Tissue Culture Plates Research Articles

Antisenescence Expansion of Mesenchymal Stem Cells Using Piezoelectric β-Poly(vinylidene fluoride) Film-Based Culture.

Regenerative therapies based on mesenchymal stem cells (MSCs) show promise in treating a wide range of disorders. However, the replicative senescence of MSCs during in vitro expansion poses a challenge to obtaining a substantial quantity of high-quality MSCs. In this investigation, a piezoelectric β-poly(vinylidene fluoride) film-based culture plate (β-CP) was developed with an antisenescence effect on cultured human umbilical cord-derived MSCs. Compared to traditional tissue culture plates (TCPs) and α-poly(vinylidene fluoride) film-based culture plates, the senescence markers of p21, p53, interleukin-6 and insulin-like growth factor-binding protein-7, stemness markers of OCT4 and NANOG, and telomere length of MSCs cultured on β-CPs were significantly improved. Additionally, MSCs at passage 18 cultured on β-CPs showed significantly better multipotency and pro-angiogenic capacities in vitro, and higher wound healing abilities in a mouse model. Mechanistically, β-CPs rejuvenated senescent MSCs by improving mitochondrial functions and mitigating oxidative and glycoxidative stresses. Overall, this study presents β-CPs as a promising approach for efficient and straightforward antisenescence expansion of MSCs while preserving their stemness, thereby holding great potential for large-scale production of MSCs for clinical application in cell therapies.

Application of Alginate-Collagen Hydrogel and Its Related Mechanisms in the Repair of Rat Cartilage Injuries

This research investigated the application of alginate-collagen hydrogel (ACH) in rat cartilage injury (CI) repair and its related mechanisms through in vitro and in vivo experiments. Alginate solution and modified collagen solution were mixed to prepare the ACH. The obtained samples were characterized and their sustained release properties were determined. L929 mouse fibroblasts were assigned into a blank control group (BC group, untreated), a positive control group (PC group, treated with 6.4% phenol), a negative control group (NC group, treated with tissue culture polystyrene), and an ACH group (treated with ACH). Cell viability was assessed using the MTT assay. Meanwhile, the cytotoxicity of the ACH and its impact on cell proliferation and cell attachment capabilities were analyzed. Thirty 4-week-old specific pathogen-free (SPF) Sprague Dawley (SD) rats were randomly grouped into a chondrocyte group (5 rats, chondrocytes collected and cultured), a cPAGE group (5 rats, chondrocytes treated with ACH), a model group (10 rats, CI model), and a mPAGE group (10 rats, CI model treated with CAH). The influence of CAH on chondrocyte proliferation and the type II collagen (COLII) expression were assessed using the cell counting kit 8 (CCK-8) assay and immunofluorescence staining. Furthermore, the impacts of CAH on chondrocyte tissue and COL-II expression were analyzed using Safranin O-Fast Green (SOFG) staining, the International Cartilage Repair Society (ICRS) score, and COL-II tissue staining. Immunoblotting was utilized to detect the TNF-α, NF-κB, matrix metalloproteinase 13 (MMP-13), and COL-II proteins in cartilage tissue. The results showed that release rate (RR) of the constructed ACH reached over 85% within 72 hours and achieved degradation equilibrium within 5 days with a weight loss (WL) rate of over 60%. L929 fibroblasts culture results showed that the ACH posed no significant cytotoxicity, and cell attachment results were not obviously different from those on tissue culture plates (P >0.05). MTT assay results demonstrated that the ACH exerted no adverse effects on L929 cell proliferation and growth, indicating a good biocompatibility. CCK-8 and COL-II results indicated that chondrocyte proliferation in cPAGE group was much higher in contrast to that in chondrocyte group (P <0.05). Besides, the animal experiments unveiled that in comparison to the CI group, the mPAGE group had more chondrocytes, upregulated COL-II, and higher ICRS scores (P <0.05). MMP-13, NF-κB, and TNF-α were downregulated, and COL-II was upshifted in the mPAGE group, with remarkable differences (P <0.05). In conclusion, ACH exhibited excellent biocompatibility and low cytotoxicity, promoted cell proliferation and growth, facilitated chondrocyte proliferation in rat CI repair, offering a valuable research foundation for CI treatment.

