Effect of Lactiplantibacillus plantarum cell-free culture on bacterial pathogens isolated from cystic fibrosis patients: in vitro and in vivo studies.

This study aimed to investigate the effects of the cell-free supernatant of Lactiplantibacillus plantarum ATCC® 10241TM on the biofilm-forming capacity of Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients. In addition, the study evaluated the in vivo potential of the cell-free supernatant to modulate inflammation and reduce lung damage in mice infected with P. aeruginosa strains or co-challenged with P. aeruginosa and the Streptococcus milleri group (SMG). The results showed that CF-derived P. aeruginosa strains can infect the respiratory tract of adult mice, inducing local inflammation and lung damage. The severity of these infections was exacerbated when P. aeruginosa was co-administered with SMG. Notably, nebulization with the cell-free supernatant of L. plantarum produced beneficial effects, reducing respiratory infection severity and inflammatory responses induced by P. aeruginosa, both alone or in combination with SMG. Reduced bacterial loads and lung damage were observed in supernatant-treated mice compared to controls. Although further mechanistic studies are necessary, the results show that the cell-free supernatant of L. plantarum ATCC® 10241TM is an interesting adjuvant alternative to treat P. aeruginosa respiratory infections and superinfections in CF patients.

Lactobacilli and Bifidobacteria: A Parapostbiotic Approach to Study and Explain Their Mutual Bioactive Influence.

Three strains of Lactiplantibacillus plantarum and three bifidobacteria (Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium subtile) were used as target strains; in addition, for each microorganism, the cell-free supernatant (CFS) was produced and used as an ingredient of the growth medium. Namely CFSs from lactobacilli were used on bifidobacteria and CFSs from bifidobacteria were used on lactobacilli. The viable count was assessed, and the data were modelled through a reparametrized Gompertz equation cast both in the positive and negative form to evaluate the parameters t-7log, which is the time after which the viable count was 7 log CFU/mL, and the t-7log*, which is the time after which the viable count was below 7 log CFU/mL; the difference between the t-7log* and t-7log defines the stability time. Statistics through a multiparametric ANOVA (analysis of variance) provided evidence for the presence of a bifidogenic and/or bioactive factor produced by bifidobacteria and active on lactobacilli, and vice versa (bioactive factor of lactobacilli with a functional effect on bifidobacteria), although further studies are required to better explain the mechanisms beyond the positive effects. In addition, the influence on the target strains can be found during the growth phase (stimulation), as well as during senescence and death phase (protective effect), with a strong strain/species dependence on both CFS production and target strain.

Dellaglioa Algida Cell-Free Supernatant Inhibits Pseudomonas Fluorescence and Pseudomonas Fragi by Destroying Cell Membranes.

The aim of this study was to examine the components of the cell-free supernatant (CFS) derived from a novel strain of psychrophilic Lactobacillus, Dellaglioa algida, and to further elucidate the impact of this CFS on various cellular processes. Specifically, we sought to understand its effects on the cell membrane, protein and DNA release, protease activity, and metabolites of Pseudomonas fluorescens and Pseudomonas fragi, thereby clarifying the antibacterial mechanism involved. The CFS components were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS), the Coomassie Brilliant Blue method, and the phenol-sulfuric acid method. The inhibitory effect of the CFS on Pseudomonas fluorescens and Pseudomonas fragi was assessed using the ethidium bromide (EB) assay, Oxford cup assay, and ultramicroassay. Additionally, we analyzed the metabolites produced by Pseudomonas fluorescens and Pseudomonas fragi when treated with the CFS. The findings reveal that the CFS of Dellaglioa algida contains 94 volatile components, with protein and sugar concentrations of 32.857 ± 0.9705 mg/mL and 98.250 ± 4.210 mg/L, respectively. The CFS induces varying degrees of damage to the cell membranes of both Pseudomonas fluorescens and Pseudomonas fragi, leading to the release of intracellular proteins and DNA. Furthermore, the CFS reduced the protease activity and metabolic capacity of Pseudomonas fluorescens and Pseudomonas fragi. These results enhance our understanding of the mechanism by which psychrophilic Dellaglioa algida inhibits Pseudomonas fluorescens and Pseudomonas fragi, confirming that its inhibitory effect predominantly occurs through damage to the biological cell membranes of Pseudomonas. Dellaglioa algida is a newly identified cold-adapted inhibitor of Pseudomonas, indicating that its CFS is an effective microbial inhibitor in cold environments. This discovery suggests potential applications in inhibiting the growth and reproduction of Pseudomonas fluorescens and Pseudomonas fragi in food, pharmaceuticals, perfumes, and other chemicals, providing a valuable new reference for industrial preservation.

