Search Results (147)

Introduction/Background: Most adrenal incidentalomas (AIs) are non-functioning adrenal tumors (NFATs) without clinically overt hormonal hypersecretion; one-third show subtle endocrine over-activity and mild autonomous cortisol secretion (MACS). One out of ten NFATs involves not a unilateral (UTs), but bilateral tumors (BTs). Bone health, as opposed to cardio-metabolic complications, is less studied in NFAs/MACS, particularly in BTs. Hence, we aimed to analyze (blood) mineral metabolism assays (MMAs), including bone turnover markers (BTMs), central Dual-Energy X-ray Absorptiometry (DXA), and 10-year fracture risk estimation (FRAX/FRAXplus) in menopausal patients with UTs vs. BTs. Methods: This was a retrospective, single-center study. The inclusion criteria were women aged ≥50 y and CT-based AI detection. The exclusion criteria were medication against osteoporosis, malignancies, bone metabolic disorders, and cs-1mg-DST >5 µg/dL. Results: The cohort [N = 129; mean age: 62.39 ± 7.9 y; and y since menopause (YSM): 13.7 ± 8] included UT (62.22%) and BT (31.78%) groups with a similar age, YSM, type 2 diabetes rate (35.23% vs. 36.59%), arterial hypertension (73.6% vs. 75.5%), BMI, fasting glycemia, and glycated hemoglobin A1c (p > 0.5 for each). The borderline significance for morning cortisol was higher in UTs vs. BTs [median (interquartile interval): 13.9 (11.16, 15.00) vs. 10.10 (8.88, 12.95) µg/dL; p = 0.05] and the MACS-positive rate (24.45% vs. 36.59%; p = 0.051). The largest tumor diameter was similar (2.26 ± 0.97 vs. 2.51 ± 0.87 cm; p = 0.175), as was cs-1mg-DST [1.27 (1.01, 1.95) vs. 1.52 (0.92, 2.78) µg/dL; p = 0.357]. MMAs, BTMs, and DXA-BMD/T scores were similar in the UT vs. BT groups. The most prevalent DXA categories were osteopenia (50.82%) and normal (41.38%). The rate of DXA bone impairment (osteoporosis + osteopenia) was 72.13% vs. 58.62%. A generally low prevalence of fragility fractures was found (3.88%; N = 5, 3/2 between the groups). Out of the 25.58% (N = 33) females who were found to be MACS-positive, 54.55% were in the UT group and 45.45% were in the BT group. Age, YSM, the rate of analyzed comorbidities, BMI, biochemical parameters, DXA/BMDs, and FRAX/FRAXplus (lumbar BMD adjustment)-based probabilities were similar between the UT and BT groups, regarding MACS-positive vs. MACS-negative groups. Diabetic patients were all MACS-positive. A higher PTH level in the MACS-positive UT vs. MACS-positive BT groups (36.32 ± 9.21 vs. 51.65 ± 9.58 pg/mL; p = 0.01) was found, with the mean 25-hydroxyvitamin D showing mild deficiency (24.21 ± 12.73 vs. 26.16 ± 9.89 ng/mL; p = 0.694). In UTs, the largest tumor diameter statistically significantly correlated with baseline ACTH (r = −0.391; p < 0.001) and cs-1mg-DST (r = 0.306; p < 0.001), while in BTs, the largest diameter of the two tumors showed a positive correlation with cs-1mg-DST (r = 0.309; p = 0.012). Conclusions: The findings from this real-life setting (similar age, YSM, and diabetes and MACS-positive rates) could help us to better understand the bone features in UTs vs. BTs, noting that ACTH/cs-1mg-DST measurements showed no difference. The study population was associated with a generally low fracture prevalence and 10-year fracture risk probabilities, which might act as a bias in this distinct clinical exploration. Whether a multifactorial algorithm is needed to provide a 360-degree perspective of the bone health assessment in these patients remains an open matter. So far, starting from the current guidelines, a patient-centered approach is mandatory. To our best knowledge, this study adds to the limited number of prior studies regarding bone impairment in bilateral tumors. Full article

