Microplate Research Articles

Empagliflozin attenuates epithelial-to-mesenchymal transition through senescence in peritoneal dialysis.

Epithelial-to-mesenchymal transition (EMT) is considered as one of the senescence processes; reportedly, antisenescence therapies effectively reduce EMT. Some models have shown antisenescence effects with the use of sodium-glucose cotransporter 2 (SGLT2) inhibitor. Therefore, our study investigated the antisenescence effects of empagliflozin as an SGLT2 inhibitor in a peritoneal fibrosis model and their impact on EMT inhibition. For in vitro study, human peritoneal mesothelial cells (HPMCs) were isolated and grown in a 96-well plate. The cell media were exchanged with serum-free M199 medium with d-glucose, with or without empagliflozin. All animal experiments were carried out in male mice. Mice were randomly classified into three treatment groups based on peritoneal dialysis (PD) or empagliflozin. We evaluated changes in senescence and EMT markers in HPMCs and PD model. HPMCs treated with glucose transformed from cobblestone to spindle shape, resulting in EMT. Empagliflozin attenuated these morphological changes. Reactive oxygen species production, DNA damage, senescence, and EMT markers were increased by glucose treatment; however, cotreatment with glucose and empagliflozin attenuated these changes. For the mice with PD, an increase in thickness, collagen deposition, staining for senescence, or EMT markers of the parietal peritoneum was observed, which, however, was attenuated by cotreatment with empagliflozin. p53, p21, and p16 increased in mice with PD compared with those in the control group; however, these changes were decreased by empagliflozin. In conclusion, empagliflozin effectively attenuated glucose-induced EMT in HPMCs through a decrease in senescence. Cotreatment with empagliflozin improved peritoneal thickness and fibrosis in PD.NEW & NOTEWORTHY Epithelial-to-mesenchymal transition (EMT) is considered one of the senescence processes. Antisenescence therapies may effectively reduce EMT in peritoneal dialysis models. Human peritoneal mesothelial cells treated with glucose show an increase in senescence and EMT markers; however, empagliflozin attenuates these changes. Mice undergoing peritoneal dialysis exhibit increased senescence and EMT markers, which are decreased by empagliflozin. These findings suggest that empagliflozin may emerge as a novel strategy for prevention or treatment of peritoneal fibrosis.

Viral adaptations to alternative hosts in the honey bee pathogen Paenibacillus larvae.

Bacteriophages (phages) that are intended to be used to treat bacterial infections are often improved using genetic engineering or experimental evolution. A protocol called "Appelmans" utilizes evolution in microtiter plates to promote the evolution of phages that can infect nonpermissive hosts. We tested a modification of the Appelmans protocol using the honey bee pathogen, Paenibacillus larvae. Three phages evolved together on four P. larvae strains following the standard Appelmans protocol and a modified version to ensure high phage diversity throughout ten rounds of passaging. The host range of 360 plaques were characterized and six new phage lysis patterns were identified. These new phage lysis patterns included plaque formation on previously nonpermissive, phage-resistant isolates that were used to identify phage types. The modified protocol did not drastically change the rate or number of new phage types observed but did prevent the phage population from being dominated by one phage that tended to rapidly raise in frequency. These findings showed how a minor modification of the Appelmans protocol influenced the development of phages for phage therapy. The method also provided improved phages for the treatment of bacterial infections in honey bees.