The antagonistic activity of Streptomyces spiroverticillatus (No. HS1) against of poplar canker pathogen Botryosphaeria dothidea.

BACKGROUND: Poplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea. RESULTS: In vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS. CONCLUSIONS: The inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.

Effects of the supernatant of Chlorella vulgaris cultivated under different culture modes on lettuce (Lactuca sativa L.) growth.

CO2 capture by microalgae is a feasible strategy to reduce CO2 emissions. However, large amounts of cell-free supernatant will be produced after microalgal harvesting, which may be harmful to the environment if it is disorderly discharged. In this study, Chlorella vulgaris (C. vulgaris) was cultivated under three common cultivation modes (autotrophic culture (AC), heterotrophic culture (HC) and mixotrophic culture (MC)), and the obtained supernatant was used as fertilizer to investigate its effect on the growth of lettuce. The biomass concentration of C. vulgaris cultivated under MC and HC was 3.25 and 2.59 times that of under AC, respectively. The contents of macronutrients in supernatant obtained from AC were higher than those of MC and HC. However, the contents of amino acids and hormones in supernatant obtained from MC and HC were higher than those of AC. The fresh shoot weight, fresh root weight and root length of lettuce treated with supernatant were significantly higher than that of control treatment. In addition, the contents of chlorophyll, soluble sugar and soluble protein in lettuce treated with supernatant were also higher than that of control treatment. However, the contents of nitrate in lettuce treated with supernatant was lower than that of control treatment. These results showed that the supernatant could promote the growth of lettuce and was a potential of fertilizer for crop planting.

Vibriocidal efficacy of Bifidobacterium bifidum and Lactobacillus acidophilus cell-free supernatants encapsulated in chitosan nanoparticles against multi-drug resistant Vibrio cholerae O1 El Tor.

BACKGROUND: Cholera is a diarrheal disease recognized for being caused by toxin-producing Vibrio (V.) cholerae. This study aims to assess the vibriocidal and immunomodulatory properties of derived cell-free supernatants (CFSs) of Bifidobacterium (B.) bifidum and Lactobacillus (L.) acidophilus encapsulated in chitosan nanoparticles (CFSb-CsNPs and CFSa-CsNPs) against clinical multi-drug resistance (MDR) isolates of V. cholerae O1 El Tor. METHODS: We synthesized CFSb-CsNPs and CFSa-CsNPs using the ionic gelation technique. The newly nanostructures were characterized for size, surface zeta potential, morphology, encapsulation efficacy (EE), stability in different pH values and temperatures, release profile, and in vitro cytotoxicity. The antimicrobial and antibiofilm effects of the obtained nanocomposites on clinical MDR isolates (N = 5) of V. cholerae E1 Tor O1 were investigated by microbroth dilution assay and crystal violet staining, respectively. We conducted quantitative real-time PCR (qRT-PCR) to analyze the relative gene expressions of Bap, Rbmc, CTXAB, and TCP in response to CFSb-CsNPs and CFSa-CsNPs. Additionally, the immunomodulatory effects of formulated structures on the expression of TLR2 and TLR4 genes in human colorectal adenocarcinoma cells (Caco-2) were studied. RESULTS: Nano-characterization analyses indicated that CFSb-CsNPs and CFSa-CsNPs exhibit spherical shapes under scanning electron microscopy (SEM) imaging, with mean diameters of 98.16 ± 0.763 nm and 83.90 ± 0.854 nm, respectively. Both types of nanoparticles possess positive surface charges. The EE% of CFSb-CsNPs was 77 ± 4.28%, whereas that of CFSa-CsNPs was 62.5 ± 7.33%. Chitosan (Cs) encapsulation leads to increased stability of CFSs in simulated pH conditions of the gastrointestinal tract in which the release rates for CFSb-CsNPs and CFSa-CsNPs were reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively at pH = 7.4. The synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL (p < 0.0001) compare to individual administration. The combined antibacterial effect of CFSb-CsNPs and CFSa-CsNPs on Bap (0.14 ± 0.05), Rbmc (0.24 ± 0.01), CTXAB (0.30 ± 0.09), and TCP (0.38 ± 0.01) gene expression was significant (p < 0.001). Furthermore, co-administration of CFSb-CsNPs and CFSa-CsNPs also demonstrated the potency of suppressing TLR 2/4 (0.20 ± 0.01 and 0.12 ± 0.02, respectively) gene expression (p = 0.0019) and reduced Caco-2 cells attached bacteria to 526,000 ± 51,46 colony-forming units/mL (11.19%) (p < 0.0001). CONCLUSION: Our study revealed that encapsulating CFSs within CsNPs enhances their vibriocidal activity by improving stability and enabling a controlled release mechanism at the site of interaction between the host and bacteria. Additionally, the simultaneous use of CFSb-CsNPs and CFSa-CsNPs exhibited superior vibriocidal potency against MDR V. cholerae O1 El Tor strains, indicating these combinations as a potential new approach against MDR bacteria.

