Water permeation drives tumor cell migration in confined microenvironments.

Cell migration is a critical process for diverse (patho)physiological phenomena. Intriguingly, cell migration through physically confined spaces can persist even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited. Here, we present an integrated experimental and theoretical approach ("Osmotic Engine Model") and demonstrate that directed water permeation is a major mechanism of cell migration in confined microenvironments. Using microfluidic and imaging techniques along with mathematical modeling, we show that tumor cells confined in a narrow channel establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, which creates a net inflow of water and ions at the cell leading edge and a net outflow of water and ions at the trailing edge, leading to net cell displacement. Collectively, this study presents an alternate mechanism of cell migration in confinement that depends on cell-volume regulation via water permeation.

Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions.

Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

AI26 inhibits the ADP-ribosylhydrolase ARH3 and suppresses DNA damage repair.

The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair, as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC50 of ∼2.41 µm in vitro Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. In addition, tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment, as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.

Fabrication and characterization of well-ordered PbS nanowires in aluminum oxide template by sulfurization and vacuum injection molding processes.

Lead (Pb) nanowire arrays were fabricated with anodic aluminum oxide (AAO) templates of 30, 100 and 300 nm in pore diameters. Through vacuum injection molding process, Pb/AAO composite was obtained, and lead sulfide (PbS) could further be synthesized after exposing to sulfur gas. AAO templates with different pore sizes were fabricated by using pure aluminum in a two-step anodization. Three types of solutions, which are 10 vol% sulfuric acid, 3 wt% oxalic acid and 1 vol% phosphoric acid, were adopted to achieve AAO of various pore sizes. Different sulfurization temperatures and time spans were applied for studying on the formation mechanism of PbS. Finally, the morphology, composition, structure and elements distribution of the as-prepared Pb and PbS nanowires were confirmed through the use of scanning electron microscopy, energy dispersive x-ray spectroscopy, element-mapping, x-ray diffraction and transmission electron microscopy analysis. The results indicated that Pb nanowires were successfully obtained after applying vacuum injection molding process with 50 kgf cm-2hydraulic pressure, and PbS nano arrays can be formed by sulfurization at 500 °C for 5 h. Furthermore, an optical property, ultraviolet-visible (UV-Vis) absorption, was also measured. The measurement of the PbS nanowires showed that a significant quantum confinement effect made the energy gap produce a blue shift from 0.41 eV to 1.65 eV or 1.72 eV.

Human DNA ligase IV is able to use NAD+ as an alternative adenylation donor for DNA ends ligation.

All the eukaryotic DNA ligases are known to use adenosine triphosphate (ATP) for DNA ligation. Here, we report that human DNA ligase IV, a key enzyme in DNA double-strand break (DSB) repair, is able to use NAD+ as a substrate for double-stranded DNA ligation. In the in vitro ligation assays, we show that the recombinant Ligase IV can use both ATP and NAD+ for DNA ligation. For NAD+-mediated ligation, the BRCA1 C-terminal (BRCT) domain of Ligase IV recognizes NAD+ and facilitates the adenylation of Ligase IV, the first step of ligation. Although XRCC4, the functional partner of Ligase IV, is not required for the NAD+-mediated adenylation, it regulates the transfer of AMP moiety from Ligase IV to the DNA end. Moreover, cancer-associated mutation in the BRCT domain of Ligase IV disrupts the interaction with NAD+, thus abolishes the NAD+-mediated adenylation of Ligase IV and DSB ligation. Disrupting the NAD+ recognition site in the BRCT domain impairs non-homologous end joining (NHEJ) in cell. Taken together, our study reveals that in addition to ATP, Ligase IV may use NAD+ as an alternative adenylation donor for NHEJ repair and maintaining genomic stability.

Super-resolution imaging identifies PARP1 and the Ku complex acting as DNA double-strand break sensors.

DNA double-strand breaks (DSBs) are fatal DNA lesions and activate a rapid DNA damage response. However, the earliest stage of DSB sensing remains elusive. Here, we report that PARP1 and the Ku70/80 complex localize to DNA lesions considerably earlier than other DSB sensors. Using super-resolved fluorescent particle tracking, we further examine the relocation kinetics of PARP1 and the Ku70/80 complex to a single DSB, and find that PARP1 and the Ku70/80 complex are recruited to the DSB almost at the same time. Notably, only the Ku70/80 complex occupies the DSB exclusively in the G1 phase; whereas PARP1 competes with the Ku70/80 complex at the DSB in the S/G2 phase. Moreover, in the S/G2 phase, PARP1 removes the Ku70/80 complex through its enzymatic activity, which is further confirmed by in vitro DSB-binding assays. Taken together, our results reveal PARP1 and the Ku70/80 complex as critical DSB sensors, and suggest that PARP1 may function as an important regulator of the Ku70/80 complex at the DSBs in the S/G2 phase.

