact of HTK on islet isolation outcomes and include pancreatic digestion efficacy, purification outcomes, and islet size distribution. Multivariable linear regression analysis, adjusted for donor age, sex, BMI, cold ischemia time, and enzyme, demonstrated similar results for the HTK group (n = 95) and the UW group (n = 157), including postpurification islet yields (HTK: 289,702 IEQ vs. UW: 283,036 IEQ; p = 0.76), percentage of digested pancreatic tissue (HTK: 66.9% vs. UW: 64.1%; p = 0.18), and islet loss from postdigestion to postpurification (HTK: 24,972 IEQ vs. UW: 39,551 IEQ; p = 0.38). Changes in islet size between the postdigestion and postpurification stages were comparable within each islet size category for HTK and UW (p = 0.14-0.99). Tissue volume distribution across purification fractions and islet purity in the top fractions were similar between the groups; however, the HTK group had significantly higher islet purity in the middle fractions (p = 0.003-0.008). Islet viability and stimulation indices were also similar between the HTK and the UW groups. In addition, we analyzed a small sample of patients transplanted either with HTK (n = 7) or UW (n = 8) preserved islets and found no significant differences in posttransplant HbA1c, β-score, and frequency of insulin independence. This study demonstrates that HTK and UW solutions offer comparable pancreas preservation for islet transplantation. More in vivo islet outcome data are needed for a complete analysis of the effects of HTK on islet transplantation.

Comparative analysis of nutritional quality of five different cultivars of sweet potatoes (Ipomea batatas (L) Lam) in Sri Lanka.

Nutritional attributes of flours obtained from five different cultivars of sweet potato roots commonly available in Sri Lanka showed significant differences in the tested parameters. The starch level ranged between 33% and 64% on the dry basis and the extractability from fresh tubers was governed by the quantity of starch. The crude fiber level ranged between 2.1% and 13.6% on dry basis and the highest level was observed in swp7 (CARI 273) and resistant starch ranged from 14.2% to 17.2%. Higher percentage of resistant starch from total starch was found in Wariyapola red (swp1) cultivar resulting in lower digestion level while higher levels of digestion was evident in cultivars with lower levels of resistant starch with high level of total starch. Low levels of calcium and significant levels of iron were found in the five cultivars studied. Crude protein level was in the range of 1.2–3.3% on dry basis and trypsin inhibitor activity level (TIA) was significantly different (P > 0.05) in the cultivars studied while heating resulted in a significantly high reduction in the TIA level than in unheated condition. Polygonal or round shaped starch granules were in the range of 16.8–23.5 μm and low level of starch digestion was shown in cultivars containing larger granules. Total amylose content lies in the range 15.4–19.6% and cultivars having higher percentage of amylose showed higher level of in vitro pancreatic digestion (Pallepola [swp4] and swp7). The starch digestibility of sweet potato flour was in the range of 36–55% and the highest digestion was observed in swp7. Orange fleshed cultivars (swp4 and swp7) were comparatively rich in nutrients and digestibility than the other three studied cultivars.

Interlocking of β-carotene in beta-lactoglobulin aggregates produced under high pressure

Vitamin A deficiency is one of the major causes of mortality and morbidity in the developing World. This deficiency can be prevented by alimentary or pharmaceutical supplementation. However, both vitamin A oxidation and isomerization should be prevented, as these phenomenons result in loss of nutritional efficacy. The aim of this study was to investigate the effect of a food protein matrix, β-lactoglobulin (β-Lg) aggregates produced by high pressure (HP), on the stabilization of β-carotene during storage and gastro-duodenal digestion and therefore on its bioavailability. In vitro gastro-duodenal digestion of β-Lg aggregates entrapping β-carotene showed that up to 12% and 33% of total β-carotene was released after peptic and pancreatic digestion, respectively. Overall, our study showed that β-Lg aggregates are efficient for caging and stabilization of β-carotene during storage and digestion. Hence, it may be an interesting approach for the protection and the delivery of vitamin A.

