Microscopy Methods Research Articles

Targeting toll-like receptor 7 as a therapeutic development strategy for systemic lupus erythematosus

Endosomal TLRs (TLR3/7/8/9) are highly analogous innate immunity sensors for various viral or bacterial RNA/DNA molecular patterns. Among them, TLR7, in particular, has been suggested to be a target for various inflammatory disorders and autoimmune diseases including systemic lupus erythematosus (SLE); but few small-molecule inhibitors with elaborated mechanism have been reported in literature. Here, we reported a well-characterized human TLR7-specific small-molecule inhibitor, TH-407b, with promising potency and negligible cytotoxicity through a novel binding mechanism. Notably, TH-407b not only effectively inhibited TLR7-mediated pro-inflammatory signaling in a variety of cultured cell lines but also demonstrated potent inflammation suppressing activities in primary peripheral blood mononuclear cells (PBMCs) derived from SLE patients. Furthermore, TH-407b showed prominent efficacy in vivo, improved survival rate and ameliorated symptoms of SLE model mice. To obtain molecular insights into the TH-407b derivatives’ inhibition mechanism, we performed the structural analysis of TLR7/TH-407b complex using cryogenic electron microscopy (cryo-EM) method. As an atomistic resolution cryo-EM structure of the TLR family, it not only of value to facilitate structure-based drug design, but also shed light to methodology development of small proteins using EM. Significantly, TH-407b has unveiled an inhibition strategy for TLR7 via stabilizing its resting/inactivated state. Such a resting state could be generally applicable to all TLRs, rendering a useful method for targeting this group of important immunological receptors.

Catalytic hydrogen combustion over supported metal catalysts; comparison and kinetic study

Catalytic H2 combustion (CHC) is an efficient way to mitigate H2 slip from industrial processes, overcoming the explosion and NOx emission risks. Although Pt-based catalysts are state-of-the-art catalysts, little is known about the CHC catalytic activity of other transition metals (TMs) and their reaction kinetics. Well-dispersed nano-sized M-γAl2O3 (M = Pt, Ru, Co, Ni, Mo) catalysts are synthesized and characterized with spectroscopic and electron microscopy methods. During the CHC reaction, a competitive reaction between O2, H2 and the catalyst is observed, which leads to partial or complete oxidation of the TMs. Stable CHC performance is attained for Ru-γAl2O3 and Co-γAl2O3 catalysts over 45 h. The kinetically controlled region is determined using a new data acquisition approach. The activation energy and pre-exponential factor are compared between M-γAl2O3 catalysts based on the Arrhenius model. This knowledge is highly valued and allows the design of supported catalysts and optimize the CHC reaction in various applications with different requirements.

Ubiquitous short-range order in multi-principal element alloys

Recent research in multi-principal element alloys (MPEAs) has increasingly focused on the role of short-range order (SRO) on material performance. However, the mechanisms of SRO formation and its precise control remain elusive, limiting the progress of SRO engineering. Here, leveraging advanced additive manufacturing techniques that produce samples with a wide range of cooling rates (up to 107 K s−1) and an enhanced semi-quantitative electron microscopy method, we characterize SRO in three CoCrNi-based face-centered-cubic (FCC) MPEAs. Surprisingly, irrespective of the processing and thermal treatment history, all samples exhibit similar levels of SRO. Atomistic simulations reveal that during solidification, prevalent local chemical order arises in the liquid-solid interface (solidification front) even under the extreme cooling rate of 1011 K s−1. This phenomenon stems from the swift atomic diffusion in the supercooled liquid, which matches or even surpasses the rate of solidification. Therefore, SRO is an inherent characteristic of most FCC MPEAs, insensitive to variations in cooling rates and even annealing treatments typically available in experiments.

Purification of Potential Antimicrobial Metabolites from Endophytic Fusarium oxysporum Isolated from Myrtus communis.