The Inhibition of Cyanobacteria Spirulina Extract on S. Mutans Biofilm as an Ingredient in Mouthwash

Background: The main cause of dental caries is Streptococcus mutans (S. mutans), to suppress the growth of cariogenic bacteria can be done by using mouthwash containing antibacterial ingredients such as chlorhexidine. Cyanobacteria spirulina extract contains compounds that have secondary metabolites which act as antibacterial and antioxidant including tannins, flavonoids, saponins and terpenoids. Objective: This study aimed to find out he inhibition of Cyanobacteria spirulina against S. mutans biofilm. Materials and Methods: The research sample consisted of 6 groups. Group K(-) was the S. mutans biofilm with aquadest, K(+) was Chlorhexidine 0.2% and four treatment groups (P1, P2, P3, P4), which were Cyanobacteria spirulina extract with a concentration of 60 , 70, 80 and 90 mg/ml. The sample was applied to a well microtiter plate, then incubated overnight at 37°C for 1 x 24 hours, then painted with 0.1% crystal violet. After that, it was rinsed with sterile distilled water, then given 0.2 mL of Tween 80 concentration of 2% and measured Optical Density with an ELISA Reader with a wavelength of 570 nm, then biofilm inhibition percentage as calculated. Results: The mean value of the percentage of S. mutans biofilm inhibition in group K(+): -131,694(SD 10,758) ; P1: -12,234(SD 2,402) ; P2: 11,076(SD 6,387) ; P3: 19.7020(SD 11,670) ; P4: 40,214(12,057) there were significant differences between P2 and P4 also between P3 and P4. Conclusion: Cyanobacteria spirulina at concentrations of 70, 80 and 90 mg/ml had S. mutans biofilm inhibition, while Chlorhexidine 0.2% and Cyanobacteria spirulina 60 mg/ml did not.

Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

Investigating the effect of resveratrol on the survival rate of 4T1 breast cancer cells by MTT method

Cancer is known as an incurable, deadly and deadly disease with pain and disability. During recent decades, changes in people's lifestyles have increased the incidence and prevalence of breast cancer worldwide. With the wide spread of this disease, research has also increased on this issue. Resveratrol is known as a natural polyphenol found in red grapes and some other foods, and with its important anti-cancer properties, it can be effective in reducing the risk of breast cancer and its treatment. Research has shown that resveratrol can have positive effects on breast cancer by reducing DNA methylation, reducing cell proliferation, and reducing cancer growth factors. In this research, resveratrol has been used against 4T1 cancer cells, which were treated with different concentrations of resveratrol after cultivation in 96-well plates and measured by the MTT method. The obtained results showed that resveratrol in all concentrations and at different times significantly reduces the survival of 4T1 cancer cells. By increasing the concentration and treatment time, the decrease in cell viability increases. This indicates strong, dose- and time-dependent effects of resveratrol on breast cancer cells.

A high-throughput sample preparation approach for the determination of nonsteroidal anti-inflammatory drugs in urine samples through membrane-based microextraction employing a green supramolecular solvent

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs with antipyretic, analgesic, and anti-inflammatory effects widely used worldwide. This study aimed at developing an environmentally friendly and high-throughput sample preparation method for the determination of six NSAIDs in urine samples followed by high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD). The experimental workflow consisted of hollow-fiber microporous membrane liquid-liquid extraction (HF-MMLLE) using a supramolecular solvent immobilized into the porous of polypropylene hollow membranes (1 cm length). These membranes were attached to a 96-well plate system to permit multiple extractions simultaneously. Under the optimized conditions, limits of detection (LODs) and limits of quantification (LOQs) ranged from 6.06 to 30.3 µg/L and from 20 to 100 µg/L, respectively. The linear ranges varied from 20 to 1000 µg/L for indomethacin and diclofenac, from 50 to 1000 µg/L for ketoprofen and ibuprofen, and from 100 to 1000 µg/L for naproxen and fenoprofen. The coefficients of determination (R²) ranged from 0.9735 to 0.9997. Intraday precision ranged from 6.13 to 20.11 %, interday precision varied from 2.87 to 20.72 %, and accuracy ranged from 92.49 to 126.89 %. Moreover, the recently proposed Sample Preparation Metric of Sustainability (SPMS) was employed to evaluate the greenness of the sample preparation technique resulting in a score of 8.0, which is highly satisfactory according to the concepts of green analytical chemistry.