Simulated digestion of proanthocyanidins in grape skin and seed extracts and the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitory activity

This study investigated the effect of in vitro gastrointestinal digestion on the stability and composition of flavan-3-ols from red grape skin and seed extracts (raw and purified, which are high in proanthocyanidins (PAs)). In addition, the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitory activities of these extracts were evaluated. The extracts were digested with a mixture of pepsin-HCl for 2h, followed by a 2h incubation with pancreatin and bile salts including a cellulose dialysis tubing (molecular weight cut-off 12kDa) at 37°C with shaking in the dark and under N2. Under gastric conditions, the mean degree of polymerisation (mDP) of seed extracts, raw (mDP≈6, p<0.05), and purified (mDP≈10, p<0.05) was stable. The mDP of the raw skin extracts increased from 19 to 25 towards the end of the digestion. The PAs were significantly degraded (up to 80%) during the pancreatic digestion, yielding low-molecular-weight compounds that diffused into the serum-available fraction (mDP≈2). The overall mass transfer coefficient (K) of the seed extracts was 10−7m2/s. After simulated gastrointestinal digestion, over 80% of ACE inhibition by raw seed and skin extracts was preserved. However, the purified seed and skin extracts lost their ability to inhibit ACE after intestinal digestion.

Pancreatic Digestive Enzyme Blockade in the Intestine Increases Survival After Experimental Shock

Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors--6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate, tranexamic acid, or aprotinin--into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multiorgan failure, which can be treated with protease inhibitors that are currently available for use in the clinic.

Microbial metabolites, but not other phenolics derived from grape seed phenolic extract, are transported through differentiated Caco-2 cell monolayers

Grape seed phenolic extract (GSE) is predicted to have health benefits, even though its bioavailability, including digestibility, permeability and ultimate metabolism, are still poorly understood. In vitro gastric and pancreatic digestion and in vitro ileal and faecal fermentation were combined with Caco-2 cell permeability studies for GSE samples. Qualitatively, there was no change in type/number of GSE compounds following gastric and pancreatic digestion and LC–MS analysis. However, the monomers were significantly (P<0.05) increased after gastric digestion, along with a significant (P<0.05) decrease in polymers. In addition, all forms of phenolic compounds decreased following pancreatic digestion. However, none of the original GSE phenolic compounds passed the Caco-2 cell monolayer, since all were recovered in the apical compartment. In contrast, the two intestinal microbiota metabolites with deprotonated molecular weights of [M–H]-165/121 and 193/175, that were found both in the ileal and faecal fermented samples, passed the Caco-2 cell monolayer.

A Proteomic Approach for Investigating Pancreatic Extracellular Matrix Digestion During Human Islet Isolation

A Proteomic Approach for Investigating Pancreatic Extracellular Matrix Digestion During Human Islet Isolation

Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential

The aim of this work was to study the potential probiotic properties of lactobacilli associated with table olives. From a total of 111 isolates from spontaneously fermented green olive brines, 109 were identified at species level by multiplex PCR amplifications of the recA gene. One hundred and seven of these were identified as Lactobacillus pentosus, one as Lactobacillus plantarum, and another as Lactobacillus paraplantarum. Repetitive bacterial DNA element fingerprinting (rep-PCR) with GTG5 primer revealed a higher variability within the L. pentosus isolates and nine different clusters were obtained. Most of them showed high autoaggregation ability, low hydrophobicity properties, and lower survival to gastric than to pancreatic digestion; however, no isolate showed bacteriocin, haemolytic or bile salt hydrolase activities. A multivariate analysis based on results from phenotypic tests led to the segregation of some L. pentosus isolates with promising potential probiotic characteristics, which are even better than probiotic reference strains. Due to the autochthonous origin of the strains, their use as starter cultures may contribute to improving natural fermentation and the nutritional characteristics of table olives.

Effects of Alcohol Extract from Soybean Meal on Pancreatic Digestive Enzyme and Bile Acid Secretion in Yellowtail Seriola quinqueradiata

Effects of Alcohol Extract from Soybean Meal on Pancreatic Digestive Enzyme and Bile Acid Secretion in Yellowtail Seriola quinqueradiata

Chicken embryo extracts enhance spleen lymphocyte and peritoneal macrophages function