The rise of microbial resistance and emerging infections pose significant health threats. Natural products from endophytic fungi offer a promising source of novel compounds with the potential as major drug leads. This research aims to screen Myrtus communis and Moringa oleifera for endophytic fungi and screen their metabolites for antibacterial and antifungal potential. Six endophytic fungal strains were isolated using a potato dextrose agar (PDA) medium. The M. communis isolates were designated MC1, MC2, and MC3, and the M. oleifera isolates were named MO1, MO2, and MO3. Preliminary bioactivity testing revealed that the MC3 isolate exhibited significant growth inhibition against multidrug-resistant bacterial and fungal pathogens, including Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Candida glabrata. The MC3 isolate was identified as Fusarium oxysporum through morphological and microscopic methods. For metabolite production, the fungal strain was cultured in potato dextrose broth (PDB) medium at 28°C for 14days in a shaking incubator. The metabolites were purified using various chromatographic techniques, HPLC and GC-MS. The GC-MS analysis of the bioactive compound containing fungal strain (F. oxysporum) revealed multiple compounds at different retention times using the NIST-20 Library. Based on RSI values and probability indices, two compounds were targeted for further purification. Structure elucidation was performed using 1D and 2D nuclear magnetic resonance (NMR) experiments on a Varian 500 NMR machine. The compounds identified were ethyl iso-allocholate (C26H44O5, exact mass 436.32) and 1-monolinoleoyl glycerol trimethylsilyl ether (C27H56O4Si2, exact mass 500.37). The MS (NIST-20) library facilitated the investigation of the in silico antimicrobial activity of these compounds against the elastase virulence protein of P. aeruginosa and protease Sapp1p from C. parapsilosis. Both the compounds were docked with druggable proteins using the Glide induced fit docking (IFD) algorithm. The ethyl iso-allocholate and 1-monolinoleoyl glycerol trimethylsilyl ether compounds showed binding scores - 10.07kcalmol-1 and - 7.47kcalmol-1 against elastase, and - 8.16kcalmol-1 and - 6.89kcalmol-1 against aspartic protease, respectively. In vitro studies confirmed the inhibitory activity of these compounds against multidrug-resistant P. aeruginosa and E. faecalis. Ethyl iso-allocholate exhibited higher bioactivity against P. aeruginosa with inhibition rates of 41%, 27%, and 35% at concentrations of 1000, 500, and 250µgmL-1, respectively. These results suggest that bioactive compounds from F. oxysporum have the potential as antimicrobial agents, warranting further research.

Microcrack characteristics of granite under circumferential strain-controlled uniaxial compression test using UDEC-GBM

Rockburst is one of the significant challenges for underground excavation stability. The post-peak behaviour of rock controls the way it fails, and the released energy is potentially involved in the rockburst. However, understanding of both microscopic and macroscopic behaviours of brittle rock in the post-peak stage is limited due to the lack of proper loading methods and quantitative microscopic analysis methods. This study proposes a circumferential strain-controlled loading algorithm in UDEC to control the failure process, which has been verified against previous literature. By employing the improved geometry generation method, the microcrack evolution is quantitatively assessed. Energy analysis reveals that up to 80% of the input energy is stored in the granite prior to complete failure, which could be released if the rock fails at this moment. Conversely, after the complete failure, intragranular cracks account for the majority of energy dissipation, indicating its importance in post-peak stability. A small portion of the peak stored strain energy needs to be extracted to achieve a controlled failure process. Furthermore, it is found that the significant increases in the formation of intergranular shear and low-angled microcracks potentially feature the complete failure of the specimen. The proposed model offers a complementary method to systematically investigate the post-peak behaviour of rock, including the evolution of microcracks and energy.

In vitro and in silico studies of the inclusion complexation of 8-bromobaicalein with β-cyclodextrins

Baicalein, a flavone derived from Scutellaria baicalensis Georgi, exhibits potent anti-inflammatory, antiviral, and anticancer properties. Its derivative, known as 8-bromobaicalein (BB), has been found to have strong cytotoxic effect on MCF-7 human breast cancer cells. However, its limited solubility in water has hindered its potential for wider applications. To address this issue, we investigated the use of cyclodextrins specifically βCD, 2,6-di-O-methyl-β-cyclodextrin (DMβCD), and hydroxypropyl-β-cyclodextrin (HPβCD) to improve the solubility of BB through inclusion complexation. During 250 ns molecular dynamics simulations, it was found that BB can form inclusion complexes with all βCDs. These complexes exhibit two distinct orientations: chromone group insertion (C-form) and phenyl group insertion (P-form). The formation of these complexes is primarily driven by van der Waals interactions. DMβCD has the highest number of atom contacts with BB and the lowest solvent accessibility in the hydrophobic cavity. These results coincide with the highest binding affinity from the MM/GBSA-based free energy calculation method. Experimental phase solubility diagrams revealed a 1:1 stoichiometric ratio (AL type) between BB and βCDs, in which BB/DMβCD showed the highest stability. The formation of inclusion complexes was confirmed by differential scanning calorimetry and scanning electron microscope methods. Additionally, the BB/DMβCD inclusion complex demonstrated significantly higher anticancer activity against MCF-7 human breast cancer cells compared to BB alone. These findings underscore the potential of DMβCD for formulating BB in pharmaceutical and medical applications.

