Iberia #25 (1948), Iberic (1949), Flights of Colors #16 (1949), and Early Dynasty (1953), was subjected to an analytical campaign that relied on both non-invasive and micro-invasive techniques. Results confirmed the use of both oil and solvent-based acrylic paints, supporting our primary research and uncovering the first-known occurrence of acrylic binders in Herrera’s Iberia #25. In all cases, the ground layer consists of a mixture of titanium white in its tetragonal form of rutile, anhydrite, and talc, while the color palette was found to be based on both traditional and modern pigments. In most areas, several paint layers appeared to be overlaid on top of one another, revealing a creative process that developed through subsequent compositions. Remnants of earlier paint layers, which appeared to have been scraped off before new ones were applied, were observed directly above the ground in some of the samples examined. In addition, Iberia #25 and Iberic, with analogous geometric and chromatic schemes, underwent technical imaging, which exposed pencil lines and notes underneath the paint layers likely indicative of the intended design and polychromy. Besides corroborating a major alteration in the current scholarship on the availability and use of acrylic-based artists’ paints in post-war Europe, this research provides new insights into Herrera’s materials, techniques, and studio practice. In addition, the results of this scientific study assisted the development of a suitable treatment plan for Iberic in preparation for its display in The Metropolitan Museum of Art’s galleries as part of the museum’s 150th anniversary exhibition Making The Met, 1870–2020.Carmen Herrera, born on May 30th, 1915, is a Cuban American abstract minimalist artist, whose first solo show was held at the Whitney Museum of American Art, New York, in 2016–2017. On this occasion, a scientific study of five paintings from the artist’s time in Paris (1948–1953) revealed her pioneering use of solvent-based acrylic paints in post-war Europe. This article presents a second phase of research into Herrera’s work aiming to shed light on her studio practice and ascertain the possible presence of other early acrylic paints in her pre-1963 artistic production. A selection of four paintings, namely The online version contains supplementary material available at 10.1186/s40494-021-00603-3. My quest is for the simplest of pictorial resolutions”. Having sold her first painting at the age of 89, Herrera achieved international success late in life and only recently began to receive proper recognition by the public and the press. Questioned by journalists about her experience as a woman artist in the post-war years, she explained that she faced some obstacles early in her career “because everything was controlled by men, not just art”.Currently 106 years old, minimalist visual artist Carmen Herrera was born in Havana, Cuba. A master of geometric abstraction, her painting compositions are often dominated by crisp lines and contrasting chromatic planes, likely influenced by her academic studies in architecture and singularly focused on the interactions between space and color. Distinctive traits of Herrera’s painting style are a drive for formal simplicity, an unafraid exploration of the concepts of balance and asymmetry, and a striking sense of color. She stated of her own work, “Carmen Herrera: Lines of Sight [Herrera’s first solo show, entitled of Sight , was helHerrera cares deeply about her materials and she would not be mistaken about this. If she didn't know or didn't recall, she would have said so”Above all, for the type of painting that I do, it was a blessing from God”1.Dana Miller, the exhibition’s curator, had numerous exchanges with Herrera between 2013 and 2016, in which the artist insisted that she began using acrylic paints during her time in Paris. Questioned multiple times about this topic, Herrera was always resolute and specific about using acrylics, even when countered with the prevailing belief that acrylic-based artists’ paints were not available in Europe in the 1940–50s; she even recalled buying them in an art supply store near her studio in the French capital. According to Miller, “Journal of Cultural Heritage [n-butyl methacrylate—polyvinyl acetate, and oil-based alkyds , in works painted during the following three years. Results corroborated Herrera’s memories and highlighted her pioneering use of solvent-based acrylic paints in post-war Europe, well before the official date of introduction to the European market of the “Cryla” brand of artist quality paints by George Rowney & Sons in 1963 [Herrera’s revelations prompted a first scientific investigation of binding media in five paintings from the artist’s time in Paris 1948–1953), carried out by some of the authors of this