Isolation and identification, genome-wide analysis and pathogenicity study of a novel PRRSV-1 in southern China.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused severe economic losses to the global swine industry. In recent years, the incidence of PRRSV-1 has been gradually increasing in China, but there are still few studies on it. In this study, clinical samples for PRRS virus isolation were collected from a pig farm in South China in 2022. We effectively isolated a strain of PRRSV utilizing PAM cells and demonstrated its consistent transmission capability on Marc-145 cells. The isolated strain was confirmed as PRRSV-1 by RT-qPCR, IFA, electron microscopy, etiolated spot purification and whole genome sequencing, the strain was named GD2022. The length of GD2022 genome is 15058nt; Based on the genome-wide genetic evolutionary analysis of GD2022, the strain was classified as PRRSV-1. Further genetic evolutionary analysis of its ORF5 gene showed that GD2022 belonged to PRRSV-1 subtype 1 and formed an independent branch in the evolutionary tree. Compared with the sequence of the classical PRRSV-1 strain (LV strain), GD2022 has several amino acid site mutations in the antigenic region from GP3 to GP5, these mutations are different from those of other PRRSV-1 strains in China. Recombination analysis showed no recombination events with GD2022. In addition, piglets infected with GD2022 displayed clinical respiratory symptoms and typical pathological changes. In this study, a strain of the PRRSV-1 virus was isolated using both PAM cells and Marc-145 and proved to be pathogenic to piglets, providing an important reference for the identification, prevention, and control of PRRSV-1.

Induction of Cell Death by Bifidobacterium infantis DS1685 in Colorectal and Breast Cancers via SMAD4/TGF-Beta Activation.

Therapeutic advancements in treatments for cancer, a leading cause of mortality worldwide, have lagged behind the increasing incidence of this disease. There is a growing interest in multifaceted approaches for cancer treatment, such as chemotherapy, targeted therapy, and immunotherapy, but due to their low efficacy and severe side effects, there is a need for the development of new cancer therapies. Recently, the human microbiome, which is comprised of various microorganisms, has emerged as an important research field due to its potential impact on cancer treatment. Among these microorganisms, Bifidobacterium infantis has been shown to significantly improve the efficacy of various anticancer drugs. However, research on the role of B. infantis in cancer treatment remains insufficient. Thus, in this study, we explored the anticancer effect of treatment with B. infantis DS1685 supernatant (BI sup) in colorectal and breast cancer cell lines. Treatment with BI sup induced SMAD4 expression to suppress cell growth in colon and breast cancer cells. Furthermore, a decrease in tumor cohesion was observed through the disruption of the regulation of EMT-related genes by BI sup in 3D spheroid models. Based on these findings, we anticipate that BI sup could play an adjunctive role in cancer therapy, and future cotreatment of BI sup with various anticancer drugs may lead to synergistic effects in cancer treatment.

Characterization and genomic analysis of Bacillus megaterium with the ability to degrade aflatoxin B1.

