Search Results (164)

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, a burden further exacerbated by HIV co-infection and the increasing prevalence of drug-resistant strains. Although a wide range of laboratory diagnostic methods are currently available, their applicability, implementation, and clinical impact vary substantially across healthcare settings with different levels of complexity and resources. This review provides a comprehensive overview of the main laboratory diagnostic methods for active and latent TB, emphasizing their clinical applicability, implementation challenges, and role within integrated diagnostic strategies. Conventional approaches, such as smear microscopy and culture, are discussed alongside modern diagnostic technologies, including automated nucleic acid amplification tests (NAATs), loop-mediated isothermal amplification (LAMP), line probe assays (LPAs), next-generation sequencing (NGS), and lateral flow assays, highlighting their strengths and limitations in distinct epidemiological and operational contexts. Unlike existing WHO guidelines and prior reviews that predominantly focus on test performance and recommendation status, this review adopts an implementation-oriented perspective, critically examining diagnostic methods in light of real-world constraints, regional disparities, and evidence gaps. Particular attention is given to limitations related to laboratory infrastructure, biosafety, workforce capacity, and sustainability, as well as to under-addressed areas such as latent TB, metagenomic approaches, and the investigation of co-pathogens. By integrating WHO guidance with contextual and operational considerations, this review aims to support rational test selection and the development of flexible, integrated diagnostic workflows tailored to local health system capacity, patient populations, and clinical scenarios, thereby strengthening the effectiveness and equity of TB diagnostic strategies. Full article

Background/Objectives: Imatinib, the first tyrosine kinase inhibitor, marks the beginning of a revolution in clinical oncology. Disrupting oncogenic kinase-dependent signaling pathways represents a key strategy for advancing targeted cancer therapies. Terpene analogues of imatinib were developed to probe the influence of terminal ring modifications on BCR-ABL inhibition and downstream oncogenic signaling. Methods: Nine novel imatinib analogues bearing bulky aliphatic moieties were designed, synthesised, and structurally characterized by ¹H/¹³C NMR spectroscopy and high-resolution mass spectrometry (HRMS). Molecular docking calculations were performed to assess the binding modes and intermolecular interactions. The cytotoxicity of the newly synthesized imatinib derivatives was evaluated across a panel of BCR-ABL+ leukemia cell lines. Results: Molecular docking analyses demonstrated conserved interactions within the ATP-binding site of BCR-ABL for all derivatives, with calculated docking scores ranging between 123 and 128, while modifications at the terminal ring introduced subtle changes in electrostatic and steric profiles. Biological evaluation using MTT-based cytotoxicity assays in BCR-ABL+ leukemic cell lines revealed enhanced antiproliferative activity compared with imatinib, with compounds 6a (flexible cyclohexyl) and 6d (rigid camphane-type (+)-isopinocampheyl) exhibiting the lowest micromolar activity in the AR-230 model (IC₅₀ values of 1.1 and 1.2 μM, respectively). Proteome-wide phosphokinase profiling demonstrated shared suppression of STAT5/3/6, RSK1/2, S6K1/p70, and Pyk2, confirming effective disruption of canonical BCR-ABL pathways. Critically, the terpene moiety dictated downstream pathway bias: 6a preferentially attenuated CREB activation, whereas 6d more effectively suppressed the PI3K/Akt oncogenic axis and strongly activated proapoptotic p53-mediated stress responses. Conclusions: Our findings establish terpene-engineered imatinib analogues as tunable modulators and promising candidates for targeting downstream BCR-ABL signaling pathways in leukemia treatment. Full article

