Invitro Analysis Research Articles

Optimization through combined compromise solution and weight determination through SHANNON-ENTROPY for multi criteria decision making in tribological testing of ultrahigh molecular weight polyethylene nano-composite in SBF environment and compression testing with it’s invitro analysis

Aluminium oxide and graphene reinforced with ultrahigh molecular weight polyethylene (UHMWPE) composites offer significant potential for prosthetic and implant applications due to their unique combination of properties. In this article, a series of aluminium oxide and graphene reinforced with ultrahigh molecular weight polyethylene (UHMWPE) composites were successfully fabricated through compression moulding. The average hardness of all the composites was observed to be higher than the UHMWPE (6.1 Hv). Composite U-15A-5 G was found to be the hardest with hardness of 10.5 Hv owing to strong bond between Al2O3 and UHMWPE. The compression strength of the U-15A-5 G material was about 81.936 MPa, which is 40.9% better than the pure UHMWPE. The compression modulus was about 330 MPa which is 22.2% higher than neat UHMWPE. Composite samples were tribo tested under simulated body fluid (SBF) lubricating environment. Using combined entropy-COCOSO method it was found that 50 mm/s speed, 200N load and 5% graphene are the optimum tribological parameters. with Cof of 0.04 and wear rate of 4.8745*10−4 mm3/Nm. Microstructure analysis indicated that long-range structured lamellar structures and microfibers were created between the crystals with the assistance of graphene layers. Apatite layer was also found on the sample. The apatite layer included phosphate and carbonate ions, which is good for in vitro bioactivity on polymeric films. This composite is a good candidate for prosthetics and implant components.

Are Sleeves Necessary in Static Computer-Assisted Implant Surgery? A Comparative InVitro Analysis.

This study aims to examine differences in trueness and precision between surgical guides with (S) and without sleeves (SL). A secondary aim was to assess the impact of the sleeve-to-bone distance. Mandible replicas (n = 120) were printed from an STL file obtained from a clinical CBCT. The mandibles were divided into sleeved (S, n = 60) and sleeveless (SL, n = 60) groups, each further divided into three categories (n = 20 each) with different heights from the guide to the implant platform: 2 mm (H2), 4 mm (H4), or 6 mm (H6). Digital planning and surgical guide design were done for a 4.1 × 10 mm implant for site #30. Post-op positions were captured using a scan body and lab scanner. Angular deviation was the primary outcome, with 3D and 2D deviations as secondary parameters. Statistical analysis included two-sample t-tests, and one-way and two-way ANOVA. Group S (2.41 ± 1.41°) had significantly greater angular deviation than Group SL (1.65 ± 0.93°; p = 0.0001). Angular deviation increased with sleeve-to-bone distance. H2 deviations were 1.48 ± 0.80° (S) vs. 1.02 ± 0.45° (SL; p < 0.05), H4: 2.36 ± 1.04° (S) vs. 1.48 ± 0.79° (SL; p < 0.05), H6: 3.37 ± 0.67° (S) vs. 2.46 ± 0.89° (SL; p < 0.05). 3D deviation at the implant platform was 0.36 ± 0.17 mm (S) vs. 0.30 ± 0.15 mm (SL; p < 0.05) and at the apex 0.74 ± 0.34 mm (S) vs. 0.53 ± 0.31 mm (SL; p < 0.01). Group SL at H2 had the smallest inter-implant distance (0.53 ± 0.37°), while Group S at H4 had the largest (1.20 ± 0.84°; p < 0.05). Sleeveless guides are more accurate than sleeved guides, and angular deviation is influenced by the distance from the guide to the implant platform.