Evaluation of biofilm formation and carbapenem resistance in Klebsiella pneumoniae isolated from clinical samples at a rural hospital in western Uttar Pradesh

ABSTRACT Introduction: Klebsiella pneumoniae commonly causes healthcare-associated infections and shows multidrug resistance. K. pneumoniae can produce biofilm. Carbapenem resistance in K. pneumoniae is due to the production of carbapenemases mainly. This study was done to evaluate the formation of biofilm and carbapenemase resistance in K. pneumoniae isolates. Material and Methods: A total of 110 K. pneumoniae isolated from various clinical samples were taken, the antibiotic susceptibility test was done by the Kirby disk diffusion method, and biofilm detection was done by the tissue culture plate method. All the carbapenem-resistant isolates were confirmed by multiplex real-time PCR (mPCR). Those found positive for any of the carbapenemase genes were tested by the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and ethylenediamine tetraacetic acid (EDTA)-modified carbapenem inactivation method (eCIM). Results: Out of 110 isolates, 66% (72/110) were carbapenem-resistant (suggestive of carbapenemase producers) by Kirby-Bauer disk diffusion but 58% (42/72) of Klebsiella isolates were confirmed for carbapenemase production by mPCR. Maximum number of carbapenemase gene were New Delhi metallo-β-lactamase (NDM) 52% (N = 22), 29% (N = 12) coproducers (NDM+OXA-48), and lowest in oxacillinase (OXA-48), 19% (N = 8). The overall sensitivity of MHT and mCIM+eCIM was 62% and 93%, and specificity was 88% and 97%, respectively. Our study showed that moderate biofilm producers were 51% (N = 56) K. pneumoniae isolates, strong biofilm producers 27% (N = 30), and 22% (N = 30) were weak/non-biofilm producers. We also found the correlation between biofilm formation and carbapenem-resistant K. pneumoniae (CR-KP) genes was statistically significant with a P value of 0.01*<0.05. Conclusion: Most isolates of K. pneumoniae demonstrated a wide range of antibiotic resistance and were biofilm producers. Our results indicated that the combination of mCIM with eCIM showed high sensitivity and specificity to detect CR-KP compared with MHT.

Mechanosensitive Differentiation of Human iPS Cell-Derived Podocytes.

Stem cell fate decisions, including proliferation, differentiation, morphological changes, and viability, are impacted by microenvironmental cues such as physical and biochemical signals. However, the specific impact of matrix elasticity on kidney cell development and function remains less understood due to the lack of models that can closely recapitulate human kidney biology. An established protocol to differentiate podocytes from human-induced pluripotent stem (iPS) cells provides a promising avenue to elucidate the role of matrix elasticity in kidney tissue development and lineage determination. In this study, we synthesized polyacrylamide hydrogels with different stiffnesses and investigated their ability to promote podocyte differentiation and biomolecular characteristics. We found that 3 kPa and 10 kPa hydrogels significantly support the adhesion, differentiation, and viability of podocytes. Differentiating podocytes on a more compliant (0.7 kPa) hydrogel resulted in significant cell loss and detachment. Further investigation of the mechanosensitive proteins yes-associated protein (YAP) and synaptopodin revealed nuanced molecular distinctions in cellular responses to matrix elasticity that may otherwise be overlooked if morphology and cell spreading alone were used as the primary metric for selecting matrices for podocyte differentiation. Specifically, hydrogels with kidney-like rigidities outperformed traditional tissue culture plates at modulating the molecular-level expression of active mechanosensitive proteins critical for podocyte health and function. These findings could guide the development of physiologically relevant platforms for kidney tissue engineering, disease modeling, and mechanistic studies of organ physiology and pathophysiology. Such advances are critical for realizing the full potential of in vitro platforms in accurately predicting human biological responses.

Anti-Müllerian hormone signaling in the ovary involves stromal fibroblasts: a study in humans and mice provides novel insights into the role of ovarian stroma.