article and previously published in the Heritage . Unfortu, carriedIberia #25 (1948), Iberic (1949), Flights of Colors #16 (1949), and Early Dynasty (1953) , catalogued as acrylic on burlap , private collection, New York; Iberic (1949), catalogued as acrylic and oil on canvas glued to pebble board , The Metropolitan Museum of Art, New York; Flights of Colors #16 (1949), catalogued as acrylic on canvas , private collection, New York; and Early Dynasty (1953), catalogued as acrylic on canvas , private collection, New York.The present study entailed the analysis of four paintings Fig. , as follIberic, recently donated to The Met, and Iberia #25, shipped to the museum for comparative technical imaging, these works could not be moved from the art gallery or conservation studio where they were on display or under treatment . As a result, scientific analysis of Flights of Colors #16 and Early Dynasty mostly relied on the removal of samples and the use of micro-invasive techniques available in The Met’s Department of Scientific Research (DSR). Samples were collected from selected areas of the four paintings under study to address various questions regarding the artist’s materials and techniques, as follows ; 18 from Iberic (9 scrapings and 9 cross sections); 6 from Flights of Colors #16 (3 scrapings and 3 cross sections); and 9 from Early Dynasty (4 scrapings and 5 cross sections). Scrapings were analyzed with Fourier-transform infrared (FTIR) spectroscopy and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) to provide a characterization of the binding media. Multi-layered samples were mounted as cross sections, photographed under visible polarized and ultraviolet (UV) illumination, and examined by means of Raman spectroscopy and scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM/EDS) to study the painting stratigraphy and gain insight into the variety of pigments and extenders used. Surface-enhanced Raman spectroscopy (SERS), performed on microscopic paint scrapings, provided complementary information on the organic colorants and lake pigments present. In addition to micro-invasive analysis, Iberic and Iberia #25 also underwent close visual examination and imaging campaigns by means of normal and raking light, IR and UV illumination, X-radiography, as well as macro-X-ray fluorescence (MA-XRF) spectroscopy. Experimental conditions for the analytical techniques employed are reported below.Except for Iberic and Iberia #25 were examined under normal and raking light, IR and UV illumination, and with the aid of a stereomicroscope. IR reflectography (IRR) was carried out by means of an Osiris InGaAs near-IR camera with a 6-element, 150-mm focal length, f/5.6 and f/45 lens. The light source was a pair of Lowel Tota lamps  on a variable transformer. Images were captured in the 900–1700 nm spectral range. Distance of the camera from the paintings was 95 cm, resulting in a field size of 42 cm with pixel dimensions of 4096 × 4096. Images were processed and stitched together by means of Photoshop. UV photography was conducted by using a Canon 5DS camera with Kodak 2E and Kodak CC40R filters. Illumination was provided by two 4-foot 40 W GE F40BLB lamps (UV-A glass mercury vapor lights with a peak at 365 nm).Iberic and Iberia #25 using a TFI Hotshot portable industrial X-ray unit, consisting of 603 head and 805D Control. The system has a 0.5-mm focal spot and 96.5-cm radiation beam. Images were recorded onto Industrex Flex XL Blue 5537 plates and digitized with a Carestream HPX-1 scanner with Industrex software. Plates were exposed at 30 kV and 5 mA for 30 s and scanned at a resolution of 50 μm, equivalent to 508 ppi. Images were processed and stitched together by means of NIP2 mosaicking software.Computed radiography was undertaken for Iberic and Iberia #25 were mapped using a Bruker M6 Jetstream instrument with the X-ray source operated at 50 kV and 0.5 mA. A selected area in Iberic and the full Iberia #25 were scanned with a 580-μm spot size, an 800-μm step size, and a dwell time of 90 ms/pixel. The spectra were processed using the Bruker M6 Jetstream software.Analysis of cross sections was performed with a FE-SEM Zeiss Σigma HD system equipped with an Oxford Instrument X-MaxN 80 silicon drift detector (SDD). Back-scattered electron (BSE) imaging, as well as EDS elemental analysis and mapping, were carried out in high vacuum at 20 kV and at an 8.5-mm working distance, on 12-nm carbon-coated samples.