THIOSTREPTON MODULATES TLR4 EXPRESSION AND INDUCES APOPTOSIS IN MDA MB 231 CELLS: AN IN VITRO AND IN SILICO ANALYSIS

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors involved in tumorigenesis, apoptosis, and metastasis. Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with a poor prognosis. Although the role of TLRs in breast cancer remains underexplored, recent studies suggest targeting TLRs in TNBC could be beneficial. In this study Thiostrepton, an antibiotic and novel inhibitor of TLR7-9 in psoriatic inflammation, was investigated for its effects on TLR3, TLR4, and TLR9 expression in TNBC cells (MDA-MB-231). Materials and Methods: The cytotoxicity of thiostrepton was assessed using the MTT assay. RT-PCR was used to measure gene expression levels of TLR3, TLR4, TLR9, Bax, Bcl-2, Nf-κB, and E-cadherin. Cell morphology changes were analyzed with Acridine Orange/Ethidium Bromide (AO/EtBr) staining. Molecular docking and dynamics simulations examined interactions between thiostrepton and the TLR4-MD-2 complex. Results: Thiostrepton led to a concentration- and time-dependent decrease in cell viability. It significantly inhibited TLR4, Bcl-2 gene expression and increased TLR3, Bax, and Nf-κB levels. The changes in Bax and Bcl-2 gene expression, along with alterations in cell morphology, demonstrated that thiostrepton promoted apoptosis in MDA-MB-231 cells. While TLR9 expression reduction was not significant, thiostrepton notably increased TLR3 expression and decreased TLR4 expression. The three independent molecular dynamics simulations demonstrated that thiostrepton binds stably to the TLR4-MD2 domain, exhibiting a high binding affinity as indicated by the binding free energy calculations. Conclusion: Thiostrepton effectively induces apoptosis and reduces cell viability in TNBC cells. In silico analysis suggest thiostrepton could modulate TLR4, highlighting its potential as a candidate for further research and therapeutic development.

In-Vitro Analysis Of Bioremediation Potential Of Bacterial Strains Isolated From Soil Contaminated With Cadmium And Chromium

In-Vitro Analysis Of Bioremediation Potential Of Bacterial Strains Isolated From Soil Contaminated With Cadmium And Chromium

Protective Effect of Aloe vera (L.) on Diabetes-Induced Oxidative Stress Linked Spermiological Co-Morbidity in Human and Rat: An In-Vitro Analysis.

Diabetes linked reproductive complications are rising problems nowadays. The study focused on the protective efficacy of Aloe vera (L.) on sperm cell damage in an oxidative stress milieu encumbered by a chronic diabetes in human and streptozotocin treated Wistar rat (Rattus norvegicus). Spermatozoa from rat's epididymal washing, and human semen after 3-4days of abstinence of mating or masturbation were collected from control and diabetes groups. Spermatozoa of human and rat were incubated for 1 or 2h at 370C in an in-vitro medium separately and considered as normo-glycemic control and diabetes sub-groups. Dose of 1 or, 2 or, 4mg/ml of Aloe vera (L.) hydro-ethanolic (40:60) extract (AVHE) was given to diabetes samples, considered as sub-sub-group for assessing its protective effect on spermiological and oxidative stress parameters. The motility, viability, plasma membrane integrity, nuclear chromatin decondensation for DNA fragmentation, acrosome cap status, and antioxidative status of human and rat spermatozoa were decreased whereas spermatozoal apoptosis was elevated significantly (p < 0.05), noted by TUNEL assay in diabetes samples compared to the duration-matched control group. Exposure of AVHE to diabetes samples resulted significant rectification (p < 0.05) in the said parameters than the unexposed diabetes group. In control group, AVHE exposure has significant protective effect from spermiological deterioration compared to unexposed control group. Identification of major phytomolecules in AVHE was done by LC-MS study. Diabetes-induced oxidative stress-mediated spermatozoal injuries can be protected by AVHE extract, raise the possibility for potentiating sperm of human for increasing the success rate of in-vitro fertilization-blastocyst implantation.

Validation of the Chicken Femur as a Model for the Human Metacarpal: An In-Vitro Analysis.