Assessment of mrkD and T1 fim Genes of K. pneumoniae and Their Association with Biofilm Formation in Community-Acquired Infections

Background: Klebsiella pneumoniae is a pathogenic bacteria that can form biofilms on abiotic and biotic surfaces. The constraints of the therapeutic choices against K. pneumoniae arise from its innate capacity to develop biofilm and harboring determinants of multidrug resistance. Objective: The current study aimed to assess the prevalence of some fimbrial genes (type 1 and type 3) of K. pneumoniae strains in community-acquired infections obtained from various clinical sources on their ability to produce biofilm. Methods: A total of 256 samples (106 male and 150 females) were taken from various clinical sources including Urine, wound swabs, sputum, burn swabs, bronchial wash, diabetic foot ulcer, and higher vaginal swabs) from outpatients in Kirkuk City hospitals. The isolates were identified on the Blood agar, MacConkey agar and Eosin Methylene Blue agar based on the cultural, morphological, in addition to biochemical assays, and confirmed using the automated VITEK 2 system. The Microtiter plate (MTP) method was employed to detect Biofilm formation. Subsequently, a polymerase chain reaction was performed to determine the presence of the T1 fim and mrkD genes. Result: Out of 256 only 38 (14.8%) isolates belonged to Klebsiella pneumoniae. Of all isolates, 38(100%) were biofilm producers. The PCR results revealed that all 38 isolates showed positive (100%) for T1 fim and 37 isolates (97.4%) for mrkD.

Association of the vitamin D metabolism gene polymorphism with the severity of coronary lesions assessed by SYNTAX score

This study aimed to determine the association of vitamin D serum blood levels and vitamin D gene polymorphism with the severity of coronary lesions in patients with stable coronary artery disease (CAD). Material and methods. 260 patients with stable CAD (average age was 58 years) were examined in the presented research. All patients were divided into two groups according to the SYNTAX score: low-risk patients with SYNTAX score ≤ 31 (n = 224) and high-risk patients with SYNTAX score > 31 (n = 36). For enzyme-linked immunosorbent assay and genetic analysis, peripheral blood was collected from the cubital vein into vacuum tubes containing coagulation activator and K3-EDTA, respectively. Serum blood level of 25-hydroxyvitamin D (DiaSource Diagnostics, Belgium) and 1,25-dihydroxyvitamin D (Immunodiagnostic Systems, Great Britain) were determined by enzyme-linked immunosorbent assay according to the manufacturers’ protocols. Genomic DNA was isolated by phenol-chloroform extraction method from whole blood. The quality and quantity of isolated DNA were assessed using NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). Five polymorphic variants in the VDR (rs2228570 and rs73123) and GC (rs7041, rs1155563 and rs2298849) genes were selected for analysis. Genotyping was performed by real-time PCR in a 96-well plate with fluorescently labeled TaqMan probes. The quality of PCR was controlled by repeated genotyping of 10 % of the analyzed samples. Results. We found no statistically significant differences in serum blood level of the studied markers in patients from low-risk and high-risk groups. One polymorphic variant in the GC gene associated with the multiple coronary lesions (rs2298849) (odds ratio 2.26, 95 % confidence interval 1.28–3.99, p = 0.006) according to an additive inheritance model was identified. In addition, we determined the association between low serum blood level of 1,25-dihydroxyvitamin D in patients with CAD with multiple lesions of the coronary vascular system with A/A – A/G genotypes of the rs2228570 polymorphism in the VDR gene, A/A genotype of the rs7041 polymorphism and A/A genotype of the rs2298849 polymorphism in the GC gene. Conclusions. Allelic variants in the vitamin D metabolism genes are associated with the degree of coronary artery lesions assessed by the SYNTAX score in patients with stable CAD. Also, serum blood level of the active form of vitamin D (1,25-dihydroxyvitamin D) is less in carriers of homozygous genotypes for the major alleles of the VDR and GC genes.