Rifampicin-resistant tuberculosis (RR-TB) remains a major threat to global TB control, primarily driven by mutations in the rpoB gene of Mycobacterium tuberculosis (Mtb). Most resistance-conferring mutations occur within the 81-base pair RIF resistance determining region (RRDR), particularly at codons S450L, H445Y/D, and D435V, which are strongly associated with high level resistance. However, increasing evidence of low-frequency and disputed variants both within and beyond the RRDR reveals a broader genetic spectrum that contributes to diagnostic uncertainty and variable phenotypic outcomes. This review summarizes current knowledge of high frequency, low frequency, and disputed rpoB mutations and their implications for molecular detection of RIF resistance. Structural analyses show that specific amino acid substitutions alter key hydrogen bonds or create steric hindrance in the RIF-binding pocket, leading to diverse resistance levels. Despite the success of molecular platforms such as Xpert MTB/RIF and line probe assays, their hotspot-based detection limits sensitivity to noncanonical variants. Lowering the minimum inhibitory concentration (MIC) breakpoint and integrating sequencing-based approaches, such as targeted and whole-genome sequencing, can enhance detection accuracy. A combined genomic and phenotypic framework will be essential to close existing diagnostic gaps and advance precision guided management of RIF-resistant and multidrug-resistant tuberculosis. Full article

The enormous applications of various nanoparticles (NPs) have raised the possibility that humans may be simultaneously exposed to mixtures of them in real life. Realistically, this situation may apply to plastic NPs, mainly derived from the breakdown of larger plastics, and to silver NPs, both of which are among the most frequently detected NPs in the envirnment due to their applications in healthcare, consumer products, and water purification. Although numerous studies have examined the toxicity of plastic and silver NPs individually, knowledge of their combined toxicity remains limited. Hence, the main objective of our study was to investigate the toxicity of high-density polyethene nanoparticles (HDPE NPs), thermally isolated from food-cooking bags, in combination with citrate-stabilised silver nanoparticles (AgNPcit) to Caco-2 and HT29MTX cells growing in 2D monoculture or in 3D triple-culture with Raji cells. Cellular uptake of NPs was quantified from the side-scatter (SSC) signal in flow cytometry; toxicity was evaluated by the neutral red assay; apoptosis was evaluated by the Annexin V method; and induction of oxidative stress was evaluated by a fluorescent method using DCFDA and DHR probes. Both cell lines took up both types of NPs; however, HT29MTX cells were more effective in the NPs’ uptake. Interestingly, HDPE NPs and AgNPcit mutually inhibited each other’s uptake, which suggests a similar mechanism of entry. Both types of NPs were toxic to both cell lines growing in monoculture; Caco-2 cells were more susceptible than HT29MTX. The toxicity was attributed to the induction of oxidative stress and associated apoptosis. In line with the mutual inhibition of the NPs’ uptake, the toxic effect of both NPs in the mixture was less than that expected as the sum of individual treatments. The toxic effects of both NPs or their mixture were less pronounced in the triple-culture Caco-2/HT29MTX/Raji, than in Caco-2 and HT29MTX growing in monoculture. Full article

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality, highlighting the urgent need for novel therapeutic strategies. Apoptin, encoded by the VP3 gene of the chicken anemia virus, selectively induces apoptosis in cancer cells while sparing normal cells. We previously engineered a recombinant oncolytic adenovirus (Ad-VP3) capable of high-level Apoptin expression in tumor cells. In this study, we evaluated the antitumor activity of Ad-VP3 in the human HCC cell line Hep3B. CCK-8, crystal violet, Hoechst 33342 staining, flow cytometry, and tumor sphere formation assays revealed that Ad-VP3 inhibited cell viability, proliferation, and stemness. Annexin V staining, JC-1/TMRM probes, and Western blot analysis demonstrated induction of apoptosis and reduction of mitochondrial membrane potential. Wound-healing, Transwell, and BioCoat invasion assays, along with Western blotting, confirmed suppression of migration and invasion. Ad-VP3 significantly inhibited the viability, proliferation, migration, and invasion of Hep3B cells in a time- and dose-dependent manner. It induced mitochondrial membrane potential loss and apoptosis, downregulated stemness-related proteins (ALDH1A1, KLF4, and Sox2), and suppressed epithelial–mesenchymal transition markers (Snail, Twist1, Slug, Vimentin, and MMP-9), indicating strong antitumor activity. The recombinant oncolytic adenovirus Ad-VP3 exerts potent antitumor effects on hepatocellular carcinoma cells by inducing mitochondrial dysfunctionmediated apoptosis and impairing stemness and metastatic potential, suggesting its promise as a novel therapeutic strategy for HCC. Full article

