Plasmodium falciparum and Plasmodium vivax. The lack of diagnostic biomarkers for P. vivax, especially those that differentiate the parasite from P. falciparum, significantly hinders P. vivax elimination. Here, we show that P. vivax tryptophan-rich antigen (PvTRAg) can be a diagnostic biomarker for diagnosing P. vivax in malaria patients. We report that polyclonal antibodies against purified PvTRAg protein show interactions with purified PvTRAg and native PvTRAg using Western blots and indirect enzyme-linked immunosorbent assay (ELISA). We also developed an antibody-antigen-based qualitative assay using biolayer interferometry (BLI) to detect vivax infection using plasma samples from patients with different febrile diseases and healthy controls. The polyclonal anti-PvTRAg antibodies were used to capture free native PvTRAg from the patient plasma samples using BLI, providing a new expansion range to make the assay quick, accurate, sensitive, and high-throughput. The data presented in this report provides a proof of concept for PvTRAg, a new antigen, for developing a diagnostic assay for P. vivax identification and differentiation from the rest of the Plasmodium species and, at a later stage, translating the BLI assay into affordable, point-of-care formats to make it more accessible.The World Health Organization (WHO) has set forth a global call for eradicating malaria, caused majorly by the protozoan parasites  Plasmodium vivax (P. vivax) elimination requires early diagnosis leading to efficient treatment. P. vivax detection methods include microscopy, rapid diagnostic tests, and nucleic acid amplification [P. vivax is the antigen P. vivax lactate dehydrogenase (PvLDH), a protein found in all Plasmodium species due to its essential role in metabolism. Therefore, diagnostic tests based on this antigen lack a differentiating protein biomarker to differentiate P. vivax from the other Plasmodium species [P. vivax with little or no identity with other Plasmodium species and the host, would be ideal for generating antibodies for immunoassay development. However, such biomarkers have been challenging to identify due to scarcity in parasite availability, either due to low parasitemia in the clinical samples or no continuous culturing of the parasite.fication . However species ,3,4. A pImmunoassay development requires polyclonal antibody development as the first step to confirm the antigenic characteristics of the target antigen. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize different epitopes of a specific antigen. Since these antibodies are directed against multiple regions/epitopes of the antigen, they are called polyclonal antibodies. Consequently, they have different antigen specificities and affinities towards the antigen , making P. vivax clinical samples , extraction of the parasite was challenging. The recent coronavirus disease (COVID-19) pandemic made correct sample collection arduous. In the era of the internet, knowledge sharing is easier, and to increase confidence in the findings of the most recurring parasite proteins, literature-based ,14,15,16 samples . Plasmodium has eight pv-fam gene families viz. pv-fam-a, pv-fam-b, pv-fam-c, pv-fam-d, pv-fam-e, pv-fam-g, pv-fam-h, and pv-fam-I [P. vivax compared to P. falciparum [P. vivax from pan Plasmodium species.pv-fam-I . The pv-lciparum . Memberslciparum ,19. PVX_E. coli BL21 DE3 pLyS, which produced the protein as inclusion bodies. These inclusion bodies were then solubilized using 0.5% N-lauryl sarcosine [The parasite protein PvTRAg was abundant in the previous studies done by our research group and literature-based data mining ,20. The The 1 mg aliquot was taken and dissolved in 400 µL of 1× phosphate-buffered saline (PBS) to make it 2.5 µg/µL, and 250 µg of the purified protein was mixed with 250 µL of complete Freund’s adjuvant (CFA) and injected intradermally into the laboratory-bred female New Zealand albino rabbit . The stu3) and 0.52 g of sodium carbonate (Na2CO3) in 50 mL Milli-Q water, pH 9.6) and incubated overnight at 4 °C on the static condition. A total of 200 µL of 1× phosphate buffer saline-tris (1× PBST) was added to the plate to remove the excess coating buffer and unbound PvTRAg and incubated for 5 min at room temperature (RT) under static conditions. The plates were washed using 200 µL of 1× PBS, pH 7.2, twice and blocked with 10% skim milk in 1× PBS for 1 h at 37 °C. Four washes of 200 µL of 1× PBST with 5 min incubation were used to remove the unbound blocking buffer. The anti-PvTRAg sera ten dilutions, ranging from 1:100 to 1:51,200 (sera: 1× PBS), were added as primary antibodies and incubated for 1 h at 37 °C under static conditions. The plate was washed four times using 200 µL of 1× PBS to remove non-specific or unbound sera/primary antibodies. The primary antibody was detected using goat-origin anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody . We added 100 µL of the secondary antibody diluents to the plate and incubated it for 1 h at 37 °C under static conditions. The plate was washed to remove the excess amount of secondary antibody using 1× PBS with 5 min of incubation four times. We added 20 µL of TMB/H2O2 to 20 mL of 1× PBS, and 100 µL of the solution was added to each well. The plate was incubated at RT for 8 min in a dark condition. The reaction was stopped by adding 50 µL of 2 M sulfuric acid, and optical density (OD) was measured at 450 nm using a multi-well plate reader .ELISA plates  were coated with purified PvTRAg (1 µg/well) suspended in 100 µL of 100 mM carbonate-bicarbonate buffer , were purified to near homogeneity by protein A-sepharose affinity chromatography. A total of 1 mL of protein A-Sepharose bead-packed column was washed using the 5-bed volume of 1× PBS. The 100 µL of rabbit polyclonal antibody or rabbit sera was diluted with 900 µL of 1× PBS and passed through the equilibrated column containing protein A. Protein A has a high affinity for IgG antibodies. The bound antibodies were eluted using 50 mM glycine buffer, pH 2.5. The elute was collected in 500 µL aliquots; the elution buffer has low pH that may degrade the eluted antibody; hence the elution buffer was mixed with 1 M tris base (pH 10) until the pH was in the 7.4 to 7.7 range. The neutralized solution was then quantified using Bradford, and 2.5 µg of each eluted fraction was run on SDS-PAGE for a quality check. The purified antibody was further taken for characterization using Western blot and indirect ELISA.2O2 as a substrate.The purified protein (PvTRAg) was electrophoresed in 12% SDS-PAGE according to the standard method described by Laemmli . The pro2O2 as the enzyme substrate. A total of 20 µL of TMB/H2O2 was added to 20 mL of 1× PBS, then 100 µL of the solution was added to each well. The plate was incubated at RT for 8 min in the dark condition. The reaction was stopped by adding 50 µL of 2 M sulfuric acid, and optical density (OD) was measured at 450 nm using a multi-well plate reader .ELISA plates were coated with purified PvTRAg (600 ng/well) suspended in 100 mM carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. The plates were then washed using 200 µL of 1× phosphate buffer saline  twice and blocked with 3% bovine serum albumin (BSA) in 1× phosphate buffer saline with 0.5% Tween 20 (1× PBST) for one h at 37 °C. After three washes using 200 µL of 1× PBST, the anti-PvTRAg sera  dilution was made in 1× PBST with 1% BSA. The plate was incubated for one h at 37 °C under static conditions. The plate was washed three times using 200 µL of 1× PBST to remove non-specific primary antibodies from polyclonal rabbit sera. The bound antibody was detected using goat anti-rabbit IgG-horseradish peroxidase conjugate (1:3000) in 1× PBST in 2% BSA. The plate was incubated at 37 °C under static conditions for one h. The unbound secondary antibody was washed using three rounds of 200 µL of 1× TBST. The interactions were developed using TMB/HPlasmodium vivax, was isolated using the saponin method from the vivax-infected patient’s whole blood sample and prepared the parasite lysate using a lysis buffer with a pH of 7.2 [The parasite pellet, H of 7.2 . SDS-PAGFor Western blot transfer, the PVDF membrane was activated by incubating the membrane in 100% methanol for 5 min in rocking conditions, followed by incubation of the PVDF membrane, tissue stacks, and SDS-PAGE in transfer buffer for 5 min. The protein bands were transferred to the PVDF membrane by setting up the tissue stacks and activating the PVDF membrane, SDS-PAGE, then tissue stacks. Transfer buffer  was used to keep the gel and membrane wet during the transfer. Blot transfer was done at 25 volts, 1 Ampere constant for 45 min using a semi-dry blot transfer machine. A total of 3% bovine serum albumin  in 1× TBST  was used as a blocking agent on the transferred PVDF membrane. The membrane was incubated for 2 h for blocking at RT on the rocker at 20 rpm. After 2 h, the blocking buffer was discarded, and the membrane was washed thrice, using 1× TBST at RT on the rocker, at 50 rpm, for 10 min. The washed membrane was then incubated in 1:3000 ratios of vivax-infected plasma samples and 1% BSA in 1× TBST solution at 4 °C overnight on the rocker at 20 rpm. The PVDF membrane was then washed thrice using 1× TBST at RT on the rocker, at 50 rpm, for 10 min. The washed membrane was then incubated in a 1:5000 ratio of anti-IgG Fc region antibody (goat anti-human IgG (H + L) secondary antibody HRP, Invitrogen) conjugated with horseradish peroxide (HRP) in 1% BSA freshly prepared in 1× TBST. The membrane was incubated for 2 h in secondary antibody dilution at RT on the rocker at 20 rpm. The membrane was rewashed to remove the excess secondary antibody, using 1× TBST, thrice for 10 min at RT, the rocker condition was 50 rpm. The blot was developed using 20 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB) in 10 mL of deionized water. The signals were read using a ChemiDoc instrument.n = 4), severe vivax malaria , healthy control , non-severe falciparum malaria , severe falciparum malaria , and dengue fever  plasma dilutions (1:150 and 1:200) were made in 1× PBST with 1% BSA. Each sample and its dilutions were plated in duplicates. The plate was incubated for 1 h at 37 °C under static conditions. The plate was washed three times using 200 µL of 1× PBST to remove non-specific primary antibodies from human plasma. The bound antibody was detected using goat anti-human IgG-horseradish peroxidase conjugate (1:3000) in 1× PBST in 2% BSA. The plate was incubated at 37 °C under static conditions for 1 h. The unbound secondary antibody was washed using three rounds of 200 µL of 1× TBST. The interactions were developed using TMB/H2O2 as the enzyme substrate. A total of 20 µL of TMB/H2O2 was added to 20 mL of 1× PBS, from which 100 µL of the solution was added in each well. The plate was incubated at RT for 8 min in a dark condition. The reaction was stopped by adding 50 µL of 0.2 M sulfuric acid, and optical density (OD) was measured at 450 nm using a multi-well plate reader, Multiskan GO .Indirect ELISA was performed using 96-well plates . ELISA plates were coated with purified PvTRAg (600 ng/well) suspended in 100 mM carbonate-bicarbonate buffer (pH 7.2) and incubated overnight at 4 °C. The plate also has two negative control (200 µL Milli-Q water), two blank wells (200 µL coating buffer), and two wells with 600 ng tetanus antigen-coated with the same coating buffer as a positive control for ELISA. The plates were washed using 200 µL of 1× phosphate buffer saline  twice and blocked with 3% BSA in 1× PBST for one h at 37 °C. After three washes using 200 µL of 1× PBST, non-severe vivax malaria . A total of 200 µL of this solution was used to immobilize antibodies on the streptavidin sensors for 10 min at 1000 rpm shaking.The rabbit polyclonal antibody was biotinylated using EZ—Link N.H.S.