Molecular Evaluation of Aminoglycoside Resistance and Biofilm Formation Potential in Escherichia coli Isolates Collected from Hospitalized Patients

Background: Resistance to antibiotics and the ability to develop biofilms, two main virulence determinants of Escherichia coli, play a crucial role in the persistence of infections. Objectives: The aim of this study was to evaluate aminoglycoside resistance and biofilm formation potential in E. coli isolates collected from hospitalized patients in the Southwest of Iran. Methods: A total of 70 E. coli clinical isolates from different specimens were collected from Ahvaz teaching hospitals affiliated with Ahvaz Jundishapur University of Medical Sciences. All the isolates were identified as E. coli using conventional microbiological tests. Susceptibility to antibiotics was determined using the Kirby–Bauer disk diffusion method. Biofilm formation was assessed using the microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants, including fimbrial genes, aminoglycoside modifying enzymes (AMEs), and 16S rRNA methylase (RMTase) genes. Results: Among aminoglycoside antibiotics, E. coli isolates showed the highest and lowest resistance rates to tobramycin (TOB; 51.4%) and gentamicin (GEN; 24.2%), respectively. Simultaneous resistance to GEN, amikacin, and TOB was observed in 28.5% of the isolates, representing the most common antibiotic resistance pattern. The prevalence of strong biofilm producers was higher in the extensively drug-resistant (XDR) phenotype group compared to the multiple drug-resistant (MDR) group (76.1% vs. 23.8%). Among the 36 isolates resistant to at least one of the aminoglycoside antibiotics, 36.1% had AME-related genes, either alone or in various combinations. Most isolates harboring AME genes were also positive for the presence of biofilm-related genes, including ecpA and fimA. Conclusions: The most frequent AME-related genes were ant(2”)-Ia and aph(3’)-Ia, followed by aac(3’)-IIa. The findings of the present study provide probable evidence that GEN is an effective aminoglycoside against biofilm-producing and antibiotic-resistant E. coli isolates.

Synthesis of Graphene@C3N4-Cu Beads Nanocomposites and their Antimicrobial Efficacy Against Drug-Resistant Bacteria and Fungi.

Increased intake of drugs worldwide and the subsequent advent of resistance to existing antibiotics have globally threatened health organizations. To combat the problem of these drug-resistant infections, as an alternative approach, graphene (GN)-related nanomaterials have attracted significant interest because of their effective anti-microbial potential. The present study shows the synthesis and characterization of nanocomposite of GN with carbon nitride viz. g- C3N4, g-C3N4-Cu, and GN@g-C3N4-Cu. Further, we investigated the anti-microbial potential of these nanocomposites against strains of Gram-negative and Gram-positive bacteria, viz., a multidrug- resistant strain of Pseudomonas aeruginosa (MDRPA), a methicillin-resistant strain of Staphylococcus aureus ATCC33593 (MRSA), and an azole-sensitive fungal strain (Candida albicans ATCC14053). The morphological characterization of GN@g-C3N4-Cu nanocomposite was executed by scanning electron microscopy, whereas the elemental analysis and their distribution were studied by energy-dispersive X-ray spectroscopy and elemental mapping methods. Furthermore, the anti-microbial and antibiofilm efficacies of g-C3N4, g-C3N4-Cu, and GN@g-C3N4-Cu nanocomposites were evaluated by disc diffusion, two-fold serial micro broth dilution, and 96 well microtiter plate methods. The ternary g-C3N4-Cu@GN, apart from the structures of g-C3N4-Cu, showed big sheets of GN. The observance of C, N, O, and Cu in the elemental analysis, as well as their uniform distribution in the mapping, indicated the successful fabrication of g-C3N4-Cu@GN. GN@g-C3N4-Cu followed by g-C3N4-Cu and (g-C3N4) exhibited significantly higher antimicrobial activity (zone of inhibition from 14.33 to 49.33 mm) against both the drug-resistant bacterial strains and azole-sensitive C. albicans. MICs of nanocomposites ranged from 32 -256 μg/ml against the tested strains. Whereas all three nanocomposites at sub-MICs (0.25 A- and 0.5 A- MICs) showed concentration- dependent inhibition of biofilm formation in MDRPA, MRSA, and C. albicans by allowing 11.35% to 32.59% biofilm formation. Our study highlights the enhanced efficiency of GN@g-C3N4-Cu nanocomposites as potential anti-microbial and antibiofilm agents to overcome the challenges of multi-drug-resistant bacteria and azole-sensitive fungi. Such kind of nanocomposites could be used to prevent nosocomial infections if coated on medical devices and food manufacturing instruments.

