Viability and stability evaluation of microencapsulated Lactobacillus reuteri in polysaccharide-based bionanocomposite.

Microencapsulation is one of the most important methods to enhance the survival of bacteria when exposed to various harsh conditions. The present study evaluated the viability of L. reuteri ATCC 23272 microencapsulated in polysaccharide-based bionanocomposite. Inulin, polydextrose, and pectin were utilized as prebiotics, and magnesium oxide nanoparticles (MgO NPs) as reinforcing agent in the microgel structure. The composition of bionanocomposite was optimized using the simplex-lattice mixture method. Bionanocomposite optimal formulation was achieved by combining 91.6 % inulin and 8.4 % pectin in the presence of MgO NPs. L. reuteri prebiotic score (1.33) and E. coli (1.08), extrusion efficiency (97.57 %), viability after drying (99.37 %), and viability in simulated gastrointestinal conditions (SGI) (91.74 %) were obtained. Not using MgO NPs in the optimal composite structure caused a decrease of 2.14 log CFU/g in SGI. During 28 days of storage of bacteria at 4 and 25 °C, respectively, a reduction of 2.56 and 3.04 log CFU/g was observed for free cells compared to encapsulated cells. SEM, FTIR, and XRD analyses were performed on ingredients and microcapsules with and without bacteria. The results exhibited that the optimal bionanocomposite could be used as a beneficial encapsulation system to improve the performance of probiotics in harsh conditions.

Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

Isolation and Preparation of Embryonic Zebrafish Retinal Cells for Single-Cell RNA Sequencing.

Recent technological advances in single-cell RNA sequencing (scRNA-Seq) have enabled scientists to answer novel questions in biology with unparalleled precision. Indeed, in the field of ocular development and regeneration, scRNA-Seq studies have resulted in a number of exciting discoveries that have begun to revolutionize the way we think about these processes. Despite the widespread success of scRNA-Seq, many scientists are wary to perform scRNA-Seq experiments due to the uncertainty of obtaining high-quality viable cell populations that are necessary for the generation of usable data that enable rigorous computational analyses. Here, we describe methodology to reproducibility generate high-quality single-cell suspensions from embryonic zebrafish eyes. These single-cell suspensions served as inputs to the 10× Genomics v3.1 system and yielded high-quality scRNA-Seq data in proof-of-principle studies. In describing methodology to quantitatively assess cell yields, cell viability, and other critical quality control parameters, this protocol can serve as a useful starting point for others in designing their scRNA-Seq experiments in the zebrafish eye and in other developing or regenerating tissues in zebrafish or other model systems.

Can polymeric nanofibers effectively preserve and deliver live therapeutic bacteria?

Probiotics and live therapeutic bacteria (LTB), their strictly regulated therapeutic counterpart, are increasingly important in treating and preventing biofilm-related diseases. This necessitates new approaches to (i) preserve bacterial viability during manufacturing and storage and (ii) incorporate LTB into delivery systems for enhanced therapeutic efficacy. This review explores advances in probiotic and LTB product development, focusing on preservation, protection, and improved delivery. Preservation of bacteria can be achieved by drying methods that decelerate metabolism. These methods introduce stresses affecting viability which can be mitigated with suitable excipients like polymeric or low molecular weight stabilizers. The review emphasizes the incorporation of LTB into polymer-based nanofibers via electrospinning, enabling simultaneous drying, encapsulation, and delivery system production. Optimization of bacterial survival during electrospinning and storage is discussed, as well as controlled LTB release achievable through formulation design using gel-forming, gastroprotective, mucoadhesive, and pH-responsive polymers. Evaluation of the presence of the actual therapeutic strains, bacterial viability and activity by CFU enumeration or alternative analytical techniques is presented as a key aspect of developing effective and safe formulations with LTB. This review offers insights into designing delivery systems, especially polymeric nanofibers, for preservation and delivery of LTB, guiding readers in developing innovative biotherapeutic delivery systems.

Antenatal counselling at the cusp of viability and parental decision-making in the zone of parental discretion: A cohort study.