Background: Recent advances in antibody discovery technologies, especially progress in de novo synthesis through machine learning, have imposed a significant production challenge for the generation of a large diversity of antibodies against nearly any target of interest. There is a demand for the rapid production of dozens of purified antibodies in 10-milligram quantities sufficient for functional screening and molecular assessment studies. Objectives: To meet this requirement, a semi-automated production methodology and workflow was developed to bridge the miniaturized high-throughput screenings (HTSs) and the conventional custom-scale workflow by taking advantage of four new technology applications. Methods: First, it exploited a novel, simple, high-titer transient expression system, “CHO4Tx^®”, which could achieve high yields in the range of 200 mg/L and above, across a variety of antibody constructs, including challenging targets. The consistently high yields from this transient CHO platform enabled the delivery of ~20 mg of crude material per 100 mL scale flask production with a throughput capacity of nineteen constructs in a single run. Secondly, we established a magnetic ProA bead in-culture antibody-capturing process, which significantly shortened the production timeline by eliminating the steps of cell centrifugation, filtration, and medium column loading. Third, we utilized the GenScript AmMag™ SA Plus semi-automation, which could handle magnetic ProA bead elution for 12 constructs within less than 1 h. Lastly, we transformed the AKTA Pure^TM system into an automated buffer exchange purification system with a capacity of processing 19 samples in a single run. Results and Conclusions: This new production platform was proven to be robust and could be applied for the routine production of antibodies of sufficient quality and quantity in support of cell-based assays and biophysical characterization. Full article

Background: Third-generation aromatase inhibitors (CYP19A1) are the mainstay of treatment for estrogen-receptor-positive breast cancer. This is because estrogen is required for cancer growth in approximately 70% of patients with this condition. Although potent and effective, aromatase inhibitors induce resistance and secondary effects, requiring treatment to be discontinued. This clinical limitation highlights the need to search for new molecules. Previous studies have led to the identification of a set of indole sulfonamide molecules that exhibit interesting activity against aromatase. Methods: Phenyl and benzyl sulfonamide derivatives with alkylated heterocycles linked by short methylene bridges were designed and synthesized. The aromatase inhibition and cytotoxicity were tested through in vitro assays. Molecular docking and dynamic simulations evaluated the interactions with the aromatase enzyme, while a target fishing strategy linked to gene associations relevant to breast cancer helped to uncover other targets. Results: All of the non-steroidal inhibitors synthesized showed significant activity. Compounds 3 and 9 demonstrated IC₅₀ values in the low micromolar range and selective action against MCF7 breast cancer cells over healthy lines. Computational studies confirmed stable and favorable aromatase binding. Target fishing identified EGFR and PTK2B as additional potential targets for a multi-target therapeutic strategy. Conclusions: Compounds 3 and 9 outperform indole-based inhibitors in their potency and selectivity, revealing strong therapeutic potential. Their binding affinity and specificity support further development. EGFR and PTK2B may enable a broader, multi-target approach. Full article

Mycoplasmas are known respiratory pathogens in tortoises, but few studies exist in snakes. To better understand the correlation with clinical signs and co-infections, samples from mycoplasma-positive snakes with and without clinical respiratory disease were analyzed. Oral swabs from 15 snakes (pythons n = 12, boas n = 3) were examined using polymerase chain reaction (PCR) and third-generation sequencing (TGS). Additionally, mycoplasma isolation assays were performed. Pathogens detected by PCR included Mycoplasmas (15/15, 100%), serpentoviruses (9/15, 60%), and Chlamydia sp. (2/15, 13%); those detected by TGS included Mycoplasmas (14/15, 93%), serpentoviruses (10/15, 67%), Chlamydia sp. (1/15, 7%), and 15 different bacterial species. Sequencing of the mycoplasma PCR products revealed a close genetic relationship to Mycoplasmopsis agassizii. TGS identified genetically distinct mycoplasmas and three different serpentoviruses. While mycoplasmas could not be successfully propagated, Brucella intermedia comb. nov. was identified in eight cultures. Respiratory disease in snakes is often multifactorial, involving various pathogens and environmental influences. This study demonstrates that comprehensive diagnostics are essential for understanding disease processes in snakes and improving the detection of diverse pathogens. Further research is needed to improve laboratory diagnostics for infectious diseases in reptiles and to better understand the roles of various pathogens in respiratory diseases in snakes. Full article

