The initial introduction of utilizing double helix structural oligonucleotides known as SNP typing with excellent specificity (STexS) in a standard PCR greatly improved the detection of single nucleotide polymorphisms (SNP) by enhancing amplification rates of primer-matching strands and interrupting mismatched strands by constant instability of kinetics regarding alignment attaching and detaching. The model was beneficial overall in detecting SNP variants consisting of large amounts of wildtype strands such as EGFR mutation genotyping for early detection of non-small cell lung cancer. While the STexS PCR is advantageous in detecting SNPs and biomarkers, limitations were yet observed. Despite the ability to detect variants 10 times more effective than a typical amplification-refractory mutation system PCR, it could only perform optimally in DNA concentrations around 101 ~ 105. To further enhance STexS specificity to perform detecting viral-RNA variants such as the infamous SARS-CoV-2, a novel improvement of the regular TaqMan Probe using Com-probes to inhibit high copy wild targets and amplify low copy mutant targets. By introducing the novel STexS II, omicron variants of SARS-CoV-2 were able to be successfully detected in high concentrations of normal genes. Currently, the dominant concept in locating SNP is through PCR. Amplification of highly specific primers matching to certain mutated genomic regions results in time-efficient and cost-effective results that are relatively reliable3. To further increase specificity, various implements are added to the PCR process. As the basis of PCR is separated into three steps; denaturation, annealing, and extension, improvements were observed in several aspects4. Supplements such as probes are designed to give fluorescent signals during annealing, enabling comparative quantitative analysis that led to qPCR5. Other approaches focused on customizing primer sequences to obtain highly specific matches and increasing the annealing temperature to block potential dimers6. The recently introduced SNP typing with excellent specificity (STexS) model also focuses on the annealing process. By implementing double helix structured oligonucleotides within the primer, small portions of matched primers were successfully discriminated from the abundant mismatched templates7. All the methods mentioned above eventually benefit the overall PCR process to detect variants.Detection of genetic alterations such as SNPs is crucial in tackling various genetic disorders and cancers. As research revealed genetic disorders and various cancers can be triggered by a single nucleotide mutation, the importance of distinguishing such diseases through methods of genotyping continues8. To make matters worse, mutated variants of SARS-CoV-2 continue to derail the overall effort to effectively diagnose and suppress9. The Coronaviridae is an RNA virus replicating by reverse transcription followed by DNA replication and transcription. The extended replication process enables the virus to have more alterations of the original template which then can form characteristics not seen in previous infections9. This gradually led to distinct variants known as Alpha, Beta, Gamma, Delta, and Omicron. While the other four subtypes have been relatively subdued through various types of vaccines, the Omicron variant continues to spread among the population. Reports indicate SARS-CoV-2 can be detected via RT-PCR 2 to 3 days prior to symptoms being observed10. Further reports show the virus to have a high transmission rate within 1 ~ 2 week after the initial exposure11. To cause the SARS-CoV-2 to further diminish, effective detection methods that can discriminate infected strands within yet inflicted strands are essential.While the STexS platform was able to perform outstandingly in a genomic environment, limitations were soon to be seen when implemented in a viral circumstance. Among the varieties of the virus, SARS-CoV-2 has been the dominant threat to public health. The ability to disseminate worldwide and inflict high casualties during a short period is concerningTo achieve such goals, this study focused on developing the previous STexS method to effectively detect and amplify mutated regions of SARS-CoV-2 while simultaneously diminishing unnecessary strands by inhibiting signal emission to increase specificity. As a result, we introduce an enhanced method utilizing a novel probe mixture named Combination and Competition probes (Com probes).1 to 108 copies. The typical ARMS PCR was able to detect variants within the 101–103 range. STexS was able to improve the detection range to 101–105. Despite the enhanced specificity, viral variants could be seen in far concentrated amounts that exceed the current detection limit. To overcome the challenge and increase specificity, methods such as TaqMan and minor groove binder (MGB) included TaqMan probes were introduced for distinct purposes12. While the original Taqman probe focused on normalizing and quantifying relatively small concentrations around 101–105, MGB included TaqMan probes that tend to perform optimally in high concentrations around 104–108. To detect variants of RNA-based viruses such as SARS-CoV-2, the spectrum must be broadened that envelops small and high concentrations13. As a result, the concept of STexS II was based on novel TaqMan probe enhancements  to be above the lowest annealing temperature to rule out unnecessary dimersase Fig. . Further14. This condenses the PCR workflow to quantify several targets simultaneously. The multiplex method of probes was possible as probes were designed to react independently with no interference within each group15. While this is the main approach for using multiplex probes, the STexS II Com probes were implemented to interfere with and block nonspecific signals, gradually increasing the gap between mutated and nonspecific signals. Each Com probe was designed to attach wildtype regions that parallel to the mutated sequence (Table 2) and high (108) concentrations  and the Korean National Culture Collection for Pathogens (NCCP). Both institutes are government affiliations that confirm integrity and regulations.Each primer was designed and manufactured to match the omicron variant mutation location Q954H and T547K. Probes were designed and manufactured to match above mentioned primers. Probe elongation was performed by adding one base to the 3' end. A Series of probes elongation was tested for optimal performance and Tm adjustments. Com probes were designed to not emit any fluorescent color only to act as a competitive combination in binding nonspecific probes. The sequence of Com probes was designed to be one base different from the main probe.Each qPCR was performed consisting of forward and reverse primers matching the Q954H and T547K region SARS-CoV-2. For quantitative measures, the main hydrolyzable fluorescent probes were attached with a FAM or JOE fluorescence in the 5' end for Fluorescence Resonance Energy Transfer (FRET) and a BHQ1 quencher in the 3' end. Com probes were added an additional phosphate in the 3' end to inhibit DNA polymerases to attach and amplify. The buffer solution of each PCR was calculated to total 20 μL using Enzynomics™ solution (cat no. RT 431M). The PCR was performed within 45 ~ 50 cycles after a 10 min reaction in 95 °C, with each cycle consisting of 10 ~ 15 s in 95 °C and 10 ~ 15 s in 60 °C in the CFX9600 Real-Time System (Biorad™). Omicron variant standard samples were performed with the same PCR platform.RNA samples of omicron variants of SARS-CoV-2 were obtained publicly through the National Culture Collection for Pathogens (NCCP). Three sample collections NCCP43326, NCCP43410, and NCCP43408 were stored for further STexS II trials.Supplementary Information 1.Supplementary Information 2.Supplementary Legends.


EVA was designed to study various speech production parameters.Objective: This paper aims to define the mean values for electroglottography tests of Brazilian Portuguese speakers on EVA.Materials and Method: The voices of 20 men and 20 women without voice-related complaints were analyzed through electroglottography so as to obtain reference values for normality. Case study: this is a descriptive cross-sectional study.Results: The mean values for normal male voices were: F0 = 127.77 Hz; F0 coefficient of variation = 2.51%; absolute jitter = 1.707 Hz; relative average perturbation = 0.0083; jitter factor = 1.34%; jitter ratio = 13.45%; QF = 0.447. The values for female voices were: F0 = 204.87 Hz; F0 coefficient of variation = 1.58%; absolute jitter = 3.30Hz; relative average perturbation = 0.0102; jitter factor = 1.60%; jitter ratio = 16.23%; QF = 0.443. Wave type for the entire sample was categorized as tilted pulse.Conclusion: Statistically significant differences were found for gender on parameters average F0 and absolute jitter. While using acoustic analysis software, users must be based on parameters inherent to the software program when analyzing the collected data. Voice assessment in speech therapy can be performed through auditory perceptual analysis, considered the gold standard in speech therapy, or through acoustic analysis, a set of measurements performed from computer-generated tracingsAcoustic analysis added objectivity to speech assessment. Additionally, it allowed increased diagnostic accuracy, the identification and documentation of short and long term therapy results, and the possibility of providing patients with visual feedbackElectroglottography (EGG) is a non-invasive test that estimates the contact area variation between vocal folds as voice is producedA center in France dedicated to studying speech and language developed a multiparameter method for objective assisted voice assessment (EVA) that uses the SESANE data processor. EVA was designed to study parameters in speech production such as sound, intensity, aerodynamic measurements, to name a few. It is equipped with a series of sensors to measure these parameters, and thus offers improved diagnostic capabilities and enhanced patient follow-up in terms of surgery, drug therapy, and phototherapy outcomeAcoustic analysis software for speech and voice differ in the way they calculate acoustic parameters, and the outcome of the measurements may be affected by linguistic variations stemming from language cultural patternsThere are no studies in the literature describing the use of EVA-based electroglottography in Brazilian Portuguese speakers.absolute jitter, relative average perturbation (RAP), jitter ratio, jitter factor, mean closed quotient (CQ), and the interpretation of the electroglottography wave types of the EGG/EVA software, so as to gather preliminary data on normal patterns of speakers of Brazilian Portuguese of both genders.The purpose of this study is to analyze mean values for fundamental frequency (F0), F0 coefficient of variation, This is a descriptive cross-sectional study. Forty native speakers of Brazilian Portuguese - 20 males and 20 females - aged between 18 and 45 years were enrolled. The selected age range aimed at excluding individuals experiencing changes in their voices and presbyphonia. The mean age of female subjects was 28 years; male patients had a mean age of 30 years.None of the subjects had voice-related complaints. Auditory perceptual analysis performed by two speech and hearing therapists did not show altered voice quality or any other communication disorder that could prevent them from performing the tests.Enrolled subjects were informed of the purpose, procedures, and publication of the test results in this study, and signed an informed consent form. This study was approved by the Research Ethics Committee of our institution and was granted permit ETIC 0488.0.203.000-10.