AIM: Safer Care Victoria updated a clinical guideline on extreme prematurity in 2020, reducing the threshold for offering resuscitation from 23 to 22 weeks gestation. The zone of parental discretion is the interval of shared decision-making between parents and doctors regarding resuscitation decisions. It is especially relevant at this periviable gestation. Our study aimed to establish current practices in antenatal counselling and steroid administration at this cusp of viability, and examine the decisions made during the zone of parental discretion. METHODS: Single centre retrospective cohort study. Sixteen thousand three hundred fifty-four admissions and emergency department presentations between January 2021 and July 2023 were retrieved from Birthing Outcomes System (BOS) and patient details were imported and manually reviewed on Microsoft Excel, with particular note to the gestation at admission/emergency department presentation and duration of admission. Eighty-seven patients were identified as present in the hospital between 21 + 0 and 22 + 6 weeks gestation. These 87 scanned records on Clinical Patient Folder (CPF) were then manually reviewed to identify if antenatal counselling occurred during this window. Thirty-six patients were included who received antenatal counselling between 21 + 0 and 22 + 6 weeks gestation (the remaining patients did not receive antenatal counselling during this window), and relevant data was subsequently extracted from the scanned medical record and analysed using SPSS software (IBM SPSS Statistics 29). RESULTS: Thirty-six women received antenatal counselling between 21 + 0 and 22 + 6 weeks. 58% decided on full resuscitation and 39% opted for comfort care if their infant was to be born between 22 + 0 and 22 + 6 weeks. All but one baby born premature were exposed to steroids, with 83.3% receiving a full course. Twenty-eight infants (62.2%) were fully steroid loaded at the time of delivery. In those fully steroid loaded, 31.1% of the time steroids were initiated prior to transfer, 50% of the time deferred until neonatal review and a decision regarding the resuscitation status of the baby, and on one occasion requested by the neonatologist before counselling. CONCLUSION: Patients at risk for premature birth who attended our hospital at the cusp of viability were generally counselled about the opportunity for resuscitation between 22 + 0 and 22 + 6 weeks gestational age, and offered steroids. Further studies are required to establish whether the content of antenatal counselling, and the timing of steroids, are consistent in this population.

3D Bioprinting a Novel Skin Co-Culture Model Using Human Keratinocytes and Fibroblasts.

3D bioprinting can generate the organized structures found in human skin for a variety of biological, medical, and pharmaceutical applications. Challenges in bioprinting skin include printing different types of cells in the same construct while maintaining their viability, which depends on the type of bioprinter and bioinks used. This study evaluated a novel 3D bioprinted skin model containing human keratinocytes (HEKa) and human dermal fibroblasts (HDF) in co-culture (CC) using a high-viscosity fibrin-based bioink produced using the BioX extrusion-based bioprinter. The constructs containing HEKa or HDF cells alone (control groups) and in CC were evaluated at 1, 10, and 20 days after bioprinting for viability, immunocytochemistry for specific markers (K5 and K10 for keratinocytes; vimentin and fibroblast specific protein [FSP] for fibroblasts). The storage, loss modulus, and viscosity properties of the constructs were also assessed to compare the effects of keratinocytes and fibroblasts individually and combined, providing important insights when bioprinting skin. Our findings revealed significantly higher cell viability in the CC group compared to individual keratinocyte and fibroblast groups, suggesting the combined cell presence enhanced survival rates. Additionally, proliferation rates of both cell types remained consistent over time, indicating non-competitive growth within the construct. Interestingly, keratinocytes exhibited a greater impact on the viscoelastic properties of the construct compared to fibroblasts, likely due to their larger size and arrangement. These insights contribute to optimizing bioprinting strategies for skin tissue engineering and emphasize the important role of different cell types in 3D skin models.

Grape pomace high-methoxyl pectin: A new prebiotic stabilizer for low-fat synbiotic yogurt gels - Optimization and characterization.