Sericin is a major polypeptidic constituent of the silk in the cocoons produced by the Bombyx mori silkworm. Certain fractions isolated from sericin exhibited antioxidant properties in a variety of reported experimental settings. In a previous study, we found that only the non-protein fraction, extracted from crude sericin, displayed antioxidative activity in cultures of murine retinal photoreceptor cells (661W), a cell line that is highly sensitive to oxidative stress associated with diseases of the retina. In the same assay, the protein fraction (purified sericin) did not show any such activity. To check these findings, in the present study, two additional different assays were employed: an in vitro assay based on the dose-dependent mitigating effects exerted by each sericin fraction on the activity of antimycin A in cultures of 661W cells and an in vivo assay based on an animal (rat) model of retinal ischemia/reperfusion injury. In both assays, nitroxide was appended as a fluorometric molecular probe, and fluorescent intensity was monitored by either flow cytometry (in vitro) or the Micron IV retinal imaging system (in vivo). The in vitro assay indicated unequivocally antioxidative capacity for the non-protein fraction and a lack of it for the purified sericin. The in vivo assay indicated that each fraction was able to act as an antioxidant. We hypothesized that the ability of purified sericin to display antioxidative activity in vivo, but not in vitro, was the result of the metabolic degradation of sericin, a process that delivered serine, an amino acid with known antioxidant properties. However, this hypothesis needs experimental confirmation. Full article

Background: Rapid and high-throughput diagnostic methods are essential for controlling the spread of infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Lateral flow immunoassay (LFIA) strips provide a cost-effective and user-friendly platform for point-of-care testing. However, the sensitivity of conventional LFIA kits is often limited by the performance of their detection probes. This study reports a highly sensitive LFIA strip for detecting the SARS-CoV-2 nucleocapsid (NP) protein using platinum-decorated poly(2-vinylpyridine) nanoparticles (Pt-P2VPs) as probes. Methods: Monoclonal antibodies against SARS-CoV-2 NP were conjugated with Pt-P2VPs and incorporated into LFIA strips. The test line was coated with anti–SARS-CoV-2 NP monoclonal antibody, and the control line with goat anti-mouse IgG. Recombinant proteins, viral strains, and nasopharyngeal swab specimens from patients were used to evaluate assay performance, with reverse transcription polymerase chain reaction (RT-PCR) as the reference standard. Diagnostic accuracy was assessed using nonparametric statistical tests. Results: Pt-P2VP-based LFIA strips enabled sensitive detection of recombinant NP and inactivated SARS-CoV-2, with minimal cross-reactivity. In 200 clinical specimens (100 PCR-negative and 100 PCR-positive), the assay achieved 74% sensitivity and 100% specificity, with strong correlation to viral RNA load. Compared with conventional LFIA kits, Pt-P2VP strips demonstrated superior sensitivity at lower viral loads. Conclusions: Pt-P2VPs represent a promising probe material for enhancing LFIA performance and may facilitate the development of rapid, sensitive, and scalable immunoassays for infectious disease diagnostics in biomedical applications. Full article