Coix seed is a good product for both medicinal and food use, which is highly susceptible to aflatoxin B1 (AFB1) contamination during field transport, storage, and processing. The aim of this study is to find microbial strains that can solve the problem of contamination of coix seed. In this study, the AFB1-degrading microorganism SX1-1 was isolated and identified as a Bacillus megaterium based on morphology, microscopy, and 16S rDNA sequencing. The optimum culture conditions for SX1-1 to degrade AFB1 were determined to be 12 h. The optimum degradation conditions were 72 h, 57°C, and an initial pH of 8.0. The highest degradation of AFB1 was observed in the fermentation supernatant of the SX1-1 strain, with a degradation rate of 97.45%. In addition, whole-genome sequencing analysis of this strain revealed the presence of a number of enzymes that could potentially degrade AFB1. Importantly, SX1-1 was able to degrade AFB1-contaminated coix seed in situ by 50.06% after co-culture. In conclusion, this strain had a high AFB1 degradation ability, and has great potential and great application as a biocontrol agent for AFB1 degradation of coix seed.

Assessment of ∆9-THC and ∆9-THCCOOH Bias, Precision, and Ionization Suppression/Enhancement between Solid Tissue Homogenate and Supernatant by LC-MS/MS.

LC-MS-MS assays are frequently utilized for screening and confirmatory purposes in the forensic toxicology laboratory. While these techniques are excellent for the targeted identification and quantitation of a wide variety of drug classes, validation and determining fit-for-purpose is a requirement for each method. In the United States, ANSI/ASB Standard 036 currently serves as a primary resource in forensic toxicology method validation, and mandates that laboratories evaluate critical performance characteristics to help ensure the production of forensically defensible results. Due to the variability of specimen quality frequently encountered in the discipline of postmortem toxicology, the [Author Information Removed] Office of the Chief Medical Examiner Forensic Toxicology Laboratory routinely analyzes solid tissue specimens as part of the medicolegal death investigation process and evaluates liver as a representative solid tissue matrix during method validation. Authentic postmortem specimens (e.g., liver, kidney, skeletal muscle, and spleen) were used to investigate the effects of analyzing solid tissue homogenate versus solid tissue supernatant on bias, precision, and ionization suppression/enhancement of ∆9-THC and ∆9-THCCOOH. Bias was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant with a single exception of the low QC concentration for Δ9-THC in liver homogenate (-29%). Within-run and between-run CV was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant. Δ9-THC and Δ9-THC-d3 exhibited significant ion suppression in both liver homogenate and supernatant, while ∆9-THCCOOH and ∆9-THCCOOH-d3 showed both ion suppression and enhancement in these matrices. Noticeable quantitative differences were observed in authentic postmortem solid tissue homogenate and supernatant specimens despite evaluating from identical tissue samplings. A brief discussion of the results is presented using a validated LC-MS-MS method for the confirmation and quantitation of ∆9-THC and ∆9-THCCOOH in postmortem casework.

Maximizing the clinical utility and performance of cytology samples for comprehensive genetic profiling - A report on the impact of process optimization through the analysis of 4,871 cytology samples profiled by MSK-IMPACT.

Comprehensive molecular profiling by next generation sequencing (NGS) has revolutionized tumor classification and biomarker evaluation. However, routine implementation is challenged by the scant nature of diagnostic material obtained through minimally invasive procedures. Here, we describe our long-term experience in profiling cytology samples with an in-depth assessment of the performance, quality metrics, biomarker identification capabilities, and potential pitfalls. We highlight the impact of several optimization strategies to maximize performance with 4,871 prospectively sequenced clinical cytology samples tested by MSK-IMPACT™. Special emphasis is given to the use of residual supernatant cell free DNA (ScfDNA) as a valuable source of tumor DNA. Overall, cytology samples were similar in performance to surgical samples in identifying clinically relevant genomic alterations, achieving success rates up to 93% with full optimization. While cell block (CB) samples had excellent performance overall, low-level cross-contamination was identified in a small proportion of cases (4.7%), a common pitfall intrinsic to the processing of paraffin blocks, suggesting that more stringent precautions and processing modifications should be considered in quality control initiatives. By contrast ScfDNA samples had negligible contamination. Finally, ScfDNA testing exclusively used as a rescue strategy delivered successful results in 71% of cases where tumor tissue from CB was depleted.

Cultivation of Diverse Novel Marine Bacteria from Deep Ocean Sediment Using Spent Culture Supernatant of Ca. Bathyarchaeia Enrichment.