High-methoxyl pectin (HMP, 72.5 % esterification degree and galacturonic acid content of 67.9 %) was extracted from grape pomace using a sequential ultrasound-microwave extraction. The extracted HMP was used to develop low-fat synbiotic set yogurts containing probiotic cells. Higher grape pomace pectin (GPP) concentrations (0.5-2 %) increased the probiotic bacterial population of Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum BB-12. Higher cell viability was observed for L. acidophilus LA-5 compared to B. bifidum BB-12. A response surface optimization showed that the presence of 8.08 Log CFU mL-1L. acidophilus LA-5 and 1.88 % HMP experimentally resulted in the best probiotic viability (10.83 ±â€¯0.11 Log CFU mL-1), overall acceptability (8.03 ±â€¯0.06), and pH (4.25 ±â€¯0.05) values. Compared to pectin-free probiotic yogurts, the optimal yogurt gels presented higher probiotic survivability, lower syneresis, and superior storage-dependent sensory attributes during 21 days of storage. However, a 14-day storage period was generally deemed suitable. The GPP-containing yogurt compared to the pectin-free sample exhibited higher colloidal stability with a larger particle size (433.8 nm vs. 272.5 nm) and lower zeta potential (-20.4 mV vs. -10.6 mV). Field emission-scanning electron (FE-SEM) and fluorescent (FLM) microscopy images confirmed a denser microstructure for GPP-enriched yogurts. The chemical interactions in the yogurt were not affected by enriching with GPP as investigated by FTIR, whereas the steady and dynamic rheological properties were significantly improved. GPP-enriched yogurt had a firmer gel structure with a larger linear region and lower G' compared to the control, indicating a semi-solid state. The GPP as a multi-functional prebiotic ingredient would be promising in designing healthier food products.

13-cis Retinoic Acid-Mediated Modulation of Human Meibomian Gland Epithelial Cells Development: Implications for In Vitro Modeling of Meibomian Gland Dysfunction.

Purpose: This study aimed to investigate the effect of 13-cis retinoic acid (13-cis RA) on human meibomian gland epithelial cells (HMGECs) and explore the potential of using this experimental model as an in vitro approach for studying meibomian gland dysfunction (MGD). Methods: First, HMGECs were cultured with 13-cis RA at different doses and times, and cell viability and proliferation rates were assessed to determine the appropriate stimulation concentration and time. Subsequently, during the proliferation stage, the expression of proliferation, inflammation, and oxidative stress genes and their products were evaluated. The meibum synthesis capacity was determined during the differentiation stage. Additionally, the peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 was used as a control to assess the impact of 13-cis RA on PPARγ. Results: 13-cis RA significantly inhibited cell viability and proliferation in a time-dose response manner. Under the stimulation of 2 and 5 µM for 48 h during the proliferation stage, a significant decrease was observed in the expression of cell proliferation markers Ki67, antioxidant SOD-2, and Nrf-2. However, the expression of the pro-inflammatory factors IL-1ß, IL-8, MMP9, and oxidative stress markers NOX-4 and reactive oxygen species increased. During the differentiation stage, it suppressed meibum synthesis and the expression of meibocyte differentiation-related proteins adipose differentiation-associated protein 4 (ADFP4), elongation of very long chain fatty acid protein 4 (ELOVL4), sterol regulatory element-binding protein 2 (SREBP-2), and PPARγ. Conclusion: 13-cis RA inhibited cell viability, promoted inflammation and oxidative stress, and suppressed meibum synthesis through the PPARγ pathway. Our study shed light on the effect of 13-cis RA on HMGECs and provided a promising direction for studying MGD in vitro.

Vitreous Tissue Cryopreservation Using a Blood Vessel Model and Cryomacroscopy for Scale-Up Studies: Observations and Mathematical Modeling.

Successful long term cryobanking of multicellular tissues and organs at deep subzero temperatures calls for the avoidance of ice cryoinjury by reliance upon ice-free cryopreservation techniques. However, the quality of the cryopreserved material is the direct result of its ability to survive a host of harmful mechanisms, chief among which is overcoming the trifecta effects of ice crystallization, toxicity, and mechanical stress. This study aims at exploring improved conditions to scale-up ice-free cryopreservation by combining DP6 as a base cryoprotective agent (CPA) solution with an array of synthetic ice modulators (SIMs). This study is conducted by integrating cryomacroscopy techniques, thermal modeling, solid mechanics analysis, and viability and contractility investigation to correlate physical effects, thermal outcomes, and cryobiology results. As an extension of previous work, this study aims at scale-up of established baseline blood vessel models, while comparing the relative toxicity and vitreous stability of 4ml and 10ml samples of DP6 containing either sucrose as a SIM, or the commercial synthetic ice blockers (X1000 and Z1000). Using that established protocol, the addition and removal of DP6+0.6M sucrose and DP6+1%X1000+1%Z1000 were both well tolerated in rabbit carotid and pig femoral artery models, when assessed for metabolic recovery and contractility. Using cryomacroscopy, it was demonstrated that DP6+0.6M sucrose provided a stable vitrification medium under marginal cooling and warming conditions that resulted in >50% survival rate. By contrast, DP6+1%X1000+1%Z1000 was subject to visible ice formation during cooling under the same thermal conditions, resulting in a significantly lower recovery of ∼20%. Thermal modeling is used in this study to verify the actual cooling and rewarming rates in the specimens, while thermo-mechanics analysis is used to explain why fractures were observed using cryomacroscopy when the specimens were contained in glass vials but not in plastic vials.