Background/Objectives: Targeting the PI3K/mTOR pathway is a promising strategy in cancer therapy, but its efficacy is often limited by apoptosis resistance. This study investigates the dual PI3K/mTOR inhibitor YYN-37, exploring its capacity to induce context-dependent cell death, particularly the non-apoptotic process of methuosis. Methods: We examined the effects of YYN-37 on HCT-116 and SJSA-1 cancer cell lines using cell viability assays, Western blot, and fluorescent tracers. Cell death mechanisms were probed with pathway-specific inhibitors. The role of VPS34 was assessed through kinase activity assays, siRNA-mediated knockdown, and rescue experiments with the agonist leucine. Proteomic profiling and an in vivo SJSA-1 xenograft model in BALB/c nude mice were utilized to evaluate broader mechanisms and anti-tumor efficacy. Results: YYN-37 induced caspase-3-dependent apoptosis in HCT-116 cells. In contrast, it triggered a reversible, cytoplasmic vacuolization in SJSA-1 cells, identified as methuosis. This vacuolization originated from endocytic pathways and was inhibited by EIPA and Baf-A1. YYN-37 directly inhibited VPS34 (IC₅₀ = 2.73 nM), and its knockdown replicated the vacuolization, which was conversely reversed by the VPS34 agonist leucine, confirming VPS34-dependency. Proteomics revealed lysosomal dysfunction in SJSA-1 cells and cell cycle alterations in HCT-116 cells. In vivo, YYN-37 treatment resulted in a 72.71% tumor growth inhibition, with histology confirming methuosis-like vacuolization. Conclusions: YYN-37 exerts potent, context-dependent anti-tumor effects by inducing apoptosis in HCT-116 cells and VPS34-mediated methuosis in SJSA-1 cells. This work establishes methuosis induction as a viable therapeutic strategy for apoptosis-resistant cancers and highlights VPS34 inhibition as a promising mechanism of action. Full article

Squaraine dyes are a class of organic compounds that exhibit some characteristics inherent to those of an “ideal photosensitizer”, such as high absorption at near-infrared-close wavelengths and to produce reactive oxygen species. The introduction of amines into their squaric ring, although known to increase the phototoxicity of squaraines, can improve dyes’ water solubility and induce bathochromic shifts compared to their unsubstituted derivatives, interesting effects in biological contexts. In this work, four new squaraines were synthesized and structurally, photophysically, and photochemically characterized (including absorption and aggregation, fluorescence, light stability, and singlet oxygen generation). Their potential as fluorescent probes for albumin detection was assessed through both in silico and in vitro approaches, as well as their suitability as potential photosensitizers for photodynamic therapy. For this last purpose, the 663 nm light-induced effects of the new dyes were evaluated against the PC-3 prostate cancer cell line, while their photocytotoxicity toward normal human dermal fibroblasts was also assessed using the MTT assay, to determine their potential tumor-selective effects. Low singlet oxygen quantum yields suggest that type I reactions predominate in generating cytotoxicity. Overall, the findings indicate that the designed squaraines exhibit moderate yet favorable interactions with albumin protein while demonstrating selective photodynamic effects toward prostate adenocarcinoma cancer cells, highlighting their potential as protein-assisted, tumor-targeted photosensitizers, providing a basis for further mechanistic studies. Full article

Drug-resistant tuberculosis (DR-TB) threatens global TB control. We investigated the prevalence and molecular characteristics of second-line drug resistance among rifampicin (RIF)- and/or isoniazid (INH)-resistant Mycobacterium tuberculosis complex (MTBC) isolates in São Paulo, Brazil, using the MTBDRsl v. 2.0 line-probe assay. MTBC isolates RIF- and/or INH-resistant by GenoType MTBDRplus or phenotypic testing (2019–2021) were subsequently tested by MTBDRsl for fluoroquinolone (FQ) and injectable drugs (capreomycin, amikacin, kanamycin) resistance. Isolates with inferred mutations underwent Sanger sequencing. Of 13,557 isolates, 728 (5.4%) were RIF- and/or INH-resistant (297 INH-R, 235 RIF-R, 196 MDR). Among them, 623 (85.6%) were tested by MTBDRsl; 582 (93.4%) showed no additional resistance, while 41 (6.6%) carried mutations. FQ resistance was detected in 38 isolates (92.7%), mostly in gyrA (n = 35). Three isolates with gyrB mutations were wild-type by sequencing. Two MDR isolates harbored the rrs a1401g mutation, and one also harbored gyrA D94G. Sequencing confirmed resistance in 38 of 41 isolates. Most MDR strains with second-line mutations (n = 32/33; 97%) were pre-XDR. Affected patients were predominantly male (68.4%), with pulmonary TB (92.1%), and unfavorable outcomes (39.5%). Second-line resistance prevalence was low overall, but FQ resistance was high among MDR isolates. Findings support integrating molecular and sequencing-based tools for accurate detection and management of DR-TB. Full article