“Mara lava a batata” twice in a row. The second utterance of the phrase was used for data analysis purposes due to its increased acoustic stability and the utterance of vowel/a/in syllable/la/, as it is assumed that there is lesser influence from the vocal tract, given that the tonic syllable is located in the more central portion of the phrase.All subjects had their voices recorded in acoustic signals and electroglottography and were asked to say the phrase Acoustic analysis software EVA was used to record and analyze speech samples. Recordings were done using a Dell Vostro 200 workstation and a professional -44 dBV AKG Acoustics C1000S condenser stereo multidirectional microphone. Two electrodes were placed on the wings of the thyroid cartilage and the informant was kept at a fixed 10 cm from the microphone to allow for proper capturing of the electroglottography signal.absolute jitter, relative average perturbation (RAP), jitter ratio, jitter factor, and closed quotient (CQ) , F0 coefficient of variation, ent (CQ) . The sofMean F0 offers a general measurement of vocal frequency which corresponds to the number of sound waves comprised within one second. The unit of measurement is Hertz (Hz).F0 coefficient of variation is the relative standard deviation compared against the mean F0. This measurement accounts for the magnitude of percent changes in comparison to the mean F0 value. For example, a standard deviation of 4.9 Hz for a mean F0 of 180 Hz results in a coefficient of variation of 2.7%. The same standard deviation for a mean F0 of 500 Hz provides for a much more significant coefficient of variation of 0.98%. The F0 coefficient of variation is the best indicator to explore the stability of the mean fundamental frequency duration, and is highly relevant in the detection of alterations such as tremor and other instabilities of neurological origin. It is measured as a percentage (%).absolute jitter) of the F0 results shows the changes in frequency between each oscillation cycle. It is calculated using absolute mean jitter and the mean F0 difference between two consecutive vibration cycles. These alterations can be accurately calculated for each cycle. It is measured in Hertz (Hz).Short term instability (RAP) measures the mean variation in three consecutive periods and denotes the mean period of the observed signal. This variable has no unit of measurement.Relative average perturbation .Jitter ratio measures the mean variation seen in a period between two consecutive vibration cycles. A high jitter ratio always signifies a relevant F0 coefficient of variation, although the opposite is not true. Indeed, the small upward or downward variations on F0 between cycles does not produce a relevant jitter ratio, but may lead to significant global F0 variations, such as vibrato. The unit of measurement is permillage (%).The closed quotient measures the ratio between the time closed (Tc) and the complete glottal cycle (Tc + To): CQ = Tc/(Tc + To). It is expressed as a percentage (%) .Figure 1The EVA software manual states that CQ normal values, based on French speakers, range between 0.4 and 0.6. Values between 0 and 0.4 suggest glottal hypoadduction and values greater than 0.6 and smaller than 1.0 suggest glottal hyperadduction.1.Pulse widening: occurs when the free border shifts uniformly towards the midline;2.Peak skewing: occurs when there is increased glottal convergence, i.e., when a vocal fold is more acutely angled and wedged;3.Bulging pulse: occurs when two knees are seen in the tracing, one going up and another going down;4.Sloping pulse: occurs when there is a slight difference in the phase angles between and upper and lower margins of the vocal fold free borders, changing the waveform to a more quadrangular or triangular shape when the angle difference between upper and lower margins is greaterIn addition to closed quotient, electroglottographic waves were qualitatively analyzed, categorized, interpreted according to waveform characteristics, and related to templates of glottal geometric variationData statistical analysis was carried out using statistical package SPSS  release 17.0. Initially, a descriptive analysis of the data was performed looking at central tendency and scatter measurements. The data followed a normal distribution. Therefore, the statistical analysis of the values between genders was done using Student's t-test with a confidence level of 95%.absolute jitter measurements.There is statistically significant difference between genders for mean F0 and peak skewing wave types.In the analysis of electroglottographic wave type according to TitzeElectroglottography looks into the contact pattern of vocal folds during the glottal cycle to assess vocal functionHuman tissues conduct electricity reasonably well when compared to airWhen vocal folds touch there is some flow of electricity, and as they move away from each other flow is significantly reduced. Only a small portion of the flow of electricity recorded shows the contact between vocal foldsThe resulting electroglottogram (EGG) shows the variation of vocal fold impedance as a function of time. Impedance also varies considerably with skin type and vertical laryngeal motion. High-pass filters are used to eliminate low frequency interference and remove the variation caused by vocal fold vibrationshimmer, frequency perturbation, jitter, and closed quotientVarious objective measurements may be gathered from the analysis of electroglottograms. Parameters such as vibration fundamental frequency, amplitude perturbation, Altuzarra & San MartinElectroglottogram tracings can be interpreted in many different ways. One may consider the configuration of the tracing curves, their amplitude, cycle periodicity, and the presence or absence of kneesElectroglottographic studiesjitter factors similar to those reported in this study  and female (CQ = 0.443) subjects enrolled in this study  when comIt is worthwhile mentioning that the statistically significant difference observed between male and female groups  for meanThe electroglottograms of all patients enrolled in this study had skewing peaks according to the categorization proposed by Titzeabsolute jitter, relative average perturbation (RAP), jitter ratio, and jitter factor.The parameters considered to assess normality among French speakers are F0 coefficient of variation, The values mentioned above  do not mMore electroglottographic research using different software programs and looking into other languages is needed to allow for a better understanding of these variables and, consequently, to improve the analyses of these values in subjects with speech and laryngeal disorders. Utter standardization is not possible, as there will always be differences between software programs for speech acoustic analysis. Therefore, when using a software pro gram for acoustic analysis, users must use as reference the parameters inherent to the program they are using to analyze the collected data samples.absolute jitter = 1.707 Hz; relative average perturbation (RAP) = 0.0083; jitter factor= 1.34%; jitter ratio = 13.45%; closed quotient (CQ) = 0.447. Female subjects - F0 = 204.87 Hz; F0 coefficient of variation = 1.58%; absolute jitter = 3.30 Hz; relative average perturbation (RAP) = 0.0102; jitter factor = 1.60%; jitter ratio = 16.23%; closed quotient (CQ) = 0.443.The mean reference values for normality found in this study for Brazilian Portuguese speakers without voice- related complaints were: male subjects - F0 = 127.77 Hz; F0 coefficient of variation = 2.51%; absolute jitter.The electroglottographic parameters that presented gender statistically significant differences were mean F0 and Peak skewing waveform was found in the electroglottograms of 100% of the subject sample of both genders.


Drosophila melanogaster. Our approach leveraged whole-fly isotopic labeling to delineate female proteins from transferred male ejaculate proteins. Our results revealed several characteristics that distinguish the FRT fluid proteome from the FRT tissue proteome: (1) the fluid proteome is encoded by genes with higher overall levels of FRT gene expression and tissue specificity, including many genes with enriched expression in the fat body, (2) fluid-biased proteins are enriched for metabolic functions, and (3) the fluid exhibits pronounced postmating compositional changes. The dynamic mating-induced proteomic changes in the FRT fluid inform our understanding of secretory mechanisms of the FRT, serve as a foundation for establishing female contributions to the ejaculate–female interactions that regulate fertility, and highlight the importance of applying proteomic approaches to characterize the composition and dynamics of the FRT environment.Fertility depends on the progression of complex and coordinated postmating processes within the extracellular environment of the female reproductive tract (FRT). Molecular interactions between ejaculate and FRT proteins regulate many of these processes, including sperm motility, migration, storage, and modification, along with concurrent changes in the female. Although extensive progress has been made in the proteomic characterization of the male-derived components of sperm and seminal fluid, investigations into the FRT have remained more limited. To achieve a comparable level of knowledge regarding female-derived proteins that comprise the reproductive environment, we utilized semiquantitative MS-based proteomics to study the composition of the FRT tissue and, separately, the luminal fluid, before and after mating in   •Drosophila FRT tissue and luminal fluid proteomes were characterized.•Whole-fly isotopic labeling was used to identify only female proteins.•Postmating responses were significantly enriched within the fluid proteome.•The fluid proteome exhibited tissue-specific expression, especially the fat body.