What is the involvement of ovarian stroma in the anti-Müllerian hormone (AMH) signaling pathway and which stromal cells are involved? Mouse and human ovaries show high expression of AMH receptor II (AMHR2) in the stromal fibroblasts surrounding the follicles and activation of the post-AMHR2 pathway by recombinant AMH was evidenced by increased phosphorylation of SMAD1,5 and 9, increased expression AMHR2 and upregulation of αSMA, suggesting fibroblast activation to initiate myofibroblast differentiation. AMH secreted by small growing follicles, regulates ovarian activity. It suppresses initial primordial follicle (PMF) recruitment and FSH-dependent growth. AMH signal transduction is mediated by AMHR2, activating intracellular SMAD proteins and other signaling cascades to induce target-gene expression. Although AMHR2 expression has been reported within the follicle unit, there is evidence suggesting it may be identified in the stroma as well. Fresh murine ovaries were extracted from BALB/c mice (6 weeks old; n = 12 and 21 days old; n = 56). Frozen-thawed ovarian fragments were obtained from 10 women, aged 18-35, who had undergone ovarian tissue cryopreservation and donated frozen ovarian tissue for research. Murine (6 weeks old) and human donor ovaries were immunostained for AMHR2 and Collagen 1α/αSMA/VCAM1, with additional vimentin staining in mice. Murine (21 days old) and human donor ovaries were used for fibroblast isolation and subsequent 7-day cultures. Prior to assessing AMH effects on isolated fibroblast culture, purity validation tests were implemented to ensure the absence of epithelial, immune, endothel, granulosa, and theca ovarian cell populations. The fibroblast culture's homogeneity was validated by RT-qPCR and western-blot assays, confirming negativity for E-cadherin, CD31, aromatase, CYP17A1, and positivity for αSMA and vimentin. Fibroblasts were then subjected to rAMH treatment in vitro (200 ng/ml) for 0-72h, with an additional time point of 96 h for human samples, followed by RT-qPCR, western blot, and immunocytochemistry (ICC) for AMHR2 expression. AMHR2 post-receptor signaling was examined by pSMAD1,5,9 levels via western blot. Activated fibroblast marker, αSMA, was assessed via western blot and ICC. Immunostaining of mouse and human ovarian tissue showed that stromal cells around follicles at all developmental stages exhibit high AMHR2 expression, while granulosa cells of growing follicles show considerably lower levels. The majority of these AMHR2-positive stromal cells were identified as fibroblasts (Collagen1α in mice and human; vimentin in mice). RT-qPCR, western blot, and immunostaining were performed on cultured mouse and human fibroblasts, confirming that they consisted of a pure fibroblast population (αSMA/vimentin positive and negative for other cell-type markers). A total of 99.81% (average 28.94 ± 1.34 cells/field in mice) and 100% (average 19.20 ± 1.39 cells/field in human samples) of these fibroblasts expressed AMHR2 (ICC). rAMH treated cultured fibroblasts showed increased pSMAD1,5 and 9 levels, demonstrating the effects of AMH on its downstream signaling pathway. pSMAD1,5 and 9 expression increased, as detected by western blot: 1.92-fold in mice (48 h, P = 0.026) and 2.37-fold in human samples (48 h, P = 0.0002). In addition, rAMH treatment increased AMHR2 protein expression, as observed in ICC (human): a 2.57-fold upregulation of AMHR2 Mean Fluorescence Intensity (MFI) (96 h, P = 0.00036), and western blot, showing a 4.2-fold time-dependent increase (48 h, P = 0.026) in mice and 2.4-fold change (48 h, P = 0.0003) in human donors. Exposure to rAMH affected AMHR2 transcription upregulation, with a 6.48-fold change (72 h, P = 0.0137) in mice and a 7.87-fold change (72 h, P < 0.0001) in humans. rAMH treatment induced fibroblast activation (αSMA positive), demonstrating the dynamic effects of AMH on fibroblast behavior. αSMA expression elevation was detected in ICC with a 2.28-fold MFI increase in humans (96 h, P = 0.000067), and in western blot with a 5.12-fold increase in mice (48 h, P = 0.0345) and a 2.69-fold increase in humans (48 h, P ≤ 0.0001). Activated AMHR2-positive stained fibroblast fractions were solely located around growing follicles, in both human and mice. In addition, a small population of AMHR2-positive stained theca cells (VCAM1 positive) was observed. N/A. Ex vivo, fibroblast gene expression might be changed by adhesion to the tissue-culture plate. Nevertheless, cultured fibroblasts (with and without rAMH) are subjected to the same conditions. Observations or significant differences can therefore be considered reliable. In addition, the presented effect of rAMH on fibroblasts is not directly linked to the known inhibitory effect of AMH on follicle activation. Clarifying the populations of AMH-responsive cells in the ovary provides a foundation for further investigation of the complex AMH signaling across the ovary. The composition of AMH-releasing and -responsive cells can shed light on the communication network between follicles and their environment, which may elucidate the mechanisms behind the AMH inhibitory effect on PMF activation. This work was financially supported by grants from the Kahn Foundation. There are no competing interests in this study. N/A.

Nicotine promotes pathogenic bacterial growth and biofilm formation in peri-implant.

Introduction. Peri-implantitis is a plaque-associated disease that leads to implant loss and arises from bacterial biofilms on the surface of the implant. Smoking is a risk factor for peri-implantitis and impedes treatment effectiveness. Additionally, aryl hydrocarbon receptor (AHR), IL-6, and IL-22 levels are related to peri-implantitis.Aim. We aimed to investigate the effects of nicotine on inflammatory response, bacterial growth and biofilm formation.Hypothesis/Gap Statement. We hypothesized that nicotine promoted pathogenic bacterial growth and biofilm formation, thereby aggravating inflammation.Methodology. The expression of AHR, IL-6 and IL-22 was measured in peri-implant sulci fluid using quantitative PCR and Western blot analyses. The cementum was incubated with bacterial suspension including Porphyromonas gingivalis, Streptococcus sanguinis and Fusobacterium nucleatum and treated with 100, 200, 250 and 300 µg ml-1 nicotine, and then, the absorbance and number of colony-forming units were detected. Biofilm formation was evaluated using the tissue culture plate method and safranin O staining. Carbohydrates and proteins were measured by the phenol-sulfuric acid method and the bicinchoninic acid method, respectively.Results. The results indicated that smoking increased the levels of AHR, IL-6 and IL-22. Functionally, nicotine promoted the growth of P. gingivalis, S. sanguinis and F. nucleatum. Additionally, it promoted the biofilm formation of these bacteria and increased the contents of carbohydrates and proteins.Conclusion. Nicotine promoted bacterial growth and biofilm build-up, suggesting that smoking may aggravate the progression of peri-implantitis.