—LC-LC—Biotin by adding 1 µg of purified rabbit polyclonal antibody and biotin in a 1:3 molar ratio. We mixed both reagents well in a polymerase chain reaction (PCR) tube and incubated the tube at RT for 45 min under shaking conditions at 200 rpm. After biotinylation, the mix was desalted using Zeba™ spin desalting columns, 7K molecular weight cut-off . The mixture was diluted to make the volume 100 µL by adding 1× PBS, it wasmixed well, and the solution was added to the Zeba column. The mixture was passed through the column by centrifuging at 2000× The biotinylated antibody immobilized on the streptavidin sensors was used to capture purified PvTRAg in 7 different dilutions starting from 1 µM and diluted six times in a 1:1 ratio with 1× PBS. Signal capturing for the PvTRAg standards was used for standard curve preparation, which was used for absolute quantification of the free native PvTRAg captured from the diluted patient plasma (1:150 dilution) sample. The immobilized sensors were equilibrated in 1× PBS for 120 s. Free native PvTRAg capturing from diluted plasma sample was given 300 s, followed by three cycles of regeneration and neutralization for 5 s each. The whole experiment was performed at 1000 rpm using BLI (Sartorius)A test bleed was collected after the first booster from the marginal ear vein to check the level of circulating antigen-specific antibodies in the sera of the immunized rabbit. The presence of anti-PvTRAg antibodies was checked by indirect ELISA using purified PvTRAg (1 µg/well) as coating protein and 10 dilutions of rabbit sera starting from a 1:100 dilution in a 1:1 ratio for serial dilution. The antiserum obtained from the immunized rabbit had anti-PvTRAg antibodies detected by the ELISA assay. However, the antibody titer was not high in the first dose; hence another booster dose was given to check if there were any further improvements in the titer value b. The anThe purification of rabbit polyclonal antibodies was done using protein A affinity chromatography. After the chromatographic experiment, the purity of the rabbit polyclonal antibody was checked on SDS-PAGE gel stained with Coomassie blue d. It wasa.Western blot using purified PvTRAg and Rabbit polyclonal antibodyWestern blot was correctly transferred, confirmed by Ponceau staining. The purified PvTRAg was run along with pure IgG as a positive control, and both the lanes gave good bands, proving the success of the antigen–antibody interaction e.b.Indirect enzyme-linked immunosorbent assay (ELISA)Indirect ELISA also confirmed the interaction of purified PvTRAg (600 ng/well) and a ratio of 1:64,000 rabbit polyclonal antibody to 1× PBS to be sufficiently apt to perform future experiments using the patient’s plasma samples b.The reactivity of the rabbit polyclonal antibody with the purified PvTRAg was analyzed by immunoblotting. The immunoblot showed that the rabbit polyclonal antibodies were specific towards PvTRAg as it recognized the recombinant purified PvTRAg protein. A clear band was observed in the parasite lysate lane at the same size where purified PvTRAg was found .n = 4), severe vivax malaria, SVM (n = 4), healthy controls, HC (n = 3), non-severe falciparum malaria, NSFM (n = 2), severe falciparum malaria, SFM (n = 2), and dengue fever, DF (n = 2) plasma dilutions were analyzed. A sequential increase in the antibody reaction from HC to NSVM to SVM plasma samples was observed. The signals were found to be lowest in NSFM, followed by SFM and DF  confirm the presence of antibodies against the pathogen protein; (b) confirm the presence of antigen of the pathogen. Out of the two cases, the latter is the more reliable and real-time status of the patient’s health. The former method may lead to false positives due to delayed clearance of the antibodies from the host system even after the infection is long gone. Therefore, a protein unique to


The obtained nanoparticles had hexagonal structures, oval shapes, and average sizes of 22.5 ± 2.2 nm and 22.2 ± 2.0 nm, respectively. The spectroscopic properties of the products were investigated by measurements of the UC emission under 1151 nm laser excitation in the temperature range between 295 to 378 K. The sample doped with Ho3+ and Er3+ ions showed unique behavior of enhancing emission intensity with the temperature. The relative sensitivity determined for the NPs containing Ho3+ and Er3+ ions, reached the maximum value of 1.80%/K at 378 K. Here, we prove that the NaYF4:Ho3+, Er3+@NaYF4 system presents unique and excellent optical temperature sensing properties based on the luminescence intensity ratios of the near-infrared bands of both Ho3+ and Er3+ ions.Recently, materials revealing the upconversion (UC) phenomenon, which is a conversion of low-energy photons to higher-energy ones, have attracted considerable attention in luminescence thermometry due to the possibility of precise and remote optical thermal sensing. The most widely studied type of luminescent thermometry uses a ratiometric approach based on changes in the UC luminescence intensity, mainly of lanthanide ions’ thermally coupled energy levels. In this work, NaYF In addition, temperature sensors are also relevant in other areas, e.g., for diagnostics of constructions, electrical installations, or even food production9.The rapid development of technology would be impossible without discoveries and inventions in multiple fields of science. The crucial aspect of various scientific studies is determining the properties of the research objects. The particularly significant parameter is temperature, which is a physical quantity important for all biological processes. Thus, temperature measurements are widely used in medicine, including early tumor detection, monitoring of brain activity, or in vivo inflammation detection10. Thus, it is important to find a non-contact system to determine the internal temperature of the object13. The appealing method for remote temperature detection is luminescence thermometry, based on changes in the emission character of previously excited material14. The ability to prepare nanoparticles (NPs), resulting from nanotechnology development, is directly responsible for the remarkable advancements in optical nanothermometry15. The NPs’ luminescence highly depends on temperature, so the remote thermal readout is possible through a simple spectroscopic analysis16. One of the most widely studied types of optical nanothermometry is the ratiometric approach, which uses changes in the luminescence intensity17. However, to measure temperature with this method, the sensor must show at least two emission peaks whose changes in the intensities are interdependent.However, it is difficult to verify the actual temperature of the material through a typical contact approach due to the interference from the introduced thermometers18. Upconversion is a process of converting low-energy photons to high-energy ones, usually from the near-infrared (NIR) to the ultraviolet or visible range19. This phenomenon occurs for materials doped with lanthanide ions (Ln3+), which have a 4f. electronic structure with many well-defined energy levels. The unique properties of the UCNPs containing Ln3+ ions result in narrow absorption and emission bands, a significant difference between the absorption and the emission wavelengths, and relatively long luminescence lifetimes23. The UCNPs can be excited by NIR radiation, which benefits medical applications. Excitation of UCNPs by NIR light results in the low autofluorescence of biological materials, a high signal-to-noise ratio, and negligible photobleaching due to the low-energy excitation inorganic structure of NPs24.The promising candidates for non-contact optical nanothermometry seem to be upconverting nanoparticles (UCNPs)25. The UCNPs with an admixture of Er3+ ions are of great interest in this area26. Researchers frequently employ the thermally-coupled transitions of Er3+ ions, specifically from the excited energy levels of 2H11/2 and 4S3/2 to the ground energy level of 4I15/2. Plenty of these studies concern materials doped with Er3+ and Yb3+ ions, excited by 980 nm radiation, in which the sensitizing Yb3+ ions transfer the absorbed energy to the emitting Er3+ ions29. However, researchers have also published findings on UCNPs that are solely doped with Er3+ and directly excited by radiation at 980 nm or 1500 nm31. Moreover, a single study reported the temperature sensing properties of Er3+ doped NPs, where the luminescence originating from non-thermally coupled bands in the NIR range, specifically 4I9/2 → 4I15/2 and 4I11/2 → 4I15/2, was utilized for temperature determination31. There are also some studies on the optical thermometry of the materials emitting from the thermally or non-thermally coupled states of the other Ln3+ ions, e.g., Ho3+34, or Tm3+ ions36, but these materials are in general excited at 980 nm because of co-doping with Yb3+ ions.Until now, the prevailing types of optical nanothermometers based on UCNPs ions have relied on the temperature-dependent emission from thermally coupled energy levels3+ ions other than Yb3+ in the proper matrix can lead to the UCNPs with properties promising for optical nanothermometry. For instance, a system in which Ho3+ ions act as sensitizers and Er3+ ions as emission centers can be excited in the NIR range with four different wavelengths 38. The observed luminescence of these UCNPs displays transition bands originating from both Er3+ and Ho3+ ions. Depending on the temperature, alterations in the emission intensity of the individual peaks of UCNPs exhibit distinct behavior. This characteristic holds great potential for enhancing temperature sensors.Using Ln3+ and Er3+ ions. We selected β-sodium-yttrium fluoride as a matrix for Ln3+ ions because β-NaYF4 is an excellent UC host material with low phonon energy (around 360 cm−1)39, reducing the multiphoton quenching processes. In addition, β-NaYF4, under the proper synthesis conditions, forms crystalline NPs of small sizes with narrow size distribution. The spectroscopic properties of the prepared NaYF4:Ho3+@NaYF4, and NaYF4:Ho3+, Er3+@NaYF4 samples were measured under 1151 nm excitation in the temperature range from 295 to 378 K. The obtained results allowed us to determine the sensitivity of this nanothermometer. Our results show that a different way of excitation than the typical one at 980 nm through Yb3+ ions can also result in a good-quality optical thermometry sensor. Moreover, the used approach allows for excitation within the second biological window, which is very convenient for biomedical applications40.This work presents a new approach to optical nanothermometry based on the properties of Ho3+, Ho3+, Er3+, Na+ and F- ions, respectively. The chlorides were placed in the dryer at 348 K for a week to remove the water . The reaction was carried out in n-octadecene (90% Alfa Aesar) and oleic acid . Ethanol (99.8% POCh S.A.) and n-hexane (≥ 99% POCh S.A.) were used to purify the post-reaction