Background: Salmonella enterica and Escherichia coli are common foodborne pathogens of global concern, particularly due to their antimicrobial resistance, notably to cephalosporins. Objective: This study aimed to evaluate a polymerase chain reaction-based lateral flow strip (PCR-LFS) assay for the detection of Salmonella spp. and E. coli harboring the bla_CTX-M gene, which confers resistance to third-generation cephalosporins. Methods: Two duplex PCRs (dPCR) were established to detect E. coli-harboring bla_CTX-M (set 1) and Salmonella-harboring bla_CTX-M (set 2). 600 bacterial isolates and raw pork mince spiked with bla_CTX-M-harboring E. coli and Salmonella were used to evaluated. Results: Both dPCR assays successfully detected bla_CTX-M-positive E. coli or Salmonella strains, while strains lacking the gene showed no amplification. Non-E. coli and non-Salmonella strains were PCR-negative unless they carried bla_CTX-M. The dPCR-LFS showed 100% validity including accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for both E. coli or Salmonella spp. harboring or lacking bla_CTX-M. The assay accurately detected target strains without cross-reactivity with other bacteria or antimicrobial resistance genes. Cohen’s Kappa coefficient indicated perfect agreement (κ = 1), reflecting the high reliability of the dPCR-LFS. The assay could detect as low as 25 CFU/mL for bla_CTX-M-positive E. coli and 40 CFU/mL for bla_CTX-M-positive Salmonella in spiked raw pork mince. Conclusions: This assay is rapid, easy to interpret, and suitable for large-scale screening in surveillance programs. Full article

The tarnished plant bug, Lygus lineolaris (TPB), (Palisot de Beauvois), and the western tarnished plant bug (WTPB), Lygus hesperus, Knight, are major agricultural pests that cause significant damage to a wide range of crops in the southeastern and southwestern United States. Flonicamid (commercial name: Carbine 50WG) is generally effective against various sap-feeding pests, including both L. hesperus and L. lineolaris. This study evaluated the toxicity of flonicamid on third-instar nymphs and adults of both Lygus species under laboratory conditions. Two bioassay methods were used: spray application to assess both contact and oral toxicity, and dipping to evaluate oral toxicity. Results showed that L. hesperus was significantly more susceptible to flonicamid than L. lineolaris across both bioassay methods. While no significant differences in toxicity were observed between spray and dipping assays, third-instar nymphs exhibited significantly higher sensitivity than adults in both species. The addition of piperonyl butoxide (PBO), a known inhibitor of cytochrome P450-monooxygenases (P450s), significantly enhanced the toxicity of flonicamid, suggesting that P450 enzyme plays a critical role in its detoxification. Sublethal exposure to flonicamid also induced increased P450 activity in both species. These findings provide valuable insights into the differences in susceptibility between L. lineolaris and L. hesperus to flonicamid and indicate that P450-mediated detoxification is critical for flonicamid metabolism. Such insights are valuable for early resistance monitoring and optimizing flonicamid application in integrated pest management programs. Full article