•Fluid proteome dynamics provided insights into FRT secretory mechanisms. Fertility depends on coordinated postmating processes within the female reproductive tract (FRT), including ejaculate–FRT protein interactions that regulate sperm motility, storage, and modification. Semiquantitative proteomics was utilized to characterize the FRT tissue and, separately, luminal fluid, before and after mating. The dynamic mating-induced changes in the FRT fluid inform our understanding of FRT secretory mechanisms and ejaculate–female interactions that regulate fertility. Our study highlights the utility of applying proteomic approaches to characterize the extracellular FRT environment. For example, interactions among sperm, seminal fluid, FRT membranes, and the luminal FRT fluid influence sperm storage and survival, including postejaculatory modifications to sperm , 4, 5, 6 insects , 27, 28  insects , 31, 32) insects , 36, 37, insects , 39, and insects ). Althouin vitro associations between oviductal fluid proteins and sperm   are also likely to contribute, as found in the male reproductive tract (Apis mellifera), spermathecal fluid proteins were found to be enriched for glycolysis and antioxidant defense (Atta colombica), spermathecal fluid proteins were enriched for proteolytic and oxidation-reductase activity (Drosophila species (simulans and mauritiana), in which conspecific sperm precedence contributes to reproductive barriers  . Althougve tract . Proteomve tract , 64, 65,ve tract , 67. In  defense  and are  defense . In addi defense . In the activity . Althougactivity , 71. In barriers , 73, idebarriers . Togethebarriers , 24.Drosophila melanogaster, the FRT fluid has not yet been characterized. However, several lines of evidence support the involvement of the FRT fluid in postcopulatory processes. First, studies of mutant lines lacking the primary FRT glandular tissues, the spermathecae and the parovaria, revealed that these tissues are necessary for proper sperm storage  is complex and enriched for metabolic proteins, (2) is encoded by genes with more restricted, tissue-enriched patterns of expression, particularly in the FRT-associated fat body, and (3) experiences widespread postmating compositional changes relative to the FRT tissue. These novel insights regarding the dynamic changes of the extracellular FRT fluid provide the basis for future studies of specific ejaculate–female interactions that contribute to postmating processes essential to fertility and potentially influence competitive fertilization success and the maintenance of species boundaries.Here, we characterize the proteome of the D. melanogaster WT LHM strain was maintained at RT (∼23 °C), with a natural light cycle on a standard yeast, cornmeal, agar, and molasses media. Experimental females were collected within 14 h of eclosion and matured in vials of 10 to 15 flies with media supplemented with live yeast for 3 to 8 days. For mated samples, 15 to 20 males were added to a vial of females, and dissections were conducted 6 h after mating . This time point was selected because that is the time of maximal postmating transcriptomic response  were dissected from etherized females in 1× PBS. Dissections were conducted to prevent any contamination from ovulated eggs or nearby gut tissue. For postmating samples, successful mating was visually confirmed by the presence of sperm in the FRT. FRTs from at least 150 females per replicate were dissected, rinsed in another PBS drop, and transferred into a 1.5-ml Eppendorf tube with 50-μl PBS. FRT fluid was isolated from the FRT tissue using an adaptation of methods used to collect FRT contents to identify the ejaculate proteins transferred during mating  ion m/z values were measured via the mass spectrometer . Data-dependent MS/MS scans  isolated and fragmented precursor ions by collision-induced dissociation  and analyzed  in the linear ion trap within a 60 s ± 10 ppm dynamic exclusion window .Proteomic analyses were conducted by the Cambridge Centre for Proteomics following standard protocols. The protein samples were quantified with an EZQ Protein Quantitation kit (Thermo Fisher Scientific), as per the manufacturer's instructions. For each sample, 15 μg of protein was separated by size on a 1.5-mm 12% SDS-PAGE gel stained with colloidal Coomassie dye and divided into ten slices . The FRTi.e., female-derived) proteins was based upon an analysis using the D. melanogaster reference genome protein annotation (r6.32) (thegpm.org). We note that restricting the search database to the longest isoforms precludes the evaluation of alternative splicing. Search parameters allowed for semispecific digestion with three missed tryptic cleavages as well as parent monoisotopic mass error of 15.0 ppm and fragment ion mass tolerance of 0.5 Da. Post-translational modifications included carbamidomethylation , oxidation , and deamidation . The samples contained a total of 2.14 million spectra resulting in the identification of 876,082 peptide–spectrum matches (PSMs). PSMs were included if their −10logP de novo identified score via the ProteomeXchange Consortium .The resulting peptide mass spectra were identified with PEAKS Studio X (Bioinformatics Solutions Inc.). Identification of unlabeled ( (r6.32) , includiapproach ) had a Pproteins . MS datap-value) were converted to p-values and corrected for multiple comparisons with the Dunn–Bonferroni correction. Proteins were considered differentially abundant if the adjusted p-value was Differential abundance analyses were conducted using the PEAKS quantitation software allowing for comparisons of abundance estimates to be fine-tuned on the direct comparison of the spectra , 87. Speas ≤0.05 .Saccharomyces cerevisiae strain was produced from the diploid yol058W/yol058W (BY4743 yeast deletion collection strain background SGD: S000005419), which is a diploid homozygous Arg deletant strain (arg1/arg1) that is also heterozygous for the Lys mutant (LYS2/lys2) (13C615N2) and arginine (13C615N4) (Cambridge Isotope Laboratories). At saturation (A600 0.7–0.8), yeast was washed twice with sterile water, pelleted, and stored at −20 °C.A double auxotrophic (Lys and Arg) mutant S2/lys2) . To obtaS2/lys2) . A 5-μl D. melanogaster embryos (fertilized eggs before hatching) were collected, washed to remove any yeast, and transferred  to vials with 4 ml heavy-yeast media  on top of a 4-ml layer of 2.5% agar to prevent media from drying out. The media were further supplemented with a small pellet of labeled yeast. Within 14 h of eclosion, flies were collected, sexed, and transferred to a new vial with heavy-labeled yeast and agar media. Males were aged at least 5 days and had mated at least once before this experiment.g for 3 min to remove any insoluble material. The samples were prepared for MS analysis as described above. Each sample was trypsin-digested, reconstituted in 0.5% formic acid, and analyzed on an Orbitrap Fusion Tribrid (Thermo Fisher Scientific) mass spectrometer with a Nanospray Flex Ion Source coupled with a Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific). Peptide filtering, elution, and separation with equivalent columns and settings as described above. The Orbitrap Fusion was operated in a positive ion mode (spray voltage 1.6 kV and source temperature 275 C) with data-dependent acquisition analysis  and a 50 s ± 10 ppm dynamic exclusion window using Xcalibur 2.0 operation software (Thermo Fisher Scientific).Labeling efficiency was determined using whole-fly samples from five females and five males. Flies were flash-frozen and solubilized in 2× Laemmli buffer with the TCEP reducing agent with alternating cycles of homogenization with a pellet pestle and heating at 95 °C. The samples were then centrifuged at 17,5006 and 15N4), Lys (13C6 and 15N2), and Arg conversion to proline (R to P [13C5] and R to P [13C5-15N1]). PSMs were included if they achieved a −10logP i.e., no detectable gene expression in the FRT .The spectra were then analyzed using PEAKS studio X (Bioinformatics Solutions Inc.) with settings for unlabeled protein identification as described above with the addition of variable modifications for SILAC Arg (13C labeled . We note the FRT ) sperm pp value cutoffs after correction for multiple testing  yeast supplemented with both heavy Arg and Lys isotopes, thus significantly improving the likelihood that all male peptides were distinguishable by labeled amino acids after tryptic digestion. In addition, independent MS/MS experiments were conducted to confirm the efficiency of heavy labeling .In total, we analyzed four biological replicates of the FRT tissue and fluid . This design provided robust replication (n = 4) for distinguishing proteomic differences between the tissue and fluid proteomes, as well as sufficient replication  for quantitative analyses of postmating proteomic changes. Robust thresholds were applied for both peptide and protein identification, including the establishment of FDRs estimated using a decoy-fusion approach . Accurac testing , 87. We p-value was <0.01. Functional enrichment analyses for proteins identified in the FRT tissues or fluid were conducted using the 8337 genes of the FRT transcriptome  using high confidence (>0.7) support for protein interactions and functional enrichment of proteins within these networks . We used a nonparametric Kruskal–Wallis test to compare the differences in the mean protein abundance or changes in protein abundance. Variance in postmating log2 fold change was analyzed with a Levene's test. All remaining data visualizations were produced with ggplot2 . The four tissue and four fluid replicates, respectively, were highly quantitatively reproducible  and formed discrete clusters based on compositional similarity (C), further supporting the distinct nature of these samples. In the following analyses, we investigate the proteomic differences between the FRT tissue and FRT fluid samples.We characterized the FRT tissue and fluid proteomes of unmated and mated females after isolating the fluid contents of the FRT lumen. Females were mated to heavy-labeled males so that postmating changes in FRT protein abundance could be distinguished from male-derived proteins transferred in the ejaculate. The distinct characteristics of the FRT tissue and fluid proteomes were initially apparent in their 1D SDS-PAGE banding patterns, which indicated that their protein composition was substantially different , A and Bmilarity C, furtheversus 756 proteins in the fluid). Consistent with the expectation that the fluid proteins should also be present in the tissues that produce them, the fluid proteome was almost entirely a subset of the tissue proteome . In addition, fluid proteins identified in the tissue exhibited significantly higher abundance levels than the remainder of the proteins in the tissue proteome . To account for the possibility that contaminating tissue material contributed to the fluid proteome, we evaluated the depth of coverage between the tissue and fluid samples. Although there were less than half as many PSMs in the fluid samples , a comparable depth of protein coverage was achieved . Thus, the fluid had a greater number of unique PSMs per protein relative to the total number of PSMs identified. This pattern supports concentrated proteomic coverage within a specific subset of proteins found in the FRT fluid.The FRT tissue proteome was significantly more complex than the FRT fluid proteome, containing over twice as many proteins (1808 proteins in the tissue B). Tissue-only proteins were significantly enriched for gene ontology (GO) annotations associated with intracellular components, such as the mitochondria, and essential cellular functions of respiration and protein transport , an enrichment of exosome- or vesicle-associated annotations was observed . Based on these complementary pieces of evidence, we contend that our methodology successfully enriched for the FRT fluid and resulted in an informative proteome with respect to fluid composition.The distinct nature of the tissue and fluid proteomes was further supported by the differences in functional enrichments B. TissueFig. 