Assessment of Neurotoxic Mechanisms of Individual and Binary Mixtures of Cobalt, Nickel and Lead in Hippocampal Neuronal Cells.

Many studies have focused on the neurotoxic effects of single metals, while investigation on the exposure to metal mixtures, which mainly occur in real-life situations, is scarce. This study sought to assess the neurotoxic effect of Ni, Co, and Pb binary mixtures and their individual effects in hippocampal neuronal cells (HT-22). Cells were exposed to Ni, Co, and Pb separately for 48 h at 37°C and 5% CO2, and cell viability was assessed. Morphological assessment of the cells exposed to binary mixtures of Co, Ni, and Pb and single metals was assessed using a microscope. Furthermore, acetylcholinesterase (AChE) activity, oxidative stress biomarkers (glutathione [GSH] and malondialdehyde [MDA] levels, catalase [CAT], and glutathione-S transferase [GST] activities) and nitric oxide [NO] levels were evaluated after treatment with the binary mixtures and single metals. Binary mixtures of the metals reduced cell viability, exerting an additivity action. The combinations also exerted synergistic action, as revealed by the combination index. Furthermore, a significant reduction in AChE activity, GSH levels, CAT and GST activities, and high MDA and NO levels were observed in neuronal cells. The additive interactions and synergistic actions of the binary mixtures might contribute to the significant reduction of AChE activity, GSH levels, GST, and CAT activities, and an increase in MDA and NO levels. The findings from this study revealed significant evidence that binary mixtures of Co, Pb, and Ni may induce impaired neuronal function and, ultimately, neurodegeneration.

Antioxidant properties of allium turcicum Özhatay & cowley plant extract, its effects on the proliferation and migration of cancer cells.

Cancer is a type of non-communicable disease that is responsible for numerous deaths worldwide. Cancer incidence and mortality rates are on the rise due to a combination of factors, such as a growing population, aging, and poor dietary habits. The Allium turcicum Özhatay & Cowley plant is an endemic plant in the area where it grows and is consumed by the public due to its various benefits. This endemic plant, which generally grows in high-altitude regions, is sold in bunches because it is costly, mixed with rock salt, crushed into powder, and consumed as a spice. The cytotoxic and growth-inhibitory effects of A. turcicum Özhatay & Cowley herb extract on human glioblastoma U373 cells, human colorectal carcinoma cell HCT-116, and healthy HUVEC cell lines were determined by the MTT method. After 24 and 48 h of application, logIC50 values in HUVEC, HCT-116, and U373 cells were defined as 3.737, 3.765; 3.513, 3.696, 4.476, and 4.104 µg/mL, respectively. We conducted a cell migration experiment to study the A. turcicum Özhatay & Cowley Extract (ATÖCE) impact on cancer cells' metastatic behavior. Our findings indicate that ATÖCE has an inhibitory effect on the migration potential of the cells used in the study. We conducted experiments using DPPH, ABTS, CUPRAC, and total phenolic content to assess the antioxidant properties of ATÖCE. The findings from the antioxidant activity experiments revealed an activity level of 0.20 ± 0.046 at IC50. Additionally, the total phenolic content was measured to be 0.26 ± 0.044 mg GAE/g.

Preparation and Biochemical Activity of Copper-Coated Cellulose Nonwoven Fabric via Magnetron Sputtering and Alginate-Calcium Ion Complexation.