Ethnopharmacological relevanceTraditionally, chicken embryo eggs are widely considered as an immunomodulatory health product in China and southeast Asia. Besides that, Compendium of Materia Medica, the most famous Chinese pharmacopoeia, has recorded that chicken embryo eggs contributed to the appetite increase and immune function enhancement, especially for the pregnant and aged. Aim of the studyThrough assessing the immune efficacy of the chicken embryo extracts on immunocompetent cells, the immunomodulatory mechanism and components were explored preliminarily. Materials and methodsThe raw material was subjected to in vitro gastric and pancreatic digestion successively, and then ligarine was added to separate the digested mixture to water-soluble fraction (WS) and lipid-soluble fraction (LS). Employing in vitro cell models, the immunomodulating activities of the extracts on immunosuppressed mouse were examined, at concentrations ranging from 10 to 500μg/ml. The cell experiments consisted of the following experiments: spleen lymphocyte proliferation and IL-2 secretion, peritoneal macrophage phagocytosis and NO production. Before cell experiments, Kjeldal method and phenol–sulfuric acid method were employed respectively to determine the total protein and polysaccharide content of WS, and gas chromatography to analyze the fatty acid composition of LS. ResultsLS significantly enhanced the spleen lymphocyte proliferation and IL-2 secretion, while peritoneal macrophage phagocytosis and NO production activity were elevated by WS. Both extracts exhibited an immunomodulatory effect with a dose–effect relationship. ConclusionThe findings confirmed the chicken embryo eggs as a potent immunopotentiator on the immunosuppressed mouse, which would be useful for the fruitful application of the chicken embryo eggs to serve as a superior immunostimulatory tonic. Furthermore, the two extracts exhibited a different bioactivity tendency which makes it a significative work to screen the bioactive component for further study.

Impaired Small-Bowel Barrier Integrity in the Presence of Lumenal Pancreatic Digestive Enzymes Leads to Circulatory Shock

In bowel ischemia, impaired mucosal integrity may allow intestinal pancreatic enzyme products to become systemic and precipitate irreversible shock and death. This can be attenuated by pancreatic enzyme inhibition in the small-bowel lumen. It is unresolved, however, whether ischemically mediated mucosal disruption is the key event allowing pancreatic enzyme products systemic access and whether intestinal digestive enzyme activity in concert with increased mucosal permeability leads to shock in the absence of ischemia. To test this possibility, the small intestinal lumen of nonischemic rats was perfused for 2 h with either digestive enzymes, a mucin disruption strategy (i.e., mucolytics) designed to increase mucosal permeability, or both, and animals were observed for shock. Digestive enzymes perfused included trypsin, chymotrypsin, elastase, amylase, and lipase. Control (n = 6) and experimental animals perfused with pancreatic enzymes only (n = 6) or single enzymes (n = 3 for each of the five enzyme groups) maintained stable hemodynamics. After mucin disruption using a combination of enteral N-acetylcysteine, atropine, and increased flow rates, rats (n = 6) developed mild hypotension (P < 0.001 compared with groups perfused with pancreatic enzymes only after 90 min) and increased intestinal permeability to intralumenally perfused fluorescein isothiocyanate-dextran 20 kd (P < 0.05) compared with control and enzyme-only groups, but there were no deaths. All animals perfused with both digestive enzymes and subjected to mucin disruption (n = 6) developed hypotension and increased intestinal permeability (P < 0.001 after 90 min). Pancreatic enzymes were measured in the intestinal wall of both groups subjected to mucin disruption, but not in the enzyme-only or control groups. Depletion of plasma protease inhibitors was found only in animals perfused with pancreatic enzymes plus mucin disruption, implicating increased permeability and intralumenal pancreatic enzyme egress in this group. These experiments demonstrate that increased bowel permeability via mucin disruption in the presence of pancreatic enzymes can induce shock and increase systemic protease activation in the absence of ischemia, implicating bowel mucin disruption as a key event in early ischemia. Digestive enzymes and their products, if allowed to penetrate the gut wall, may trigger multiorgan failure and death.

Analysis of Genetic Determination of &lt;I&gt;Vibrio cholerae&lt;/I&gt; Tweenase Activity

Studied are Vibrio cholerae of different serogroups on the presence of cef (CHO cell elongating factor) gene and activity against tweens and tributyrin using HDS-agar, prepared on the basis of bakery yeast pancreatic digest. Determined is the fact that all cef-positive strains hydrolyze tweens 20, 40, 60, 80, 85 and all but toxigenic V.cholerae O139, hydrolyze tributyrin. In contrast, non-toxigenic cef-negative strains of O139 serogroup, are active only against the latter. Apparently, the tweenase activity of Vibrio cholerae is provided, partly, by Cef, and the ability to hydrolyze tributyrin is the result of combined activity of Cef and other ferments. Shown is the efficiency of HDS-agar application to determine these characteristics.