Comparison of Foldscope to optical microscope to identify basic histology

Introduction: The teaching of basic histology is crucial in the training of healthcare professionals. Traditionally, the optical microscope is used, but it has limitations such as cost and the need for specific infrastructure. The Foldscope (FS), a low-cost and easy-to-handle tool, emerges as a promising alternative. Objective: To validate the efficacy of the FS in teaching basic histology for healthcare training, comparing it with the traditional method of binocular optical microscopy. Method: We used a kit of prepared histology slides, first examined with an optical microscope and then with the FS. We used a smartphone for image capture, ensuring comparative accuracy between the methods. Results: The FS could observe structures seen with a 10x objective in conventional microscopy but with less sharpness and some peripheral distortions. The applicability of the FS proved effective in observing structures of various tissues, such as the thyroid, arteries, veins, and neurons, although it has limitations for teaching muscular and testicular tissues. Conclusion: The FS is an innovative educational tool that bridges the gap between theory and practice in histology teaching. It is a complementary resource to the optical microscope, effectively identifying structures with about 100x magnification. However, it does not completely replace the optical microscope due to its limitations in magnification and image quality.

Multiplex PCR for gastrointestinal parasites in stool: Benchmarking against direct microscopy and simplex PCR

PurposeTo develop and validate a multiplex conventional PCR assay to simultaneously detect Cryptosporidium spp., Entamoeba histolytica, and Giardia lamblia in diarrheal samples as a rapid, cost-effective, and sensitive diagnostic tool for prevalent co-infections for improved diagnostic accuracy and efficiency in resource-limited settings. MethodsStool samples collected from patients with gastrointestinal symptoms after taking written consent, processed via wet mount, iodine mount, and PCR assays. Cohen's kappa statistical analysis was done to test agreement. ResultAmong 240 patients, 28.75% showed intestinal protozoa via Microscopy; Single-plex and multiplex PCR demonstrated 100% concordance, detecting 27.9%; confirmed by sequencing. Highest parasite positivity was observed in transplant and immunocompromised patients, with moderate to almost perfect agreement between microscopy and molecular methods. ConclusionMultiplex-conventional PCR offers superior sensitivity and specificity over microscopy and 100% concordance with single-plex PCR, enabling rapid, cost-effective diagnosis of multiple parasites from single stool sample. Its adoption could revolutionize parasitic infection management in routine diagnostics.

Aging drives cerebrovascular network remodeling and functional changes in the mouse brain

Aging is frequently associated with compromised cerebrovasculature and pericytes. However, we do not know how normal aging differentially impacts vascular structure and function in different brain areas. Here we utilize mesoscale microscopy methods and in vivo imaging to determine detailed changes in aged murine cerebrovascular networks. Whole-brain vascular tracing shows an overall ~10% decrease in vascular length and branching density with ~7% increase in vascular radii in aged brains. Light sheet imaging with 3D immunolabeling reveals increased arteriole tortuosity of aged brains. Notably, vasculature and pericyte densities show selective and significant reductions in the deep cortical layers, hippocampal network, and basal forebrain areas. We find increased blood extravasation, implying compromised blood-brain barrier function in aged brains. Moreover, in vivo imaging in awake mice demonstrates reduced baseline and on-demand blood oxygenation despite relatively intact neurovascular coupling. Collectively, we uncover regional vulnerabilities of cerebrovascular network and physiological changes that can mediate cognitive decline in normal aging.

Scanning Plasmon-Enhanced Microscopy for Simultaneous Optoelectrical Characterization.

Scanning microscopy methods are crucial for the advancement of nanoelectronics. However, the vertical nanoprobes in such techniques suffer limitations such as the fragility at the tip-sample interface, complex instrumentation, and the lack of in operando functionality in several cases. Here, we introduce scanning plasmon-enhanced microscopy (SPEM) and demonstrate its capabilities on MoS2 and WSe2 nanosheets. SPEM combines a nanoparticle-on-mirror (NPoM) configuration with a portable conductive cantilever, enabling simultaneous optical and electrical characterization. This distinguishes it from other current techniques that cannot provide both characterizations simultaneously. It offers a competitive optical resolution of 600 nm with local enhancement of optical signal up to 20,000 times. A single gold nanoparticle with a 15 nm radius forms pristine, nondamaging van der Waals contact, which allows observation of unexpected p-type behavior of MoS2 at the nanoscale. SPEM reconstructs the NPoM method by eliminating the need for extensive statistical analysis and offering excellent nanoscale mapping resolution of any selected region. It surpasses other scanning techniques in combining precise optical and electrical characterization, interactive simplicity, tip durability, and reproducibility, positioning it as the optimal tool for advancing nanoelectronics.