Alginate-based materials have gained significant recognition in the medical industry due to their favorable biochemical properties. As a continuation of our previous studies, we have introduced a new composite consisting of cellulose nonwoven fabric charged with a metallic copper core (CNW-Cu0) covered with a calcium alginate (ALG-Ca2+) layer. The preparation process for these materials involved three main steps: coating the cellulose nonwoven fabric with copper via magnetron sputtering (CNW → CNW-Cu0), subsequent deposition with sodium alginate (CNW-Cu0 → CNW-Cu0/ALG-Na+), followed by cross-linking the alginate chains with calcium ions (CNW-Cu0/ALG-Na+ → CNW-Cu0/ALG-Ca2+). The primary objective of the work was to supply these composites with such biological attributes as antibacterial and hemostatic activity. Namely, equipping the antibacterial materials (copper action on representative Gram-positive and Gram-negative bacteria and fungal strains) with induction of blood plasma clotting processes (activated partial thromboplastin time (aPTT) and prothrombin time (PT)). We determined the effect of CNW-Cu0/ALG-Ca2+ materials on the viability of Peripheral blood mononuclear (PBM) cells. Moreover, we studied the interactions of CNW-Cu0/ALG-Ca2+ materials with DNA using the relaxation plasmid assay. However, results showed CNW-Cu0/ALG-Ca2+'s cytotoxic properties against PBM cells in a time-dependent manner. Furthermore, the CNW-Cu0/ALG-Ca2+ composite exhibited the potential to interact directly with DNA. The results demonstrated that the CNW-Cu0/ALG-Ca2+ composites synthesized show promising potential for wound dressing applications.

Nonconventional Imaging for Viable Bacteria Detection: A Review.

The first attempts of bacteria observation started with the use of glass lenses to generate magnified images of specimens. This technique is constrained by the principal limit to the resolution of any optical system. Besides optical microscopy, other imaging techniques emerged to reveal more levels of details. The more the achievable resolution, the more complex the imaging systems, and at the same time, the more potentially cell-killing or DNA-damaging they may become. This article provides a state of the art of nonconventional sensor techniques that have been used in applications related to bacteria imaging, for the purpose of comparing the information they provide and determine their suitability or find out if their combination can yield new results without compromising the ability to keep the cells alive.

Prolonged incubation of frozen-thawed equine spermatozoa for in vitro fertilization: A preliminary study using low temperature and INRA96 medium.

A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p < .001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p < .05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p < .001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF.

Viability of Lactobacillus acidophilus in acidophilus milk during frozen storage and its potential to lower cholesterol: an In vivo study.

The viability and functional activity of lactic acid bacteria (LAB), such as Lactobacillus acidophilus in a fermented milk product is important. One of the functional activities of the LAB in fermented milk is the ability of the LAB to positively impact the human health. This study aimed to determine the effect of frozen storage on the fermented milk, namely acidophilus milk, based on the nutritional milk quality, the viability of L. acidophilus, and its potential to lower cholesterol levels in Wistar rats. The parameters measured was including milk quality (mainly pH, protein content, lactic acid levels, and syneresis), L. acidophilus bacterial viability in frozen storage (for 1, 2, and 3 months), and some biological assays to evaluate the potential of milk in lowering blood cholesterol levels in Wistar rats. The result of this study suggests that acidophilus milk quality can be maintained in frozen storage for two months, and it had lactic acid levels of 1.07%, pH of 4.08, and protein levels of 3.33%. Giving acidophilus milk to Wistar rats for 15 days could reduce the cholesterol level continuously until 30 days of treatment. Therefore, this study proves that acidophilus milk quality can be maintained very well in frozen storage, and its functional properties to lower the cholesterol level of Wistar rats can be achieved after two weeks of consumption.

A model of task-level human stepping regulation yields semistable walking.

A simple lateral dynamic walker, with swing leg dynamics and three adjustable input parameters, is used to study how motor regulation affects frontal-plane stepping. Motivated by experimental observations and phenomenological models, we imposed task-level multi-objective regulation targeting the walker's optimal lateral foot placement at each step. The regulator prioritizes achieving step width and lateral body position goals to varying degrees by choosing a mixture parameter. Our model thus integrates a lateral mechanical template, which captures the fundamental mechanics of frontal-plane walking, with a lateral motor regulation template, an empirically verified model of how humans manipulate lateral foot placements in a goal-directed manner. The model captures experimentally observed stepping fluctuation statistics and demonstrates how linear empirical models of stepping dynamics can emerge from first-principles nonlinear mechanics. We find that task-level regulation gives rise to a goal-equivalent manifold in the system's extended state space of mechanical states and inputs, a subset of which contains a continuum of period-1 gaits forming a semistable set: perturbations off of any of its gaits result in transients that return to the set, though typically to different gaits.

A quinoline-2-thione derivative as a novel chemotherapy drug candidate displays anti-tumor activity in vitro and in vivo.