Background: Kidney transplant recipients (KTRs) exhibit a significantly diminished immune response to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) vaccines compared with the general population, primarily due to ongoing immunosuppressive therapy. This study evaluated the immunogenicity of a third SARS-CoV-2 mRNA vaccine dose in KTRs and assessed the association between antibody response and protection against SARS-CoV-2 breakthrough infection. Additionally, the clinical and immunological correlates of post-vaccination SARS-CoV-2 infection were examined. Methods: A prospective cohort of 135 KTRs received a third vaccine dose approximately six months following the second dose. Plasma samples were collected at baseline (pre-vaccination), six months after the second dose, and six weeks following the third dose. Humoral responses were assessed using SARS-CoV-2-specific Immunoglobulin G (IgG) titers and virus neutralization assays against wild-type (WT) and viral strains, including multiple Omicron sub-lineages. Results: After the third vaccine dose, 74% of the KTRs had detectable SARS-CoV-2-specific IgG antibodies, compared with 48% following the second dose. The mean IgG titers increased approximately ten-fold post-booster. Despite this increase, neutralizing activity against the Omicron variants remained significantly lower than that against the WT strain. KTRs who subsequently experienced a SARS-CoV-2 breakthrough infection demonstrated reduced neutralizing antibody activity across all variants tested. Additionally, individuals receiving triple immunosuppressive therapy had a significantly higher risk of SARS-CoV-2 breakthrough infection compared with those on dual or monotherapy. A multivariate machine learning analysis identified age and neutralizing activity against WT, Delta, and Omicron BA.2 as the most robust correlates of SARS-CoV-2 breakthrough infection. Conclusions: A third SARS-CoV-2 mRNA vaccine dose significantly improves SARS-CoV-2-specific IgG levels in KTRs; however, the neutralizing response against Omicron variants remains suboptimal. Diminished neutralizing capacity and intensified immunosuppression are key determinants of SARS-CoV-2 breakthrough infection in this immunocompromised population. Full article

Polyomaviruses have the potential to cause significant morbidity not only in transplant medicine, but also in other forms of disease or variants of immunosuppression. In kidney transplant recipients or recipients of human stem cell transplants, the BK-Virus is the major proponent of manifestations such as BKPyV-associated nephropathy or hemorrhagic cystitis. As no polyomavirus-specific drug with proven in vivo effects has been developed so far, methods to screen for such drugs are important. This work describes the establishment of a virus-secreting cell line. By infecting a pre-established monkey kidney cell line (COS-1) with a non-rearranged human BK polyomavirus isolated from a kidney transplant patient suffering from BKPyV-associated nephropathy, a continuously replicating cell type with consistent virus secretion could be established and was termed COSSA. Measurements of BKPyV replication, virion production, and secretion were performed both intracellularly and in the cell supernatant. Viral proteins such as VP1 and LTAg were accurately tracked by confocal microscopy, as well as by immunoblot and qPCR. An intracellular flow cytometry (FACS) assay detecting VP1 protein was established and revealed an expanded range of positive intracellular signals. The viruses produced proved to be infectious in human tubular epithelial cell lines. Long-range sequencing of the COSSA genome using Oxford Nanopore Technology revealed a total of five distinct BKPyV integration events. One integration of a partial BKPyV genome was located upstream of the epidermal growth factor receptor gene. The second and third, both truncated forms of integration, were close to histocompatibility gene locuses, while the fourth was characterized by a ninefold and the fifth by a fourfold tandem repeat of the BKPyV genome. From both of the repeat forms, virus replicates were derived showing deletions/duplications on early and late genes and inversions within the non-coding control region (NCCR). This pattern of repetitive viral genome integration is a potential key driver of enhanced viral replication and increased virion assembly, ultimately supporting efficient virus egress. Quantitative PCR analysis confirmed the release of approximately 10⁸/mL viral units per 48 h from 2 × 10⁵ COSSA cells into the culture supernatant. Notably, the NCCR region of the most frequent copies of circular virus and the integrated tetrameric tandem repeat exhibited a rearranged configuration, which may contribute to the observed high replication dynamics. The establishment of a consistent methodology to generate and secrete BKPyV from a cell line is expected to significantly facilitate antiviral drug development. Full article