−1. The output laser power (10 or 25 mW), number of scans, and integration time were adjusted to prevent damage from overheating and according to the response of the various areas and samples examined. Spectra were interpreted by comparison with published data and library databases available at The Met’s DSR.Analysis was conducted using a Bruker Senterra Raman spectrometer equipped with an Olympus 50× long working distance microscope objective and a charge-coupled device (CCD) detector. A continuous wave diode laser, emitting light at 785 nm, was used as the excitation source, and two holographic gratings (1800 and 1200 rulings/mm) provided a spectral resolution of 3–5 cmAnalysis was carried out by means of the same Raman instrument described above, in association with an Olympus 20× long working distance microscope objective and a Spectra Physics Cyan solid state laser providing excitation at 488 nm. A two-step procedure was applied to the samples prior to analysis in order to account for the possible presence of both free dyes and lake pigments . An outp−1 and obtained as the sum of 128 or 256 scans, depending on the response of the various areas or samples examined. Spectra were interpreted by comparison with published data and library databases available at The Met’s DSR.Analysis was performed with a Hyperion 3000 FTIR spectrometer equipped with a mercury cadmium telluride (MCT) detector. Each sample was crushed in a Spectra Tech diamond anvil cell and analyzed through a 15× objective in transmission mode. Spectra were collected at a resolution of 4 cmAnalysis was conducted on an Agilent 6890 gas chromatograph equipped with a Frontier PY-2020iD Double-Shot vertical furnace pyrolyzer fitted with an AS-1020E Auto-Shot autosampler. The GC was coupled to a 5973N single quadrupole mass selective detector (MSD). Samples of 30–50 µg were weighed out in deactivated pyrolysis sample cups (PY1-EC80F Disposable Eco-Cup LF) on a Mettler Toledo UMX2 Ultra microbalance. Samples were then either pyrolyzed without derivatization or derivatized with tetramethylammonium hydroxide (TMAH) before pyrolysis. Derivatization took place in the same cups as follows: 3–4 µL of 25% TMAH in methanol (both from Fisher Scientific), depending on the sample size, were added directly to the sample in each cup with a 50-µL syringe and, after 1 min, loaded onto the autosampler. The interface to the GC was held at 320 °C and purged with helium for 30 s before opening the valve to the GC column. The samples were then dropped into the furnace and pyrolyzed at 550°C for 30 s. The pyrolysis products were transferred directly to a DB-5MS capillary column (30 m × 0.25 mm × 1 µm) with the helium carrier gas set to a constant flow of 1.5 mL/min. Injection with a 30:1 split was used, in accordance with the sample size. The GC oven temperature program was: 40°C for 1 min; 10°C/min to 320°C; isothermal for 1 min. The Agilent 5973N MSD conditions were set as follows: transfer line at 320°C, MS Quad 150°C, MS Source 230°C, electron multiplier at approximately 1770 V; scan range 33–550 amu. For samples run with TMAH, the detector was turned off until 3 min to avoid saturation by excess of derivatizing agent and solvent. Data analysis was performed on an Agilent MSD ChemStation D.02.00.275 software by comparison with the NIST 2005 spectral libraries.Cross sections were prepared by embedding each sample within a double layer of methyl methacrylate resin (Technovit® 2000 LC). Each layer of resin was cured under UV light for 20 min. Excess resin was ground off and the surface was finely polished using CarbiMet 2 and Micro-mesh abrasive paper of various grits to expose the sample’s stratigraphy. Cross sections were examined by means of a Zeiss Axio Imager M2m microscope, equipped with an Axiocam HRc digital camera and providing 50×, 100×, 200×, 400×, and 500× magnification. Photographs were collected using the AxioVision 4.X.X software.A summary of the results obtained from the scientific analysis of the four paintings under study is reported in Table Iberia #25 by means of IRR did not reveal any discernible underdrawing  in combination with oil and Pinaceae resin , and in which the detection of unsaturated fatty acids suggests incomplete drying. A small amount of phthalic acid, di(3-ethylphenyl)ester, was also identified, possibly serving as a plasticizer, as is Pinaceae resin. The red paint in S7, fluorescent under UV illumination, is a different kind of oil paint, possibly linseed, with an elevated azelaic acid content , reflecting accelerated drying, and also containing Pinaceae resin. The fluorescing pink paint in S5 is similar: an oil paint, possibly linseed, with increased azelaic acid amount . Unsaturated fatty acids and Pinaceae resin are also present. Conversely, sample S9, removed from an area of orange paint, has a binder composed of n-BMA with oil, possibly linseed, with hindered drying that decreased the azelaic acid amount . Unsaturated fatty acids and Pinaceae resin were also detected. As the stratigraphy of this sample is also unknown, this could possibly be another example of the miscibility of early solvent-based acrylic paints with oil