Ovarian cancer is the fifth most prevalent cancer in women. Chemotherapy is a major treatment option for patients with advanced ovarian cancer (OC). Quinoline-2-thione and its derivatives are potential candidates for tumor therapy. In this study, we investigated the anticancer activity of the quinoline-2-thione derivative KA3D against ovarian cancer. The effect of KA3D on the viability of ovarian cancer cells was evaluated using MTT assay, and its effects on apoptosis and the cell cycle were detected using flow cytometry. Western blotting was performed to identify apoptosis-and cell cycle-related proteins altered by KA3D treatment. A xenograft model was used to verify the inhibitory effect of KA3D in vivo. H&E staining, biochemical indicator detection, and blood cell counts were used to observe the toxicity and side effects of KA3D. KA3D treatment impeded cell viability, induced apoptosis, and impeded the G2 phase of the cell cycle in ovarian cancer cells. Mechanistically, we found that KA3D enhanced the expression of proapoptotic molecules such as BAX and Caspase 3, while antiapoptotic proteins such as BCL2 were inhibited. The G0/G1 phase-related protein cyclin D1 was reduced and the G2 phase-related protein cyclin B1 was upregulated. In vivo, KA3D displayed potent anticancer activity, with no apparent toxicity in BABLC/c nude mice bearing SKOV3 cells. KA3D demonstrated remarkable chemotherapeutic drug efficacy in terms of significant cancer suppression in vitro and in vivo with low toxicity.

Synthetic free fatty acid receptor (FFAR) 2 agonist 4-CMTB and FFAR4 agonist GSK13764 inhibit colon cancer cell growth and migration and regulate FFARs expression in in vitro and in vivo models of colorectal cancer.

INTRODUCTION: Free fatty acid receptors (FFARs) are G protein-coupled receptors that divide into 4 subtypes; FFAR2 and FFAR3 are activated by short-chain fatty acids, while FFAR1 and FFAR4 - by long-chain fatty acids. Recent studies show the potential involvement of FFARs in the pathophysiology of colorectal cancer (CRC). A decrease in FFAR2 and FFAR4 gene expression is observed in patients with CRC. The aim of our study was to evaluate the anti-cancer effect of FFAR2 and FFAR4 stimulation by selective synthetic agonists in in vitro and in vivo models of CRC. MATERIALS AND METHODS: FFAR2 agonist, 4-CMTB, and FFAR4 agonist, GSK137647 were used. Cell viability (CCD 841 CoN and SW-480) was determined after 48 h incubation with tested compounds using MTT assay. Real-time qPCR and Western Blot were used to identify changes in FFARs expression. Migration and invasion were characterized by commercially available tests. Colitis-associated CRC (CACRC) mouse model was induced by azoxymethane and dextran sodium sulfate. RESULTS: 4-CMTB and GSK137647 significantly reduced cancer cell growth as well as migration and invasion capacities. Both synthetic compounds increased FFAR2 and FFAR4 expression in SW-480 cells. Neither 4-CMTB nor GSK137647 influenced the course of AOM/DSS-induced CACRC in mice, however, 4-CMTB elevated FFAR2 protein expression in mouse tissues. CONCLUSION: We presented that stimulation of FFAR2 and FFAR4 may inhibit CRC cell viability and migration and that the FFAR2 and FFAR4 expression decreased in CRC can be restored by treatment with respective agonists, indicating new promising pharmacological targets in CRC treatment.

Randomised-crossover clinical trial on the substantivity of a single application of a gel containing chlorhexidine and o-cymen-5-ol on the oral biofilm and saliva.