Magnetically Integrated Tumor-Vascular Interface System to Mimic Pro-angiogenic Endothelial Dysregulations for On-Chip Drug Testing.

The tumor-vascular interface is a critical component of the tumor microenvironment that regulates all of the dynamic interactions between a growing tumor and the endothelial lining of the surrounding vasculature. In this paper, we report the design and development of a custom-engineered tumor-vascular interface system for investigating the early stage tumor-mediated pro-angiogenic dysfunctional behavior of the endothelium. Using representative endothelial cells and triple negative breast cancer cell lines, we established a biomimetic interface between a three-dimensional tumor tissue across a mature, functional endothelial barrier using a magnetically hybrid-integrated tumor-vascular interface system, wherein vasculature-like features containing a monolayer of endothelial cell culture on porous microfluidic channel surfaces were magnetically attached to tumor spheroids generated on a composite polymer-hydrogel microwell plate and embedded in a collagen matrix. Tumor-mediated endothelial microdynamics were characterized by their hallmark behavior such as loss of endothelial adherens junctions, increased cell density, proliferation, and changes in cell spreading and corroborated with endothelial YAP/TAZ nuclear translocation. We further confirm the feasibility of drug-mediated reversal of this pro-angiogenic endothelial organization through two different signaling mechanisms, namely, inhibition of the vascular endothelial growth factor pathway and the Notch signaling pathway, thereby demonstrating the utility of the tumor-vascular interface platform for rapid, early stage prediction of antiangiogenic drug efficacy. Overall, our work emphasizes the importance of our strategic engineering approach for identifying some unique, physiologically relevant aspects of the tumor-vascular interface, which are otherwise difficult to implement using standard in vitro approaches.

Characterization of Clonal Groups of Antibiotic-Resistant Biofilm-Forming Staphylococcus aureus Strains Isolated from Patients with Urinary Tract Infections in Isfahan, Iran

Background: Over the past decades, the role of biofilm-forming Staphylococcus aureus strains in urinary tract infections (UTIs) has garnered significant attention. Objectives: This study aimed to determine the epidemiological characteristics and diversity of S. aureus strains isolated from patients with UTIs in Isfahan, Iran, in 2017, with regard to their antimicrobial resistance, biofilm formation, and phylogenetic profiles. Additionally, the study investigated potential relationships among these factors statistically to develop efficient control and treatment approaches. Methods: All patients with symptomatic UTIs who had positive urine cultures for S. aureus during the study period at the laboratory of a referral hospital in Isfahan were included. All isolates were identified using specific primers for the nucA gene. Their biofilm formation capacity was evaluated using a combination of the microtiter plate and Congo-red agar methods. Antibiotic susceptibility testing was performed using the disk diffusion method. The presence of genes involved in biofilm formation and resistance to cefoxitin, aminoglycosides, and fluoroquinolones was detected using polymerase chain reaction (PCR). Staphylococcal cassette chromosome mec (SCCmec) typing, agr typing, and phene plate (PhP) typing were employed to investigate the diversity of collected strains. Results: Results showed that 19%, 57%, and 24% of confirmed S. aureus strains were strong, intermediate, and non-biofilm formers, respectively. The highest rate of resistance was against nalidixic acid (77%), followed by streptomycin (73%). The icaD and icaA genes had the highest frequency among biofilm-producing strains. gyrA (44%) and grlA (35%) were the most frequent genes among fluoroquinolone-resistant strains, while aph(3′)-IIIa was the most prevalent aminoglycoside-modifying enzyme gene. The majority of bacterial strains harbored SCCmec type III and agr type I. PhP typing of strains revealed the presence of 8 common types (CTs) and 14 single types (STs), with CT2 being the dominant type. Conclusions: The present investigation revealed various biofilm production capacities, antimicrobial resistance profiles, and clonal lineages in S. aureus isolated from patients with UTIs. These findings provide further insights into the epidemiology and pathogenicity of S. aureus strains in Iran, thereby improving the quality of surveillance and therapeutic protocols.