1B; . In contB; e.g., ). Althoui.e., bursa, oviduct, seminal receptacle, spermathecae, and parovaria) and FRT-associated fat body may contribute to the fluid composition, we compared the fluid proteome with our recent comprehensive analysis of gene expression across the FRT tissues . In addition, FRT gene expression of fluid proteins was significantly greater than that of tissue-only proteins . Protein abundances in both the tissue and fluid, respectively, were also significantly correlated with gene expression across the FRT tissues . We note, however, that the relationship was more robust between the tissue proteome and FRT transcriptome, consistent with a more direct relationship between gene expression and intracellular protein abundance. The weaker relationship with the fluid proteome may be due, in part, to the impact of secretory mechanisms, which further decouples extracellular protein composition from FRT gene expression.To evaluate how the FRT tissues . We then determined which tissues were represented among the set of genes with tissue-specific expression . For both the tissue and fluid proteomes, the proportions of specific genes from each of the five FRT tissues and the fat body were significantly different from the proportions across the FRT transcriptome . In particular, the tissue-only proteome was derived from a significantly higher proportion of fat body–specific genes  and lower proportion of seminal receptacle–specific genes  than all FRT tissue-specific genes. This pattern was even stronger in the fluid proteome that had an even greater overrepresentation of fat body–specific genes  and underrepresentation of seminal receptacle–specific genes  than the tissue-only proteins . Thus, in contrast to the FRT-associated fat body, the glandular spermatheca and parovaria appear to contribute a relatively small number of tissue-specific products to the FRT fluid. This pattern supports the hypothesis that the tightly associated FRT fat body, which was found to have a distinct transcriptomic profile relative to the remainder of FRT tissues, contributes a substantive number of proteins to the FRT lumen . However, there were a substantial number of significant protein abundance differences . We found that the tissue-biased proteins were part of a protein network with significantly more interactions than expected , including hubs of proteins with intracellular functions such as ribosomal  and muscle proteins  but were enriched for metabolic processes , specifically glycolysis/gluconeogenesis . The enrichment of metabolic pathways in the FRT fluid was consistent with previous insect FRT fluid analyses . Thus, a major axis of variation was due to postmating changes in fluid composition. To confirm this pattern, we compared the distribution of protein abundance changes and found that the fluid proteome exhibited a significantly greater variance in postmating response relative to the tissue proteome . The difference in postmating response was further reflected by the fact that 37% of the fluid proteome exhibited an absolute LogFC >1 compared with only 9% of the tissue proteome. We next compared protein abundance changes between postmating tissue and fluid proteomes and found no correlation . Together, these analyses indicate that the FRT fluid exhibits a pronounced postmating response at 6 h that is distinct from the more limited changes in the FRT tissue.To investigate how the FRT tissue and fluid proteomes changed in response to mating, we compared samples from unmated females with those 6 h after mating, the time of maximal postmating transcriptomic response , 83. A PFig. 4A), whereas no proteins in the tissue proteome were found to be significantly different (B). Among the differentially abundant fluid proteins, there were nearly four times as many proteins that increased in abundance (241 proteins) rather than decreased in abundance (67 proteins) after mating, an observation consistent with increased postmating secretory activity. We note that there was no significant enrichment for signal sequences among differentially abundant proteins , suggesting that they may be secreted via alternative pathways. Proteins with greater postmating abundance had numerous significant functional enrichments relating to translation, similar to the enrichments observed in the whole fluid proteome  exhibited significant changes in abundance A, whereaifferent B. Among proteome . There w2 = 8.58, adjusted p < 0.01) or decreased in abundance . However, we observed no differences in codon bias between proteins that increased, decreased, or did not change in abundance after mating . We also compared protein abundance changes with gene expression changes at the same postmating time points , we identified an enrichment of vesicle proteins in the fluid. However, we did not observe an enrichment of proteins with secretion signals. One possible explanation for this discrepancy is that the secreted products have greater regional specificity with lower overall abundance, making them recalcitrant to proteomic identification with current techniques. The secreted proteins are also often heavily glycosylated, which could have precluded their identification. Alternative mechanisms, such as apocrine and holocrine secretion, could also account for the observed fluid composition and dynamics. In particular, the enrichments of intracellular cytoplasmic protein contents, such as translational machinery among identified fluid proteins, support the contribution of apocrine or holocrine secretion to the fluid. We note that the support for these alternative secretory pathways has been found in other invertebrate male reproductive systems D. melanogaster as spatially discrete clusters of cells surrounding the spermatheca and parovaria, possesses dedicated roles in the establishment and dynamics of the FRT fluid. The spatial and functional heterogeneity of abdominal fat body populations is supported by recent single-cell analyses demonstrating that the fat body cells form clusters with stereotypical expression profiles and disparate postmating responses . Such pis clade . NonetheIt is somewhat surprising that the prominent postmating changes in the fluid did not correspond to contemporaneous changes in the FRT tissue proteome. This pattern may reflect the "poised state" of the FRT allowing for a rapid response to mating , potentiGiven its central role in mediating ejaculate–female interactions that determine reproductive outcomes, the FRT fluid should be a priority of future investigations. This is especially true in relation to intraspecific and interspecific compositional variation and of how such variation corresponds to reproductive outcomes, including fertility, paternity, and female postmating responses. In particular, expanding on the time points characterized here to establish a timeline of FRT tissue and fluid proteome changes after mating would allow for a more refined interpretation of protein changes corresponding to physiological events of interest. In addition, it is important to similarly characterize the presence and temporal dynamics of the nonprotein composition of the fluid, including ions, metabolites, amino acids, prostaglandins, and hormones.via the ProteomeXchange Consortium (FRT tissue and fluid: PXD025085 and isotopic labeling: PXD025072). The analysis code is available on GitHub at https://github.com/CEMcDonoughGoldstein/FRT.TissueFluid.Proteome.Raw mass spectrometry data are available This article contains The authors declare no competing interests.


Infection with the varicella-zoster virus (VZV) causes chickenpox and shingles, which lead to significant morbidity and mortality globally. The detection of serum VZV-specific antibodies is important for the clinical diagnosis and sero-epidemiological research of VZV infection, and for assessing the effect of VZV vaccine immunization. Over recent decades, a variety of methods for VZV antibody detection have been developed. This review summarizes and compares the current methods for detecting VZV antibodies, and discussed future directions for this field. Chicksex, UK) ,11,12. HLaboratory testing is essential for the diagnosis and surveillance of VZV-induced diseases. Virus isolation was once the “gold standard” for the diagnosis of VZV infection ,14. HoweHerein, we review the existing serological assays for the detection of anti-VZV antibodies and compare their advantages and limitations. This paper could help clinicians and technicians to choose the appropriate serological method for diagnosing VZV infection or assessing the efficacy of VZV vaccines.The FAMA test, which was initially developed by Williams et al. , is the The FAMA test can be performed using chemically fixed VZV-infected cells. The fixed-cell FAMA was developed from the classic FAMA to increase throughput and efficiency and has been used to study human immunity to VZV ,30,31,32To avoid the use of infectious viruses, one study has reported a simple and safer FAMA using HEK293T cells transfected with a plasmid encoding VZV glycoprotein E (gE) to replace VZV-infected cells, which is called the gE FAMA  have, in recent years, shown promise in achieving high-throughput quantitative analysis of anti-VZV neutralizing antibodies.IFAs for the detection of anti-VZV antibodies include the anti-complement immunofluorescence (ACIF) assay  and the TRFIA, which is also called dissociation enhanced-lanthanide fluorescence immunoassay (DELFIA), was first used to detect anti-VZV antibodies in 2006 . As showTRFIA uses EU3+ as the fluorescent probe; it has a long decay time, and antibody concentrations can be measured after other fluorescent substances with short half-lives are decayed, thus eliminating the interference of non-specific fluorescence. Coupled with the narrow emission light crest of lanthanum fluorescence, the background of TRFIA is further reduced. TRFIA has sensitivity and specificity equivalent to those of the Merck gp-ELISA. Furthermore, compared with FAMA, this method is easy to use and has a relatively short testing time. However, TRFIA requires special equipment and is only used in a few specialist testing centers.IAHA was once commonly used to detect anti-VZV antibodies for the evaluation of the immune status of the population against VZV and the immune effect of chickenpox vaccination . IAHA isLA is a modified version of IAHA. According to the procedure of LA  , but furLFIA, a simple point-of-care testing (POCT) based on antigen and antibody immune responses, has been used for the detection of VZV antibodies . The repIn recent decades, a variety of serological methods for detecting anti-VZV antibodies have been established for the clinical auxiliary diagnosis of VZV infection; these methods have facilitated VZV-related epidemiological studies and vaccine studies, as well as risk assessments of healthcare workers. FAMA is the most widely recognized and commonly used method due to its relatively high sensitivity and specificity. However, standard live-cell FAMA is labor-intensive with low throughput, and is susceptible to subjective judgment. ELISA is the most accessible method, but commercial ELISA kits may yield false-positive or false-negative results and are not reliable in evaluating serum conversion after vaccination for chickenpox, which generates lower antibody levels compared to the wild-type VZV infection. Neutralization tests can directly evaluate the immune protection effect, but have a low sensitivity for detecting anti-VZV antibody. IFA and LA are very sensitive but have limitations in determining positive reactions, which make them less than ideal for both chickenpox susceptibility screening and detecting seroconversion to VZV vaccines. TRFIA and CLIA showed good specificity and sensitivity but require further validation. LFIA is an easy-to-use POCT without the requirement for experienced technicians and equipment, but it only provides qualitative or semi-quantitative results.The existing VZV serological tests mainly detect IgG antibodies, but the analysis of different antibody subtypes is also of value in the diagnosis of VZV infection. For example, it has been reported that the IgG3 subtype was the main subtype in the recovery period of chickenpox, and the IgGl subtype was the main subtype in the recovery period of shingles . Meanwhi(1)Biosensor techniques, such as lateral flow assays and electrochemical assays, are evolving rapidly, and have shown promising prospects in making inexpensive, easy-to-use diagnostic tools  for quicker sensitive and specific detection of anti-VZV antibodies. These biosensor-based assays would not require elaborate instrumentation and/or a laboratory set-up and would therefore be more accessible to researchers, clinicians, and the general public, thus better meeting the needs of large-scale population screening. They could be useful complements to conventional laboratory tests.(2)For national institutions or testing centers involved in disease control and prevention, it is better to combine high-precision detection equipment with different assay strategies to develop standardized methods for the automated measurement of anti-VZV antibodies, to not only achieve high sensitivity, accuracy, and reliability but also to enable high-throughput, objective, and stable testing.(3)It is hoped that the methods developed in the future will be able to detect different subtypes of anti-VZV antibodies with similarly high sensitivity, which would be beneficial for clinical diagnosis, increasing our understanding of the role of antibody responses in the prevention of VZV infection, and may provide insights into ways to improve the effectiveness of VZV vaccines.While current methods to detect anti-VZV antibodies are numerous and relatively mature, researchers can still strive to make improvements to obtain better detection efficiency and measurement accuracy, as well as greater convenience. Some potential directions for future development are as follows:


The nonlocal spintransport signal was found to strongly depend on temperature and disappearat a temperature below the Curie temperature of the FGT flakes, whichstemmed from the temperature-dependent ferromagnetism of FGT. Thespin injection efficiency was estimated to be about 1%, close to thatof conventional nonlocal spin valves with transparent contacts betweenferromagnetic electrodes and the graphene channel. In addition, thespin transport signal was found to depend on the direction of themagnetic field and the magnitude of the current, which was due tothe strong perpendicular magnetic anisotropy of FGT and the thermaleffect, respectively. Our results provide opportunities to extendthe applications of van der Waals heterostructures in spintronic devices.Withrecent advances in two-dimensional (2D) ferromagnets withenhanced Curie temperatures, it is possible to develop all-2D spintronicdevices with high-quality interfaces using 2D ferromagnets. In thisstudy, we have successfully fabricated nonlocal spin valves with Fe On the other hand, 2D materialsoffer new opportunities to fabricate novel devices.10 The combination of spintronicsand 2D materials started with fabricating local11 and nonlocal12 spin valves (NLSVs)with graphene as the transport channel. Then, it quickly expandedto devices to manipulate spins,16 enhance spin transport,21 and increase the spin injection efficiency.22 With the help of h-BN to maintain the materialand structure integrity, the spin relaxation length has been increasedto 30.5 μm at room temperature in graphene.17 Multilayer graphene has also been employed to transportspin current since its outer layers can screen scattering potentials.24 The study of 2D spintronics entered a new era with the discoveryof 2D ferromagnets,29 making devices composed of all-2D materials possible. Fe3GeTe2 (FGT) was discovered as a vdWs ferromagnetic metalwith a high Curie temperature of up to 220 K (bulk state) and strongperpendicular magnetic anisotropy.30 Moreover,the resistivity of FGT is more similar to that of multilayer graphenethan those of the traditional 3d transition ferromagnetic metals,27 and the reduced conductivity mismatch may enhancethe spin injection efficiency.32 Therefore, FGT canbe used to inject and detect spin currents in a multilayer graphenechannel. In addition, FGT has been utilized to build vertical magnetictunnel junctions with all-2D vdWs materials.37 For example, the FGT/BN/FGT heterostructures showeda tunneling magnetoresistance as high as 160% due to the high-qualityinterfaces between vdWs materials.35 Recently,2D vdWs ferromagnetic metals, Fe5GeTe2 and Fe3GaTe2, with the Curie temperature near38 or above39 room temperaturehave been discovered. The tunnel magnetoresistance of up to 85% hasbeen realized in Fe3GaTe2/WSe2/Fe3GaTe2 heterostructures at room temperature.40 In addition, 2D Fe5GeTe2 and cobalt have been employed to fabricate NLSVs at room temperature.41 However, whether an all-2D vdWs NLSV can befabricated and more importantly whether their performance can be improvedby the high-quality interfaces have not been investigated yet.Devicesbased on spintronics and two-dimensional (2D) van der Waals(vdWs) materials are potential candidates for developing next-generationintegrated circuits.42 The charge current was injected from one FGTflake, and the spin transport signal was detected using the otherFGT flake. The nonlocal spin transport signal was observed, and thespin injection efficiency was estimated to be about 1%. The dependenceof the spin transport on the direction of the magnetic field and themagnitude of the current was also investigated.In this study, NLSVs with two FGT electrodes and a multilayer graphenechannel were fabricated using a deterministic transfer method.235 and thus their magnetic moments could be manipulated separately.The atomic force microscopy (AFM) images in Figure S1 indicate that the thicknesses of the graphene channel, FGT1,and FGT2 were 10 nm (∼29 layers), 23 nm (∼28 layers),and 16 nm (∼20 layers), respectively. Two Cr/Au (10 nm/70 nm)electrodes (E1 and E2) were fabricated using electron-beam lithographyand subsequent electron-beam evaporation. The distance between theinjector (FGT1) and the detector (FGT2) was about 3 μm. Note S1 andFigure S2). The Hall resistances (xyR) as a function of the applied magnetic field at different temperaturesare shown in 30 Note that the magnetic field in this study wasalways applied perpendicular to the sample plane unless otherwisespecified. The nearly square hysteresis loops of xyR of FGT1 and FGT2 indicate that the reversal of the magneticmoment in FGT1 or FGT2 was realized through the coherent rotationof a single domain. The kinks observed near the coercive fields inthe hysteresis loops of xyR of FGT2 mightrelate to defects or local thickness variations in FGT2, which changedthe coercive field locally. The hysteresis loops narrowed with theincrease in temperature (up to 200 K) , indicating that the Curie temperatures of FGT1 and FGT2 were around200 K, which agreed with the previous reports.30I) is injected from FGT1 to E1, spins accumulateunderneathFGT1 and diffuse to both sides of the graphene channel.24 When the spin current reaches FGT2, a voltagedrop VNL develops between FGT2 and E2;the nonlocal resistance RNL can be definedas RNL = VNL/I.24 Different fromconventional NLSVs,43 the magnetization in this studywas perpendicular to the sample plane.30 Due to thedifferent coercivities in FGT1 and FGT2, when the magnetic field wasswept between −1000 and 1000 mT, the magnetic configurationbetween FGT1 and FGT2 changed from parallel to antiparallel and thenparallel again, as indicated by the vertical arrows in VNL and RNL, i.e., ΔVNL and ΔRNL, occurred during the field sweep,44 as shown in 12 This happened becausepart of the injected charge current “leaked” to thedetector and induced magnetoresistance and Hall resistance that variedwith the perpendicular magnetic field. Meanwhile, the magnetoresistanceand the Hall resistance were superimposed on the spin signal. Second,ΔRNL was surprisingly weak evenwhen the applied current reached 0.8 mA , whose spin signal was much enhanced already with a current ofonly 0.3 mA, as shown in Figure S5a. (2)Part of the spins was absorbed by FGT, similar to that observed previously;45 and (3) there was still a conductivity mismatchbetween FGT and graphene, so the backflow of spin current could notbe avoided. Third, the magnetic fields where the nonlocal resistancesabruptly decreased , much lower than theCurie temperature,30 indicating that amore significant thermal effect was induced in the device. To verifythe reliability of our results, the injector and the detector wereswitched with each other, and then the same experiments were performed.We observed similar phenomena, except for different background signals,as shown in Figure S6. This implied that,unlike the previous study,12 the nonlocalspin signal in our experiment had strong temperature dependence, stemmingfrom the temperature-dependent ferromagnetism of FGT.30As shown in d 0.8 mA 2b. Threed 0.8 mA 1b, whichd 0.8 mA S4, whosed 0.8 mA S4, whosed 0.8 mA S4, whosed 0.8 mA S4, whosed 0.8 mA S4, whosed 0.8 mA S4, whosed 0.8 mA S4, whosed 0.8 mA S4, whose12 whereas at a higher temperature (80 K), thecoercive fields of both FGT1 and FGT2 decreased below 200 mT, andthe “normal” nonlocal resistance similar to that in xyR of FGT2. Increasing the temperature from 2 to 40 K, theloop narrowed monotonically  to observe theHanle effect.43 However, even with themagnetic field and the current reaching 3000 mT and 0.8 mA, respectively,no Hanle precession signal was detected. We argue that the absenceof the Hanle effect could be attributed to the wide FGT electrodesand spin absorption by the FGT electrodes. The widths of the FGT electrodeswere either comparable to  the channel length. Hence, the injected spins, coming from thedifferent portions of FGT, did not have the same phase and counteractedeach other, weakening the Hanle precession signal. In addition, spinabsorption by the FGT electrodes shortened the effective spin relaxationtime, further making the Hanle precession signal difficult to be detected.To extract more information from the NLSVs, we also performedspinprecession experiments. We applied a magnetic field in the irection S7 to obsRNL is the change of the nonlocal resistance, σG is the conductivity of the graphene channel, PJ is the spin injection efficiency, λG is the spin relaxation length of thegraphene channel, and W and L arethe width and the length of the graphene channel between the injectorand the detector, respectively.22 Sincethe Hanle effect was absent in our experiment, the spin relaxationlength could not be determined. Hence, we assumed a typical valueof λG (∼1.5 μm) formultilayer graphene by referring to our previous study.47 Moreover, ΔRNL was ∼1.5 mΩ . Graphene flakes were first exfoliated froma natural graphite crystal (HQ graphene) onto a Si/SiO2 (500 μm/300 nm) substrate. Then, a multilayer graphene stripwith the appropriate size was located by an optical microscope . Subsequently, FGT flakes were exfoliatedfrom an FGT bulk (HQ graphene) onto polydimethylsiloxane (PDMS) films,and two FGT flakes with different thicknesses on two PDMS films wereidentified under the optical microscope via their different opticalcontrasts, respectively. Afterward, the two FGT flakes were transferredand stacked onto the chosen graphene strip with the help of an accuratetransfer platform . After that, the heterostructurewas spin-coated with poly(methyl methacrylate) (PMMA) solution andmoved outside of the glove box for further fabrication. Finally, Cr/Au(10 nm/70 nm) electrodes were fabricated by electron-beam lithography,followed by electron-beam evaporation. After the fabrication, thesamples were quickly moved into a high-vacuum chamber for thermalannealing , which improved the coupling between FGT and graphene.NLSVs were fabricatedin a glove box in an Ar atmosphere  to fabricate STEM samples. Then, a monochromated Cs-correctedhigh-resolution STEM instrument  was employed tocharacterize the interfacial structure of the graphene/FGT heterostructures.The thicknesses of the samples were measured by an AFM instrument using AC mode after performing electronicmeasurements.4.3NLSV devices were moved into a physical property measurement system for measurements. During the measurements,an alternating current at 13 Hz was applied by a Keithley 6221 currentsource, and the voltage was measured by an SR830 lock-in amplifier.A current of 1 μA was applied to the devices during the anomalousHall-effect measurements to avoid the thermal effect.