Salmonella Detection in Food Using a HEK-hTLR5 Reporter Cell-Based Sensor.

The development of a rapid, sensitive, specific method for detecting foodborne pathogens is paramount for supplying safe food to enhance public health safety. Despite the significant improvement in pathogen detection methods, key issues are still associated with rapid methods, such as distinguishing living cells from dead, the pathogenic potential or health risk of the analyte at the time of consumption, the detection limit, and the sample-to-result. Mammalian cell-based assays analyze pathogens' interaction with host cells and are responsive only to live pathogens or active toxins. In this study, a human embryonic kidney (HEK293) cell line expressing Toll-Like Receptor 5 (TLR-5) and chromogenic reporter system (HEK dual hTLR5) was used for the detection of viable Salmonella in a 96-well tissue culture plate. This cell line responds to low concentrations of TLR5 agonist flagellin. Stimulation of TLR5 ligand activates nuclear factor-kB (NF-κB)-linked alkaline phosphatase (AP-1) signaling cascade inducing the production of secreted embryonic alkaline phosphatase (SEAP). With the addition of a ρ-nitrophenyl phosphate as a substrate, a colored end product representing a positive signal is quantified. The assay's specificity was validated with the top 20 Salmonella enterica serovars and 19 non-Salmonella spp. The performance of the assay was also validated with spiked food samples. The total detection time (sample-to-result), including shortened pre-enrichment (4 h) and selective enrichment (4 h) steps with artificially inoculated outbreak-implicated food samples (chicken, peanut kernel, peanut butter, black pepper, mayonnaise, and peach), was 15 h when inoculated at 1-100 CFU/25 g sample. These results show the potential of HEK-DualTM hTLR5 cell-based functional biosensors for the rapid screening of Salmonella.

Mechanomemory of pulmonary fibroblasts demonstrates reversibility of transcriptomics and contraction phenotypes

Fibroblasts are cells responsible for producing extracellular matrix (ECM) components, which provides physical support for organs. Although these mesenchymal cells are responsive to mechanical cues in their environment, the permanence of these mechanophenotypes is not well defined. We investigated the mechanomemory of lung fibroblasts and determined how switching culture conditions modulate cell responses and function. Primary murine lung fibroblasts were isolated and cultured on 2D tissue culture plates or within 3D collagen hydrogels and were then passaged within the same or opposite culture condition to assess changes in gene expression, protein production, fibroblast subpopulation, contractile behavior, and traction forces. Compared to fibroblasts isolated on 2D tissue culture plates, fibroblasts within 3D hydrogels exhibited a decreased activation phenotype including reduced contraction profiles, diminished cell traction forces and decreased αSMA gene expression. Cells initially isolated via 2D culture and then cultured in 3D hydrogels exhibited a reversal in activation phenotype as measured by gene expression and contraction profiles. Bulk RNAseq identified groups of genes that exhibit reversible and non-reversable expression patterns. Overall, these findings indicate that lung fibroblasts have a mechanical memory that is altered by culture condition and can be reversible through precondition of cells within a softer 3D microenvironment.

Effect of viscosity of gelatin methacryloyl-based bioinks on bone cells

The viscosity of gelatin methacryloyl (GelMA)-based bioinks generates shear stresses throughout the printing process that can affect cell integrity, reduce cell viability, cause morphological changes, and alter cell functionality. This study systematically investigated the impact of the viscosity of GelMA-gelatin bioinks on osteoblast-like cells in 2D and 3D culture conditions. Three bioinks with low, medium, and high viscosity prepared by supplementing a 5% GelMA solution with different concentrations of gelatin were evaluated. Cell responses were studied in a 2D environment after printing and incubation in non-cross-linked bioinks that caused the gelatin and GelMA to dissolve and release cells for attachment to tissue culture plates. The increased viscosity of the bioinks significantly affected cell area and aspect ratio. Cells printed using the bioink with medium viscosity exhibited greater metabolic activity and proliferation rate than those printed using the high viscosity bioink and even the unprinted control cells. Additionally, cells printed using the bioink with high viscosity demonstrated notably elevated expression levels of alkaline phosphatase and bone morphogenetic protein-2 genes. In the 3D condition, the printed cell-laden hydrogels were photo-cross-linked prior to incubation. The medium viscosity bioink supported greater cell proliferation compared to the high viscosity bioink. However, there were no significant differences in the expression of osteogenic markers between the medium and high viscosity bioinks. Therefore, the choice between medium and high viscosity bioinks should be based on the desired outcomes and objectives of the bone tissue engineering application. Furthermore, the bioprinting procedure with the medium viscosity bioink was used as an automated technique for efficiently seeding cells onto 3D printed porous titanium scaffolds for bone tissue engineering purposes.