Myrtle Essential Oil-Loaded Alginate Nanoparticles: A Dual Therapeutic Approach for Cytotoxicity Against Skin Cancer Cells and Antibacterial Effects on Common Pathogens

Background & Objectives: The skin, being the body’s largest organ, is not only susceptible to one of the most prevalent forms of cancer but also vulnerable to a myriad of pathogens, including bacteria. Myrtle essential oil (EO) has been shown to possess both anticancer and antimicrobial properties. This study aimed to investigate the efficacy of alginate nanoparticles containing myrtle EO on melanoma (A375) and epidermoid carcinoma (A431) cell lines, as well as on some common bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Materials & Methods: Initially, myrtle EO was analyzed using Gas Chromatography-Mass Spectrometry. Subsequently, alginate nanoparticles were prepared via the ionic-gelation method and characterized by dynamic light scattering, Zeta potential, and attenuated total reflectance-Fourier transform infrared spectroscopy. Encapsulation efficacy was then determined using UV-Vis spectrometry. Both cytotoxicity assessment (MTT assay) and evaluation of antibacterial effects (microdilution assays) were conducted using the 96-well plate format. Results: The major compounds identified in myrtle EO were α-pinene, 1,8-cineole, linalool, linalool acetate, and geranyl acetate. The alginate nanoparticles exhibited a size of 160 ± 9 nm, a SPAN of 0.96, and a Zeta potential of -26 ± 2 mV. The encapsulation efficacy was determined to be 78.4%. It was found that the nanoparticles demonstrated cytotoxicity against A375 and A431 cells with IC50 values of 211 μg/mL and 308 μg/mL, respectively. The most potent antibacterial effect was observed against S. aureus (IC50: 266 μg/mL). Conclusion: The alginate nanoparticles containing myrtle EO, which exhibited notable cytotoxicity against melanoma and epidermoid carcinoma cells as well as potent antibacterial effects, show promise for further investigation in vivo studies.

An In Vitro Orbital Flow Model to Study Mechanical Loading Effects on Osteoblasts.

Flow induced by an orbital shaker is known to produce shear stress and oscillatory flow, but the utility of this model for studying mechanical loading effects in osteoblasts is not well defined. To test this, osteoblasts derived from the long bones of adult male C57BL/6J mice were plated on 6-well plates and subjected to orbital shaking at various frequencies (0.7, 1.4, and 3.3 Hz) for 30 and 60 min in serum-free differentiation media. The shear stress on cells produced by 0.7, 1.4, and 3.3 Hz shaking frequencies were 1.6, 4.5, and 11.8 dynes/cm2, respectively. ALP activity measured 72 h after shaking (orbital flow) showed a significant increase at 0.7 and 1.4 Hz, but not at 3.3 Hz, compared to static controls. Orbital flow-induced mechanical stress also significantly increased (25%) osteoblast proliferation at a 0.7 Hz flow compared to static controls. Additionally, expression levels of bone formation markers Osf2, Hif1a, Vegf, and Cox2 were significantly increased (1.5- to 3-fold, p < 0.05) in cells subjected to a 0.7 Hz flow compared to non-loaded control cells. We also evaluated the effect of orbital flow on key signaling pathways (mTOR, JNK, and WNT) known to mediate mechanical strain effects on osteoblasts. We found that blocking mTOR and WNT signaling with inhibitors significantly reduced (20-30%) orbital flow-induced ALP activity compared to cells treated using a vehicle. In contrast, inhibition of JNK signaling did not affect flow-induced osteoblast differentiation. In conclusion, our findings show that the flow produced by an orbital shaker at a lower frequency is an appropriate inexpensive model for studying the molecular pathways mediating mechanical strain effects on primary cultures of osteoblasts in vitro.

Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture.

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.

PhagoScreener: A novel phagogram platform based on a capillary-wave microbioreactor

Due to the overuse of antibiotics, the number of multidrug-resistant pathogen bacteria is rising in recent years posing a serious threat to human health. One promising alternative for treatment is the application of phage therapy using highly selective bacteriophages. Because of their selectivity, individual screens called phagograms for each patient are required to select phages from a phage library. Phagograms are mostly performed via bacterial cultivation on double layer agar plates and phage addition causing bacterial lysis. However, these assays are work-intensive and have a low ability for parallelization and automation. Hence, highly parallelizable and automatable microbioreactors in the lowest microliter scale could offer an economic solution increasing the throughput of phagograms. This paper demonstrates the applicability of a novel capillary-wave microbioreactor (cwMBR) to perform phagograms. Due to its small volume of only 7 µL and the open-droplet design, it can be easily automated and parallelized in future. Furthermore, the ability of online biomass measurement makes the cwMBR a perfect phagogram platform in the future. Herein, phagograms with E. coli and different concentrations of the phages MM02 and EASG3 were performed as proof of concept for phagograms in the cwMBR. Thereby, the cwMBR was able to measure differences in lysis kinetics of different phages. Furthermore, the phagograms were compared to those in conventional microtiter plate readers revealing the cwMBR as ideal alternative for phagograms as it combines favorable mixing conditions and a phage repellent hydrophilic glass surface with online biomass measurement in an open-droplet design for future parallelization and automation.

High-throughput analysis of topographical cues for the expansion of murine pluripotent stem cells

The expansion of pluripotent stem cells (PSCs)in vitroremains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markersOct4, Nanog, andSox2and the differentiation markerLmnAas well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1µm pillars (1µm spacing, square arrangement), 2µm wells (c-c (x, y) = 4, 4µm), and 5µm pillars (c-c (x, y) = 7.5, 7.5µm). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion.

The effect of soaking heat-polymerized acrylic resin denture base in avocado seed extract (Persea americana Mill.) on the inhibition of denture-plaque microorganisms biofilm growth

Background Heat polymerized acrylic (HPA) resins are known to have high porosity that contributes to increased surface roughness and microcrack formation in stress areas. This facilitates the attachment and growth of polymicrobial biofilms contributing to increased antimicrobial resistance. Many research had been carried out on avocado seeds, but no research that studies the effect of avocado seeds on denture-plaque microorganism biofilm on HPA resin has been found. Methods This study used 144 samples (n=144), namely HPA resin discs covered with mono-species and polymicrobial biofilms consisting of Candida albicans, Candida glabrata, Actinomyces odontolyticus, Streptococcus gordonii, and Staphylococcus aureus. The discs were soaked for 8 hours in the 5%, 10%, 15%, 20% avocado seed extract, positive control (alkaline peroxide), and negative control (aquadest). Each disc was shaken with a vortex mixer for 1 minute, and 100 μL was added into 96-well microplates with three times repetition and incubated for 24 hours. The inhibition values were determined from the percentage inhibition value formula which required absorption values from a microplate reader (595 nm). Results In this research, it was found that the MBIC50 of avocado seed extract against the mono-species of C. albicans (5%), C. glabrata (5%), A. odontolyticus (15%), S. gordonii (15%), S. aureus (10%), while against the biofilm was 20%. There was a significant effect of soaking HPA resin in avocado seed extract of 5%, 10%, 15%, 20% on the inhibition of mono-species and polymicrobial biofilms of denture-plaque microorganisms with a value of p&lt;0.001 (p&lt;0.05). Conclusion The MBIC50 of avocado seed extract in polymicrobial biofilm group was higher than that in the mono-species biofilm groups. Although alkaline peroxide showed higher inhibition value than that of the MBIC50 in polymicrobial biofilm group, 20% avocado seed extract was effective in inhibiting polymicrobial biofilm because it was able to inhibit more than 50% polymicrobial biofilm.