Permanent-magnet small ball-end magnetorheological polishing method can be used to polish the small part with complex structure. However, the material removal rate of this method is low, which is difficult to improve the output and reduce the cost. In this research, the effect of magnetorheological fluid temperature on the material removal rate is theoretically analyzed by measuring the effect of temperature on the flow properties of magnetorheological fluid, establishing the hydrodynamic model of polishing zone and solving the material removal parameters. It is found that with the increase of the magnetorheological fluid temperature, the polishing relative velocity increases accordingly, which can promote the improvement of material removal rate. But the shear stress decreases accordingly, which inhibits the improvement of material removal rate. The verification experiment results show that the promoting effect can exceeds the inhibitory effect, so that the material removal rate increases with the increase of magnetorheological fluid temperature. When the magnetorheological fluid temperature increases to 60 °C, the material removal rate is improved by 108.4% and the polished surface roughness Sa can reach 14.9 nm. Therefore, increasing the magnetorheological fluid temperature can significantly improve the efficiency of permanent-magnet small ball-end magnetorheological polishing and obtain high quality polished surface. Thus, it is suitable for polishing these parts. However, the size of the polishing wheel of the commonly used wheel-type MR polishing equipment is too large, which cannot polish the complex structure surface of the small part. Therefore, the small MR polishing tool is needed to be designed and used. Chen et al.4 designed a permanent-magnet small ball-end polishing head (diameter 4 mm) and successfully polished a Ψ-shaped small-bore complex component. The minimal transition fillet curvature radius of the curved surfaces of the component was less than 3 mm. The surface accuracy PV of polished surface reached 0.332 μm, and the surface roughness Ra reached 10.7 nm. However, limited by the size of the polishing head, the volume of the permanent magnetic material of the polishing head is small, leading to the relatively low magnetic induction intensity (no more than 0.44 T). Besides, the linear velocity of the polishing head at high rotation speed is low, which limits the improvement of polishing relative speed. These factors result in low material removal rate during polishing. The processing cost is high and the output is low. Therefore, it is urgent to improve the efficiency of the permanent-magnet small ball-end MR polishing.The small part with complex structure plays an important role in all kinds of precision equipment. These parts are mostly made of hard and brittle materials that are difficult to process. The polished surface quality and profile accuracy of these parts are required highly. Magnetorheological (MR) polishing method has the advantages of high machining accuracy, no tool wear and no subsurface damage, and the roughness of polished surface can reach nanoscale7, Non-resonant vibration8 and chemical action12 are introduced into the MR polishing process, which can significantly improve the material removal rate and polished surface quality. However, there are few studies on improving the material removal rate by changing the MR fluid temperature. MR fluid is a typical non-Newtonian fluid. Its flow properties are closely related to temperature14. Hemmatian et al.15 studied the temperature dependency of MR fluids’ properties. It was found that the effect of temperature on the viscosity and shear stress of MR fluid diminished with increasing magnetic field. Wang et al.16 and Sherman et al.17 studied the temperature-dependent material properties of the components of MR fluids. It was found that the viscosity of MR fluid depended on the viscosity of carrier fluid. The viscosity of the carrier fluid decreased as the temperature increased, and the carrier fluid with a higher viscosity was more sensitive to the temperature variation. Chen et al.18 analyzed the influence of temperature on the rheological properties of MR fluid. It was found that within 100 °C, the viscosity of MR fluid decreased with the increase of temperature. The shear stress of MR fluid was affected by the change of viscosity and would decrease with the increase of temperature. Wang et al.19 used a parallel disk shear stress testing device to measure the temperature-dependent mechanical properties of MR fluids. It was found that with the increase of MR fluid temperature, the reduction in viscous stress was much more evident than that in yield stress. The viscous stress component dominated the change of total stress in a particular temperature range. Bahiuddin et al.20 used the extreme learning machine (ELM) method to develop a new constitutive model of MR fluids with temperature-dependent prediction parameter. It accurately predicted the shear and yield stresses of MR fluids under specific temperature, shear rate and magnetic field. In conclusion, changing the MR fluid temperature can influence the flow properties and mechanical properties, so as to affect the polishing removal efficiency.Ultrasonic vibrationAiming at the permanent-magnet small ball-end polishing method, the influence law of temperature on MR fluid flow properties and material removal parameters are studied, and the verification experiment is carried out in this research. The experiment results show that increasing the MR fluid temperature can greatly improve the polishing removal efficiency.In this research, the solid particles in water-based MR fluid are carbonyl iron powder (CIP) and cerium oxide abrasive particle. When the polishing head is stationary, the MR fluid flowing into the polishing zone is affected by the applied magnetic field of the polishing head. The MR fluid is adsorbed on the surface of the polishing head to form an "iron ball" with solid-like state, as shown in Fig. In a certain temperature range, increasing the MR fluid temperature can reduce the initial viscosity of MR fluid, which can reduce the flow resistance of MR fluid film. It is speculated that this can increase the flow velocity of MR fluid film, which can increase the relative velocity when abrasive particles shear the workpiece surface. Moreover, the number of abrasive particles flowing through the workpiece surface per unit time also increases, which can improve the shear frequency between abrasive particles and the workpiece surface, so as to improve the polishing removal efficiency.21. When the temperature exceeds 80 °C, the magnetic field of NdFeB declines irreversibly or even disappears. Based on the laboratory room temperature and the above factors, 20–60 °C is selected as the temperature range of MR fluid. When the temperature rises from 20 to 60 °C, the maximum magnetic induction intensity of the polishing head decreases from 0.439 to 0.418 T. Thus, it is also necessary to consider the influence of the changed magnetic induction intensity of the polishing head on the flow properties of the MR fluid.MR fluid is a non-Newtonian fluid, and the flow properties of MR fluid mainly depend on the initial viscosity and the yield stress. When subjected to an applied magnetic field, the magnetization intensity of MR fluid also affects the flow properties. Therefore, it is necessary to study the influence of temperature on initial viscosity, yield stress and magnetization intensity of MR fluid. In the polishing process, the heated MR fluid firstly transfers heat to the iron ball, and then the iron ball transfers heat to the polishing head. This increases the temperature of the polishing head. The permanent magnetic material of the polishing head is sintered NdFeB. When the temperature increases by 1 °C, the magnetic induction intensity of NdFeB reduces by about 0.11–0.12%22. The viscosity variation curve in the range of 20–60 °C is shown in Fig. η0 is the initial viscosity of MR fluid (Pa s), t is the MR fluid temperature (°C), ϕ is the volume percentage of solid particles in MR fluid.In the absence of magnetic field, the relationship between the initial viscosity and temperature can be expressed by Eq. 22. The vWhen the MR fluid temperature increases from 20 to 30 °C, 40 °C, 50 °C and 60 °C respectively, the initial viscosity of MR fluid decreases by 40.4%, 64.5%, 78.9% and 87.4% respectively. The results indicate that the initial viscosity of MR fluid is very sensitive to temperature variation. With the increase of temperature, the initial viscosity of MR fluid decreases remarkably. The flow resistance of MR fluid film decreases, which is conducive to increasing the polishing relative velocity between abrasive particles and workpiece surface, so as to improving the material removal rate.τ is shear stress (Pa), τ0 is yield stress (Pa), η0 is initial viscosity (Pa s), −1).After the yielding flow is generated, the flow state of MR fluid can be expressed by Bingham model, as shown in Eq. .2\documeThe shear rate vs. shear stress curve of MR fluid was measured by Anton Paar MCR301 rheometer, and the magnetic induction intensity of the applied magnetic field was depended on the input current of the magneto-rheological device of the rheometer. The curve was measured repeatedly by changing the temperature and input current. A typical shear rate vs. shear stress curve is shown in Fig. 15 and reference19. In order to simply the calculation, it is considered that the yield stress is only relate to the magnetic induction intensity of the polishing head. The mathematical model of yield stress is obtained by fitting, as shown in Eq. (τ0 is the yield stress of MR fluid (kPa), B0 is the magnetic induction intensity of the polishing head (mT).It can be seen from Fig. n in Eq. . The curIn the polishing process, the magnetic induction intensity of the polishing head decreases with the increase of the MR fluid temperature, resulting in the reduce of the yield stress. According to Eq. , under tThe magnetization intensity of CIP is related to temperature. Increasing the temperature can change the magnetization intensity of CIP, thus affecting the flow properties of MR fluid. Therefore, it is necessary to study the effect of temperature on CIP magnetization intensity. The magnetization intensity curve of CIP in the range of 20–60 °C is measured by the Physical Property Measurement System (PPMS), as shown in Fig. It can be seen from Fig. B is the total magnetic induction intensity (T), H is the applied magnetic field strength of polishing head (A/m), M is the magnetization intensity of CIP (A/m), μ0 is the permeability of vacuum (N/A2).Moreover, the total magnetic field in the polishing zone consists of an applied magnetic field of polishing head and an induced magnetic field generated by CIP. It can be seen from Eq.  that theB-H curves of CIP at different temperatures are obtained by solving the Eq. , η0 is the initial viscosity (Pa s), −1), −1), τ0 is the yield stress (Pa).Doriern in Eq. . In thisEquation  can be cCombine Eqs. , 3) and and3) anu, v and w respectively.The MR fluid in the polishing zone is divided into many small three-dimensional meshes. The flow parameters of each node of the meshes are soled by the finite difference method, so as to obtain the flow state of the whole polishing zone. In order to simplify the calculation, a smooth and stationary plane is regarded as the workpiece surface. The hydrodynamic analysis model and the coordinate system of MR fluid are shown in Fig. The MR fluid is an incompressible fluid with constant density;The flow of MR fluid between the upper and lower walls is laminar flow and meets the no-slip condition, hence the flow velocity of the MR fluid at the boundary is equal to the velocity of the solid surface;The gravity and inertia force of the MR fluid are ignored;The partial derivatives of flow velocity and stress in X and Z directions are ignored;The hydrodynamic pressure across the film thickness remains constant.The following assumptions are made for the MR fluid.y1 = 0, u1 = v1 = w1 = 0; y2 = d, u2 = U2, v2 = V2, w2 = W2. The boundary conditions are substituted into the velocity expression, fluid dynamic pressure expression and apparent viscosity expression of MR fluid to obtain Eqs. , K is coefficient of correction (m2/kPa), τ is the shear stress (kPa), V is the polishing relative velocity (m/s).In the process of MR polishing, the shear stress plays a major role in material removaln in Eq. .11\documIgnoring the coefficient of correction, the product of shear stress and polishing relative velocity can reflect the distribution zone of the material removal rate on the polished surface. Taking the MR fluid temperature at 20–60 °C as examples, the product of shear stress and polishing relative velocity on the polished surface were calculated respectively, as shown in Fig. It can be seen from Fig. The polishing experimental equipment and MR fluid circulation process are shown in Fig. The processing parameters are as follows: MR fluid temperature is 20–60 °C, polishing head rotation speed is 7000 rpm, polishing gap is 0.1 mm. When polishing the plane glass workpiece, the polishing time of each pit is 5 min. When polishing the glass rod workpiece, the workpiece rotation speed is 120 rpm and the polishing time of each pit is 20 min. In the polishing process, the water of the MR fluid is replenished every 10 min to keep the mixture ratio of MR fluid stable. A Zygo NewView 8200 white-light interferometer was used to measure the three-dimensional shape and the section contour of the polished pit. The shape of teardrop-shaped polished pits at different MR fluid temperatures is shown in Fig. With the MR fluid temperature increases from 20 to 60 °C, the maximum removal depth of teardrop-shaped polished pits increases from 13 to 54 μm. Under different MR fluid temperatures, the shape of polished pits is similar with the distribution zone shape of material removal rate in Fig. MRR is the material removal rate (mm3/min), t is the MR fluid temperature (°C).It can be seen from the Fig. R2 of the fitting equation exceeds 0.99, showing that the equation is in good agreement with the change curve of material removal rate. Under the same processing conditions, the material removal rate at different MR fluid temperatures can be predicted by this equation. Keeping the process parameters unchanged, a section of smooth surface was polished on the rough surface at the MR fluid temperature of 60 °C, as shown in Fig. The coefficient of determination It can be seen from Fig. In the range of 20–60 °C, the initial viscosity decreases with the increase of MR fluid temperature, which can improve the fluidity of MR fluid film. The magnetization intensity of CIP is very stable and hardly affects the material removal rate. The magnetic field intensity of the polishing head decreases with the increase of temperature, resulting in decreasing the yield stress of MR fluid.Increasing the MR fluid temperature can increase the polishing relative velocity, but also reduce the shear stress. The increase of polishing relative velocity has a promoting effect on improving the material removal rate, but the decrease of shear stress has an inhibitory effect. Experiment results show that the promoting effect can exceed the inhibitory effect, so as to improve the material removal rate.The material removal rate continues to increase with the increase of MR fluid temperature, and the convergence of surface shape accuracy in the material removal process can be maintained well at different temperatures. When the MR fluid temperature is 60 °C, the polishing removal efficiency is improved by 108.4%, and the polished surface roughness Sa can reach 14.9 nm.Through theoretical analysis and experimental verification, it is found that increasing the MR fluid temperature can improve the efficiency of permanent magnet small ball-end MR polishing. The research results are summarized as follows.In conclusion, the polishing removal efficiency can be improved by increasing the MR fluid temperature. This method can be applied to practical production to shorten processing time and increase production.