Field Application of an Innovative Approach to Assess Honeybee Health and Nutritional Status.

Environment, forage quality, management practices, pathogens, and pesticides influence honeybee responses to stressors. This study proposes an innovative approach to assess colony health and performance using molecular diagnostic tools by correlating hemolymph proteins with common measures of colony strength, prevalent honeybee pathogens (Varroa destructor and Nosema spp.), and essential trace elements (iron, zinc and copper). Colonies were selected from four apiaries located in different environmental and foraging conditions in the province of Bologna (Italy). Hemolymph samples were taken from June to October 2019. The Varroa infestation of the colonies was estimated by assessing the natural mortality of the mites, while the bees were tested for Nosema spp. spores using a microscopic method. Hemolymph proteins were quantified and separated using SDS-PAGE, and colony performance was assessed by determining adult bees, total brood, honey, and pollen reserves. The biomarkers measured proved to be useful for monitoring changes in performance and trophic conditions during summer and early autumn. Significant correlations were found between hemolymph proteins and colony performance measures. A positive correlation between pollen reserves, vitellogenin, and hexamerin 70a highlights the importance of these proteins for successful overwintering. In October, Varroa infestation was negatively correlated with total proteins, vitellogenin, apolipophorin II, transferrin, and hexamerin 70a, with negative implications for overwintering; furthermore, Varroa infestation was also negatively correlated with iron content, potentially affecting iron homeostasis.

Macroscopic brain dynamics beyond contralateral primary motor cortex for movement prediction

This study investigates the complex relationship between upper limb movement direction and macroscopic neural signals in the brain, which is critical for understanding brain-computer interfaces (BCI). Conventional BCI research has primarily focused on a local area, such as the contralateral primary motor cortex (M1), relying on the population-based decoding method with microelectrode arrays. In contrast, macroscopic approaches such as electroencephalography (EEG) and magnetoencephalography (MEG) utilize numerous electrodes to cover broader brain regions. This study probes the potential differences in the mechanisms of microscopic and macroscopic methods. It is important to determine which neural activities effectively predict movements. To investigate this, we analyzed MEG data from nine right-handed participants while performing arm-reaching tasks. We employed dynamic statistical parametric mapping (dSPM) to estimate source activity and built a decoding model composed of long short-term memory (LSTM) and a multilayer perceptron to predict movement trajectories. This model achieved a high correlation coefficient of 0.79 between actual and predicted trajectories. Subsequently, we identified brain regions sensitive to predicting movement direction using the integrated gradients (IG) method, which assesses the predictive contribution of each source activity. The resulting salience map demonstrated a distribution without significant differences across motor-related regions, including M1. Predictions based solely on M1 activity yielded a correlation coefficient of 0.42, nearly half as effective as predictions incorporating all source activities. This suggests that upper limb movements are influenced by various factors such as movement coordination, planning, body and target position recognition, and control, beyond simple muscle activity. All of the activities are needed in the decoding model using macroscopic signals. Our findings also revealed that contralateral and ipsilateral hemispheres contribute equally to movement prediction, implying that BCIs could potentially benefit patients with brain damage in the contralateral hemisphere by utilizing brain signals from the ipsilateral hemisphere. In conclusion, this study demonstrates that macroscopic activity from large brain regions significantly contributes to predicting upper limb movement. Non-invasive BCI systems would require a comprehensive collection of neural signals from multiple brain regions.

Metal-Organic Framework on Fullerene (MOFOF) as a Hierarchical Composite by the Integration of Coordination Chemistry and Supramolecular Chemistry.