The fabrication and thermal properties of bismuth-aluminum oxide nanothermometers.

Bismuth (Bi) nanowires, well controlled in length and diameter, were prepared by using an anodic aluminum oxide (AAO) template-assisted molding injection process with a high cooling rate. A high performance atomic layer deposition (ALD)-capped bismuth-aluminum oxide (Bi-Al2O3) nanothermometer is demonstrated that was fabricated via a facile, low-cost and low-temperature method, including AAO templated-assisted molding injection and low-temperature ALD-capped processes. The thermal behaviors of Bi nanowires and Bi-Al2O3 nanocables were studied by in situ heating transmission electron microscopy. Linear thermal expansion of liquid Bi within native bismuth oxide nanotubes and ALD-capped Bi-Al2O3 nanocables were evaluated from 275 °C to 700 °C and 300 °C to 1000 °C, respectively. The results showed that the ALD-capped Bi-Al2O3 nanocable possesses the highest working temperature, 1000 °C, and the broadest operation window, 300 °C-1000 °C, of a thermal-expanding type nanothermometer. Our innovative approach provides another way of fabricating core-shell nanocables and to further achieve sensing local temperature under an extreme high vacuum environment.

Fabrication of device with poly(N-isopropylacrylamide)-b-ssDNA copolymer brush for resistivity study.

In this study, we grafted bromo-terminated poly(N-isopropylacrylamide) (PNIPAAm) brushes onto thin gold films deposited on silicon, and then reacted with NaN3 to produce azido-terminated PNIPAAm brushes. A probe sequence of single-stranded DNA (ssDNA) with a 4-pentynoic acid succinimidyl ester unit was grafted onto the azido-terminated PNIPAAm brushes through a click reaction, resulting in the formation of block copolymer brushes. The PNIPAAm-b-ssDNA copolymer brushes formed supramolecular complexes stabilized by bio-multiple hydrogen bonds (BMHBs), which enhanced the proton transfer and thereby decreased the resistivity of the structures. In addition, the optimal operation window for DNA detection ranges from 0 to 0.2 M of NaCl concentration. Therefore, the specimens were prepared in the PBS solution at 150 mM NaCl concentration for target hybridization. The supramolecular complex state of the PNIPAAm-b-ssDNA copolymer brushes transformed into the phase-separated state after the hybridization with 0.5 ng/µL of its target DNA sequence owing to the competition between BMHBs and complementary hydrogen bonds. This phase transformation of the PNIPAAm and probe segments inhibited the proton transfer and significantly increased the resistivity at 25 °C. Moreover, there were no significant changes in the resistivity of the copolymer brushes after hybridization with the target sequence at 45 °C. These results indicated that the phase-separated state of the PNIPAAm-b-ssDNA copolymer brushes, which was generally occurred above the LCST, can be substantially generated after hybridization with its target DNA sequence. By performing the controlled experiments, in the same manner, using another sequence with lengths similar to that of the target sequence without complementarity. In addition, the sequences featuring various degrees of complementarity were exploited to verify the phase separation behavior inside the PNIPAAm-b-ssDNA copolymer thin film.

The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response.

Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response.

New Flexible Silicone-Based EEG Dry Sensor Material Compositions Exhibiting Improvements in Lifespan, Conductivity, and Reliability.

This study investigates alternative material compositions for flexible silicone-based dry electroencephalography (EEG) electrodes to improve the performance lifespan while maintaining high-fidelity transmission of EEG signals. Electrode materials were fabricated with varying concentrations of silver-coated silica and silver flakes to evaluate their electrical, mechanical, and EEG transmission performance. Scanning electron microscope (SEM) analysis of the initial electrode development identified some weak points in the sensors' construction, including particle pull-out and ablation of the silver coating on the silica filler. The newly-developed sensor materials achieved significant improvement in EEG measurements while maintaining the advantages of previous silicone-based electrodes, including flexibility and non-toxicity. The experimental results indicated that the proposed electrodes maintained suitable performance even after exposure to temperature fluctuations, 85% relative humidity, and enhanced corrosion conditions demonstrating improvements in the environmental stability. Fabricated flat (forehead) and acicular (hairy sites) electrodes composed of the optimum identified formulation exhibited low impedance and reliable EEG measurement; some initial human experiments demonstrate the feasibility of using these silicone-based electrodes for typical lab data collection applications.