Current work portrays the flow of Marangoni convection Magneto hydrodynamics Casson fluid with carbon nanotubes under the effect of transpiration and radiation. The carbon nanotube particles namely water-single wall carbon nanotubes are inserted in the fluid to enhance better thermal efficiency. This type of flow problems is applicable for real life situations such as drying of silicon wafers, glues, crystal growth and heat exchangers and so on. The ordinary differential equations (ODEs) form of the result is yield to convert partial differential equations of the given equation by using similarity variables. Then this resulting ODEs are solved analytically, firstly using momentum equation to get solution domain and then by using this domain the energy equation solved to get the temperature profile in terms of Laguerre polynomial. Additionally, mass transpiration is also solved to get the concentration profile in terms of Laguerre polynomial. By using the different controlling parameters, the results can be discussed. And the effect of this parameters are discussed by using graphical arrangements. The newness of the present work is to explain the physically flow problem on the basis of chemically radiative thermosolutal Marangoni convective fluid. Napolitano3 addressed the original work of this field. Temperature and concentration dependent surface tension is respectively called as Thermocapillary and destillocapillary effects5. The application of Marangoni convection can be found in the fields of crystal growth, soap films and crystal growth. Chamkha et al.6 worked on Marangoni convection problem and he come to concluded that surface driven flows may build layers along the interfaces as well as buoyancy effect brought on by gravity and the external pressure gradient. When employing the arc length as coordinates, Napolitano et al.7 addressed the issue that non-Marangoni boundary layers in bulk fluids do not explicitly depend on the geometry of the interface. Only a few research and initiatives have been made to comprehend the fundamental laws of nature and the issues surrounding Marangoni convection.Basically in the interfaces of liquid–liquid or liquid–gas we found the layers of Marangoni convection, these layers are normally called as dissipative layers and these layers plays a great role in industrial applications. Gibbs8. The effects of heat transmission on MHD and radiation are examined by Aly and Ebaid9. Marangoni boundary layer nanofluid led him to conclude that a magnetic parameter causes a fluid's velocity to slow down and its temperature to rise. The double-diffusive convection in an open cavity was studied by Arbin et al.10 Nayak11 investigated the magnetohydrodynamics viscoelastic fluid under the impact of chemical reaction effect with porous medium. See some other examples related to Marangoni convection are seen in15.The magnetohydrodynamics thermosolutal Marangoni convection over a flat surface in the presence of a heat source/sink parameter was addressed by Mudhaf and Chamkha16. Arshad et al.20 investigated flow problems in the presence of different nanofluids with various aspects such as heat source/sink parameter, radiation and so on. See some more references on nanofluid in22. Similarly, carbon nanotubes take a lot of attention in many fields such as chemistry, physics, medicine, biology, and Engineering. These are the few examples for the importance of nanofluids in many fields25. Additionally, the fluid known as Casson fluid is quite interesting, and it is used to describe non-Newtonian phenomena. The researcher Casson addressed this flow model in 1995. This flow model is useful in many real life applications. See some of the recent works of this model in28.Recent advancements in nanotechnology helps to conduct innovative techniques to develop applications of nanofluids in many fields. The term nanofluid is initially addressed by Choi29. There are many equations and derivations are available to describe the fluid flow process through porous medium and also the effect of thermal radiation. See some more articles published on porous medium and thermal radiation are given in 32.Effect of Porous medium and thermal radiation take places major role in the fluid flow because these effects in the fluid flow is used in many industrial and real life applications namely metallurgic processes, geophysical and allied areas33.The current study is investigating Casson fluid flow in the presence of carbon nanotubes with thermal radiation and mass transpiration. It is prompted by the aforementioned studies. In this problem we use the new method to provide a similarity variable on the impact of chemically radiative thermosolutal Marangoni convective fluid flow, the partial differential equations of the governing equations are converted into ordinary differential equations. The novelty of the present work explains that the momentum energy and mass equation solved analytically to get the solution domain and the solution in terms of Laguerre polynomial. The impact of different parameters is examined with the help of graphical scenario. This work is also important in many industrial applications such as welding machines, metallurgical process, geosciences, space technology and so on. The current issue is persuasively argued in the work of Mahabaleshwar et al.Flow of a Casson fluid with thermosolutal Marangoni convection thermal radiation and transpiration is analyzed in this study. The particles of carbon nanotubes are immersed inside the fluid to get better thermal efficiency. Temperature gradients and solute concentrations define surface tension. Figure x axis. The governing equations can be defined as follows by taking into account the aforementioned premises (See35).Let us assume the surface of the fluid move towards Here the Casson fluid term is used for characterize the non-Newtonian fluid. Magnetic term and porous medium term is used for many scientific and technological phenomena. Effect of Porous medium and thermal radiation take places major role in the fluid flow because these effects in the fluid flow is used in many industrial and real life applications. Heat source/sink in the fluid flow influences the characteristics of heat transfer as there is a substantial amount of difference in the temperature between the surface and the fluid. Also the combination of mass transfer and heat source/sink helps in overcoming the problem of boundary layer separation.38)The surface tension along with heat and mass boundaries is given by By using Eqs.  and 10)10) in Eq45)Consider Eq. ’s solutiAlthough, from the governing B. Cs On applying Eqs. , 21 in EThen the velocity can be required asFor the purpose of solving temperature and concentration equation we introduce the following new variable for temperature and concertation respectively as followsUsing Eqs.  and 26)26) respeThe B. Cs are also reducing toing Eqs.  and 28)29\documeThis article portrays the Casson fluid flow with Marangoni convection with Carbon nanoparticles are immersed in the fluid flow to enhance the thermal efficiency of the fluid. Analytical results are examined with the help of different controlling parameters namely Casson fluid parameter, inverse Darcy number, Chandrasekhar’s number, Marangoni number and so on. The significant effect of Prandtl number, Schmidt number, chemically reaction coefficient and heat source/sink parameter on temperature, concentration and heat source/sink parameter is discussed as follows. The graphical scenario can be disused as follows.Figure Impact of The effect of mentclasspt{minimaEffect of the physical solution is directly affected by the Presence of porous media, prevents heat loss/gain and also accelerates the heat source/sink. Chemical reaction term thinning the thermal boundary, Marangoni convection results in more induced flows.The future perspectives of the present work motivate to explain the physically flow problem on the basis of chemically radiative thermosolutal Marangoni convective fluid also helps to conduct flow problems with porous media.33.If 0In Eqs.  and 17\d8.If  In Eqs.  and 17 \14,If  In Eqs.  and 17\dFollowing conditions explain the comparison pf present work with previous works.The investigation of results from the 2-D Casson fluid with mass transpiration, thermal radiation and chemically reaction parameter. The ODEs of equations are yielded when we mapped PDEs equation with similarity variables. These ODE equations are solved exactly then the momentum equation is solved to get solution domain, this domain is used in energy and concentration equation to get the temperature profile and concentration profile. The outlook of the present work explains the importance of porous media, thermal radiation, Marangoni convection, thermal radiation and heat source/sink parameter in the physically modelling of the flow. The outcomes we discovered using the graphical scenario are as follows.