There is a synergy between coordination chemistry and supramolecular chemistry that has led to the development of innovative hierarchical composites with diverse functionalities. Here, we present a novel approach for the synthesis and characterization of a metal-organic framework on fullerene (MOFOF) composites, achieved through the integration of coordination chemistry and supramolecular chemistry principles. The hierarchical nature of the MOFOF harnesses the inherent properties of metal-organic frameworks and fullerenes. The two-step synthesis procedure involves controlled assembly of fullerenes as tube-like nanostructures (fullerene nanotube: FNT), their surface functionalization, and the on-surface growth of the MOF (in this case, ZIF-67). The method permits the precise tuning of morphology, effective distribution of MOF-on-FNT, and tight compositional control. The materials were comprehensively structurally characterized using electron microscopy, spectroscopic techniques, and other methods to elucidate the unique features and interactions within the MOFOF composites. The main findings reveal that the novel synthesis and characterization of MOFOF composites demonstrate the successful integration of coordination chemistry and supramolecular chemistry for the designing and fabricating of advanced hierarchical composites with tailored properties, including micro- and mesopore channels, interfacial facets, and defect sites. These properties are expected to lead to numerous potential applications such as gas storage and separation, catalysis, sensing, energy storage, and environmental remediation. However, only the capability of acid vapor sensing was tested and is described here.

How to achieve nanometer flat surfaces: Pulsed electrochemical machining of bulk metallic glass

The Bulk Metallic Glass (BMG) Vitreloy 105 (Zr52.5Ti5Ni14.6Cu17.9Al10) as well as two crystalline reference materials of the same composition were machined with Pulsed Electrochemical Machining (PECM). One reference material was nano crystalline with an average grain size of 11 nm and the second one was a fine grain crystalline sample with an average grainsize of 110 nm. The goal was to achieve maximum surface quality in terms of roughness by utilizing a readily available industrial process. Samples were characterized by tactile profilometer, atomic force microscopy and electron microscopy methods. The results show superior surface quality of the amorphous samples over the reference materials with an average roughness depth as low as 1 nm. The observed dissolution mechanism of the BMG differs from the reference materials -as in contrast to the crystalline alloys that show a passive layer formation - no such surface layer other than the native oxide could be observed. The PECM procedure is found to be a promising post-processing step for BMGs with a resulting surface that can face even the most challenging surface quality requirements.

Phase retrieval via conjugate gradient minimization in double-plane lensless holographic microscopy

Optimization-based phase retrieval method for digital lensless holographic microscopy in the double-plane recording configuration is proposed. In our method the phase retrieval is framed as an optimization problem that can be efficiently and rigorously tackled with gradient decent tools. This is done with the conjugate gradient method that possesses excellent theoretical features such as global and fast convergence (compared to steepest descent) and relatively low computational cost (compared to second order optimizers). The proposed method is extensively tested with simulations and experimental measurements that show superiority of our method over the Gerchberg-Saxton algorithm, especially in terms of reconstruction of problematic low frequency components of viable phase information.

Advancing cellular insights: Super-resolution STORM imaging of cytoskeletal structures in human stem and cancer cells

Fluorescence microscopy is an important tool for cell biology and cancer research. Present-day approach of implementing advanced optical microscopy methods combined with immunofluorescence labelling of specific proteins in cells is now able to deliver optical super-resolution up to ∼25 nm. Here we perform super-resolved imaging using standard immunostaining protocol combined with easy stochastic optical reconstruction microscopy (easySTORM) to observe structural differences of two cytoskeleton elements, actin and tubulin in three different cell types namely human bone marrow-derived mesenchymal stem cells (MSCs), human glioblastoma (U87MG) and breast cancer (MDAMB-231) cells. The average width of the actin bundle obtained from STORM images of stem cells is observed to be larger than the same for U87MG and MDAMB-231 cells. No significant difference is however noticed in the width of the tubulin within the same cells. We also study the functional effect on the 2D migration potential of MDAMB-231 cells silenced for NICD1 and β-catenin. Although similar migration speed is observed for cells with the above two conditions compared to their control cells, easySTORM images show that widths of the actin in MDAMB-231 cells in β-catenin silenced is significantly lower than the same in control cells. Such minute differences however are not observable in widefield images. The outcome of our easySTORM investigation should benefit the researchers carrying out detailed investigations of the cellular structure and potential therapeutic applications.

Leveraging Methods in Microscopy for Exploring Structure-Property Relations of Ultrahigh Aspect Ratio Multiwalled Carbon Nanotubes Subject to Ball Milling and Compounding with Polymers

Leveraging Methods in Microscopy for Exploring Structure-Property Relations of Ultrahigh Aspect Ratio Multiwalled Carbon Nanotubes Subject to Ball Milling and Compounding with Polymers

Antibiofilm and Antivirulence Potentials of 3,2'-Dihydroxyflavone against Staphylococcus aureus.