Evaluating the antibacterial, antibiofilm, and anti-toxigenic effects of postbiotics from lactic acid bacteria on Clostridium difficile.

The most common cause of healthcare-associated diarrhea is Clostridium difficile infection (CDI), which causes severe and recurring symptoms. The increase of antibiotic-resistant C. difficile requires alternate treatments. Postbiotics, metabolites produced by probiotics, fight CDI owing to their antibacterial capabilities. This study aims to evaluate the antibacterial, antibiofilm, and anti-toxigenic potential of postbiotics in combating CDI. GC-MS evaluated postbiotics from Bifidobacterium bifidum and Lactobacillus plantarum. Disk diffusion and broth microdilution determined C. difficile antibacterial inhibition zones and MICs. Microtiter plates assessed antibiofilm activity. MTT assay evaluated postbiotics anti-viability on HEK293. ELISA testing postbiotic detoxification of toxins A and B. Postbiotics were examined for tcdA and tcdB genes expression using real-time PCR. The most identified B. bifidum and L. plantarum postbiotic compounds were glycolic acid (7.2%) and butyric acid (13.57%). B. bifidum and L. plantarum displayed 13 and 10 mm inhibition zones and 2.5 and 5 mg/ml MICs against C. difficile. B. bifidum reduced biofilm at 1.25 mg/ml by 49% and L. plantarum by 31%. MTT assay showed both postbiotics had little influence on cell viability, which was over 80%. The detoxification power of postbiotics revealed that B. bifidum decreased toxin A and B production more effectively than L. plantarum, and also their related tcdA and tcdB genes expression reduction were statistically significant (p < 0.05). Postbiotics' ability to inhibit bacterial growth, biofilm disruption, and toxin reduction makes them a promising adjunctive for CDI treatment and a good solution to pathogens' antibiotic resistance.

Enabling Modular Click Chemistry Library through Sequential Ligations of Carboxylic Acids and Amines

AbstractHigh‐throughput synthesis and screening of chemical libraries play pivotal roles in drug discovery. Click chemistry has emerged as a powerful strategy for constructing highly modular chemical libraries. However, the development of new click reactions and unlocking new clickable building blocks remain exceedingly challenging. Herein, we describe a double‐click strategy that enables the sequential ligations of widely available carboxylic acids and amines with fluorosulfuryl isocyanate (FSO2NCO) via a modular amidation/SuFEx (sulfur‐fluoride exchange) process. This method provides facile access to chemical libraries of N‐fluorosulfonyl amides (RCONHSO2F) and N‐acylsulfamides (RCONHSO2NR′R′′) in near‐quantitative yields under simple and practical conditions. The robustness and efficiency of this double click strategy is showcased by the facile construction of chemical libraries in 96‐well microtiter plates from a large number of carboxylic acids and amines. Preliminary biological activity screening reveals that some compounds exhibit high antimicrobial activities against Gram‐positive bacterium S. aureus and drug‐resistant MRSA (MIC up to 6.25 μg ⋅ mL−1). These results provide compelling evidence for the potential application of modular click chemistry library as an enabling technology in high‐throughput medicinal chemistry.