Background/Objectives: Lercanidipine is a third-generation dihydropyridine calcium channel blocker. In addition to their well-established cardiovascular effects, calcium channel blockers are increasingly recognized for their therapeutic potential in various cancers. This study aimed to investigate the potential anticancer effects of lercanidipine on cancer cell lines—particularly in combination with cisplatin—by assessing parameters such as cell viability (MTT assay), proliferation, MAPK pathway activity, caspase enzyme levels, and TNF-α expression. Methods: In this study, the effects of lercanidipine, both alone and in combination with cisplatin, on cell viability were evaluated using the MTT assay in MCF-7, SH-SY5Y, PC3, and HEK293 cell lines. To assess intracellular signaling and apoptotic pathways, MAPK inhibition, as well as caspase-3 and caspase-8 activities, were measured using ELISA. Additionally, to evaluate the anti-inflammatory potential, TNF-α levels in LPS-stimulated RAW264.7 cells were analyzed via. Results: The study revealed that lercanidipine showed significant cytotoxic effects, particularly in SH-SY5Y and PC3 cancer cell lines, while it did not induce a 50% loss of viability in healthy HEK293 cells. When combined with cisplatin, lercanidipine enhanced cytotoxicity by 2.7-fold in neuroblastoma (SH-SY5Y) cells, 1.6-fold in breast cancer (MCF7) cells, and 1.9-fold in prostate cancer (PC3) cells. MAPK activity was inhibited by 83.6% at 20 μM lercanidipine, while dose-dependent increases in caspase-3 and caspase-8 activities were observed. Additionally, lercanidipine decreased TNF-α levels in LPS-stimulated RAW264.7 cells, indicating its potential anti-inflammatory effect. Conclusions: In conclusion, lercanidipine demonstrated selective anticancer effects in cancer cell lines and showed synergistic cytotoxicity when combined with cisplatin. It also significantly inhibited MAPK signaling, activated apoptotic caspases, and reduced TNF-α levels, suggesting potential anti-inflammatory activity. These findings highlight lercanidipine’s potential for repurposing as an adjunct in cancer therapy. Full article

Background/Objectives: Over the past decade, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli have emerged as a significant public health concern due to their potential to spread beyond clinical settings and healthy carriers, in various environments, including in animal source foods. This study seeks to investigate the molecular characteristics of ESBL-producing E. coli strains isolated from food of animal origin, with a focus on chromosomal typing, plasmid typing, and the description of the associated resistance genes’ genetic environment. Methods: Ninety-seven food of animal origin samples were tested for E. coli isolates resistant to cefotaxime. The resulting isolates were then subjected to antimicrobial susceptibility testing and PCR analysis to detect beta-lactamase genes. Additional assays, encompassing mating-out procedures, molecular typing utilizing Pulsed-Field Gel Electrophoresis, Multilocus Sequence Typing Analysis, and Oxford Nanopore Technology Lite whole plasmid sequencing, were also conducted. Results: E. coli was detected in 26 raw food specimens, generating a percentage of 27%. Fourteen of the current isolates (14%) were resistant to third generation cephalosporins, producing CTX-M-1, CTX-M-15, CTX-M-55, and SHV-12 beta-lactamases. The respective genes were accompanied by Insertion Sequences ISEcp1 and IS26, facilitating their transfer. Among plasmids harboring ESBL genes, representatives belonging to incI1 incompatibility group prevailed (5/8), followed by IncY and IncX3. Most plasmids proved conjugative. Diversity of molecular fingerprints of ESBL producing E. coli was revealed. Conclusions: To the best our knowledge, this study is the first to describe the molecular characteristics of E. coli isolates producing ESBLs sourced from foods of animal origin in Greece. The prevalence of ESBLs in our confined food collection is primarily associated with the very successful IncI1 plasmids, which were not linked to a specific E. coli genetic background. This lack of association confirms that horizontal plasmid transfer plays a more significant role than clonal dissemination in the spread of ESBL-mediated cephalosporin resistance. Full article