Background: The aim of this study was to evaluate the chicken femur as a laboratory model for the human metacarpal by comparing the bone microarchitecture and mechanical properties of chicken femurs to human cadaveric metacarpals. Methods: Sixteen fresh chicken femora and 20 fresh frozen cadaveric human metacarpals were imaged using a micro computed tomography scanner. The bones were then mechanically tested using four-point-bending and torsional testing. Results: There were no significant differences in macroscopic features between chicken femora and human metacarpals, including overall length, external radius, internal radius, cortical width and cross-sectional area of the diaphyseal cortex (p > 0.05). There were no significant differences in the trabecular number and spacing in the distal metaphysis of both groups (p > 0.05). The diaphysis and proximal metaphysis did not share any microarchitectural similarities. Four-point bending tests resulted in significantly higher yield forces, ultimate force, failure points and stiffness in human metacarpals (p < 0.05). Torsion tests resulted in significant higher ultimate torque and torsional rigidity in human metacarpals (p < 0.05). Conclusions: The chicken femur has structural and biomechanical differences to the fresh frozen human metacarpal despite the similarity in their macroscopic features.

Phosphoribosyl pyrophosphate amidotransferase: Novel biomarker and therapeutic target for nasopharyngeal carcinoma.

Cancer cells show a dynamic metabolic landscape, requiring a sufficient supply of nucleotides to proliferate. They are highly dependent on de novo purine biosynthetic pathways for their nucleotide requirements. Phosphoribosyl pyrophosphate amidotransferase (PPAT), catalyzing the first step of de novo purine biosynthesis, is highly expressed in various cancers. We observed an increased expression of PPAT in nasopharyngeal carcinoma (NPC). Moreover, our ribonucleic acid sequencing analysis showed high PPAT expression in Epstein-Barr virus-positive NPC, which was supported by invitro analysis. Through a gene knockdown study, we showed that the suppression of PPAT expression reduced the proliferation and invasion of NPC cells. We also demonstrated the regulation of PPAT by glutamine, a cosubstrate for PPAT. A glutamine antagonist, 6-diazo-5-oxo-L-norleucine, blocked glutamine-mediated induction of PPAT and reduced NPC cell proliferation. Immunohistochemical analysis of PPAT in NPC tissues revealed increased expression of PPAT with disease progression, which was significantly associated with poor prognosis. In summary, this study highlighted the biological function of PPAT in NPC, establishing its potential as a novel prognostic biomarker for aggressive NPC and a promising therapeutic target.

In-Vitro Analysis of Polyethylene Liner Wear Performance at 3 and 5 Million Cycles

The purpose of this study is to compare the wear properties of UHMWPE acetabular liners after undergoing 3 million (3 Mc) and 5 million (5 Mc) cycles of in-vitro wear testing. The results will serve as a reference for the design of in-vitro testing for hip prostheses and as a control for clinical revision removals. Wear tests were conducted on three different sizes of acetabular liners (28 mm, 32 mm, and 36 mm internal diameters) using a hip simulator to determine the amount of wear after 3 and 5 million cycles. The analysis included the number, size, and shape of abrasive particles. After 3 and 5 million cycles of wear, the amount of wear on the acetabular liner increased as the inner diameter increased. The abrasive particles had an average equivalent circular area diameter (ECD) of 0.27 μm and 0.29 μm, and 94.4% and 90.1% of the aspect ratio (AR) less than 4. The amount of wear on the acetabular liner after 3 Mc wear can indicate the wear performance of the product. The number of particles increased and the percentage of fibrous particles was higher after 5 Mc wear compared to 3 Mc wear.

Identification and Design of Novel Potential Antimicrobial Peptides Targeting Mycobacterial Protein Kinase PknB

Antimicrobial peptides have gradually gained advantages over small molecule inhibitors for their multifunctional effects, synthesising accessibility and target specificity. The current study aims to determine an antimicrobial peptide to inhibit PknB, a serine/threonine protein kinase (STPK), by binding efficiently at the helically oriented hinge region. A library of 5626 antimicrobial peptides from publicly available repositories has been prepared and categorised based on the length. Molecular docking using ADCP helped to find the multiple conformations of the subjected peptides. For each peptide served as input the tool outputs 100 poses of the subjected peptide. To maintain an efficient binding for relatively a longer duration, only those peptides were chosen which were seen to bind constantly to the active site of the receptor protein over all the poses observed. Each peptide had different number of constituent amino acid residues; the peptides were classified based on the length into five groups. In each group the peptide length incremented upto four residues from the initial length form. Five peptides were selected for Molecular Dynamic simulation in Gromacs based on higher binding affinity. Post-dynamic analysis and the frame comparison inferred that neither the shorter nor the longer peptide but an intermediate length of 15 mer peptide bound well to the receptor. Residual substitution to the selected peptides was performed to enhance the targeted interaction. The new complexes considered were further analysed using the Elastic Network Model (ENM) for the functional site’s intrinsic dynamic movement to estimate the new peptide’s role. The study sheds light on prospects that besides the length of peptides, the combination of constituent residues equally plays a pivotal role in peptide-based inhibitor generation. The study envisages the challenges of fine-tuned peptide recovery and the scope of Machine Learning (ML) and Deep Learning (DL) algorithm development. As the study was primarily meant for generation of therapeutics for Tuberculosis (TB), the peptide proposed by this study demands meticulous invitro analysis prior to clinical applications.Graphical