Staphylococcus aureus, particularly drug-resistant strains, poses significant challenges in healthcare due to its ability to form biofilms, which confer increased resistance to antibiotics and immune responses. Building on previous knowledge that several flavonoids exhibit antibiofilm activity, this study sought to identify a novel flavonoid capable of effectively inhibiting biofilm formation and virulence factor production in S. aureus strains including MRSA. Among the 19 flavonoid-like compounds tested, 3,2'-dihydroxyflavone (3,2'-DHF) was identified for the first time as inhibiting biofilm formation and virulence factors in S. aureus with an MIC 75 µg/mL. The antibiofilm activity was further confirmed by microscopic methods. Notably, 3,2'-DHF at 5 µg/mL was effective in inhibiting both mono- and polymicrobial biofilms involving S. aureus and Candida albicans, a common co-pathogen. 3,2'-DHF reduces hemolytic activity, slime production, and the expression of key virulence factors such as hemolysin gene hla and nuclease gene nuc1 in S. aureus. These findings highlight the potential of 3,2'-DHF as a novel antibiofilm and antivirulence agent against both bacterial and fungal biofilms, offering a promising alternative to traditional antibiotics in the treatment of biofilm-associated infections.

Development of tungsten trioxide nano-flakes intercalated on tannic acid-functionalized reduced graphene oxide for flexible acebutolol sensors

Acebutolol (ACE) is commonly used to treat hypertension and high blood pressure. Large doses of ACE can have adverse effects with potentially life-threatening consequences. It is, therefore, essential to develop a simple, low-cost, reliable, and reproducible device for detecting ACE in biofluids. This study explores the potential of unique two-dimensional nano-flakes, such as tungsten trioxide (WO3). Graphene oxide (GO) typically exhibits lower electrical conductivity than pristine graphene due to the presence of oxygen-containing functional groups that interfere with the π-conjugated structure. Functionalizing GO with tannic acid (TA) can partially reinstate the π-conjugation and limit the amount of oxygen, resulting in enhanced electrical conductivity. Ultrasonic techniques were utilized to create WO3 NFs@TA-rGO, and a range of spectroscopic and microscopic methods were applied to examine the formation of the resulting WO3 NFs@TA-rGO nanocomposites. Under optimal conditions, modified sensors resulted in lower limits of detection (0.0055 μM) and good sensitivity (0.40 μA μM−1 cm−2). They also exhibited a broad linear range spanning from 0.009 to 568.6 μM. Fabricated sensors have significant anti-interference properties with high specificity and excellent storage stability (RSD = 4.3 %), reproducibility (RSD = 3.9 %), and repeatability (RSD = 3.3 %). Ultimately, the sensor's efficacy was confirmed through the successful detection of ACE in biological samples (with recoveries ranging from 99.1 to 99.6 %). Lastly, this study highlights the substantial potential of ACE detection and extends its applications in biomedical diagnostics and pharmaceutical research.

Tribological performance and mechanism of graphite, hBN and MoS2 as nano-additives in palm kernel oil-based lubricants: A comparative study

Layered nanoparticles have received considerable interest in tribological applications owing to their outstanding tribological performances, including in vegetable oil-based lubricants. Despite the advantages of vegetable oil-based lubricants over petroleum-based lubricants, the limited manufacturing scale of vegetable oil-based lubricants restricts their more comprehensive implementation in various industries. In order to gain further insights into the exquisite nano-additive properties of layered nanoparticles, this research was conducted to explore the tribological performance of palm kernel oil-based lubricants combined with three different layered nanoparticles, namely graphite, hexagonal boron nitride (hBN), and molybdenum disulfide (MoS2), using a four-ball tribotester. The four-ball tribotester analysis indicated that the wear scars lubricated with graphite, hBN, and MoS2 nanolubricants exhibited a lower coefficient of friction (COF) and wear scar diameter (WSD) than the base lubricant. The findings also showed that the MoS2 nanolubricant had the best friction reduction and anti-wear performance, with a 24.9% drop in COF and a 12.3% drop in WSD. It was followed by the hBN and graphite nanolubricants. To fully understand the lubricating mechanisms involved, the worn surfaces of the tested balls were characterised using various microscopic and spectroscopic methods. Accordingly, the structural changes that occurred during sliding were shown to affect the formation of protective tribofilm. In summary, the findings in this study suggest that the formulated layered nanolubricants possessed exceptional tribological performance, which was contributed by the inherent properties of the individual nanoparticles and the formation of effective tribofilms.