Most microorganisms resist pure cultivation under conventional laboratory conditions. One of the primary issues for this un-culturability is the absence of biologically produced growth-promoting factors in traditionally defined growth media. However, whether cultivating microbes by providing spent culture supernatant of pivotal microbes in the growth medium can be an effective approach to overcome this limitation is still an under-explored area of research. Here, we used the spent culture medium (SCM) method to isolate previously uncultivated marine bacteria and compared the efficiency of this method with the traditional cultivation (TC) method. In the SCM method, Ca. Bathyarchaeia-enriched supernatant (10%) was used along with recalcitrant organic substrates such as lignin, humic acid, and organic carbon mixture. Ca. Bathyarchaeia, a ubiquitous class of archaea, have the capacity to produce metabolites, making their spent culture supernatant a key source to recover new bacterial stains. Both cultivation methods resulted in the recovery of bacterial species from the phyla Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota. However, our SCM approach also led to the recovery of species from rarely cultivated groups, such as Planctomycetota, Deinococcota, and Balneolota. In terms of the isolation of new taxa, the SCM method resulted in the cultivation of 80 potential new strains, including one at the family, 16 at the genus, and 63 at the species level, with a novelty ratio of ~ 35% (80/219). In contrast, the TC method allowed the isolation of ~ 10% (19/171) novel strains at species level only. These findings suggest that the SCM approach improved the cultivation of novel and diverse bacteria.

Effect of macrophages combined with supernatant of mesenchymal stem cell culture and macrophage culture on wound healing in rats.

Wound healing is an orderly sequence of events restoring the integrity of the damaged tissue. It consists of inflammatory, proliferation, and remodeling phases. The objective of the current study was to investigate the effect of local transplantation of cultured macrophage loaded with mesenchymal stem cell/macrophage culture supernatants on wound healing. Sixty-four healthy adult male Wistar rats were randomized into 4 groups of sixteen animals each: 1) SHAM group. 2) MAC-MSC/SN group: One-milliliter application of a mixture comprising mesenchymal stem cell and macrophage culture supernatants in a 1:1 ratio was administered locally to the wound bed. 3) MAC group: Local transplantation of macrophage cells cultured in the wound bed. 4) MAC + MAC-MSC/SN group: Local transplantation of cultured macrophage in combination with mesenchymal stem cell/ macrophage culture supernatants in the wound bed. An incisional wound model was used for biomechanical studies, while an excisional wound model was used for biochemical, histopathological, and planimetric assessments. The wound area was significantly reduced in the MAC + MAC-MSC/SN group compared to other groups (P < 0.05). Biomechanical measurements from the MAC + MAC-MSC/SN group were significantly higher compared to other experimental groups (P < 0.05). Biochemical and quantitative histopathological analyses revealed a significant difference between MAC + MAC-MSC/SN and other groups (P < 0.05). MAC + MAC-MSC/SN showed the potential to improve wound healing significantly. This appears to work by angiogenesis stimulation, fibroblast proliferation, inflammation reduction, and granulation tissue formation during the initial stages of the healing process. This accelerated healing leads to earlier wound area reduction and enhanced tensile strength of the damaged area due to the reorganization of granulation tissue and collagen fibers.

Biogenic amine tryptamine in human vaginal probiotic isolates mediates matrix inhibition and thwarts uropathogenic E. coli biofilm.

Probiotics offer a promising prophylactic approach against various pathogens and represent an alternative strategy to combat biofilm-related infections. In this study, we isolated vaginal commensal microbiota from 54 healthy Indian women to investigate their probiotic traits. We primarily explored the ability of cell-free supernatant (CFS) from Lactobacilli to prevent Uropathogenic Escherichia coli (UPEC) colonization and biofilm formation. Our findings revealed that CFS effectively reduced UPEC's swimming and swarming motility, decreased cell surface hydrophobicity, and hindered matrix production by downregulating specific genes (fimA, fimH, papG, and csgA). Subsequent GC-MS analysis identified Tryptamine, a monoamine compound, as the potent bioactive substance from Lactobacilli CFS, inhibiting UPEC biofilms with an MBIC of 4 µg/ml and an MBEC of 8 µg/ml. Tryptamine induced significant changes in E. coli colony biofilm morphology, transitioning from the Red, Dry, and Rough (RDAR) to the Smooth and White phenotype, indicating reduced extracellular matrix production. Biofilm time-kill assays demonstrated a four-log reduction in UPEC viability when treated with Tryptamine, highlighting its potent antibacterial properties, comparable to CFS treatment. Biofilm ROS assays indicated a significant elevation in ROS generation within UPEC biofilms, suggesting a potential antibacterial mechanism. Gene expression studies with Tryptamine-treated samples showed a reduction in expression of curli gene (csgA), consistent with CFS treatment. This study underscores the potential of Tryptamine from probiotic Lactobacilli CFS as a promising antibiofilm agent against UPEC biofilms.