Butyrate and tributyrin reduce LPS-induced inflammatory cytokine production from human visceral fat.

The current obesity crisis has resulted in many people with excess adipose tissue suffering from chronic inflammation. This inflammation is largely due to the release of cytokines and chemokines from visceral fat. The aim of this study was to identify potential anti-inflammatory agents that might alleviate obesity-induced chronic inflammation. To identify agents that might alleviate this obesity-induced chronic inflammation we have developed a simple protocol for incubating intact pieces of human visceral adipose tissue in 35 mm tissue culture plates, in the presence of low-dose lipopolysaccharide (LPS) and co-incubating these samples with potential anti-inflammatory agents. RNA-Seq analysis was performed to identify enriched gene expression signatures among the most significantly differentially expressed genes. From this screen, we have identified the short-chain fatty acid (SCFA) sodium butyrate and its triacylglyceride form, tributyrin, as effective agents, significantly reducing the production of LPS-induced inflammatory cytokines and chemokines from all adipose tissue samples tested. As well, these agents appear to be non-toxic at the concentrations tested. RNA-Seq analysis has revealed that IL36γ is one of the most upregulated genes in response to LPS and one of the most downregulated when sodium butyrate is added to human fat samples stimulated with LPS. IL-36γ ELISAs confirmed this holds true at the protein level as well. These studies suggest that the short-chain fatty acid, sodium butyrate, and its triacylglyceride form, tributyrin, might alleviate the chronic inflammation that is associated with many individuals with obesity.

Mentha longifolia assisted nanostructures: An approach to obliterate microbial biofilms.

Microbes maneuver strategies to become incessant and biofilms perfectly play a role in scaling up virulence to cause long-lasting infections. The present study was designed to assess the use of an eco-friendly formulation of functionalized silver nanoparticles generated from Mentha longifolia leaf extract (MℓE) for the treatment of biofilm-producing microbes. Nanoparticles synthesized using MℓE as a reducing agent were optimized at different strengths of AgNO3 (1 mM, 2 mM, 3 mM, and 4 mM). Synthesis of M. longifolia silver nanoparticles (MℓAgNPs) was observed spectrophotometrically (450 nm) showing that MℓAgNPs (4 mM) had the highest absorbance. Various techniques e.g., Fourier transforms Infrared spectroscopy (FTIR), Dynamic light scattering (DLS), zeta potential (ZP), X-ray Diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscope (TEM) were used to characterize MℓAgNPs. In the present study, the Kirby-Bauer method revealed 4mM was the most detrimental conc. of MℓAgNPs with MIC and MBC values of 0.62 μg/mL and 1.25 μg/mL, 0.03 μg/mL and 0.078 μg/mL, and 0.07 μg/mL and 0.15 μg/mL against previously isolated and identified clinical strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, respectively. Moreover, the MℓAgNP antibiofilm activity was examined via tissue culture plate (TCP) assay that revealed biofilm inhibition of up to 87.09%, 85.6%, 83.11%, and 75.09% against E. coli, P. aeruginosa, K. pneumonia, and S. aureus, respectively. Herbal synthesized silver nanoparticles (MℓAgNPs) tend to have excellent antibacterial and antibiofilm properties and are promising for other biomedical applications involving the extrication of irksome biofilms. For our best knowledge, it is the first study on the use of the green-synthesized silver nanoparticle MℓAgNP as an antibiofilm agent, suggesting that this material has antibiotic, therapeutic, and industrial applications.