Lateral Flow Assays (LFAs) are ubiquitous test platforms due to their affordability and simplicity but are often limited by low sensitivity and lack of flow control. The present work demonstrates the combination of LFAs with centrifugal microfluidic platforms that allows for enhancement of LFAs’ sensitivity via the increase in the dwell time of the analyte at the test line as well as by passing a larger sample volume through the LFA strip. The rate of advancement of the liquid front in the radially positioned NC strip is retarded by the centrifugal force generated on spinning disc; therefore, the dwell time of the liquid front above the test line of LFA is increased. Additionally, integrating a waste reservoir enables passive replenishment of additional sample volume increases total probed volume by approximately 20% (from 50 μL to 60 μL). Comprehensive analysis, including COMSOL multiphysics simulation, was performed to deduce the importance of parameters such as channel height (100–300 μm), disc spin rate (0–2000 rpm), and reaction kinetics (fast vs. slow binding kinetics). The analysis was validated by the experimental observation of the slower-reacting CD79b protein on the test strip. For slower-reacting targets like CD79b, fluorescence intensity increased by ~40% compared to the static LFA. A new merit number, TRc (Transport Reaction Constant), is introduced, which refines the traditional Damköhler number (Da) by including the thickness of the liquid layer (such as the height of the microchannel), which affects the final sensitivity of the assays and is designed to reflect the role channel height plays for surface-based assays (in contrast to the bulk assays). Full article

Antibody-free nucleic acid lateral flow assays (AF-NALFA) are an established approach for rapid detection of amplified pathogens DNA but can yield inconsistent signals across targets. Since AF-NALFA depends on dual hybridization of probes to single-stranded amplicons (ssDNA), site-specific thermodynamic (Gibbs free energy-ΔG) at probe-binding regions may be crucial for performance. This study investigated how site-specific-ΔG and sequence complementarity at probe-binding regions determine Test-line signal generation, comparing native and synthetic amplicons and assessing the effects of local secondary structures and mismatches. Asymmetric PCR-generated ssDNA amplicons of Listeria monocytogenes, Mycobacterium leprae, and Leishmania amazonensis were analyzed in silico and tested in AF-NALFA prototypes with gold-labeled thiol probes and biotinylated capture probes. T-line signals were photographed, quantified (ImageJ version 1.4k), and statistically correlated with site-specific-ΔG. While native ssDNA from M. leprae and L. amazonensis failed to produce AF-NALFA T-line signals, L. monocytogenes yielded strong detection. Site-specific-ΔG below −10 kcal/mol correlated with reduced hybridization. Synthetic oligos preserved signals despite structural constraints, whereas ~3–4 mismatches, especially at capture probe regions, markedly impaired T-line intensity. The performance of AF-NALFA depends on the synergism between thermodynamic accessibility, site-specific-ΔG-induced site constraints, and sequence complementarity. Because genomic context affects hybridization, target-specific thermodynamic in silico evaluation is necessary for reliable pathogen DNA detection. Full article

Tamoxifen is a well-established selective estrogen receptor modulator (SERM) widely used in breast cancer treatment, yet its efficacy varies across tumor types. To enhance its antitumor potential, we previously synthesized and investigated novel ferrocene-linked (T5, T15) derivatives. This publication is a close continuation of this work, introducing a new indene-based (T6) derivative. Objectives: The main aim of this study was to further broaden our knowledge of the mechanism behind the increased antitumor effect of the ferrocene-linked drugs (T5 and T15) and compare it with a new, indene-based tamoxifen derivative, T6. The indene moiety was selected as a rigid, hydrophobic aromatic unit to probe pharmacological effects independent of ferrocene’s redox activity. Methods: The compounds were tested on MCF7, MDA-MB231 and PANC1 cells. Cell viability was assessed with the AlamarBlue assay and the xCELLigence SP system. Reactive oxygen species (ROS) production was measured with the ROS Glo assay. Flow cytometry and RT-qPCR experiments were conducted to assess apoptosis and ROS regulation as well. Results: The modified compounds demonstrated an increased cell-viability-decreasing effect in breast (MCF7, MDA-MB-231) and pancreatic (PANC1) cancer cell lines, influencing both estrogen-receptor-dependent and -independent pathways. T6 led to G2/M phase arrest in PANC1 cells. Beyond cell cycle disruption, these derivatives significantly elevated ROS levels, contributing to apoptosis. Conclusions: Our findings suggest that these structural modifications retain tamoxifen’s pharmacophore properties while expanding its mechanism of action, particularly through universal interactions independent of the ER status of tumor cells. The enhanced antitumor effects highlight the potential of these derivatives as promising candidates for improved cancer therapies. Full article