Metabolic flux increases glycoprotein sialylation: implications for cell adhesion and cancer metastasis.

This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ≈ 31% showed no change in sialylation, ≈ 29% exhibited a modest increase, whereas ≈ 40% experienced an increase of greater than twofold. Increased sialylation of specific glycoproteins resulted in changes to the adhesive properties of SW1990 pancreatic cancer cells (e.g. increased CD44-mediated adhesion to selectins under physiological flow and enhanced integrin-mediated cell mobility on collagen and fibronectin). These results indicate that cancer cells can become more aggressively malignant by controlling the sialylation of proteins implicated in metastatic transformation via metabolic flux.

Wastewater-based epidemiology to monitor 68 NPS/conventional drug use in Taipei metropolitan area in Taiwan during and after COVID-19 pandemic.

Amidst far-reaching COVID-19 effects and social constraints, this study leveraged wastewater-based epidemiology to track 38 conventional drugs and 30 new psychoactive substances (NPS) in northern Taiwan. Analyzing daily samples from four Taipei wastewater plants between September 2021 and January 2024-encompassing club reopenings, holidays, Lunar New Year, an outbreak, and regular periods-thirty-one drugs were detected, including 5 NPS. Tramadol, zolpidem tartrate, CMA, and MDPV were newly detected in Taiwanese sewage with frequency of 1.4 %- 89.0 %. Conventional drug use typically increased post-pandemic, aside from benzodiazepines and methadone. Methamphetamine showed 100 % frequency, indicating ongoing daily consumption despite COVID-19 measures. Methamphetamine and morphine's consumption dipped then rose around club reopening, hinting at limited access. The consumption trend of methadone appeared to compensate for the use of morphine. Ketamine and NPS demonstrated similar patterns throughout the entire period. NPS as party drugs seemed influenced by an unstable supply chain and complexities in implementation. Benzodiazepines, commonly abused alongside synthetic cathinones in Taiwan exhibited an opposing trend to NPS while aligned with acetaminophen, suggesting elevated stress and anxiety levels during the pandemic. No significant differences were observed in drug consumption between weekdays and weekends, potentially indicating that COVID-19 measures blurred the traditional distinctions between these timeframes. ENVIRONMENTAL IMPLICATION: New psychoactive substances refer to chemically modified variants of controlled drugs designed to mimic the effects of the original drugs while evading modern detection methods, categorizing them as hazardous materials. The study presents a sewage monitoring project conducted from 2021 to 2024, collecting samples from four WWTPs to analyze NPS and conventional drug trends during and after the COVID-19 pandemic. The findings uncovered connections between drug consumption patterns and pandemic-related policies. In light of the persistent drug abuse and their environmental presence, the results bear critical importance for both environmental and public health. We provide a thorough assessment of these relationships and prioritize areas for future research.

Mucin 16 is a functional selectin ligand on pancreatic cancer cells.

Selectins promote metastasis by mediating specific interactions between selectin ligands on tumor cells and selectin-expressing host cells in the microvasculature. Using affinity chromatography in conjunction with tandem mass spectrometry and bioinformatics tools, we identified mucin 16 (MUC16) as a novel selectin ligand expressed by metastatic pancreatic cancer cells. While up-regulated in many pancreatic cancers, the biological function of sialofucosylated MUC16 has yet to be fully elucidated. To address this, we employed blot rolling and cell-free flow-based adhesion assays using MUC16 immunopurified from pancreatic cancer cells and found that it efficiently binds E- and L- but not P-selectin. The selectin-binding determinants are sialofucosylated structures displayed on O- and N-linked glycans. Silencing MUC16 expression by RNAi markedly reduces pancreatic cancer cell binding to E- and L-selectin under flow. These findings provide a novel integrated perspective on the enhanced metastatic potential associated with MUC16 overexpression and the role of selectins in metastasis.