products.Yttrium, holmium, and erbium chloride hydrates , sodium oleate , and ammonium fluoride  were used as a source of Y4:7.5%Ho3+,7.5%Er3+@NaYF4 preparation:β-core4:7.5%Ho3+,7.5%Er NPs, 110 mL of n-octadecene and oleic acid mixture (1:1), 4.6750 mmol of yttrium chloride, 0.4125 mmol of both holmium and erbium chlorides were purified at 373 K under vacuum for 2.5 h. 11.0 mmol of sodium oleate (2× excess) and 33.0 mmol of ammonium fluoride (1.5× excess) were separately added to the heated mixture under nitrogen flow and purified at 373 K under vacuum for 30 and 5 min, respectively. The mixture was heated at 573 K with vigorous stirring, under nitrogen flow for 1 h, and cooled down. The post-reaction product was purified five times by sequential dispersing in n-hexane and precipitating by ethanol . The obtained NPs were dispersed in n-hexane and air-dried for 24 h.To obtain 5.5 mmol of β-NaYFα-shell4 NPs, 300 mL of n-octadecene and oleic acid mixture (1:1) and 15 mmol of yttrium chloride were purified at 373 K under vacuum for 3 h. 22.5 mmol of sodium oleate (1.5× excess) and 60.0 mmol of ammonium fluoride were separately added to the heated mixture under nitrogen flow and purified at 373 K under vacuum for 45 and 10 min, respectively. The mixture was heated at 473 K with vigorous stirring, under nitrogen flow for 1 h, and cooled down. The post-reaction mixture was centrifuged , the product was precipitated by adding ethanol and purified three times by sequential disperse in n-hexane and precipitated by ethanol . The obtained NPs were air-dried for 36 h.To obtain 15 mmol of α-NaYFβ-core@β-shell4:7.5%Ho3+,7.5%Er3+@β-NaYF4  NPs, 1 mmol of β-NaYF4:7.5%Ho3+,7.5%Er3+ NPs and 7 mmol of α-NaYF4 NPs were added to 32 mL of n-octadecene and oleic acid mixture (1:1) and purified at 373 K under vacuum for 3 h. Then the mixture was heated at 573 K with vigorous stirring, under nitrogen flow for 2 h and 15 min, and cooled down. The post-reaction product was purified four times by sequential dispersing in n-hexane and precipitating by ethanol . The obtained NPs were dispersed in n-hexane or air-dried for 48 h.To obtain β-NaYFNaYFNaYF4:7.5%Ho3+@NaYF4 preparation:4:7.5%Ho3+@β-NaYF4 (briefly NaYF4:Ho3+@NaYF4) NPs were prepared via the above-mentioned multi-stage procedure under similar synthesis conditions. 5.0875 mmol of yttrium chloride and 0.4125 mmol of holmium chloride were used for the core preparation. The other reagents were added in the same amounts. The previously prepared α-NaYF4 NPs were also used for the NaYF4:Ho3+@NaYF4 synthesis.The β-NaYFCu Kα1 = 1.5406 Å) and a LynxEye strip detector . The reference data was taken from JCPDS (00-016-0334). The images of synthesized NPs, based on which the average sizes and size distributions were determined, were recorded on the high-resolution transmission electron microscope Hitachi HT7700 with an accelerating voltage of 120 kV.The purity of the products obtained at individual synthesis stages was determined by Thermogravimetric Analysis (TGA) on Netzsch TG 209 Libra in the temperature range from 298 to 878 K under nitrogen flow see Fig. . The cry4:Ho3+@NaYF4, and NaYF4:Ho3+, Er3+@NaYF4 NPs were obtained using a 10 ns pulsed optical parametric oscillator OPO (EKSPLA/NT342/3/UVE) as the laser source with energy 0.5 mJ. Emissions from the oven were focused on the entrance slit of a spectrograph (Andor SR-303i-A) equipped with a cooled CCD camera (Andor Newton). All spectra were corrected from the spectral response of the equipment. The QuantaMasterTM 40 spectrophotometer equipped with an Opolette 355LD UVDM tunable laser (with a repetition rate of 20 Hz) and a PIXIS:256E digital CCD camera with an SP-2156 imaging spectrograph (Princeton Instruments) was used to measure the dependencies of the energy transitions intensities on the laser energy. The luminescence rise and decay lifetimes were recorded with a Mixed Domain Oscilloscope—200 MHz—Tektronix MDO3022. These measurements were carried out for solid samples at room temperature.Measurements from 295 to 378 K were carried out in a tubular electric furnace (Gero RES-E 230/3), where the sample temperature was controlled via a type K thermocouple in contact with it. The temperature-dependent UC emission spectra of the NaYF4:Ho3+@NaYF4, and NaYF4:Ho3+, Er3+@NaYF4 NPs crystallized as a single hexagonal phase, with the 3+ ions with Ho3+ or Er3+ ions did not affect the unit cell parameters, as all ions are of similar size , as evidenced by broad peaks in the measured diffractograms. The obtained results agree with the TEM images in Fig. The prepared NaYFmentclass2pt{minimmentclass2pt{minim3+ and Er3+ ions added to the core compared to Y3+ ions in the shell. Ultimately, the presence of the core@shell structure of the prepared samples was confirmed by the observed increase in their sizes compared with the core-only NPs  with narrow size distributions Fig. b,c. In t3+ only, as well as Ho3+ and Er3+ ions, revealed inquire and not entirely apparent results. The excitation spectrum .The UC luminescence spectra of the prepared NPs were measured under 1151 nm excitation in the temperature range from 295 to 378 K , which is a ratio of the luminescence intensity from the upper (IU) and lower (IL) states, was used43:The parameters characterizing prepared optical temperature sensors were determined using the ratiometric approach. To describe the properties of the NaYF4:Ho3+, Er3+@NaYF4 sample, resulted from the overlapped emission from the energy levels of both Ho3+ and Er3+ ions. In that case, the LIRs were calculated as a ratio of the shorter wavelength peaks’ intensities (Is) to the intensities of the peaks with the longer wavelength (Il):The luminescence spectra of the NaYFSR) of the material to the temperature changes, determined as the rate of LIR changes with the temperature44:The most important parameter related to temperature-dependent luminescence, especially for the application in optical temperature sensors, is the relative sensitivity  or only Ho3+ ions (NaYF4:Ho3+@NaYF4 NPs).The LIR and SR dependencies were estimated for the NPs’ luminescence peaks with the different temperature behaviors and are shown in Fig. 3+ ions, we selected the emission peaks at 489 and 544 nm, 648 and 898 nm, as well as 898 and 970 nm %/K for the NIR peaks at 898 and 970 + 982 nm. In contrast, all determined SR values of the NaYF4:Ho3+@NaYF4 sample were below 1.00%/K. The results indicate that the co-doping with Er3+ ions to a system based on the Ho3+ ions significantly improves the temperature sensing properties, particularly in the NIR range. The prepared NPs exhibit minimum temperature uncertainty around 1.08 K  are determined from the dependencies of the luminescence intensities IUC on the excitation power densities P, or in the case of the pulsed laser excitation, from its energies EDetermining the number of photons involved in populating the excited states of the emitting ions is crucial for explaining the mechanism behind the observed spectroscopic properties of the NPs. The photon's numbers . The n coefficients for the NaYF4:Ho3+, Er3+@NaYF4 NPs were mainly below 2.0, suggesting that the observed UC was primarily influenced by processes that necessitated the absorption of only 2 photons. The exception was green emission, which resulted from 3 photons process (n = 2.14).Luminescence peaks recorded at similar wavelengths showed significant differences in the es Figs.  and S4. 3+ and Er3+ transitions  or decay (D) time, tp is the time when the lifetime trend changes from rise to decay and I is the intensity at time t  processes between Ho3+ ions, as evidenced by the visible rise times of the registered transitions  indicate that the 5I6 and 5I4 Ho3+ levels are likewise populated. The 5F5, 5S2, and 5F3 states related to the emission at 648, 544, and 489 nm are also populated similarly, slower than the previously mentioned, as evidenced by the congruous rise times close to 40 μs. In the case of the NaYF4:Ho3+, Er3+@NaYF4 sample, the UC luminescence at 1250 nm results from quick processes since the rise time of the 5I6 → 5I8 transition is not visible. The other emissions, which have rise times below 20 μs, are connected with the energy transfer processes between Ho3+ and Er3+ ions. In the recorded lifetime profiles of the Ho3+ ion transitions, the Er3+ ion influence is visible, especially for the red emission  process  and 970 nm (5F5 → 5I7) is from this energy level. The Ho3+ ions, previously populated to the 5I4 level, absorb another photon by the quick ESA process, whereby the Ho3+ ions are excited to their 5F3 energy levels. Hence, the sample presented emissions at 489 nm and after relaxation at 544 nm.The irradiation of the NaYFess Fig. a. The in4:Ho3+, Er3+@NaYF4 NPs, most of the energy absorbed by Ho3+ ions is transferred to Er3+ ions (see Fig. 3+ ions are excited to their 4F9/2 levels from where an additional emission is possible giving a peak at 672 nm. Further, intense emission at 982 nm occurs after the relaxation to the 4I11/2 state. Another photon absorbed within the Er3+ ion leads to a population of the 2H11/2, 4S3/2 Er3+ ions levels and an additional emission band at 523 nm. The emission of Ho3+ ions at 890 and 743 nm results from energy back transfer from the excited Er3+ ions and is weaker than in the case of a system containing only Ho3+ ions48. The emission of Ho3+ ions in NaYF4:Ho3+, Er3+@NaYF4 sample probably also consists of the processes occurring in sample NaYF4:Ho3+@NaYF4 sample, however, they are less intense because the pre-excited Ho3+ ions transferred most of the energy to Er3+ ions.In the case of the NaYFsee Fig. b. The Er3+ ions or both Ho3+ and Er3+ ions. The prepared UCNPs exhibited an oval shape and average sizes of approximately 22.5 nm.Using the precipitation reaction in the oleic acid/octadecene solution, we successfully obtained core@shell UCNPs based on sodium yttrium fluorides doped with either Ho4:Ho3+@NaYF4 and NaYF4:Ho3+, Er3+@NaYF4 samples showed UC emission under 1151 nm pulsed laser excitation. We registered the emission of the products in the 295 to 378 K temperature range to determine their temperature-sensing properties.The NaYF3+ and Er3+ ions revealed unusual behavior manifested by increased luminescence intensity with the temperature increase. This observation can be attributed to the specificity of the UC mechanism based on energy transfers from Ho3+ to Er3+ ions. Upon the research, we discovered that the NaYF4:Ho3+, Er3+@NaYF4 NPs have great potential as a temperature sensor based on the excitation and emission in the range of biological windows. This sample shows intense NIR luminescence from Ho3+ ions at 899 and 970 nm and Er3+ ions at 982 nm. The relative sensitivity determined for these peaks reached the maximum value of 1.80%/K at 378 K. This optical temperature sensor based on the NIR UC emission of the system containing Ho3+ and Er3+ ions has been reported for the first time. The possibility of excitation within the second biological window and detecting temperature changes in emission intensity around the first biological window make our UCNPs promising candidates for biomedical applications. However, the obtained UCNPs presented high sensitivities not only in the NIR range, which generally makes them excellent candidates for temperature sensing applications not only limited to biological ones.The NPs containing HoSupplementary Information.