Tuberculosis (TB) remains a major global health challenge, particularly in resource-limited settings where access to rapid and reliable diagnostics is limited. Conventional diagnostic methods, such as smear microscopy and culture, are either time-consuming or lack adequate sensitivity. This study optimized recombinase polymerase amplification (RPA) using 16 primer combinations targeting IS6110 highly specific to the Mycobacterium tuberculosis complex (MTC). A novel naked-eye assay, TB-GoldDx, was developed by integrating RPA combined with gold nanoparticles (AuNPs), enabling equipment-free diagnostics. TB-GoldDx demonstrated a detection limit of 0.001 ng of MTB H37Rv DNA (~210 bacilli) per 25 µL reaction. Among 100 bacterial strains, it achieved 95.83% sensitivity and 100% specificity among 100 bacterial strains, comprising 72 MTB isolates and 28 nontuberculous bacterial species. In 140 sputum samples, the assay showed 81.43% sensitivity and 58.57% specificity versus acid-fast bacilli (AFB) smear microscopy, with sensitivity improving to 95.45% in high-load AFB 3+ specimens. Compared to a commercial line probe assay (LPA), TB-GoldDx exhibited slightly higher sensitivity (84.78% vs. 82.61%) but lower specificity (54.05% vs. 78.38%). Delivering rapid, visual results in under an hour, TB-GoldDx offers a low-cost, easily deployable solution for point-of-care tuberculosis detection, especially in underserved regions, reinforcing global End TB efforts. Full article

Background/Objectives: Cancer is one of the leading causes of death worldwide, and skin cancer is especially prevalent and lethal in Brazil. Despite advancements in treatment, there is still a need for new anticancer agents that are effective, selective, and less toxic. This study aimed to evaluate the cytotoxic and therapeutic potential of the peptide PEPAD. Methods: The cytotoxicity of PEPAD was assessed by MTT assay in murine melanoma (B16F10-Nex2), human melanoma (SK-MEL-28), breast (MCF-7), and cervical (HeLa) cancer cell lines. Selectivity was evaluated in healthy cells (RAW 264.7 and FN1). Morphological changes were analyzed by microscopy. Cell migration was assessed using scratch assays. Apoptotic features were evaluated using MitoTracker Deep Red, NucBlue, CaspACETM labeling, and flow cytometry. Immunogenic cell death was investigated by calreticulin and HMGB1 release. Molecular dynamics simulations explored peptide structure and interaction with lipid membranes. Results: PEPAD showed IC₅₀ values of 7.4 µM and 18 µM in B16F10-Nex2 and SK-MEL-28 cells, respectively, and >60 µM in MCF-7 and HeLa cells. Low toxicity was observed in healthy cells (IC₅₀ > 56 µM), indicating high selectivity. Apoptotic morphology and reduced cell migration were observed. Flow cytometry and fluorescence probes confirmed apoptosis and mitochondrial swelling. Calreticulin and HMGB1 release indicated immunogenic cell death. Simulations showed that PEPAD maintains a stable α-helical conformation and interacts with membranes. Conclusions: These findings highlight PEPAD’s selective cytotoxicity and its potential as an anticancer agent with apoptotic and immunogenic properties, making it a promising candidate for therapeutic development. Full article