Commensal bacterial outer membrane protein A induces interleukin-22 production.

Interleukin (IL)-22 promotes host-microbiota homeostasis. We sought to identify microbiota metabolite(s) that drive intestinal IL-22 production. We observed that exposing Peyer's patch cells (PPCs), ex vivo, to fecal supernatants (FSs) recapitulates fermentable fiber- and microbiota-dependent IL-22 production, and cellular sources thereof, thus supporting the use of this model. An interrogation of FSs generated from mice fed the fermentable fiber inulin (FS-Inu) revealed that its IL-22-inducing activity is mediated by heat-labile protein. Fractionation of FS-Inu by ion-exchange chromatography, and subsequent proteomic analysis of IL-22-inducing fractions, indicates that outer membrane protein A (OmpA) might be a microbial driver of IL-22 expression. Concomitantly, recombinant OmpA from Parabacteroides goldsteinii, which is enriched by an inulin diet, induces IL-22 production and expression of the IL-22-dependent genes REG3γ and -ß, in PPCs and mice. Thus, OmpA is one bacterial inducer of IL-22 expression, potentially linking diet, mucosal immune homeostasis, and gut health.

Contribution of Osteoblast and Osteoclast Supernatants to Bone Formation: Determination Using a Novel Microfluidic Chip.

We fabricated a microfluidic chip (osteoblast [OB]-osteoclast [OC] chip) that could regulate the mixture amounts of OB and OC supernatants to investigate the effect of different supernatant distributions on osteogenesis or osteoclastogenesis. Computer-aided design was used to produce an OB-OC chip from polydimethylsiloxane. A pressure controller was assembled and different blends of OB and OC supernatants were correctly determined. OB and OC supernatants were placed on the upper panels of the OB-OC chip after differentiation for an in vitro evaluation. We then tested the changes in osteogenesis using MC3T3-E1 cells in the middle chambers. We observed that a 75:25 distribution of OB and OC supernatants was the most potent in osteogenesis. We then primed the osteogenic differentiation of MC3T3-E1 cells using an OB-OC mixed supernatant or an OB supernatant alone (supernatant ratios of 75:25 or 100:0, respectively). These cells were placed on the calvarial defect sites of rats. Microcomputed tomography and histological analyses determined a significantly higher bone formation in the group exposed to the OB-OC supernatant at a ratio of 75:25. In this study, we demonstrate the applicability of an OB-OC chip to evaluate the effect of different supernatant distributions of OB and OC. We observed that the highest bone-forming potential was in MC3T3-E1 cells treated with conditioned media, specifically the OB-OC supernatant at a ratio of 75:25.

Moderate concentration of Lactobacillus metabolites does not adversely affect mouse sperm.

Many couples in contemporary societies suffer from infertility of unexplained origins (idiopathic). A promising treatment strategy within this context involves the administration to women of preparations containing lactic acid bacteria (Lactobacillus) and/or their metabolites. Recent investigations underscore the role of lactobacilli in sustaining female fertility and enhancing the effectiveness of assisted reproductive techniques. There have also been reports describing the effect of lactobacilli on sperm functions, but our knowledge in this domain remains uncertain. In this study, the effect of supernatant from Lactobacillus rhamnosus culture on mouse sperm viability and motility was tested. The protective properties of lactobacilli metabolites against hydrogen peroxide-induced DNA damage were also verified. It was shown that the metabolites have no effect on viability, motility, and genome integrity of spermatozoa, but in excessive concentrations they become toxic. The obtained results imply that probiotic and/or postbiotic preparations taken by women should not adversely affect the sperm of their partners, provided the dose is correctly selected.

Cytotoxic activity and apoptosis induction by supernatant of Lentilactobacillus buchneri on HT-29 colon cancer cells.