Secoiridoids from the stem barks of Fraxinus rhynchophylla with pancreatic lipase inhibitory activity

Pancreatic lipase digests dietary fats by hydrolysis, which is a key enzyme for lipid absorption. Therefore, reduction of fat absorption by the inhibition of pancreatic lipase is suggested to be a therapeutic strategy for obesity. From the EtOAc-soluble fraction of the stem barks of Fraxinus rhynchophylla (Oleaceae), four secoiridoids such as ligstroside (1), oleuropein (2), 2″-hydroxyoleuropein (3) and hydroxyframoside B (4) were isolated. The inhibitory activity of these compounds on pancreatic lipase was assessed using porcine pancreatic lipase as an in vitro assay system. Compound 4 showed the strongest inhibition on pancreatic lipase, which followed by compounds 1–3. In addition, compound 4 exerted inhibitory effect on pancreatic lipase in a mixed mechanism of competitive and noncompetitive manner. Taken together, F. rhynchophylla and its constituents might be beneficial to obesity.

A Model to Evaluate Toxic Factors Influencing Islets during Collagenase Digestion: The Role of Serine Protease Inhibitor in the Protection of Islets

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.

Inhibition of acetylcholinesterase by green and white tea and their simulated intestinal metabolites

By 2034 it is forecast that 5% of the global population will be aged 85 years or over--approximately two and half fold increase on present day figures--which will inevitably lead to an increase in age-associated disorders such as Alzheimer's disease. There is mounting evidence that green tea (Camellia sinensis) possesses numerous health-promoting properties, and may potentially be beneficial to those suffering from Alzheimer's and other diseases, including cardiovascular disease and cancer. These beneficial properties are largely attributed to the high polyphenol content, particularly the catechins. In this study, we measured acetylcholinesterase inhibition by white and green teas and their simulated intestinal digests. We found that the potency with which the white and green tea extracts inhibited acetylcholinesterase varied through the simulated digestion procedure. Initially, in the undigested extract form, potency was high with IC₅₀ values of 7.20 μg mL⁻¹ and 8.06 μg mL⁻¹ for green and white tea respectively.However, this decreased significantly after gastric digestion but activity was recovered after pancreatic digestion which could be related to relative increases in the levels of caffeine and specific phenolic components. Of the pure tea compounds tested, EGCG was the most potent with an IC₅₀ of 0.0096 μmol mL⁻¹ but its breakdown product; γ-valerolactone was the least potent analyte. Particularly interesting were the results of caffeine,which exhibited a strong inhibitory activity and pyrogallol, which recorded a much stronger potency than its parent compound gallic acid, suggesting a pro-drug-like relationship. Overall, the results indicate that further research is necessary to determine the full potential of digestion of tea and its metabolites and how inter-individual variation may indicate that some sections of society could potentially benefit more from drinking tea as a strategy to prevent the development of dementia. We have also shown the activities of a number of metabolites,however, further research is required to determine their potential bioavailability.

The Methods of Reconstruction of Pancreatic Digestive Continuity After Pancreaticoduodenectomy: A Meta‐Analysis of Randomized Controlled Trials

Pancreatic fistula (PF) is an important factor responsible for the considerable morbidity associated with pancreaticoduodenectomy (PD). There have been many techniques proposed for the reconstruction of pancreatic digestive continuity to prevent fistula formation but which is best is still highly debated. We carried out a systematic review and meta-analysis to determine the effectiveness of methods of anastomosis after PD. A full literature search was conducted in the Cochrane Controlled Trials Register Databases, Medline, and other resources irrespective of language. Randomized controlled trials (RCTs) were considered for inclusion. Analyses were carried out using RevMan software. In all, ten RCTs that included a total of 1,408 patients were included. The meta-analysis showed that the PF, postoperative complications, biliary fistula, mortality, reoperation, and length of hospital stay were not statistically different between the pancreaticogastrostomy (PG) and pancreaticojejunostomy (PJ) groups. The PF, postoperative complications, mortality, and reoperation were not statistically different between the duct-to-mucosa PJ and PJ groups. Binding PJ significantly decreased the PF and postoperative complications compared with conventional PJ. The PF, postoperative complications, and mortality were not statistically different between ligation of the pancreatic duct without anastomosis versus PJ. No pancreatic reconstruction technique after PD was found to be applicable to all kinds of pancreatic remnants in our systematic review and meta-analysis. Some new approaches such as binding PJ and modified PG will be considered for study in the future.