Moiety-linkage map reveals selective nonbisphosphonate inhibitors of human geranylgeranyl diphosphate synthase.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS). Current bisphosphonate drugs (e.g., Fosamax and Zometa) are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease, and tumor-induced osteolysis, but they are often less potent in blood and soft-tissue due to their phosphate moieties. The discovery of nonbisphosphonate inhibitors of FPPS and/or GGPPS for the treatment of bone diseases and cancers is, therefore, a current goal. Here, we propose a moiety-linkage-based method, combining a site-moiety map with chemical structure rules (CSRs), to discover nonbisphosphonate inhibitors from thousands of commercially available compounds and known crystal structures. Our moiety-linkage map reveals the binding mechanisms and inhibitory efficacies of 51 human GGPPS (hGGPPS) inhibitors. To the best of our knowledge, we are the first team to discover two novel selective nonbisphosphonate inhibitors, which bind to the inhibitory site of hGGPPS, using CSRs and site-moiety maps. These two compounds can be considered as a novel lead for the potent inhibitors of hGGPPS for the treatment of cancers and mevalonate-pathway diseases. Moreover, based on our moiety-linkage map, we identified two key residues of hGGPPS, K202, and K212, which play an important role for the inhibitory effect of zoledronate (IC50 = 3.4 µM and 2.4 µM, respectively). This result suggests that our method can discover specific hGGPPS inhibitors across multiple prenyltransferases. These results show that the compounds that highly fit our moiety-linkage map often inhibit hGGPPS activity and induce tumor cell apoptosis. We believe that our method is useful for discovering potential inhibitors and binding mechanisms for pharmaceutical targets.

Designable Poly(methacrylic Acid)/Silver Cluster Ring Arrays as Reflectance Spectroscopy-Based Biosensors for Label-Free Plague Diagnosis.

A hole array was fabricated via photolithography to wet the bottoms of holes using oxygen plasma. Amide-terminated silane, a water immiscible compound before hydrolysis, was evaporated for deposition on the plasma-treated hole template surface. The silane compound was hydrolyzed along the edges of circular sides of the hole bottom to form a ring of an initiator after halogenation. Poly(methacrylic acid) (PMAA) was grafted from the ring of the initiator to attract Ag clusters (AgCs) as AgC-PMAA hybrid ring (SPHR) arrays via alternate phase transition cycles. The SPHR arrays were modified with a Yersinia pestis antibody (abY) to detect the antigen of Yersinia pestis (agY) for plague diagnosis. The binding of the agY onto the abY-anchored SPHR array resulted in a geometrical change from a ring to a two-humped structure. The reflectance spectra could be used to analyze the AgC attachment and the agY binding onto the abY-anchored SPHR array. The linear range between the wavelength shift and agY concentration from 30 to 270 pg mL-1 was established to obtain the detection limit of ~12.3 pg mL-1. Our proposed method provides a novel pathway to efficiently fabricate a ring array with a scale of less than 100 nm, which demonstrates excellent performance in preclinical trials.

Scalability of Sartobind® Rapid A Membrane for High Productivity Monoclonal Antibody Capture.

Improved upstream titres in therapeutic monoclonal antibody (mAb) production have shifted capacity constraints to the downstream process. The consideration of membrane-based chromatographic devices as a debottlenecking option is gaining increasing attention with the recent introduction of high-capacity bind and elute membranes. We have evaluated the performance and scalability of the Sartobind® Rapid A affinity membrane (1 mL) for high-productivity mAb capture. For scalability assessment, a 75 mL prototype device was used to process 100 L of clarified cell culture harvest (CH) on a novel multi-use rapid cycling chromatography system (MU-RCC). MabSelect™ PrismA (4.7 mL) was used as a benchmark comparator for Protein A (ProtA) resin studies. Results show that in addition to a productivity gain of >10×, process and product quality attributes were either improved or comparable to the benchmark. Concentrations of eluate pools were 7.5× less than that of the benchmark, with the comparatively higher bulk volume likely to cause handling challenges at process scale. The MU-RCC system is capable of membrane operation at pilot scale with comparable product quality profile to the 1 mL device. The Sartobind® Rapid A membrane is a scalable alternative to conventional ProtA resin chromatography for the isolation and purification of mAbs from harvested cell culture media.

Essence of the Giant Reduction of Power Density in Osmotic Energy Conversion in Porous Membranes: Importance of Testing Area.

Harvesting osmotic energy through nanofluidic devices with diverse materials has received considerable attention in recent years. Often, a small testing area on a membrane was chosen to assess its power performance by calculating power density as output power per effective area. Since the choice of this testing area is arbitrary, and it is usually quite small, the result obtained can be too optimistic. There is a need to come up with a common standard so that the performance of a device/membrane can be assessed reasonably. In this study, we systematically investigate the power density as a function of testing area in nanoporous anodic-aluminum-oxide membranes. Through changing the aperture size of substrates, we clearly show that the obtained power density decreases drastically with increasing testing area. For instance, the power density acquired from the testing area of µm2-scale can be five orders of magnitude larger than that from the pristine membrane of cm2-scale. We also advance simulations by building a 3D model to simulate osmotic-driven ion transport in the multichannel system. The result of modeling agrees with our experimental observation that the power density decreases with increasing number of channels, and the ionic concentration profile reveals that the concentration polarization becomes serious as the number of channels increases. Our result highlights the importance of effective area on testing the power performance in nanofluidic devices.