The Ti3C2Tx was prepared by two etching methods, including a mild LiF/HCl mixture and HF solution, which was directly used as the pH-sensitive materials. Both etched Ti3C2Tx showed a typical lamellar structure and exhibited enhanced potentiometric pH responses compared with a pristine precursor of Ti3AlC2. The HF-Ti3C2Tx disclosed the sensitivities of −43.51 ± 0.53 mV pH–1 (pH 1–11) and −42.73 ± 0.61 mV pH–1 (pH 11–1). A series of electrochemical tests demonstrated that HF-Ti3C2Tx exhibited better analytical performances, including sensitivity, selectivity, and reversibility, owing to deep etching. The HF-Ti3C2Tx was thus further fabricated as a flexible potentiometric pH sensor by virtue of its 2D characteristic. Upon integrating with a solid-contact Ag/AgCl reference electrode, the flexible sensor realized real-time monitoring of pH level in human sweat. The result disclosed a relatively stable pH value of ~6.5 after perspiration, which was consistent with the ex situ sweat pH test. This work offers a type of MXene-based potentiometric pH sensor for wearable sweat pH monitoring.The level of hydrogen ions in sweat is one of the most important physiological indexes for the health state of the human body. As a type of two-dimensional (2D) material, MXene has the advantages of superior electrical conductivity, a large surface area, and rich functional groups on the surface. Herein, we report a type of Ti M represents transition metals , X represents carbon and/or nitrogen, and Tx represents the functional groups on the surface of the MXene [MXenes, a family of two-dimensional 2D) materials, were discovered by Drexel University in 2011 [D materiahe MXene ,4,5, enehe MXene ,7 and sehe MXene ,9.2 [2 [3) [The concentration of hydrogen ions in physiological fluids such as sweat, saliva, and urine in the human body is closely related to various physiological diseases . Sweat m2 ,34 and I2 [2 ,36,37,382 [2 [3) ,40,41,423C2Tx was directly employed as a pH-sensitive material to fabricate a solid-contact potentiometric pH sensor. The Ti3C2Tx was prepared by etching the precursor of Ti3AlC2. After etching, the Ti3C2Tx contains surface-abundant functional groups that could be worked as hydrogen ion-sensitive sites. The HF-etched Ti3C2Tx disclosed a sensitivity up to −43.51 ± 0.53 mV pH–1 in a wide range (pH 1–11) and also a reversible response with a sensitivity of −42.73 ± 0.61 mV pH–1 (pH 11–1). In particular, a reversible pH response was shown for this material. Based on the flexible characteristic of the D Ti3C2Tx, it was further integrated into a wearable device with an Ag/AgCl solid reference electrode. The flexible Ti3C2Tx-based pH sensor was successfully applied for on-body sweat pH monitoring.In this work, the MXene of Ti3AlC2 (99%) was purchased from Jilin Yiyi Technology Co., Ltd. . Hydrofluoric acid  and silver chloride  were purchased from Macklin . Lithium fluoride , boric acid (99.5%), acetic acid (99.5%), sodium hydroxide , N-methyl-2-pyrrolidone , and ferric chloride  were obtained from Innochem . Potassium chloride , sodium chloride , lithium chloride , magnesium chloride hexahydrate , Nafion solution , polyvinyl chloride , tetrahydrofuran , tridodecylmethyl ammonium chloride , and bis (2-ethylhexyl) sebacate  were purchased from Sigma-Aldrich . Potassium tetrakis(pentafluorophenyl)borate  and ammonium chloride  were purchased from Alfa Aesar . Hydrochloric acid , phosphoric acid  and sulfuric acid  were purchased from Guangzhou Chemical Reagent Factory . All aqueous solutions were prepared with ultrapure water . The triacid mixture consisted of 0.04 M phosphate, boric acid and acetic acid. Britton–Robinson buffers (B-R buffer) with different pH were prepared by mixing different volumes of the triacid mixture and 0.2 M sodium hydroxide.Ti3C2Tx by using the Phenom nano SEM . The crystal structure characterization was recorded by X-ray diffraction patterns (XRD) using a Miniflex 600  by scanning in the 2θ range of 5−80° with Cu Kα radiation. The valence states of the materials were analyzed by X-ray photoelectron spectroscopy (XPS) using a Thermo Scientific K-Alpha .Scanning electron microscopy (SEM) was carried out to examine the morphology and size of the Ti3C2Tx was prepared according to a previous report [3C2Tx was synthesized by adding 2 g LiF into 40 mL 6 M HCl solution, followed by stirring until the powder was completely dissolved. Then, 1 g of Ti3AlC2 was slowly added into the above solution (~10 min). The mixture was kept stirred at 35 °C for 24 h. After etching, the sediment was washed with deionized water by centrifugation until the pH value of the supernatant was higher than pH 6. Then, the sediment was dried in a vacuum oven for 12 h to obtain MILD-Ti3C2Tx. The HF-Ti3C2Tx was synthesized by slowly adding 1 g Ti3AlC2 into 20 mL HF solution, followed by stirring for 24 h at 35 °C. After etching, the sediment was washed with deionized water by centrifugation until the pH value of the supernatant was higher than pH 6. Then, the sediment was dried in a vacuum oven for 12 h to obtain the HF-Ti3C2Tx.The Tis report . Typical3C2Tx or 10 mg HF-Ti3C2Tx into 800 μL NMP and 200 μL Nafion solution. Nafion solution was used as a binder to adhere the sensing materials on the surface of the electrode. Glassy carbon electrodes (GCE) with a 5 mm diameter were used as the substrate electrode. The GCE was well-polished and washed separately in deionized water and ethanol, then dried by N2 blowing. The Ti3C2Tx-based pH electrode was further prepared by depositing 10 μL of the above ink on the GCE, and then the GCE was dried in a vacuum oven at 60 °C for 1 h. The Ti3C2Tx-based pH electrode was conditioned in pH = 1 B-R buffer solution for 2 h before use. The purpose of this conditioning step was to promote the proton transport in the Ti3C2Tx sensing material, which is similar to the conditioning step for the solid-contact ion-selective electrodes. The controlled experiment of Ti3AlC2-based pH electrode followed the same procedure as Ti3C2Tx-based pH electrodes.pH sensing material inks were obtained by dispersing 10 mg MILD-Ti2 plasma treatment for 5 min. The microwell pattern of Ag electrodes was fabricated by the magnetron sputtering deposition technique AJA Orin5  with respective 30 nm Cr and 200 nm Ag layers. Then, the electrodes were insulated by spin-coating a thin layer of polydimethylsiloxane (PDMS) and then dried in an oven at 90 °C for 40 min. The Ti3C2Tx working electrode was fabricated on the obtained Ag-coated PET electrode in the same way as the GCE. The reference electrode was fabricated as follows.Firstly, a polyethylene terephthalate (PET) membrane (8 cm × 8 cm) was cleaned by ultrasonication in acetone, ethanol, and deionized water, followed by O3 solution for 10 s for partial oxidation of Ag to AgCl and was cleaned with deionized water. Then, the reference membrane solution was further coated on the Ag/AgCl electrode. The reference membrane solution was prepared by dissolving KTPFB (0.9 wt%), TDMA-Cl (1.1 wt%), DOS (68 wt%) and PVC (30 wt%) in THF. Then, 40.4 mg KCl and 15.3 mg AgCl powder were added into 250 µL of the above solution. In addition to the reference membrane layer, a reference protective layer was further coated. The reference protective layer cocktail was prepared by dissolving 4 g of PVC (33.1 wt%) and DOS (66.9 wt%) in 50 mL THF. The solid Ag/AgCl reference electrode was obtained by depositing 10 µL of reference membrane solution and 20 µL of reference protective layer cocktail, respectively. The dropping time interval of the two solutions was ~12 h.First, the Ag/AgCl electrode was prepared by immersing the Ag-coated PET electrode into 0.3 M FeCl3AlC2- or Ti3C2Tx-modified GCE. The reference electrode was a saturated calomel electrode (SCE). For the flexible pH sensor, a solid Ag/AgCl electrode was used as the reference electrode. The electromotive force (EMF) between the working and reference electrodes was recorded in different pH solutions in the range of pH 1–11. The B-R buffer solution was prepared by mixing 0.04 M three acid and 0.2 M NaOH with a tunable pH range of 2–11. A pH = 1 solution was prepared by diluting concentrated sulfuric acid.The potentiometric measurements for sensitivity and selectivity were performed by using a multi-channel potentiometer EMF6  at room temperature based on a two-electrode system. The working electrode was Ti–1 to 10–5 M.The selectivity was evaluated by two methods. One was the continual addition of interfering ions, and the other was the separation solution method. The former involved adding each interfering ion (10 mM) into a pH 7 buffer solution to record the EMF. The separation solution method involved measuring potentiometric response curves for each interfering ion from 103C2Tx-based pH sensor was worn on the forehead of a healthy male volunteer. A homemade mini-potentiometer with an input resistance of 1013 Ω was connected to the sensor. The data during the on-body test was recorded based on a mobile APP. Before the test, the sensor was calibrated by potentiometric tests in B-R buffer solutions . After the on-body test, the volunteer ran again, and the sweat was collected, which was tested by a simple pH strip for comparison. In addition, the flexible pH sensor was calibrated again to examine the stability of the sensor.For the on-body sweat pH test, the fabricated flexible HF-Ti3C2Tx was carried out according to the established etching method . Briefly, two etching reagents of mild LiF/HCl and HF acid were used to exfoliate the pristine Ti3AlC2  patterns in MILD-Ti3C2Tx and HF-Ti3C2Tx was due to expanded interlayer spacing after the successful removal of Al from Ti3AlC2 [3AlC2 disappeared in the XRD spectra of MILD-Ti3C2Tx and HF-Ti3C2Tx, suggesting that the Al layer was etched [The preparation of Ti Ti3AlC2 a. After d by XRD b. Typica Ti3AlC2 . In addis etched .3AlC2, MILD-Ti3C2Tx, and HF-Ti3C2Tx were examined by scanning electronic microscopy (SEM)  c–e. The tructure c. After tructure d,e. The  etching , which f3AlC2, MILD-Ti3C2Tx and HF-Ti3C2Tx were analyzed by X-ray photoelectron spectroscopy (XPS). XPS survey spectra of Ti3AlC2, MILD-Ti3C2Tx, and HF-Ti3C2Tx are shown in 3AlC2 in which the O element originated from the oxidation of the sample during the long-term storage. After etching, the F element could be clearly distinguished in both MILD- and HF-Ti3C2Tx. The high-resolution Ti 2p XPS spectra were further examined in 3AlC2, three double peaks were located at 463.6 and 460.5 eV , 459.3 and 454.7 eV , and 457.8 and 453.4 eV . As presented in the Ti 2p spectra of MILD-Ti3C2Tx and HF-Ti3C2Tx, three double peaks were assigned to Ti-C, Ti(II), and Ti(III), respectively [3AlC2 disappeared, indicating that the Al atom was etched. F 1s XPS spectra further demonstrated the F element introduction after etching  signals of the Ti3AlC2 electrode show an irregular decrease. It has a poor reversible response and potential stability. The slope for Ti3AlC2 (−22.14 ± 5.18 mV pH–1 between pH 1 and 11) significantly deviates from Nernstian sensitivity. Since Ti3AlC2 is a raw material without etching, poor electronic conductivity resulted in difficulties in efficient proton-to-electron transduction. In addition, fewer functional groups on the surface of Ti3AlC2 could cause difficulties for the proton association, leading to low sensitivity. However, the MILD-Ti3C2Tx electrode reveals a reversible response and much-improved reproducibility, and the sensitivity for MILD-Ti3C2Tx increased to −37.91 ± 0.63 and −36.26 ± 0.25 mV pH−1 for the forward and reverse pH tests, respectively  and −42.73 ± 0.61 mV pH−1   f. These 3AlC2 changes apparently, while relatively small changes are observed for MILD-Ti3C2Tx. In addition, we further