paints.Py-GC/MS analysis highlighted the use of different binders in the specimens examined. Sample scrapings S1 and S2, removed from two distinct black fields in the proper left bottom quadrant, appear to contain acrylic and oil binders. The black paint in S1 fluoresces under UV illumination and is mostly composed of sin Fig. . While m in 1963 –12. In c in 1963 . In S1, Iberic did not uncover any compositional changes or underlying composition, yet it confirmed results from visual examination under normal light of a rather thinly painted surface  [Iberic, but not for those in Iberia #25, points to the possible use of different types of paints by the artist in these two works. In addition, as observed for Iberia #25, in this case, too, Ba and Cd are detected in different particles, suggesting that Ba is most likely mixed in the cadmium paints, likely in the form of a filler.Microscopic examination of the samples under UV illumination reveals, in some cases, the presence of abundant particles with a bright greenish-yellow fluorescence. These are visible, for example, in cross sections S5, S6, S7, S9, and S10  . The lum1-xZnxS) –18. As a1-xZnxS) –23. The Iberic for binding media analysis. The major component identified by Py-GC/MS in all these specimens was n-BMA. In addition, an oil-based binding medium was detected, as well as markers for Pinaceae resin, possibly indicating turpentine, commonly used as a solvent for early acrylics, or constituting an early enamel. Moreover, all three samples contain trace amounts of phthalic anhydride along with various phthalates, possibly present as plasticizers in the oil component. Traces or small amounts of styrene were also found. Interestingly, all samples were found to contain α-nicotine, indicating that the painting had been exposed to tobacco smoke.5As detailed in a previous publication by some of the authors , three sn-BMA and phthalic acid. Traces of styrene and Pinaceae resin were also identified in this specimen. Analysis of samples S14-18, on the other hand, showed primarily n-BMA, with smaller amounts of methyl methacrylate, hexyl acrylate, dodecyl acrylate, and hexyldecyl acrylate, as well as relatively small quantities of drying oil . A trace of Pinaceae resin, but no styrene, was identified in this acrylic coating. The additional acrylic monomers do not necessarily reflect a different formulation, but rather the disparity in the proportion of the acrylic phase of the sample. This would appear to indicate that the black passages were selectively saturated by the application of a modified oil coating, while for the red and orange passages an acrylic coating was used. Because of the similarity in composition of the acrylic binder in the paints and the acrylic coating, it could be postulated that Herrera herself may have selectively applied these coatings. An additional sample (S13) was removed from a gummy residue found on the surface, which was identified as an adhesive from pressure sensitive tape derived from a 2-ethylhexyl acrylate polymer [Five samples, namely S12, S14, S15, S16, and S18, are scrapings of surface coating from areas of the composition that appear to have an organic medium applied on top of the black, red, and orange paint. The coating in S12 was found to be composed primarily of oil modified to accelerate its drying , as well as  polymer , 25.Flights of Colors #16, namely S2, S4, and S6, each from one of the three main hues in the painting—pink, orange, and light brown, respectively. All three samples show the same preparation as the two paintings discussed above and contain multiple paint layers. In cross section S2  contains oil, possibly walnut or linseed mixed with poppy oil , and unsaturated fatty acids.As far as the binding media are concerned, two samples removed from pink (S1) and orange (S3) fields were found to be oil-based paints, possibly walnut or linseed mixed with poppy oil, or linseed oil with added heavy metal palmitates . In the pink paint, unsaturated fatty acids, indicating incomplete drying, and Pinaceae resin are also present. The latter was also detected in the orange paint, along with traces of Early Dynasty, too, results confirmed the presence of a preparatory layer based on rutile, anhydrite, and talc. The paint stratigraphy was evaluated through visual examination and the analysis of five cross sections. Among these, S2  and from the top blue paint (S3), are oil paints, possibly linseed, with an elevated azelaic acid amount, indicating accelerated drying . Pinaceae resin was also identified in these samples. The red paint in sample S8 is also an oil paint, although it contains elevated levels of palmitic acid, presumably from the addition of heavy metal palmitates .Iberia #25 (1948), Iberic (1949), Flights of Colors #16 (1949), and Early Dynasty (1953). Questions