Background/Objectives: Circulating high-sensitivity C-reactive protein (hsCRP) is a well-established biomarker of low-grade systemic inflammation; levels above 3 mg/L indicate high cardiovascular risk. Although cross-sectional studies have shown associations between antioxidant vitamin intake and hsCRP levels, prospective data remain limited. This study aims to investigate the associations of dietary intake of vitamins A, C, and E with the 8-year risk of elevated serum hsCRP levels (>3 mg/L). Participants/Methods: This prospective study included 7695 adults from population-based cohorts. Serum hsCRP was assayed at the 4- and 8-year follow-ups; levels above 3 mg/L were considered elevated. Dietary intake of vitamin A, retinol, β-carotene, and vitamins C and E was assessed at baseline and at the 4-year follow-up using a food frequency questionnaire. A multivariable Cox proportional hazards regression was conducted with adjustments for potential confounders. Results: When vitamin intake was categorized into quintiles, vitamin C intake demonstrated an inverse association, whereas β-carotene intake exhibited a U-shaped association with the risk of elevated serum hsCRP concentrations. Hazard ratios (HRs) [95% confidence intervals (CIs)] for the third and fourth quintiles of vitamin C intake were 0.72 [0.53, 0.98] and 0.70 [0.49, 0.98], respectively, compared with the first quintile. The HR [95% CI] for the third quintile of β-carotene intake was 0.69 [0.50, 0.95] compared with the first quintile. However, excessive consumption of vitamin E increased the risk of elevated hsCRP levels; HR (95% CI) was 1.62 [1.19, 2.21] for participants consuming >120% of adequate intake (AI) relative to those with 80–119% of AI. In stepwise analysis to identify a best-fit model, significant variables included the presence of diabetes or hypertension, calorie intake, age, body mass index, sex, educational level, moderate or vigorous physical activity, and vitamin C intake. Conclusion: These findings suggest that dietary intake of vitamins A and C may help prevent elevated hsCRP levels in the general adult population. Further epidemiological studies are warranted to confirm these potential causal associations. Full article

Background/Objectives: The pulmonary administration of antibiotics can be advantageous in treating pulmonary infections by promoting high intrapulmonary drug concentrations with reduced systemic exposure. However, limited benefits have been observed for pulmonary administration versus other administration routes due to its rapid clearance from the lung. Here, the effects of structural modifications on the epithelial permeability and antibacterial potency of a third-generation cephalosporin were investigated to improve the understanding of drug properties that promote intrapulmonary retention and how they may impact efficacy. Methods: Ceftazidime was modified by attaching 18 hydrophobic, hydrophilic, and mucus-binding motifs to the carboxylic acid distant from the beta-lactam by amidation. Epithelial permeability was investigated by drug transport assays using human bronchial epithelial air–liquid interface cultures. Antibacterial potency was determined by microtiter MIC assays with B. pseudomallei, P. aeruginosa, E. coli, and S. aureus. Results: A 40–50% reduction in the transepithelial transport rate was exhibited by two PEGylated ceftazidime analogs (mPEG8- and PEG5-pyrimidin-2-amine-ceftazidime) and n-butyl-ceftazidime. An increase in the transport rate was exhibited by four analogs bearing small and hydrophobic or negatively charged motifs (n-heptane-, phenyl ethyl-, glutamic acid-, and 4-propylthiophenyl boronic acid-ceftazidime). The antibacterial potency was reduced by ≥10-fold for most ceftazidime analogs against B. pseudomallei, P. aeruginosa, and E. coli but was retained by seven ceftazidime analogs primarily bearing hydrophobic motifs against S. aureus. Conclusions: The covalent conjugation of PEGs with MW > 300 Da reduced the epithelial permeability of ceftazidime, but these modifications severely reduced antibacterial activity. To improve the pulmonary retention of antibiotics with low membrane permeability, this work suggests future molecular engineering studies to explore high-molecular-weight prodrug strategies. Full article