evaluated the selectivity using the separation solution method according to –1 to 10–5 M, the precursor of Ti3AlC2 responds to all interference ions, in which the sensitivities for some interfering ions are even higher than the target H+ (−20.77 ± 7.42 mV pH−1), for example (−28.52 ± 6.66 mV dec−1 for K+ and −31.66 ± 4.85 mV dec−1 for NH4+)  d. The MIing ions f, which 3C2Tx shows the best potentiometric pH response performances compared with MILD-Ti3C2Tx and pristine Ti3AlC2. The purpose of the etching step is to remove Al atoms in Ti3AlC2. The precursor of Ti3AlC2 is nearly an insulator. After etching, the Ti3C2Tx becomes more conductive [3C2Tx. These groups could play the role of association sites that exchange with protons and couple with the transition metal redox transition of Ti3+/2+ , resulting in a potentiometric pH response. Furthermore, the Ti3C2Tx owns an exfoliated structure after etching, which could promote proton transport in the interlayer of Ti3C2Tx. We also compared the Ti3C2Tx-based pH sensor with literature results (3C2Tx is better than PANI (pH 4–9) and comparable to metal oxides. Regarding the sensitivity, it is lower than the RuO2 and IrOx but competitive to PANI and non-precious metal oxides . The PANI/LGG-MXene discloses a high sensitivity up to −57.03 mV pH–1 but only in a narrow range  [The above results demonstrated that the deeply-etched HF-Tinductive , which i results . It was .g., WO3  and ZnO .g., WO3 ). For ex.g., WO3  compositpH, 5–9) . In addi3C2Tx disclosed the best sensitivity, reproducibility and selectivity. In this section, a flexible pH sensor based on HF-Ti3C2Tx was fabricated and applied for on-body sweat pH monitoring. The Ti3C2Tx-based pH working electrode (WE) and reference electrode (RE) of Ag/AgCl were designed  was further coated on the Ag/AgCl (solid RE of Ag/AgCl/KCl). The bending tests for the WE are shown in The above potentiometric pH response tests have identified that HF-Tidesigned a–d. The Finally, on-body pH sweat monitoring was examined b–f. One 3C2Tx-based wearable solid-contact potentiometric pH sensor for sweat pH monitoring. It has been demonstrated that the deeply etched HF-Ti3C2Tx disclosed the best pH analytical performances compared with the precursor of Ti3Al2 and MILD-Ti3C2Tx owing to its high conductivity and abundant surface functional groups for proton association. The HF-Ti3C2Tx-based pH sensor revealed a sensitivity of −43.51 ± 0.53 mV pH–1 (pH 1–11) and −42.73 ± 0.61 mV pH–1 (pH 11–1), which is comparable to representative transition-metal oxide-based pH sensors. The sensor also revealed reversible and reproducible characteristics, which was proved by nearly overlapped potentiometric pH response curves upon forward and reverse tests. In addition, the HF-Ti3C2Tx also showed good selectivity, which was confirmed by both the continual addition method and the separation solution method. Furthermore, this fabricated flexible pH sensing device has realized on-body sweat pH monitoring. The online monitoring pH value is consistent with the ex situ results, suggesting its reliability. The 2D Ti3C2Tx could be recognized as a new type of potentiometric pH sensor that can be applied as advanced wearable pH devices toward health monitoring.In summary, we have developed a Ti


Alloys with minor B and B-free alloy exhibited similar superplastic elongations of 400–1000% in a temperature range of 700–875 °C and strain rate sensitivity coefficient m of 0.4–0.5. Along with this, a trace boron addition provided a stable flow and effectively reduced flow stress values, especially at low temperatures, that was explained by the acceleration of the recrystallization and globularization of the microstructure at the initial stage of superplastic deformation. Recrystallization-induced decrease in yield strength from 770 MPa to 680 MPa was observed with an increase in boron content from 0 to 0.1%. Post-forming heat treatment, including quenching and ageing, increased strength characteristics of the alloys with 0.01 and 0.1% boron by 90–140 MPa and insignificantly decreased ductility. Alloys with 1–2% B exhibited an opposite behavior. For the high-boron alloys, the refinement effect of the prior β-grains was not detected. A high fraction of borides of ~5–11% deteriorated the superplastic properties and drastically decreased ductility at room temperature. The alloy with 2% B demonstrated non-superplastic behavior and low level of strength properties; meanwhile, the alloy with 1% B exhibited superplasticity at 875 °C with elongation of ~500%, post-forming yield strength of 830 MPa, and ultimate tensile strength of 1020 MPa at room temperature. The differences between minor boron and high boron influence on the grain structure and properties were discussed and the mechanisms of the boron influence were suggested.The decrease of superplastic forming temperature and improvement of post-forming mechanical properties are important issues for titanium-based alloys. Ultrafine-grained and homogeneous microstructure are required to improve both processing and mechanical properties. This study focuses on the influence of 0.01–2 wt.% B (boron) on the microstructure and properties of Ti-4Al-3Mo-1V (wt.%) alloys. The microstructure evolution, superplasticity, and room temperature mechanical properties of boron-free and boron-modified alloys were investigated using light optical microscopy, scanning electron microscopy, electron backscatter diffraction, X-ray diffraction analysis, and uniaxial tensile tests. A trace addition of 0.01 to 0.1 wt.% B significantly refined prior  However, obtaining parts from titanium alloys with traditional methods at low temperatures is a difficult and energy-consuming process due to their high strength, low elasticity modulus, and high sensitivity to processing parameters . Superplse ratio ,5. Contrβ-Ti grains solidify first and the eutectic-originated TiB phase could not stimulate β-Ti phase nucleation in the liquid phase with B content below ~2% [Alloying with 0.05–0.2 B  improves the processing and mechanical properties of titanium-based alloys ,7,8,9. Below ~2% . Larson elow ~2%  that theelow ~2% . The Zenelow ~2% . A traceelow ~2% ,27 and telow ~2% . The graelow ~2% .α phase nucleation near TiB particles [α/β interfaces and reasonably assume the ‘boron solute pinning mechanism’ for α-phase colonies.For wrought alloys, boron provides globularization of the microstructure during heat and thermomechanical treatments. The proposed mechanism is stimulation of the articles ,29. The articles  observe The TiB phase has an equivalent density to titanium but five times greater strength . NotablyThus, minor boron refines the grain structure in the as-cast state as well as after thermo-mechanical processing and increases the homogeneity of the microstructure in the ingots and thermomechanically treated products ,32,33,34A conventional Ti-4Al-3Mo-1V alloy is widely applied due to its high strength, excellent corrosion resistance, and good creep resistance. The alloy exhibits superplasticity in a temperature range of 825–875 °C similar to Ti-6Al-4V ,36. In c3. The ingots were subjected to homogenization annealing in a furnace with a vacuum atmosphere at 800 °C for 1 h and subsequently heated to the β-field and water-quenched.The boron-free reference Ti-Al-Mo-V alloy and five alloys with boron additions in a range of 0.01–2 wt.% were prepared . The ing2O + 3% HF + 5% HNO3) for 20–30 min to remove the α-phase layer from the surface. The final thickness of the hot-rolled sheets was 1.0 ± 0.1 mm.Quenched ingots of the 0B, 0.01B, 0.05B, and 0.1B alloys were hot-rolled with a total reduction of 90% at a temperature of 750 ± 10 °C. A low ductility did not allow us to roll the high-boron alloys at 750 °C. Ingots of the 1B and 2B alloys (with 1–2% B) were successfully rolled at a higher temperature of 900 ± 10 °C. The sheets were treated in Kroll’s reagent  technique using an HKL NordlysMax EBSD detector . The scanning area was 50 × 50 μm2 and scanning step was 0.15 μm. The EBSD data, i.e., grain and subgrain sizes , were analyzed using HKL CHANNEL 5 software . Grain boundaries with a misorientation angle (θ) of 2° < θ < 15° were classified as low-angle grain boundaries (LAGBs), and grain boundaries with θ ≥ 15° were classified as high-angle grain boundaries (HAGBs).The as-cast grain structure of the alloys was analyzed using a Neophot-30 optical (light) microscope (LM) with polarized light. To analyze the phase composition and grain size of the thermomechanical treated alloys, a Vega 3-LMH scanning electron microscope (SEM)  equipped with the EDS X-MAX 80 energy dispersive spectrometer  was used. The microstructural parameters of the 2O2 (30%) 10 mL, and Kroll reagent 5 mL) on a Struers LaboPol-5 . To identify grain structure in an as-cast state, the samples were etched with Weck’s reagent .Samples for microstructural analysis were prepared by mechanical grinding and polishing on a CHEM MD cloth in a solution  software and a TTTI3 database were used.XRD analysis was performed using a D8 ADVANCE X-ray diffractometer  in a Cu-Kα radiation. To calculate the equilibrium 3 were cut parallel to the rolling direction and annealed for 30 min at the superplastic deformation temperatures before testing.The superplastic deformation behavior was characterized by uniaxial tension tests at a constant rate and a step-by-step decrease in the strain rate at temperatures of 875 °C, 775 °C and 700 °C on a Walter Bai LFM-100  universal testing machine in a furnace with an argon atmosphere. Samples with the gauge section size dimensions of 14 × 6 × 1 mm3. Before cutting the samples, the sheets were pre-strained for 100% at a temperature of 875 °C with a constant strain rate of 1 × 10−3 s−1. Then, two types of treatment were processed: (1) air-cooling from the deformation temperature, and (2) water-quenching from the deformation temperature and further ageing at 480 °C for 16 h. Before testing, the samples were treated in Kroll’s reagent for ~20 min for α-case removal. Three samples per point were tested.The mechanical properties of the alloys at room temperature were determined on a Zwick Z250 testing machine  using samples with a gauge section size of 6 × 0.8 × 25 mmα and β phases. Due to a high boron content in the 1B and 2B alloys, the clear peaks of the TiB phase were revealed. The peaks of low intensity attributed to titanium boride were found in the alloy with 0.1% B, and this phase was not observed in the alloys with 0.01–0.05% B.The boron content for the studied alloys was chosen in a hypoeutectic concentration range for (0.01–1%) B and at the eutectic point for 2% B according to Thermo Calc simulated polythermal cross section of the multicomponent Ti-Al-Mo-V-B phase diagram (for Ti-4Al-3Mo-1V-(0–4%) B nominal composition) a. XRD phβ phase grains from 700 ± 70 μm to 490 ± 40 μm  of 2.1–3.1 μm and width (⊥L) of 0.6–0.7 μm and a low aspect ratio (⊥/L∥L) of 0.2–0.3. In contrast, the morphology of TiB particles in the eutectic 2B alloy was characterized by a near-spherical shape with an aspect ratio (⊥/L∥L) equal to 1 and an average size of 1.0 μm.The microstructure of the alloys studied after thermomechanical treatment was characterized by elongated grains of the 5–0.8 µm , Table 3α and β phases was observed (α phase with HCP structure (including α and transformed β) were 1.0 µm/0.6 µm for 0B and 1.7 µm/1.1 µm for 0.1B alloy.Annealing at 875 °C for 30 min, which led to the microstructure before the onset of superplastic deformation, did not influence on the morphology and fraction of borides. Meanwhile, the microstructure of boron-free and boron-bearing alloys were different . The micobserved a–c. The observed d–f. EBSDobserved . The fraobserved a,b, the observed c,d and