Differential expression and clinical significance of IGF2BP3 in peritoneal dialysate of patients with varying duration of peritoneal dialysis.

This study aims to investigate the differential expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the peritoneal dialysate among patients with different durations of peritoneal dialysis and its association with the angiogenic marker vascular* endothelial growth factor (VEGF), the fibronectin (FN), and various clinical indicators. A cohort of 122 peritoneal dialysis patients was categorized into short-term (≤1 year, n = 33), mid-term (>1 and ≤5 years, n = 55), and long-term (>5 years, n = 34) groups based on dialysis duration. We utilized enzyme-linked immunosorbent assay (ELISA) and western blot assays to quantify the levels of IGF2BP3, VEGF, and FN in the dialysate. Our findings showed a progressive increase in IGF2BP3 levels with the duration of PD, with the long-term group exhibiting significantly higher levels than both the short-term and mid-term groups (p < 0.001). A positive correlation between IGF2BP3 and VEGF (r = 0.386, p = 0.013), as well as between IGF2BP3 and FN (r = 0.340, p = 0.030), was observed. IGF2BP3 levels also correlated positively with serum creatinine, calcium, and phosphorus levels. Invitro analysis further confirmed that IGF2BP3 expression is enhanced in human peritoneal mesothelial cells under high-glucose conditions (p < 0.05). The study highlights the potential of IGF2BP3 in PD effluent as a biomarker for monitoring PF progression, with its expression significantly correlated with the duration of PD (Pearson r = 0.897, p < 0.001). In conclusion, our results underscore a correlation between elevated IGF2BP3 levels and PD duration, suggesting the clinical significance of IGF2BP3 as a biomarker for PF progression.

Research of the Potential Radioprotective Activity of Trimethyl Glycine and N-Acetyl-L-Cysteine with Quantitative Analysis Using Real-Time Polymerase Chain Reaction (QRT-PCR)

The irradiation exposure could affect the cellular genome and specifically target genes, such as TP53 gene. The TP53 gene is known as a “guardian of the genome” and is a tumor suppressor gene. Its role is to keep the cell of malignant transformation. After ionizing radiation exposure, the TP53 could mutate and the cell could continue to active proliferation (tumor cellular transformation). It has been noticed that different type of substances could have radioprotective effect and to protect the cell of malignant transformation, apoptosis, genome injuries or necrosis. Some of the natural metabolites that are proven antioxidants, show significant ability of radiation protection of the cells. The aim of that study is to analyze the possible radioprotection ability of two origin metabolites (amino acids)–trimethyl glycine (betaine) and N-acetyl-L-cysteine, applied together and in combination, first before irradiation (preventive treatment) and then 2 hours after irradiation (as a therapeutic agents). The research work is done as invitro analysis to peripheral blood cell cultures. Conclusion: The conclusion of the study is that both amino acids showed good radiation protection activity after in-vitro performed analysis. The most significant results showed the combination of both natural metabolites.

Invitro analysis of Syzygium aromaticum for its anti-cancer potential

Invitro analysis of Syzygium aromaticum for its anti-cancer potential

Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by invitro analysis of hiPSC differentiation and invivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

Fibroblast growth factor signalling regulates the development of tooth root.