Isolation of pure primary term human cytotrophoblasts and their differentiation into syncytiotrophoblast-like cells as an ex vivo model of the human placenta

The placenta plays a fundamental role in fetal growth and maintenance of pregnancy. Its cellular components include a large multinucleated syncytiotrophoblast (STB) and its progenitor, cytotrophoblasts (CTBs), both of which perform vital functions in the human placenta. Primary cytotrophoblasts isolated from term human placentas that spontaneously fuse and differentiate into syncytiotrophoblast-like cells in vitro have been utilized to investigate the functions of the syncytiotrophoblast and placenta with multiple modifications. Although recent advances have enabled the use of trophoblast stem cell-derived organoids as a model for villous trophoblasts, primary CTBs offer several advantages, including spontaneous differentiation, easy access to materials (from term-delivered human placentas), and simple methodology. Here, we present a precise step-by-step process for isolating pure CTBs from term human placenta based on previously reported placenta digestion, density centrifugation, and CTB purification using anti-HLA-A, B, C antibody. Subsequently, we provide a method to improve CTB viability and differentiation into STB-like cells using epidermal growth factor (EGF) and a ROCK inhibitor (Y-27632) that ensures long-term and stable cultures without altering their proliferation. Because these cells can grow on standard tissue culture plates, this model can be easily utilized for various placental investigations, including innate immune responses, drug resistance, and STB metabolism. Employing this approach considerably enhances our understanding of placental functions, which are key to maternal and offspring health.

Exploring Genetic and Phenotypic Factors Contributing to Urethral Catheter Biofilm Formation in Hospitalised Patients in Jordan

Background The biofilm phenomenon represents a prevalent mode of microbial life in nature which is characterized by cells irreversibly attaching to surfaces or each other and getting embedded in a matrix of extracellular polymeric substances. Object This study aimed to identify and characterize the genes associated with the common bacterial species responsible for biofilm formation in the catheters of hospitalized patients. Method Different bacterial strains were collected from catheterised patients at three local Jordanian hospitals for biofilm formation. The isolates were identified using Gram stain and Remel Rapid test. Biofilm formation was detected using the Tube method and tissue culture plate method. Result The presence of fimA and csgD genes was detected by polymerase chain reaction (PCR). Gram-negative bacteria species were isolated on the urethral catheters and the result shows the majority of the isolates were E. coli (40%), followed by K. pneumonia (27%). In comparison, the least was Citrobacter sp (2.6%). Similarly, some Gram-positive bacteria were also identified such as Staphylococcus aureus (4%) and Staphylococcus epidermidis (2.6%). K. pneumonia is strongly associated with biofilm formation (45%) followed by E. coli (33%). Conclusion Biofilm-associated genes, fimA and csgD were detected in all biofilm-positive samples containing the F plasmid, while csgD was detected in all biofilm-negative samples. Biofilm formation tends to be a prevalent process in patients included in this study and may cause dangerous complications in the patients especially in the elderly due to prolonged catheterization periods.

Biofilm-Producing and Specific Antibiotic Resistance Genes in Pseudomonas aeruginosa Isolated from Patients Admitted to a Tertiary Care Hospital, Bangladesh

Pseudomonas aeruginosa is one of the organisms well-known for producing biofilm. Biofilms are responsible for persistent infections and antimicrobial resistance. The aim of this was to investigate P. aeruginosa for its ability to form biofilm. Genes that were responsible for the production of biofilms and biofilm-specific antimicrobial resistance were detected. The association between antibiotic resistance and biofilm was investigated. This cross-sectional study was conducted from July 2017 to December 2018. A total of 446 samples (infected burns, surgical wounds, and ETA) were collected from admitted patients at Dhaka Medical College and Hospital, Bangladesh. P. aeruginosa was isolated and identified by biochemical tests and PCR. Biofilm production by the tissue culture plate (TCP) method was followed by the detection of biofilm[1]producing genes (pqsA, pslA, pslD, pslH, pelA, lasR) and biofilm-specific antibiotic resistance genes (ndvB, PA1874, PA1876, PA1877) by PCR. The antibiotic susceptibility test was carried out by the disc diffusion method; for colistin agar dilution, the MIC method was followed. Among 232 (52.02%) positive strains of P. aeruginosa, 24 (10.30%) produced biofilms in TCPM. Among biofilm-producing genes, pqsA was found the highest number of isolates (79.17%), which was followed by pslA and pelA (70.83%). Other were found in lesser extent. Among the biofilm-specific antibiotic resistance genes, 16.67% of the isolates had ndvB, and 8.33% had PA1874 and PA1877. Biofilm-forming strains were significantly resistant to colistin in comparison to non-biofilm-forming ones. In conclusion, detection of biofilm-forming genes may be a good tool for the evaluation of biofilm production, which will help in prompt and better management of chronic or device-associated infections. Bangladesh J Microbiol, Volume 40, Number 2, December 2023, pp 60-65

Enhancing liquid biopsy potential: The impact of the R3CE 3D culture platform.