aobserved e was domobserved f,g and sobserved h,i were observed j. The mem ~0.5 in a strain rate range of 5 × 10−4 to 5 × 10−3 s−1. Elongation to failure of ~1000% was received at a constant strain rate of 1 × 10−3 s−1 (m shifted to the lower strain rates of (2–4) × 10−4 s−1, and elongation to failure decreased to 500–600%. At a temperature of 700 °C, the deformation was characterized by a lower coefficient of m ~0.4 and elongations of 400–500%. Due to the trace boron addition of 0.01–0.1%, the elongation to failure changed insignificantly at 875 °C and slightly increased at 700–775 °C. Moreover, alloys with minor boron addition exhibited significantly smaller flow stresses at the initial stage of deformation than that of the B-free alloy. The differences in the deformation behavior of the alloys were most pronounced at a temperature of 775 °C. At the initial stage (in a range of ε = 0.1–0.7), the deformation of B-free alloy was accompanied by strain softening but a steady flow was observed for the alloys with 0.01–0.1% B .Alloys with 1 and 2% B were characterized by lower values of the strain rate sensitivity coefficient 2B alloy c. The 1BYS, ultimate tensile strength UTS, and elongation to fracture δ) were determined after deformation at a temperature of 875 °C with a constant strain rate of 1 × 10−3 s−1  for a strain of 0.69 (100% of engineering strain)  . Increasβ grains. The mean grain size decreased more than three times (β phase field [β-phase field, Zener pinning force should be stronger and finer grains are expected. Meanwhile, grains were coarser at a high borides fraction. Thus, the refinement effect could not be explained by the Zener pinning mechanism. Similarly, the effect of segregation of boron at the periphery of β dendrites during their growth in a liquid phase should be stronger with higher B content. The microstructure and boride morphology suggested the same origin of the TiB phase precipitation via eutectic transformation for 0.1% B and 1–2% B. A similar B concentration in eutectic point was observed for Ti-B alloys [β-grains via trace boron additions is required. The possible explanation is as follows. First, considering the widely acceptable constitutional supercooling effect, trace boron addition refines the β-phase dendrites, and the change of the crystal growth type from dendritic to eutectic bicrystal with colonies of two phases eliminates this effect. At a high boron content and a high fraction of β phase of eutectic origin, the effect disappeared due to extra-rapid simultaneous growth of TiB and β colonies. At minor concentrations, boron atoms in the liquid may stimulate the nucleation of the β phase due to a decrease of the liquid–solid interphase energy or a decrease in the critical size of nuclei due to B atomic segregations [β-phase grains are solidified before the eutectic-originated TiB phase [β-phase during solidification of the studied alloy. Even a small fraction of such phases is enough to stimulate inoculation. The formation of the thermodynamically stable borides simplified the high-boron 1B and 2B alloys and no inoculation effect was observed.A trace boron addition, up to 0.1%, provided an effective refinement of the prior ee times . The grase field ,44 or B se field . In addise field  and Ni-Cse field . For these field . Thus, dB alloys  and sligB alloys . The solegations ,38. A seegations . Second,egations , Zr in Megations . This meiB phase . MeanwhiiB phase  as Larsoα/β interfaces, which were recently found in [Trace boron alloying also significantly changed the as-annealed microstructure of the thermomechanical processed alloys. The B-free alloy demonstrated an inhomogeneous structure with a mixture of the elongated and equiaxed grains and a high fraction of low-mobile LAGBs. Alloying with 0.1% B provided a globular microstructure in the alloy. A similar effect was found due to alloying of Ti-Al-Mo-V alloy with both Fe and B . Thus, bfound in , may alsfound in ,37. The found in . The borDue to a high fraction of borides, an increase in B content to 1% increased room temperature strength but weakens superplastic properties and decreased ductility at both elevated and room temperatures. The same influence of boron on the room temperature properties was observed for Ti-6Al-4V alloy . The autThe influence of 0.01–2 wt.% B on the microstructure, superplasticity, and mechanical properties at room temperature of the Ti-4%Al-3%Mo-1%V alloy was studied. The boron effect on the microstructure and properties of the alloys was strongly different for the ranges of 0.01–0.1% B and 1–2% B. The main conclusions are summarized as follows.β grains from ~700 µm for the B-free alloy to ~210 µm for the alloy with 0.1% B. Grain refinement was not shown and the mean grain size was ~670–750 µm for the alloys with 1 and 2% B.The TiB particles of eutectic origin were formed after solidification in the studied B range. In as-cast alloys, the mean size of borides increased from 0.7 to 2.5 µm with an increase in B from 0.01 to 0.1% and insignificantly changed in a range of 0.1–2% B. The volume fraction of the TiB phase increased from ~0.1% at 0.01% B to ~11% at 2% B, which agreed with the Thermo Calc simulation. Trace boron addition significantly reduced the mean size of prior α phase and 0.5–0.8 µm for the β phase. For the B-free and minor B alloys with TiB volume fraction below ~1.1%, the strain rate sensitivity coefficient m was ~0.5, and the elongation to failure was ~500–1000% at temperatures of 775–875 °C and a strain rate of 1 × 10−3 s−1, but B influenced deformation behavior. Due to the facilitation of recrystallization and globularization effects, the 0.01–0.1% B decreased flow stress values at the initial stage of superplastic deformation and provided a stable flow. The effect was significant at the low deformation temperature of 775 °C. Along with this, owing to the stimulation recrystallization effect, a trace B addition decreases post-forming strength properties and insignificantly influenced ductility at room temperature; yield strength decreased from 770 MPa for the B-free alloy to 733 MPa for 0.01% B, and to 680 MPa for 0.1% B. Quenching and aging increased the strength properties by 90–140 MPa and slightly decreased ductility for 0.1% B. The modifying of Ti-Al-Mo-V alloy with 0.01% B provided both uniform and stable flow during superplastic deformation and good mechanical properties at room temperature.After thermomechanical treatment with a final hot rolling, the alloys exhibited similar elongated grains of a mean size in a range of 0.9–1.2 µm for the m of 0.5, elongation to failure of ~500%, room temperature yield strength of 830 MPa, and ultimate strength of 1020 MPa, but low elongation at fracture of 1.1%. An increased boron content with the eutectic concentration of 2 wt.% increased the TiB particles fraction to ~11% and provided the particles with a mean size of ~1 μm after thermomechanical processing. The high-boron eutectic alloy demonstrated a non-superplastic behavior at elevated temperatures and a brittle fracture at room temperature, with a strong decrease in the ultimate tensile strength to 780 MPa.The alloy with 1% B and ~5.2% of TiB whiskers of a mean thickness ~0.7 µm and a mean length of ~3 µm exhibited superplasticity at elevated deformation temperature of 875 °C with


Consequently, the presence of bismuth appears to have a detrimental effect on the machining properties of the alloy when magnesium is present in the composition.The present study comprises an investigation involving thermodynamic analysis, microstructural characterisation, and a comparative examination of the solidification sequence in two different aluminium alloys: EN AW 6026 and EN AW 1370. These alloys were modified through the addition of pure indium and a master alloy consisting of indium and bismuth. The aim of this experiment was to evaluate the potential suitability of indium, either alone or in combination with bismuth, as a substitute for toxic lead in free-machining aluminium alloys. Thermodynamic analysis was carried out using Thermo-Calc TCAL-6 software, supplemented by differential scanning calorimetry (DSC) experiments. The microstructure of these modified alloys was characterised using SEM–EDS analysis. The results provide valuable insights into the formation of different phases and eutectics within the alloys studied. The results represent an important contribution to the development of innovative, lead-free aluminium alloys suitable for machining processes, especially for use in automatic CNC cutting machines. One of the most important findings of this research is the promising suitability of indium as a viable alternative to lead. This potential stems from indium’s ability to avoid interactions with other alloying elements and its tendency to solidify as homogeneously distributed particles with a low melting point. In contrast, the addition of bismuth does not improve the machinability of magnesium-containing aluminium alloys. This is primarily due to their interaction, which leads to the formation of the Mg Conversely, with a higher Si/Mg ratio and a lower magnesium content, more low melting point eutectics are formed, resulting in improved machinability. In one of our previous studies [2Sn, Mg2, and Mg3Bi2 phases, which decrease the ductility of the EN AW 6060 alloy. Bismuth exhibits a higher affinity to magnesium than to silicon, resulting in the formation of the intermetallic α-Mg3Bi2 phase during solidification. This phase manifests as angular or hexagonal particles within the α-Al crystal grains or as a binary eutectic (α-Al + α-Mg3Bi2) with a high melting point [The 6XXX series of aluminium alloys are precipitation-hardening alloys known for their moderate strength, favourable strength-to-density ratio, good ductility, weldability, and corrosion resistance. However, machining these alloys can be challenging due to the tendency to form long, continuous strips or curls that may wrap around the workpiece or cutting tool, causing disruptions in the machining process ,2,3,4. T studies , we alsong point . MT < 350 °C) could potentially improve machinability. We also performed detailed thermodynamic analyses and microstructural characterizations to clarify the solidification sequence and microstructure evolution. Some patented alloys incorporate tin, bismuth, and indium as substitutes for lead in various combinations, with the indium content not exceeding 0.4 wt.