Fibroblast growth factor (FGF) signalling plays a crucial role in the morphogenesis of multiple tissues including teeth. While the role of the signal has been studied in tooth crown development, little is known about root development. Of several FGF ligands involved in hard tissue formation, we suggest that FGF18 regulates the development of murine tooth roots. We implanted FGF18-soaked heparin beads into the lower first molar tooth buds at postnatal day 6 (P6), followed by transplantation under the kidney capsule. After 3 weeks, FGF18 significantly facilitated root elongation and periodontal tissue formation compared to the control. Insitu hybridisation showed that Fgf18 transcripts were initially localised in the dental pulp along Hertwig's epithelial root sheath at P6 and P10 and subsequently in the dental follicle cells at P14. Fgf receptors were expressed in various dental tissues during these stages. Invitro analysis using the dental pulp stem cells revealed that FGF18 inhibited cell proliferation and decreased expression levels of osteogenic markers, Runx2, Alpl and Sp7. Consistently, after 1 week of kidney capsule transplantation, FGF18 application did not induce the expression of Sp7 and Bsp, but upregulated Periostin in the apical region of dental mesenchyme in the grafted molar. These findings suggest that FGF18 facilitates molar root development by regulating the calcification of periodontal tissues.

Impact of Auxiliary Features on Retention of Short Dental Crowns: An In-Vitro Analysis of Box and Groove Preparations.

BACKGROUND Several auxiliary features have been proposed to achieve sustainable retention for short-prepared dental crowns; however, achieving retention is challenging. This study aimed to assess the impact of increased total occlusal convergence and auxiliary preparation factors like box and groove on the retention form of short tooth preparations. MATERIAL AND METHODS Eighty resin machine-milled dies with a height of 3 mm and a deep chamfer margin of 1 mm were prepared to mimic the short-prepared molar. Initially, 2 teeth were prepared following the guidelines, and the total occlusal convergence was kept at 10° and 20°, respectively. Auxiliary features such as the proximal box and buccal groove were prepared on separate 20° dies. Eighty dies were prepared with 10 samples each for 10°, 20°, 20° with proximal box and 20° with buccal groove for zirconia (n=40) and metal crowns (n=40). Cementation was done with glass ionomer luting cement, and a pull-off test was conducted. Data were analyzed using one-way analysis of variance and post hoc fisher least significant difference test (P<0.05). RESULTS The highest mean was observed in the proximal box group with the metal crown (14.59), and the lowest in the group with 20° zirconia crowns (9.12). Within groups, the highest retentive values were found for the 20° taper with proximal box preparation; the lowest was for the 20° taper group. CONCLUSIONS Within the study limitations, it could be concluded that incorporating a proximal box or buccal groove in short tooth preparations with an increased total occlusal convergence improved retentive values.

An invitro analysis of the accuracy of static and robot-assisted implant surgery.

Robot-assisted implant surgery (RAIS) is purported to improve the accuracy of implant placement. The objective of this study was to compare RAIS with static computer-assisted implant surgery (sCAIS) in a controlled environment. A total of n = 102 implants were placed in the same modified typodont (n = 17 repeated simulated implant surgeries with each n = 3 implants per group) using robot-assisted or static computer-assisted implant surgery. The final implant positions were digitized utilizing cone-beam tomography and compared with the planned position. The angular deviation was the primary outcome parameter. 3D deviations at the implant platform level and the apex were secondary outcome parameters. Accuracy in terms of trueness and precision were assessed. Means, standard deviation, and 95%-confidence intervals were analyzed statistically. The overall angular deviation was 2.66 ± 1.83° for the robotic system and 0.68 ± 0.38° for guided surgery using static guides (p < .001), the 3D-deviation of the implant platform at crest level was for sCAIS 0.79 ± 0.28 mm and RAIS 1.51 ± 0.53 mm (p < .001) and at the apex for sCAIS 0.82 ± 0.26 mm and for RAIS 1.97 ± 0.79 mm (p < .001), respectively. Robotically guided implant surgery was less accurate in terms of trueness (planned vs. actual position) and precision (deviations among implants) than traditional static computer-assisted implant surgery in this invitro study.

The influence of guide stabilizers and their application sequences on trueness and precision of surgical guides in free end situations: An invitro analysis.