e15032 Background: The ability to expand and preserve tumor cells ex vivo has been an important aspect of cancer research, offering insights into tumor biology and the advancement of personalized therapies. However, expanding rare tumor cells, such as circulating tumor cells (CTCs) and needle biopsies, remains challenging. Methods: We present the R3CE (Reproducible, Rapid, Rare Cell Expansion) 3D culture platform, a novel system that enhances the conventional tissue culture plate with a coating of polyelectrolyte multilayer. This approach can reliably cultivate various tumor specimens, including CTCs, needle biopsies, and surgical tissues, into tumor spheroids. Results: Our platform achieved over a 90% success rate (n = 305) in producing tumor spheroids with uniform size and preserved mutational profiles across diverse cancer types such as breast, lung, and colorectal cancers. In comparison to traditional tissue biopsies, liquid biopsies are less invasive and contain CTCs that offer a more comprehensive representation of heterogeneous tumor cell populations. We show that the proliferative capacity of cultured CTCs, rather than the initial CTC counts, have a strong correlation with lung adenocarcinoma prognosis (n = 124), highlighting the clinical potential of the R3CE platform (1). Furthermore, we have successfully employed cultured CTCs for drug screening for various cancers (breast cancer, cholangiocarcinoma, colon cancer, EMPD, gastric cancer, hepatoma, lung cancer, mucosal melanoma, pancreatic cancer, and urinary bladder cancer, n = 80), resulting in measurement of ex vivo response to chemotherapy and targeted drugs. For instance, R3CE-cultured CTCs have been used in predicting response to neoadjuvant chemotherapeutic drugs in breast cancer, as evidenced by an ROC curve analysis with an AUC of 0.963, 100% sensitivity, 87.5% specificity, P=0.02. Also in an exploratory analysis of immunotherapy efficacy, involving co-culture of R3CE-cultured CTCs (n = 6) and tumor tissue-derived organoids (n = 6) with immune cells such as CAR-T and autologous NK cells, has shown promising outcomes for selected cancers. Conclusions: The R3CE 3D culture platform represents a potentially transformative tool above and beyond CTC enumeration, significantly enhancing 1) the expansion of tumor cells ex vivo and, more importantly, informing phenotypic and genotypic characterization of CTCs, thereby holding promise for advancing cancer care and treatment efficacy. 1. Che et al., Lung Cancer, 2023; 178:198-205.

Phenotypic Investigation and Detection of Biofilm-Associated Genes in Acinetobacter baumannii Isolates, Obtained from Companion Animals.

Bacteria of the genus Acinetobacter, especially Acinetobacter baumannii (Ab), have emerged as pathogens of companion animals during the last two decades and are commonly associated with hospitalization and multidrug resistance. A critical factor for the distribution of relevant strains in healthcare facilities, including veterinary facilities, is their adherence to both biotic and abiotic surfaces and the production of biofilms. A group of 41 A. baumannii isolates obtained from canine and feline clinical samples in Greece was subjected to phenotypic investigation of their ability to produce biofilms using the tissue culture plate (TCP) method. All of them (100%) produced biofilms, while 23 isolates (56.1%) were classified as strong producers, 11 (26.8%) as moderate producers, and 7 (17.1%) as weak producers. A correlation between the MDR and XDR phenotypes and weak or moderate biofilm production was identified. Moreover, the presence of four biofilm-associated genes bap, blaPER, ompA, and csuE was examined by PCR, and they were detected in 100%, 65.9%, 97.6%, and 95.1% of the strains respectively. All isolates carried at least two of the investigated genes, whereas most of the strong biofilm producers carried all four genes. In conclusion, the spread and persistence of biofilm-producing Ab strains in veterinary facilities is a matter of concern, since they are regularly obtained from infected animals, indicating their potential as challenging pathogens for veterinarians due to multidrug resistance and tolerance in conventional eradication measures. Furthermore, considering that companion animals can act as reservoirs of relevant strains, public health concerns emerge.

Chromatin Condensation Delays Senescence in Human Mesenchymal Stem Cells by Safeguarding Nuclear Damages during In Vitro Expansion

Human mesenchymal stem cells (hMSCs) are multipotent cells that differentiate into adipocytes, chondrocytes, and osteoblasts. Owing to their differentiation potential, hMSCs are among the cells most frequently used for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, which necessitates in vitro expansion. Conventionally, this is often performed on rigid surfaces such as tissue culture plates (TCPs). However, during in vitro expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, rendering them unsuitable for clinical use. Although multiple approaches have been attempted to maintain hMSC stemness during prolonged expansion, finding a suitable culture system remains an unmet need. Recently, a few research groups have shown that hMSCs maintain their stemness over long passages when cultured on soft substrates. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. Furthermore, it has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay replicative senescence in hMSCs during long-term expansion on TCPs. However, the mechanism by which chromatin condensation/decondensation influences nuclear morphology and DNA damage, which are strongly related to the onset of senescence, remains unknown. To answer this question, we cultured hMSCs for long duration in the presence of epigenetic modifiers, histone acetyltransferase inhibitor (HATi), which promotes chromatin condensation by preventing histone acetylation, and histone deacetylase inhibitor (HDACi), which promotes chromatin decondensation, and investigated their effects on various nuclear markers related to senescence. We found that consistent acetylation causes severe nuclear abnormalities, whereas chromatin condensation by deacetylation helps to safeguard the nucleus from damage caused by in vitro expansion.