Background and Objectives: Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer and the third most deadly cancer in the world. According to recent experimental reports, probiotics and their derivatives protect CRC patients from treatment-related side effects. Therefore, the present study aimed to investigate the cytotoxic impact of the cell-free supernatant (CFS) of Lentilactobacillus buchneri on the HT-29 cancer cell line. Materials and Methods: In the current study, we used the L. buchneri CFS, which was well isolated and identified in our previous investigation from traditional yogurt in the Arak region of Iran. The apoptosis induction in HT-29 cancer cells was assessed by cell cytotoxicity, flow cytometry, and qRT-PCR. Results: L. buchneri CFS inhibited the proliferation of HT-29 cancer cells in a time- and dose-dependent manner. The apoptotic effect of CFS was further supported by the flow cytometry data, which showed that the maximum incidence of apoptosis was observed in HT-29 cancer cells treated with the IC50 concentration of CFS after 72 hours. CFS of L. buchneri also exerted the up-regulating effect on the expression of pro-apoptotic genes including BAX, CASP9, and CASP3. L. buchneri CFS at an IC50 dose induced cell cycle arrest in the G0/G1 phase in HT-29 cells. Conclusion: This study indicates that L. buchneri CFS can prevent colorectal cancer (CRC) development in patients by inducing cancer cell apoptosis. This finding suggests that the CFS of L. buchneri could be used as a therapeutic agent for the control of CRC.

Lyophilized cell-free supernatants of Limosilactobacillus fermentum T0701 exhibited antibacterial activity against Helicobacter pylori.

Helicobacter pylori is a prominent gastrointestinal pathogen associated with various gastrointestinal illnesses. It presents substantial health risks due to its antibiotic resistance. Therefore, it is crucial to identify alternative treatments for H. pylori infections. Limosilactobacillus spp exhibit probiotic properties with beneficial effects in humans; however, the mechanisms by which it counteracts H. pylori infection are unknown. This study aimed to evaluate the potential of Limosilactobacillus fermentum T0701 lyophilized cell-free supernatants (LCFS) against H. pylori. The LCFS has varying antimicrobial activities, with inhibition zones of up to 10.67 mm. The minimum inhibitory concentration and minimum bacterial concentration of LCFS are 6.25-25.00 mg/mL and 6.25 mg/mL to > 50.00 mg/mL, respectively, indicating its capability to inhibit H. pylori. There is morphological damage observed in H. pylori treated with LCFS. Additionally, H. pylori adhesion to AGS cells (human gastric adenocarcinoma epithelial cells) reduces by 74.23%, highlighting the LCFS role in preventing bacterial colonization. Moreover, LCFS exhibits no cytotoxicity or morphological changes in AGS cells, and with no detected virulence or antimicrobial resistance genes, further supporting its safety profile. L. fermentum T0701 LCFS shows promise as a safe and effective non-toxic agent against H. pylori, with the potential to prevent gastric colonization.

Supernatant fluid from endobronchial ultrasound-guided transbronchial needle aspiration for rapid next-generation sequencing.

INTRODUCTION: There is an increasing demand to optimize the workflow and maximize tissue available for next-generation sequencing (NGS) for non-small cell carcinoma. We looked at transbronchial needle endobronchial ultrasound-guided bronchoscopy with transbronchial needle aspiration samples and evaluated the performance of supernatant (SN) fluid processed from a dedicated aspirate collected for NGS testing. MATERIALS AND METHODS: Nineteen samples were collected and processed using a new workflow. Five aspirates were collected in formalin. One additional dedicated pass was collected fresh and centrifuged. The resulting cell pellet was added to formalin for cell block (CB) processing. DNA and RNA were extracted from concentrated SN for targeted testing using the Oncomine Precision Assay (Thermo Scientific, Waltham, MA). NGS results from the corresponding CB samples were used as "controls" for comparison. RESULTS: Thirty-one mutations were detected in SN (Table 1). The most frequently mutated genes were TP53 (35%), EGFR (23%), KRAS (13%), CTNNB1 (6%), and ERBB2 (6%). There was 100% concordance between the mutations detected in SN and corresponding CBs with comparable variant allele frequencies. Turnaround time of NGS results was 1 day for SN compared to 4-10 days for CB. CONCLUSIONS: We were able to demonstrate the usefulness of SN for reliable rapid molecular results. We successfully incorporated the workflow for tissue handling and processing among our clinical, cytopathology, and molecular teams. Molecular results were available at the same time as the cytologic diagnosis, allowing for timely reporting of a comprehensive diagnosis. This approach is particularly useful in patients with advanced disease requiring urgent management.