To evaluate the impact of guide stabilizers and their application sequences on implant placement accuracy of guided implant surgery in multiple teeth loss at free end. In this study, 96 implants were placed in the regions of #34, #36, and #37 of 32 identical mandibular models. The influence of using guide stabilizers or not (group A and group B) and various guide stabilizers application sequences (group B: #34 → #36 → #37; group C: #36 → #34 → #37; group D: #37 → #34 → #36) on implant placement trueness and precision was investigated. Data were analyzed using T-tests and one-way ANOVA. Group B showed significant benefits in enhancing implant placement precision. Compared to group A, it resulted in reducing 3D-deviation at crest and 2D deviation in vestibular-oral direction at both crest and apex. Furthermore, group D demonstrated greater improvement in global implant placement precision by reducing 2D deviation in mesial-distal direction at both crest and apex. Among the three different stabilizer application sequences, group D exhibited the highest level of implant placement precision. In cases of missing teeth at distal free end, the use of guide stabilizers and their application sequences does not have a significant impact on implant placement trueness. However, they do improve implant placement precision compared to methods that do not utilize guide stabilizers. Specifically, applying a guide stabilizer first at the furthest implant site to change teeth loss classification from free end to edentulous space with posterior support is the most reliable sequence.

Invitro analysis of antifungal effects of botanicals on sclerotinia sclerotiorum causing white mold disease

White mold, Sclerotinia sclerotiorum, is a devastating fungal plant pathogen that has affected many crop species worldwide. Using chemicals to control the disease has been practiced over the years, whose prolonged application has negatively impacted the environment, thus finding an organic solution is crucial. The analysis quantifies the effect of 5 different local plants that have been proven to have fungicidal properties; Artemisia vulgaris L., Azadirachta indica L., Zingiber officinale Roscoe, Allium sativum L., and Lantana camara L. Poisoned food technique was used to study the inhibition effect, carried out by inoculating and growing the fungus on PDA media infused with botanical extracts. The data of mycelial mat diameter was recorded till the control plates were fully occupied. The growth-inhibiting capacity was found as 100% by Allium sativum, 28% &amp; 43% by Azadirachta indica, 22.44% &amp; 44% by Zingiber officinale, and 16.33% &amp; 8.55% by Lantana camara at 10% and 20% concentrations respectively. Only a slight difference between the overall inhibition effect of the two concentrations was found with 37.22% inhibition by 20% concentration and 33.38% inhibition by 10% concentration. No inhibition effect was observed from Artemisia vulgaris which could be due to heat neutralization of the active constituent during sterilization. Further research needs to be conducted using the botanical with different sterilization techniques. This in-vitro study identified garlic as a critical antifungal alternative to conventional fungicides. Field experiments need to be done to prove its effectiveness.

Evaluating Enterococcus faecium9 N-2 as a probiotic candidate from traditional village white cheese.

In this study, various functional and probiotic attributes of the Enterococcus faecium 9 N-2 strain isolated from village-style white cheese were characterized, while also assessing its safety. To achieve this, we conducted an invitro analysis of several key probiotic properties exhibited by the 9 N-2 strain. Notably, this strain demonstrated robust resilience to low pH, high bile salt concentrations, lysozyme, pepsin, pancreatin, and phenol. Furthermore, this strain displayed exceptional auto-aggregation capabilities and moderate co-aggregation tendencies when interacting with Escherichia coli. The cell-free supernatant derived from strain 9 N-2 exhibited significant antimicrobial activity against the tested pathogens. The strain exhibited resistance to gentamicin, meropenem, and bacitracin, while remaining susceptible to vancomycin and various other antibiotics. Furthermore, it was found that E. faecium 9 N-2 possessed the capacity to produce the phytase enzyme. When all the results of this study are evaluated, it is thought that 9 N-2 strain has superior probiotic properties, and therefore it can be used as probiotic in food, medicine, and animal feed in the future. In addition, further invivo tests should be performed to fully understand its effects and mechanisms of action and to confirm its safety and probiotic effects. Further research and clinical trials are also needed to identify new strains with potential probiotic properties.

Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1+ VP2)/VP3= 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1+ VP2)/VP3= 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. Invitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.