% ,22. In oThe studied free-machining alloys were made from the commercially produced alloy EN AW 6026 . These methods were utilized to determine phase equilibria and characteristic solidification temperatures.When the temperature in the furnace reached 750 °C, indium or the Bi–In master alloy was introduced. Temperature control was achieved using a K-type thermocouple, so that a constant casting temperature of about 750 °C was maintained during all casting operations. Before casting, the melt was manually stirred for about 1 min to prevent the settling of indium or BiIn phases, which possess significantly higher densities than the base alloy. The casting process itself was conducted within a steel mould. The chemical composition of the alloys was determined using an ARL 4460 OES Thermo Scientific spectrometer , except for the indium content, which was analysed via X-ray fluorescence spectroscopy (XRF), utilizing the Precious Metals Database. Microstructural analysis was performed using a Thermo Scientific Quattro S field emission scanning electron microscope (FEG SEM) equipped with an EDS SSD UltimThe chemical composition of all synthesized alloys is listed in 3Bi2, which formed experimentally in the alloy EN AW 6026 with bismuth addition and in the alloy EN AW 6060 with tin and bismuth addition [Equilibrium isopleth diagrams and non-equilibrium Scheil–Gulliver solidification models were created using Thermo-Calc software for all synthesised alloys. However, in this article, we present the diagram only for the EN AW 6026-In alloy. These diagrams allow us to identify individual microstructural phases and compare the equilibrium and non-equilibrium states with the actual solidification sequence of the alloy. The Thermo-Calc software calculates these diagrams based on thermodynamic laws, a TCAL-6 database of experimentally determined parameters and the assumption of equilibrium solidification . Table 5addition ,20.15Si24, Mg2Si, β-Si, and Al13Cr4Si4. Due to the altered solubility of the elements in the Al15Si24 and Mg2Si phases, a transformation of the phases occurs at temperatures below 400 °C 4 and Al5Cu2Mg8Si6 from Mg2Si. w 400 °C a, leadin13Cr4Si4, Al15Si24, Al9Fe2Si2, β-Si, Mg2Si, Al5Cu2Mg8Si6, and elemental indium particles. Scheil’s calculation predicts the formation of these phases, with the difference that HCP_A3 is present ) and (α-Al + Al13Fe4), respectively. Small indium particles were detected in some Al13Fe4 phases ). The melting point of the α-Al solid solution was determined to be 671.9 °C.Based on the mass fractions of the individual elements SEM–EDS,  of consttal form . Other e4 phases . Scheil’13Fe4, and bright BiIn phases in the microstructure is evident ). The DSC curve ), (α-Al + Al13Fe4), and α-Al solid solution.The microstructure of the alloy EN AW 1370-BiIn with marked EDS analysis sites is shown in e4 phase  also detSC curve  shows cl15Si2(FeMn)3, Mg2Si, Al5Cu2Mg8Si6, and In. The EDS analysis confirmed that indium solidified in elemental form  eutectic. Additional endothermic peaks were observed at temperatures of 557.4 °C and 575.6 °C, corresponding to the predicted melting of the (α-Al + Mg2Si) and (α-Al + Al15Si2(FeMn)4) eutectics, respectively. The liquidus temperature of the α-Al solid solution was determined to be 646.8 °C.The alloy EN AW 6026 contains magnesium and silicon as the main alloying elements, together with other trace alloying elements such as Fe, Cu, Mn, Cr, Zn, and Ti. 15Si2(FeMn)4, Mg2Si, Al5Cu2Mg8Si6, Mg3Bi2, and indium particles. Morphologically, the Al5Cu2Mg8Si6 phases exhibited similar characteristics to those of the alloy EN AW 6026-In. EDS mapping was carried out to determine the specific distribution of each alloying element, focusing on Mg3Bi2 and In particles. Indium was found not to react with any of the alloying elements. However, in the case of Mg3Bi2 phase particles, an overlap between Bi and Mg was observed  and (α-Al + Mg2Si) eutectics. The most notable peaks corresponded to the melting of the (α-Al + α-Mg3Bi2) and (α-Al + Al15Si2(FeMn)4) eutectics at temperatures of 589.9 °C and 655.2 °C, and the α-Al solid solution melted at a temperature of 663.4 °C.observed . Due to ng curve  did not ng curve  occurredMT < 350 °C), which was found in both alloys as indium particles. This article examines the microstructural and thermodynamic characterisation of laboratory-prepared aluminium alloys EN AW 1370 and EN AW 6026 with the addition of pure indium and an In–Bi master alloy, respectively. The aim of the experiments was the formation of a low-melting phase ) at 641.8 °C and binary (α-Al + Al13Fe4) at 653.8 °C, while the α-Al solid solution exhibited a melting point of 671.9 °C.The microstructure of the alloy EN AW 1370-In consisted of α-Al, Al13Fe4, and a BiIn phase. Thermodynamic calculations suggest the possibility of a ternary eutectic ). On the DSC heating curve, the melting of the BiIn phase was found to occur at 113.7 °C, which is very close to the BiIn phase melting point (109.7 °C) in the equilibrium Bi–In phase diagram. The melting points of the eutectics ) and (α-Al + Al13Fe4) were detected at 646.4 °C and 658.5 °C, respectively, while the α-Al solid solution exhibited a melting point of 683.7 °C.The microstructure of the alloy EN AW 1370-BiIn consisted of α-Al, Al5Cu2Mg8Si6), (α-Al + Mg2Si), and (α-Al + Al15Si2(FeMn)4) binary eutectics as well as particles of elemental indium. Unfortunately, the DSC method could not reliably detect the melting or solidification of indium particles, due to their small number and the low enthalpy of indium. On the DSC heating curve, the initial prominent endothermic peak was seen at 532.4 °C, indicating melting of the (α-Al + Al5Cu2Mg8Si6) eutectic. Other endothermic peaks were observed at temperatures of 557.4 °C and 575.6 °C, indicating the melting of the (α-Al + Mg2Si) and (α-Al + Al15Si2(FeMn)4) eutectics, while the α-Al solid solution exhibited a melting point at 646.8 °C.The microstructure of the alloy EN AW 6026-In exhibited a higher degree of complexity and comprising an α-Al matrix, (α-Al + Al5Cu2Mg8Si6), (α-Al + Mg2Si), (α-Al + Mg3Bi2), and (α-Al + Al15Si2(FeMn)4) binary eutectics; and indium in elemental form. EDS mapping demonstrated that indium formed particles in elemental form and did not interact with other elements, while bismuth reacted with magnesium to form the Mg3Bi2 phase and prevent the formation of the BiIn phase. The DSC heating curve did not exhibit any endothermic peaks indicating the presence of the BiIn phase nor any prominent endothermic peaks corresponding to the melting of the indium particles. Instead, phase changes were observed at temperatures of 536.1 °C, 545.9 °C, 589.9 °C, and 655.2 °C and were attributed to the melting of the following eutectics (α-Al + Al5Cu2Mg8Si6), (α-Al + Mg2Si), (α-Al + α-Mg3Bi2), and (α-Al + Al15Si2(FeMn)4), while the α-Al solid solution melted at 663.4 °C.The microstructure of the alloy EN AW 6026-BiIn consisted of the α-Al matrix; (α-Al + Al


RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3′ untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections. Antisense oligonucleotides (ASOs) are short, single-strand nucleic acid analogues having a length of 14–30-mer that bind target RNAs and modulate their functions in a sequence-dependent manner. ASO-based therapeutic approaches have demonstrated potent efficacy in treating various diseases, including viral infections . Since tASOs primarily function through two mechanisms: RNase H-mediated cleavage/degradation of or steric blocking of target RNAs . ASOs wiGiven the ASO property, antiviral gapmer ASOs have the potential advantages of eliminating viral RNAs from infected cells and being flexibly synthesized into mutant viral RNA sequences. Currently, gapmer ASOs modified with locked nucleic acids (LNA gapmers) hold great promise for a new class of antiviral therapeutics to induce RNase H-mediated viral RNA cleavage a 3,18].,18.3,18]Flavivirus in the family Flaviviridae, including other clinically significant flaviviruses such as dengue virus (DNV), yellow fever virus (YFV), West Nile virus (WNV), and zika virus (ZKV) ) at 37 °C in 5% COhttp://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi accessed on 29 November 2022) [Complete genome sequences of JEV strains belonging to either of five genotypes were obtained from National Center for Biotechnology Information (NCBI) GenBank: SH 53 , FU , JaGAr 01 , GP78 , JKT6468  and Muar strain . JEV RNA secondary structures of 3′ UTR were predicted using the RNAfold program (er 2022) .LNA gapmer 1 targeting two different regions with the same sequence in the 3′ UTR of the JaGAr 01 strain was designed and synthesized by QIAGEN (ID: LG00204874) for a preliminary test. LNA gapmers 2 to 9 were designed based on the sequences and predicted structures of the 3′ UTR of the JaGAr 01 strain, referring to those of other strains . Control5 cells/well in a 24-well plate and incubated at 37 °C in 5% CO2 for 24 h to form approximately 95% confluent cell monolayer. SK-N-SH cells seeded at a density of 1.4 × 105 cells/well in a 24-well plate were grown for 72 h to more than 95% confluent. Cells were infected with JEV at the MOI of 0.1 and 0.05 for in vitro screening in Vero cells and other experiments, respectively, for 1 h with tilting every 15 min. The virus solution was diluted with maintenance medium: EMEM and MEMα containing 2% FBS for Vero and SK-N-SH cells, respectively. LNA gapmer transfection was performed with lipofection with Lipofectamine RNAiMAX . In brief, during the inoculation of the virus, an 80 µL mixture of LNA gapmer from 0.15 to 15 µM and 3 µL Lipofectamine RNAiMAX was prepared in Opti-MEM (Thermo Fisher Scientific). To obtain final concentrations from 0.05 to 5 µM of LNA gapmers to be tested, the LNA mixture was diluted three times with EMEM and MEMα with 5% FBS for Vero and SK-N-SH cells, respectively. After removing the virus solution, cells were transfected with 230 µL of the LNA mixture prepared above for 4 h. Following the transfection, cells were washed once with a medium with 5% FBS and then maintained in a maintenance medium with 2% FBS. The supernatants were collected at 29 and 24 h post-infection (hpi) of JaGAr 01 for plaque assay in a screening test with Vero cells and other experiments, respectively. The supernatants of SK-N-SH cells infected with four other JEV strains were obtained at 48 hpi. Virus titers were expressed as the number of plaque-forming units (PFU) per milliliter. A schematic of the transfection is shown in Vero cells were seeded at a density of 1.0 × 105 cells/well density in GM: EMEM supplemented with 10% FBS and 0.5% antibiotics. Following the removal of GM, over 95% confluent Vero cells were infected with 10-times serial dilutions of viral culture supernatants, which were prepared in the maintenance medium with 2% FBS and 0.5% antibiotics, for 1 h at 37 °C in 5% CO2 tilting every 15 min. Following the infection, cells were washed once with the maintenance medium and then overlaid with 1 mL of a semisolid medium (1% methylcellulose (Wako), 2% FBS, and 0.5% penicillin/streptomycin in EMEM). After incubation for 72 h, 500 µL of 10% formalin neutral buffer solution (Wako) was added to each well to fix cells, and 12-well plates were placed under UV light for 30 min. After the cell fixation, cells were washed with distilled water and stained with 0.5% crystal violet  for the counting of plaque numbers and calculating virus titers as PFU/mL. Vero cells were seeded on 12-well plates at 2.0 × 10E. coli RNase H  were mixed and incubated in 1 × RNase H reaction buffer at 37 °C for 120 min. The 20 µL reaction mixture was collected at 0, 5, 10, 30, 60, and 120 min after the reaction. The collected reaction mixture was immediately quenched by adding 1 µL of 0.5 M EDTA  at pH 8.0 and denatured with an equal amount of 21 µL formamide (Wako). The samples were immediately used for a subsequent procedure or stored at −80 °C until use. They were mixed with an equal volume of NovexTM TBE-Urea sample buffer (Thermo Fisher Scientific), heated at 70 °C for 4 min, and immediately placed on ice. Aliquots having a volume of 20 µL and 12 µL microRNA marker (New England Biolabs) were submitted to a Mini-PROTEAN 15% TBE-urea gel with ten wells  and electrophoresed at 200 V for 30 min. The gel was stained by SYBR® Green II (Thermo Fisher Scientific), and images were captured using the Gel DocTM EZ system (Bio-Rad). The band intensities were quantified using ImageJ software .A target 17-mer JEV RNA sequence complementary to 3′ UTR stem-loop-targeted LNA gapmers was synthesized with HPLC grade by Ajinomoto Bio-Pharma Services GeneDesign, Inc. LNA gapmers (1.5 µM), synthesized JEV RNA (6 µM), and JEV RNA sequences targeted by LNA gapmers or encoding envelope protein  were ampGAPDH mRNA levels were measured using a primer set as previously reported [TM Reagent (Thermo Fisher Scientific) per well and stored at −80 °C until use. Then, RNA was extracted using the RNeasy Mini Kit (QIAGEN). RNA concentrations were quantified