Sialofucosylated podocalyxin is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells.

Selectin-mediated interactions in the vasculature promote metastatic spread by facilitating circulating tumor cell binding to selectin-expressing host cells. Therefore, identifying the selectin ligand(s) on tumor cells is critical to the prevention of blood-borne metastasis. A current challenge is to distinguish between structures expressed by circulating tumor cells that can bind selectins in vitro from the functional ligands whose depletion suppresses selectin-dependent binding under flow in vivo. Interestingly, podocalyxin (PODXL), which can bind E- and L-selectin, is upregulated in a number of cancers, including those of the breast, colon, and pancreas. In this work, we show that metastatic pancreatic cancer cells overexpress PODXL compared with nonmalignant pancreatic epithelial cells. We further demonstrate via tissue microarray that 69% of pancreatic ductal adenocarcinomas stain positive for PODXL. In cases of focal expression, positive staining is restricted to the invasive front of primary tumors. By combining immunoblot, immunodepletion, short-hairpin RNA-mediated gene silencing, and flow-based adhesion assays, we evaluated the functional role of sialofucosylated PODXL in selectin-mediated adhesion under flow. Our data indicate that sialofucosylated PODXL is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells, as specific depletion of this molecule from the cell surface significantly interferes with selectin-dependent interactions. Cumulatively, these data support a correlation between sialofucosylated PODXL expression and enhanced binding to selectins by metastatic pancreatic cancer cells and offer additional perspective on the upregulation of PODXL in aggressive cancers.

Roles of amino acids in the Escherichia coli octaprenyl diphosphate synthase active site probed by structure-guided site-directed mutagenesis.

Octaprenyl diphosphate synthase (OPPS) catalyzes consecutive condensation reactions of farnesyl diphosphate (FPP) with five molecules of isopentenyl diphosphates (IPP) to generate C(40) octaprenyl diphosphate, which constitutes the side chain of ubiquinone or menaquinone. To understand the roles of active site amino acids in substrate binding and catalysis, we conducted site-directed mutagenesis studies with Escherichia coli OPPS. In conclusion, D85 is the most important residue in the first DDXXD motif for both FPP and IPP binding through an H-bond network involving R93 and R94, respectively, whereas R94, K45, R48, and H77 are responsible for IPP binding by providing H-bonds and ionic interactions. K170 and T171 may stabilize the farnesyl carbocation intermediate to facilitate the reaction, whereas R93 and K225 may stabilize the catalytic base (MgPP(i)) for H(R) proton abstraction after IPP condensation. K225 and K235 in a flexible loop may interact with FPP when the enzyme becomes a closed conformation, which is therefore crucial for catalysis. Q208 is near the hydrophobic part of IPP and is important for IPP binding and catalysis.

Effect of Varying Plasma Powers on High-Temperature Applications of Plasma-Sprayed Al0.5CoCrFeNi2Ti0.5 Coatings.

In this work, the microstructure and mechanical properties of atmospheric plasma-sprayed coatings of Al0.5CoCrFeNi2Ti0.5, prepared using gas-atomized powders at varying spray powers, are studied in as-sprayed and heat-treated conditions. Gas-atomized powders had spherical shapes and uniform element distributions, with major FCC phases and metastable BCC phases. The metastable BCC phase transformed to ordered and disordered BCC phases when sufficient energy was applied during the plasma-spraying process. During the heat treatment process for 2 hrs, disordered BCCs transformed into ordered BCCs, while the intensity of the FCC peaks increased. Spraying power plays a significant role in the microstructure and mechanical properties of plasma sprayed because at a high power, coatings exhibit better mechanical properties due to their dense microstructures resulting in less defects. As the plasma current was increased from 500 A to 700 A, the coatings' hardness increased by approximately 21%, which is directly proportional to the decreased wear rate of the coatings at high spraying powers. As the coatings experienced heat treatments, the coatings sprayed with a higher spraying power showed higher hardness and wear resistances. Precipitation strengthening played a significant role in the hardness and wear resistances of the coatings due to the addition of the titanium element.