The coffee industry generates a large amount of waste that is difficult to treat due to its chemical composition, namely, the presence of caffeine and its derivatives, as well as recalcitrant molecules such as tannins (mainly condensed tannins or polymeric procyanidins), which make it an undervalued waste product. Procyanidins are compounds beneficial to human health and can be found in nature in fruit, grain, seeds, and beverages, among other foods. The zero-waste approach has allowed for the valorization of by-products from the food industry. Currently, coffee pulp is the target of research on extraction, purification, and alternative use. Research on the fungal degradation of procyanidins has emerged as an avenue for the efficient use of these by-products. In this study, the degradation and biotransformation of procyanidin is evaluated and comprises three steps: first, the extraction and partial purification of procyanidins from coffee pulp; second, the production of the potential procyanidin-degrading enzyme by submerged fermentation with Aspergillus niger GH1; third, enzymatic extracellular extract evaluation using a model system with commercial procyanidin C1. The biodegradation/biotransformation results reveal the formation of new compounds, including a final compound with an m/z of 289, possibly a monomeric molecule such as catechin or epicatechin. Identification of the compounds by HPLC-MS confirmed procyanidin C1 depletion under the described assay conditions, which could be used to understand biodegradation pathways proposed for future study. Furthermore, these results confirm that A. niger GH1 is able to degrade and biotransform procyanidin C1. Full article

Chemotherapy resistance is a significant barrier to effective cancer treatment. A key mechanism of resistance at the single-cell level is the overexpression of drug transporters in the ABC family, particularly P-glycoprotein (P-gp), which leads to multidrug resistance (MDR). Inhibitors of these transporters can help re-sensitize cancer cells to chemotherapeutics. This study evaluated elacridar (GG918 and GF120918), a potent third-generation P-gp inhibitor, for its ability to reverse MDR in paclitaxel (PAC)-resistant ovarian cancer cell lines. Sensitive and PAC-resistant cells were cultured in two-dimensional (2D) and three-dimensional (3D) models. MDR1 gene expression was analyzed using Q-PCR, and P-gp protein expression was examined via Western blot and immunofluorescence. Drug sensitivity was evaluated with MTT assays, and P-gp activity was analyzed by flow cytometry and fluorescence microscopy. Elacridar effectively inhibited P-gp activity and increased sensitivity to PAC and doxorubicin (DOX) in 2D cultures but not cisplatin (CIS). In 3D spheroids, P-gp activity inhibition was observed via Calcein-AM staining. However, no re-sensitization to PAC occurred and limited improvement was observed for DOX. These findings suggest that elacridar effectively inhibits P-gp in both 2D and 3D conditions. However, its ability to overcome drug resistance in 3D models is limited, highlighting the complexity of tissue-specific resistance mechanisms. Full article

Actinobacillus pleuropneumoniae is a major swine pathogen, classified into 19 serotypes based on capsular polysaccharide (CPS) loci. This study aimed to improve the diagnostic method to differentiate between serotypes 9 and 11, which are challenging to distinguish using conventional serological and molecular methods. A novel qPCR assay based on locked nucleic acid (LNA) probes was developed and validated using a collection of reference strains representing all known 19 serotypes. The assay demonstrated specificity in detecting the nucleotide variation characteristic of the serotype 9 reference strain. However, the analysis of a clinical isolate collection identified discrepancies between LNA-qPCR and serological results, prompting further investigation of the cps and O-Ag loci. Subsequent nanopore sequencing and whole-genome sequencing of a collection of 31 European clinical isolates, previously identified as serotype 9, 11, or undifferentiated 9/11, revealed significant genetic variations in the cps and O-Ag loci. Ten isolates had a cpsF sequence identical to that of the serotype 11 reference strain, while six isolates had single-nucleotide polymorphisms that were unlikely to cause significant coding changes. In contrast, 15 isolates had interruptions in the cpsF gene, distinct from that found in the serotype 9 reference strain, potentially leading to a serotype 9 CPS structure. In the O-Ag loci, differences between serotypes 9 and 11 were minimal, although some isolates had mutations potentially affecting O-Ag expression. Overall, these findings suggest that multiple genetic events can lead to the formation of a serotype 9 CPS structure, hindering the development of a single qPCR assay capable of detecting all cpsF gene mutations. Our results suggest that, currently, a comprehensive analysis of the cpsF gene is necessary to accurately determine whether the capsule of an isolate corresponds to serotype 9 or 11. Although such analyses are feasible with the advent of third-generation sequencing technologies, their accessibility, cost, and time to result limit their use in routine diagnostic applications. Under these circumstances, the designation of the hybrid serovar 9/11 remains a valid approach. Full article