using Nano drop (Thermo Fisher Scientific) and normalized to 20 ng/µL. Real-time RT-PCR was performed with the Thermal Cycler Dice® Real Time System III (Takara) using One Step TB Green® PrimeScriptTM PLUS RT-PCR Kit (Takara). The total reaction volume was 25 µL with 12.5 µL of 2 × One Step TB Green RT-PCR Buffer, 0.5 µL of PrimeScript PLUS RTase Mix, 0.4 µM each of the forward and reverse primers, 1.5 µL of TaKaRa Ex Taq HS Mix, and 2 µL of the RNA. The amplification condition was 5 min at 42 °C and then 10 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The expression levels of JEV RNA were normalized to human GAPDH mRNA levels. The relative expression levels of JEV RNA were analyzed using the comparative Ct method. The primer set to amplify LNA gapmer target region on JaGAr 01 strain genomic RNA was designed using Primer 3 Plus. As a reference gene, human reported . To use reported . Cells w5 cells/well on 96-well plates in GM: MEMα containing 10% FBS and cultured for 72 h. For lipofection, a 240 µL mixture of LNA gapmers at the final concentration from 0.05 to 1 µM and 3 µL of Lipofectamine RNAiMAX was prepared with an identical procedure to that described above. Cells were transfected with 40 µL of the transfection mixture for 4 h. After transfection, cells were washed with MEMα containing 5% FBS once and incubated in 100 µL MEMα with 2% FBS for 18 h. After incubation, the CCK-8 assay was conducted according to the manufacturer’s instruction. A quantity of 10 µL of CCK-8 solution was added to each well; 96-well plates were incubated for 2 h. Then, the absorbance was measured using a microplate reader, Multiskan GO (Thermo Fisher Scientific), at 450 nm. The cell viability was calculated and represented by the percentage of the negative control treated with neither LNA gapmers nor Lipofectamine RNAiMAX as 100%.SK-N-SH cells were plated at 0.4 × 10https://gggenome.dbcls.jp/ja/ accessed on 29 November 2022). The number of complementary regions to LNA gapmer 7, 8, and 9 and control LNA gapmer sequences with or without mismatches or gaps in the human genome ) and human RNA (RefSeq human RNA release 210 (Jan 2022)) was analyzed using GGGenome (http://www.viprbrc.org/ accessed on 2 May 2022) and from the four JEV strains determined in this study . They were composed of 136, 3, 146, 4, and 3 strains of the five genotypes I to V, respectively. The conservation of the JEV RNA sequences complementary to the LNA gapmers was analyzed using Clustal Omega .A total of 292 JEV RNA sequences that LNA gapmers 7, 8, and 9 target were collected using ViPR . In RNA cleavage assay and cytotoxic assay, the Shirley−Williams test was performed using Statcel-the Useful Addin Forms on Excel-4th ed . In quantitative real-time RT-PCR, delta Ct values were compared using Student’s t-test and one-way ANOVA followed by a post hoc Tukey–Kramer multiple comparison test in GraphPad Prism 8. The


S. pombe telomerase holoenzyme by the LARP7 family protein Pof8, where it acts in a catalytic-independent manner to protect the telomerase RNA and facilitate holoenzyme assembly. Here, we show that Bmc1 and Pof8 are required for the formation of a distinct U6 snRNP that promotes 2′-O-methylation of U6, and identify a non-canonical snoRNA that guides this methylation. We also show that the 5′ γ-monomethyl phosphate capping activity of Bmc1 is not required for its role in promoting snoRNA guided 2′-O-methylation, and that this role relies on different regions of Pof8 from those required for Pof8 function in telomerase. Our results are consistent with a novel role for Bmc1/MePCE family members in stimulating 2′-O-methylation and a more general role for Bmc1 and Pof8 in guiding noncoding RNP assembly beyond the telomerase RNP.Splicing requires the tight coordination of dynamic spliceosomal RNAs and proteins. U6 is the only spliceosomal RNA transcribed by RNA Polymerase III and undergoes an extensive maturation process. In humans and fission yeast, this includes addition of a 5′ γ-monomethyl phosphate cap by members of the Bin3/MePCE family as well as snoRNA guided 2′-O-methylation. Previously, we have shown that the Bin3/MePCE homolog Bmc1 is recruited to the   Pre-mRNA splicing, comprised of intron excision and subsequent exon ligation, relies on dynamic RNA-RNA and RNA-protein interactions in the spliceosome (reviewed in ). The spSchizosaccharomyces pombe, transcription of U6 by RNAP III is associated with the addition of a 5′ γ-monomethyl phosphate cap catalyzed by enzymes of the Bin3/MePCE (methyl phosphate capping enzyme) family   family . U6 cont) family , as well) family . U6 also) family ,13. More) family . Most in) family ,20, seveS. pombe LARP7 protein Pof8 associates with the Bin3/MePCE homolog Bmc1 and a fission yeast-specific protein, the telomerase holoenzyme component 1 (Thc1)  ,34, whic1 (Thc1) ,36. We h1 (Thc1) ,20,34. TS. pombe is linked to particular intron features including strength of 5′ splice site sequences. Together, these data point towards an intricate network of post-transcriptional processing events that are critical for normal U6 biogenesis, and provide the first direct evidence for a function of the Bin3/MePCE family in promoting U6 snRNP maturation.In this work, we set out to examine the role of Bmc1 in U6 biogenesis and spliceosome function. We describe a new RNP containing the U6 snRNA and the telomerase components Bmc1, Pof8 and Thc1, and show that this complex is required for wild type levels of 2′-O-methylation in the U6 ISL and U6 snRNP assembly. We also show that while Bmc1’s 5′ capping catalytic activity is not required for its function in promoting 2′-O-methylation of U6, an intact Pof8-Lsm2-8 interaction is. Finally, we show that Bmc1 deletion influences the splicing of some introns under wild-type and heat-shock conditions, consistent with previous work linking human LARP7 to splicing robustness. Using our transcriptome-wide pre-mRNA splicing data set, we also show that increased intron retention upon heat shock in Strains were grown at 32°C in yeast extract with supplements (YES) or Edinburgh Minimal Media (EMM), as indicated. Tag integration and knockouts were generated as described in  primer . A list Native protein extractions and immunoprecipitations were carried out as described in . Protein32P γ-ATP-labeled DNA probes for northern blotting are provided in 32P γ-ATP-labeled probe, 50 mM Tris–HCl pH 7.4, 60 mM NaCl, then transferred to 55°C for 20 min to allow the probe to anneal. Reverse transcription was carried out with 1.5 mM (high concentration) or 0.1 mM (limiting concentration) dNTP mix and 2.5 U AMV-RT  and 1 h incubation at 42°C. cDNA products were separated on 8% TBE–urea sequencing gels, dried, and exposed to Phosphor screens overnight. Relative 2′-O-methylation was calculated by determining the ratio of each RT stop relative to the total signal in each lane . 2′-O-methylations were also detected by RNase H  digestion of 2 μg with 25 pmol chimeric RNA-DNA probes, as per  as per . For nat1 μg TURBO DNase-treated RNA was reverse transcribed with the iScript cDNA reverse transcription kit  or 5 U AMV-RT  and gene-specific reverse primers. qRT-PCR was performed with the SensiFAST SYBR No-Rox kit  and 1 μM of each primer, with settings outlined in . For sem2, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail ), frozen in liquid nitrogen, and ground to fine powder with a mortar and pestle. Powder was thawed on ice and spun in a JA 25.50 rotor (Beckman) for 16 min at 15000 rpm and the supernatant was subsequently spun in a 70.1 Ti rotor (Beckman) for 45 min at 50 000 rpm to pellet ribosomes and heavy molecular weight complexes. Supernatants were flash frozen and stored at −80°C. For native snRNP gels, glycerol with xylene cyanol and bromophenol blue was added to 30 μg cell extract  and fractionated on 4% 19:1 acrylamide: bis-acrylamide native gels (15 cm × 18 cm) for 220 min at 240 V and 4°C, then transferred to nylon membranes for northern blotting. For glycerol gradients, cell extracts from 1.0 g frozen cell powder were layered on an 11 ml 10–30% glycerol gradient  and spun in an SW41Ti rotor (Beckman) for 20 hours at 30 900 rpm. Fractions were collected starting from the top of the gradient and RNA and proteins were extracted with phenol: chloroform: isoamyl alcohol (25:24:1) and TCA precipitation, respectively.Pellets from 1 L yeast cultures were resuspended to 1 g/ml in AGK400 buffer  was heated and cooled from 50°C to 65°C at a rate of 2°C per minute without collecting data, then re-heated and cooled while monitoring absorbance at 260 nm at 1°C intervals. Absorbance at 260 nm at each temperature point was normalized to absorbance at 50°C and absorbance curves were fitted with an equation for one site specific binding with a Hill slope to determine Tm values. RNA sequences are provided in UV melt curves were recorded on a Cary BIO 100 spectrometer with a 6 × 6 temperature-controlled cell holder. 2 μl 10 mM U4 and modified or unmodified U6 RNA oligos in 96 μl buffer  and stranded libraries were prepared by Genome Québec. cDNA libraries were sequenced on a NovaSeq6000 with 150 bp paired-end reads. Reads were aligned to the fission yeast genome (ASM294v2) with Bowtie2 . Intron s v2.3.0  and sequs v2.3.0 . Intron s v2.3.0 .S. pombe and ciliates . We. We20). ciliates .As an additional means to confirm U6 snRNP formation, we fractionated native cell extracts on glycerol gradients and compared protein and RNA sedimentation in wild type and knockout yeast strains Figure . A substTogether, these data point towards the existence of a new U6-containing complex that also shares components with the telomerase holoenzyme, providing a surprising link between two seemingly disparate fission yeast noncoding RNA pathways.To gain further insight into the role of the Bmc1-containing U6 snRNP, we examined our Bmc1 RIP-Seq dataset , which rInitial attempts at identification of the U6 A64-methylating snoRNA using box C/D snoRNA consensus sequences and base pairing rules  yielded We then tested how disruption of the Bmc1-containing U6 snRNP might impact spliceosome assembly by fractionating cell extracts in a glycerol gradient and assaying protein and snRNA distribution. In wild-type cells, we noted a lowly abundant, lighter sedimenting complement of U6 that does not appear to co-sediment with U4 . We observed an increase in intron retention following heat shock, and confirmed their further impaired splicing in the context of the Bmc1 deletion  Figure . We notes Figure , in lines Figure ,. Additis Figure .This work represents the first report of an MePCE homolog with a role in splicing and U6 snRNP assembly, beyond 5′ methyl phosphate cap addition of U6. In our efforts to investigate functions for Bmc1 beyond telomerase, we revealed an unanticipated overlap between components of the yeast telomerase holoenzyme and a U6-containing snRNP. While it is surprising that Bmc1, Pof8, Thc1, and Lsm2-8 interact with 2 very distinct non-coding RNAs produced by different polymerases, both RNAs possess uridylate-rich sequences recognized by Lsm2-8 and highly structured regions, including stem loops in U6 and pseudoknots in telomerase, that act as scaffolds to recruit other RNP components. These common features may provide an explanation as to why these divergent RNAs share a common set of protein binding partners. Such RNP plasticity is not unique to fission yeast telomerase and U6, but may represent a shared feature of LARP7 and MePCE family proteins. Mammalian LARP7 and MePCE are particularly well-studied for their roles in capping and stabilizing the 7SK snRNP ,68,69, tS. cerevisiae, lack both a LARP7 and MePCE homolog . While atalysis . It remagkad563_Supplemental_FilesClick here for additional data file.