BACKGROUND: No clinical trials have evaluated the antimicrobial activity and substantivity of gel formulations containing chlorhexidine (CHX) and cymenol. OBJECTIVE: To compare the in situ antimicrobial effect and substantivity of a new 0.20% CHX + cymenol gel (test) with the current 0.20% CHX gel formulation (control) on salivary flora and dental plaque biofilm up to seven hours after a single application. METHODS: A randomised-crossover clinical trial was conducted with 29 orally healthy volunteers participating in the development of Experiments 1 (saliva) and 2 (dental plaque biofilm). All subjects participated in both experiments and were randomly assigned to receive either the test or control gels. Samples were collected at baseline and five minutes and one, three, five, and seven hours after a single application of the products. The specimens were processed using confocal laser scanning microscopy after staining with the LIVE/DEAD® BacLight™ solution. Bacterial viability (BV) was quantified in the saliva and biofilm samples. The BV was calculated using the DenTiUS Biofilm software. RESULTS: In Experiment 1, the mean baseline BV was significantly reduced five minutes after application in the test group (87.00% vs. 26.50%; p < 0.01). This effect was maintained throughout all sampling times and continued up to seven hours (40.40%, p < 0.01). The CHX control followed the same pattern. In Experiment 2, the mean baseline BV was also significantly lower five minutes after applying the test gel for: (1) the total thickness of biofilm (91.00% vs. 5.80%; p < 0.01); (2) the upper layer (91.29% vs. 3.94%; p < 0.01); and (3) the lower layer (86.29% vs. 3.83%; p < 0.01). The reduction of BV from baseline was observed for the full-thickness and by layers at all sampling moments and continued seven hours after application (21.30%, 24.13%, and 22.06%, respectively; p < 0.01). Again, the control group showed similar results. No significant differences between test and control gels were observed in either saliva or dental plaque biofilm at any sampling time. CONCLUSIONS: A 0.20% CHX + cymenol gel application demonstrates potent and immediate antimicrobial activity on salivary flora and de novo biofilm. This effect is maintained seven hours after application. Similar effects are obtained with a 0.20% CHX-only gel.

A Novel Approach to Identifying Hibernating Myocardium Using Radiomics-Based Machine Learning.

Background To assess the feasibility of a machine learning (ML) approach using radiomics features of perfusion defects on rest myocardial perfusion imaging (MPI) to detect the presence of hibernating myocardium. Methodology Data of patients who underwent 99mTc-sestamibi MPI and 18F-FDG PET/CT for myocardial viability assessment were retrieved. Rest MPI data were processed on ECToolbox, and polar maps were saved using the NFile PMap tool. The reference standard for defining hibernating myocardium was the presence of mismatched perfusion-metabolism defect with impaired myocardial contractility at rest. Perfusion defects on the polar maps were delineated with regions of interest (ROIs) after spatial resampling and intensity discretization. Replicable random sampling allocated 80% (257) of the perfusion defects of the patients from January 2017 to September 2022 to the training set and the remaining 20% (64) to the validation set. An independent dataset of perfusion defects from 29 consecutive patients from October 2022 to January 2023 was used as the testing set for model evaluation. One hundred ten first and second-order texture features were extracted for each ROI. After feature normalization and imputation, 14 best-ranked features were selected using a multistep feature selection process including the Logistic Regression and Fast Correlation-Based Filter. Thirteen supervised ML algorithms were trained with stratified five-fold cross-validation on the training set and validated on the validation set. The ML algorithms with a Log Loss of <0.688 and <0.672 in the cross-validation and validation steps were evaluated on the testing set. Performance matrices of the algorithms assessed included area under the curve (AUC), classification accuracy (CA), F1 score, precision, recall, and specificity. To provide transparency and interpretability, SHapley Additive exPlanations (SHAP) values were assessed and depicted as beeswarm plots. Results Two hundred thirty-nine patients (214 males; mean age 56 ± 11 years) were enrolled in the study. There were 371 perfusion defects (321 in the training and validation sets; 50 in the testing set). Based on the reference standard, 168 perfusion defects had hibernating myocardium (139 in the training and validation sets; 29 in the testing set). On cross-validation, six ML algorithms with Log Loss <0.688 had AUC >0.800. On validation, 10 ML algorithms had a Log Loss value <0.672, among which six had AUC >0.800. On model evaluation of the selected models on the unseen testing set, nine ML models had AUC >0.800 with Gradient Boosting Random Forest (xgboost) [GB RF (xgboost)] achieving the highest AUC of 0.860 and could detect the presence of hibernating myocardium in 21/29 (72.4%) perfusion defects with a precision of 87.5% (21/24), specificity 85.7% (18/21), CA 78.0% (39/50) and F1 Score 0.792. Four models depicted a clear pattern of model interpretability based on the beeswarm SHAP plots. These were GB RF (xgboost), GB (scikit-learn), GB (xgboost), and Random Forest. Conclusion Our study demonstrates the potential of ML in detecting hibernating myocardium using radiomics features extracted from perfusion defects on rest MPI images. This proof-of-concept underscores the notion that radiomics features capture nuanced information beyond what is perceptible to the human eye, offering promising avenues for improved myocardial viability assessment.