Biofilm-producing and specific antibiotic resistance genes in Pseudomonas aeruginosa isolated from patients admitted to a tertiary care hospital, Bangladesh

ObjectivesBiofilms are responsible for persistent infections and antimicrobial resistance. Pseudomonas aeruginosa was investigated with its ability to form biofilm by detecting genes responsible for producing biofilms and biofilm-specific antimicrobial resistance. The association between antibiotic resistance and biofilm was investigated. MethodsThis cross-sectional study was conducted from July 2017 to December 2018. A total of 446 samples (infected burn, surgical wounds, and endotracheal aspirate) were collected from admitted patients of Dhaka Medical College and Hospital, Bangladesh. P. aeruginosa was isolated and identified by biochemical tests and polymerase chain reaction. Biofilm production by tissue culture plate method followed by detection of biofilm-producing genes (pqsA, pslA, pslD, pslH, pelA, lasR) and biofilm-specific antibiotic resistance genes (ndvB, PA1874, PA1876, PA1877) by polymerase chain reaction were done. Antibiotic susceptibility test was carried out by disk diffusion method; for colistin agar dilution method of minimal inhibitory concentration was followed. ResultsAmong 232 (52.02%) positive strains of P. aeruginosa, 24 (10.30%) produced biofilms in tissue culture plate. Among biofilm-producing genes, pqsA was the highest (79.17%). pslA and pelA were 70.83%, pslD 45.83%, pslH and lasR 37.5%. Among biofilm-specific antibiotic resistance genes, 16.67% were ndvB, and 8.33% were PA1874 and PA1877. Biofilm-forming strains were significantly resistant to colistin. ConclusionsDetection of biofilm-forming genes may be a good tool for the evaluation of biofilm production, which will help in prompt and better management of chronic or device-associated infections.

SIS derivative as a novel delivery system for ibuprofen

Ibuprofen is a commonly used analgesic due to its effectiveness, but its half-life is relatively short. Therefore, finding an appropriate delivery system is a significant challenge. In this study, we designed a new controlled release carrier for ibuprofen using a biocompatible poly(styrene-b-isoprene-b-styrene) triblock copolymer. For this purpose, the polyisoprene block was modified with 2-mercaptoethanol or ethanolamine to introduce hydroxyl groups. These linkers allowed the incorporation of the drug through a covalent bond. The modified copolymers were characterized using spectroscopic techniques, including 1H NMR, FTIR-ATR, and UV-Vis, which confirmed successful synthesis. The thermal method revealed that the new delivery system has a lower glass transition temperature than its precursor, verifying the incorporation of the hydrophobic drug. Furthermore, enzymatic release of ibuprofen was studied under physiological conditions and was found to be slow and sustained over time. The in vitro biocompatibility of the release system was evaluated using RAW264.7 monocytes. It was found that cells were able to adhere and proliferate onto the matrices. Additionally, the cells growing on ibuprofen-substituted SIS produced low levels of nitric oxide, a marker of cytotoxicity, when compared to cells growing on tissue culture plates. Taken together, the findings indicate that the polymer may serve as a promising delivery system for ibuprofen with potential biological applications.

Pseudomonas aeruginosa from a Tertiary Teaching Hospital: A Study of AmpC and Biofilm-producing Clinical Isolates

Abstract Introduction: Opportunistic Pseudomonas aeruginosa is one of the most prevalent bacteria with a broad spectrum of human-associated infections. The World Health Organization (WHO) claims that Pseudomonas aeruginosa infection is a typical healthcare-associated disease of critical or high concern. It is well known for being inherently resistant to many antibiotics and has the ability to produce biofilm. In the present study, we aimed to investigate P. aeruginosa with reference to biofilm and AmpC β-lactamases producing isolates in a tertiary care hospital. Materials and Methods: An observational cross-sectional study was performed at the Department of Microbiology, KIMS, KVV, Karad, and Krishna Hospital &amp; Medical Research Center, Karad. (KH &amp; MRC). A total of 180 isolates were subjected to this test to find out which produced the AmpC β -lactamase enzyme by the cefoxitin-cloxacillin double-disc synergy test method and biofilm production by tissue culture plate assay method. Results: Our study found a total of 97 isolates (53.89%) of AmpC production and biofilm production accounted for 151 (83.89%). Conclusion: AmpC- beta-lactamases are responsible for increasing the resistance of P. aeruginosa to several beta-lactam antibiotics and this study also showed that the clinical isolates of P. aeruginosa had a higher propensity to form biofilm and that there was a direct association between biofilm formation and antibiotic resistance.