ROS induced by chemodynamic therapy compensates for the incomplete cure of osteosarcoma cells. The AG-encapsulated NPs could mediate synergistic chemodynamic and photothermal therapy with self-sufficient H2O2, providing a novel therapeutic strategy for osteosarcoma.Osteosarcoma is a malignant bone cancer that usually occurs in children and adolescents. Although chemotherapy, radiotherapy and other methods have been used to treat osteosarcoma, these therapeutic regimens fail to cure this disease completely. Herein, doxorubicin-encapsulated iron–gallic acid (FeGA-DOX) nanoparticles (NPs) were fused with agarose hydrogels (AG) for synergistic therapy of osteosarcoma. Under near-infrared laser irradiation, the local temperature of FeGA-DOX NPs was increased. Therefore, tumour cells were killed using photothermal therapy, and AG dissolved to release FeGA-DOX into the cells. Doxorubicin generates hydrogen peroxide, which is then converted to reactive oxygen species (ROS)  Osteosarcoma is one of the most common cancers in children and adolescents that mainly occurs in the metaphyseal region of long bones, especially the limbs and shoulders, and often leads to metastasis . The cli2O2 and glutathione levels (H. in situ cell toxic reactive oxygen species (ROS) in cancer cells from this intracellular overexpressed H2O2 , with the assistance of NPs, has emerged as one of the novel treatment methods for cancers . Upon la2O2. Together with overexpressed H2O2 in tumor site, FeGA reacts with the produced H2O2 to generate abundant ROS under a mild acidic tumour microenvironment for performing CDT by the Fenton reaction, which induced tumour cell death. By this method, the growth of osteosarcoma tumours in mice could be efficiently suppressed without severe side effects. This study presents a novel synergistic CDT and PTT strategy for osteosarcoma treatment.Gallic acid (GA) is a natural polyphenolic compound that is present in rhubarb, eucalyptus, dogwood and other plants . Additio2+ ions will be significantly inhibited in serum. In contrast, FeGA-DOX has excellent and stable Fenton-like catalytic activity in water and serum. Therefore, FeGA-DOX with good catalytic activity and physiological environment stability has broad biological application prospects.The prepared FeGA-DOX + AG NPs with controlled release in thermal response are shown in in vitro photothermal-enhanced CDT. As shown in 2O2 levels in the K7M2wt cells, according to confocal laser scanning microscope images : Group I: saline; Group II: NIR; Group III: FeGA-DOX + AG; Group IV: FeGA + AG + NIR and Group V: FeGA-DOX + AG + NIR. Tumour volume increased rapidly in the saline and NIR treatment groups within nearly 2 weeks of treatment, as shown in 2O2 in the tumour microenvironment to generate ROS to destroy mitochondria, thus improving the chemotherapy sensitivity and tumour killing ability. Hence, in the most effective treatment group, the growth curve of tumour volume in Group V was almost completely inhibited during treatment. Most importantly, no significant changes in the body weight of the treated mice were observed in five groups, which suggest no significant systemic toxicity in all treatment methods (We tested FeGA-DOX + AG-mediated antitumour activity in the K7M2wt tumour-bearing mice. A tumour model was established by subcutaneously injecting 1 × 10 methods . This re methods  showed sIn vivo experiments showed that our unique combination therapy achieved high biosafety, increased the ROS content in tumours and enhanced the antitumour activity.Additionally, the FeGA-DOX treatment did not cause systemic toxicity risk and system damage in mice, as shown in 2O2, which can be converted to ROS via a Fenton reaction under acidic conditions by FeGA. Thus, the synergistic effect of CDT/PTT was realised with the assistance of FeGA-DOX + AG treatment. The strategy overcame the limitation of single CDT or PTT and showed outstanding therapeutic outcomes on osteosarcoma tumour-bearing mice with satisfactory biocompatibility results. Hence, this novel approach can integrate the advantage of CDT/PTT by this H2O2 self-sufficient AG-encapsulated FeGA-DOX, exhibiting potential in clinical applications.In this study, we developed a novel strategy using injectable AG for performing local CDT/PTT with FeGA-DOX NPs to enable osteosarcoma tumour suppression in mice. Upon NIR laser irradiation, the FeGA-DOX NPs emit much heat to induce cell apoptosis by hyperthermia. Meanwhile, a local temperature rise could promote the intratumoural release of FeGA-DOX. It is well known that doxorubicin can promote the generation of H


The International Headache Society has been offering multiple award opportunities for young researchers and clinicians for many years, with the aim of supporting the development of careers in headache science and medicine.In order to assess the outcomes of the International Headache Society award grants, a questionnaire was sent to all previous recipients, investigating a series of aspects related to their work, both during and after award completion.Of 44 total questionnaires sent, 36 were returned. Eighty-one percent of the recipients reported to have remained in the headache field since the award, half of them held a current academic position and over three-quarters had stayed in contact with the host institution. The totality of questionnaire responders stated that the grant had had a significantly positive impact on their careers.The International Headache Society grants have assisted many young researchers in building an academic and clinical career in the field of headache, throughout the years. One of the leading purposes of the International Headache Society (IHS) is to advance headache science and education worldwide. Over the years, the IHS has created and funded a number of training and grant programs, which have been directed at young trainees who are in the process of developing a career in headache research and clinical practice. These awards have typically been undertaken by young investigators who have traveled to large centers with a strong track record of clinical knowledge and research in headache. Different schemes are in place in order to fulfil different purposes, with some awards being directed to clinical and basic scientists alike, and others being offered to neurology trainees or neurologists in order to enhance their clinical skills in headache.IHS Fellowship is a one-year award for a total of £50,000, which was first started in 2011. The scope of the fellowship is to support impactful research from young investigators who are within six years of completing their training . This grant scheme is open to applicants from both a basic or clinical background.The Trainee/Pioneer Program enables young neurologists or neurology trainees from low, lower-middle and upper-middle income countries as defined by the World Bank , the program is held in the same region as the congress, and winning scholars are also funded to attend this. When taking place in alternate years with respect to IHC, the scholars receive funding to attend a regional headache conference. Funding for this scheme has been €5,000 up to 2016 and €6,000 from 2017. The Allied Specialties Scholarship, which was pioneered in 2021, facilitates clinical training or a research project through a scholarship directed at specialists in fields that are allied to headache medicine, such as nurses and physiotherapists.Finally, the With the aim of assessing the outcomes of the different grant schemes, a structured questionnaire was sent via email on behalf of the IHS Education Committee, Science and Research Committee, and Junior’s Group, to all awardees of the above-mentioned grants . All questionnaire recipients had obtained a grant between 2011 to 2020 and all had been actively completed.The questionnaire asked for the recipient’s personal details  and details of the grant . It also included six yes/no questions aimed at understanding the impact the grant had on the recipients’ careers and their ongoing presence in the headache field. These were: 1) Are you still working in the headache/neurology field?; 2) Are you a member of your national headache society?; 3) Are you a member of any IHS or national society Committees/Special Interest Groups?; 4) Have you published any peer-review articles that were related to your stay?; 5) Are you still in contact and collaborating with the headache center you visited?; 6) How have you benefited from the IHS grant? If the recipient answered yes, they were further asked to elaborate details in their answer.The turnover for questionnaire completion was high, over 80% in all three groups and the main results of the questionnaire assessment have been summarized in With regards to IHS Fellowships, of the total 20 awarded since the start of the scheme, five are currently ongoing. The remaining 15 recipients were sent the questionnaires and 13 completed it . Of these, currently 77% have obtained an academic position, which in over half the cases involves headache. Ten former IHS fellows have published at least one first author paper in collaboration with the host institution, and several of these publications have been accepted in high impact journals . Figure The Trainee Program has been awarded to 12 people, and two of these awards are currently ongoing. Eight of the remaining ten recipients who completed this scheme responded to the questionnaire. Of these, all of them have remained active in the headache field, with at least five running independent headache clinics in their country of origin. The large majority of former trainees have been participating in the activities of the IHS or their local headache society in the country of origin, when these exist. Even if this training scheme is mostly clinical in nature and shorter than a fellowship, relevant first author publications have arisen from it as well .A total of 24 Short-Stay Scholarships have been awarded over the years; five of these were originally awarded in 2020 and had to be rescheduled for the current year due to the COVID-19 pandemic. Of the 19 recipients who were contacted, 15 completed the questionnaire. A high proportion of them are still active in the headache field and the large majority has kept some form of contact with the host institution, usually in the context of discussions of complex clinical cases or for scientific collaborations. Importantly, two scholars have been instrumental in setting up a national headache society in their home country, upon returning from the program.Finally, two Allied Specialties Scholarships were awarded in 2021 but deferred to 2022 due to the COVID-19 pandemic. Therefore, questionnaires were not sent out to these recipients.Considering all grants schemes together, a total of 29 awarded recipients (81% of questionnaire completers) have remained in the headache field, however, this was much higher for the trainee program (100%) than for the fellowship program (62%). Looking at individual cases, the main reason for former fellows leaving the field was due to the difficulty in pursuing a scientific career in academia altogether, particularly in the case of those with a basic science background. In the case of clinicians, the headache field was mostly abandoned while undertaking a neurology residency program.The majority of grant recipients have remained in contact or in active collaboration with their host institution; two have since stayed in the same laboratory/research group following the end of the award period, and another three have stayed in the country where the fellowship took place for further career opportunities.Importantly, all the completers of the questionnaire, regardless of the type of award received, judged it to have positively impacted and shaped their careers. The awards were explained to have helped them in several ways through their careers, from increasing knowledge to allowing their growth as scientists and researchers. By opening further funding and collaboration opportunities, the grants were described as having directly determined the work of the awardees, their current lines of research as well as relevant academic and scientific positions obtained after the grant. In several cases, the grant was even instrumental in shaping life choices such as changing country of residence after the end of the award.A limitation of our study is certainly the small sample size due to the fact that the awards are limited in number because of monetary resources, and have started relatively recently. Further, their duration can be long, with several recipients currently involved in a grant and being thus unable to take part in the questionnaire. Repeating this study in a few years might help address these issues and provide further information on the long-term effects of the awards. A further source of bias resides in the fact that the awardees who benefitted the most might have been more inclined to respond. Finally, it is important to acknowledge that, although these schemes have benefitted people from a large variety of countries, they cannot fully counteract the inequality in opportunities between different regions of the world.In conclusion, this short questionnaire survey helped to consolidate the importance of grant schemes created by the IHS in order to promote the careers of young scientists and clinicians in the field of headache. It also shows that improvement needs to be made for future opportunities, particularly in facilitating basic scientists and clinicians who struggle to remain in the academic field. Some possibilities could include the addition of targeted training programs focused on academic development, the creation of specific networking events to connect past awardees with newly awarded recipients, and finally a continuation of support from the host institutions to former grant holders through virtual mentoring programs.The IHS grants have been instrumental in allowing young neurologists and researchers in building an academic and clinical career in the field of headache, throughout the years.