investigated included the identification of binding media, as well as pigments, colorants, and extenders in the ground and paint layers. Careful inspection of the paint stratigraphy was also carried out to uncover unknown details of Herrera’s studio practice.Expanding on a preliminary study of Carmen Herrera’s pioneering use of solvent-based acrylic paints in post-war Europe , this arIberia #25, dated to 1948. Particularly striking is the artist’s use of both oil and early solvent-based acrylics in the earlier paintings (Iberia #25 and Iberic), but only oil in the later works (Flights of Colors #16 and Early Dynasty). This finding appears to confirm the availability of solvent-based acrylic paints to Herrera in Paris as early as 1948. It also allows one to pose the question as to whether her use of different paint binders for specific colored passages in the early paintings was a thoughtful and intentional selection, or just a case of availability. From the color notations and color changes that we observed in Iberia #25 and Iberic, it is clear that Herrera was very deliberate about her polychromy choices. Similarly, the surface sheen or lack thereof of each color field was probably also important to her, which would explain the presence of selective coating in Iberic. While the working properties of Herrera’s historic paints is not known, solvent-based acrylic paints were derived initially to mimic the solubility of oil paints, thinnable in turpentine, while decreasing the drying time. Working in the living room of her Paris apartment, faster drying paints would have been attractive to Herrera. In her early works, many of the oil paints exhibited high azelaic acid contents, indicating that their drying time was accelerated. These results could indicate that the working properties of solvent-based acrylics and oils with elevated azelaic acid were similar enough that Herrera might not have made a distinction in using them, but rather employed paints that best suited her compositional color scheme. The artist’s exclusive use of oil paints in the later works would also seem to corroborate this hypothesis.As in the authors’ previous study , resultsIberic—featured in the authors’ former study—underwent a more in-depth documentation along with Iberia #25, characterized by analogous geometric design and chromatic arrangement. Interestingly, investigation of these two works uncovered similarities in the choice of materials and, in the case of Iberic, exposed pencil lines and notes below the paint layers that are indicative of the intended geometric and chromatic scheme in specific areas. These findings clearly show how Herrera has carefully planned her compositions starting from the early phases of the creative process, relying at the same time on later adjustments and alterations.In all cases, the ground layer consists of a mixture of titanium white in the form of rutile, anhydrite, and talc. The color palette was found to be based on a relatively broad array of pigments, including bone or ivory black, Prussian blue, cobalt blue, cerulean blue, ultramarine blue, yellow and red ochers, cadmium-based pigments, a purpurin lake, viridian, and zinc white. Microscopic examination of cross sections revealed how Herrera’s creative process typically developed through subsequent compositions. In most instances, multiple layers are indeed present below the thick surface paint in monochromatic areas, sometimes of a similar color as the uppermost paint, but with different chemical composition. In some cases, remnants of earlier paint layers, which appeared to have been scraped off before new ones were applied, were observed right above the ground preparation. This frequently encountered practice of overpainting may be indicative of the artist changing her mind and altering the original color scheme, although it is also possible that, in some cases, she intentionally overlapped different colors to obtain particular hues and visual effects. Additionally, upon incorporation into The Met’s collection, Iberic in preparation for display in The Met’s galleries as part of the 150th anniversary exhibition Making The Met, 1870–2020.Besides corroborating a major update in the current scholarship regarding the availability and use of solvent-based acrylic artists’ paints in post-war Europe, this research provides new insights into Herrera’s materials and studio practice. In addition, the results of this scientific study assisted the development of a suitable treatment plan for Additional file 1: Figure S1. From left to right, sampling sites for Iberia #25 (1948), Iberic (1949), Flights of Colors #16 (1949), and Early Dynasty (1953).Additional file 2: Figure S2. Combined elemental distribution maps for Cd Kα (red) and Se Kα (blue) obtained by MA-XRF for Iberia #25  and for a selected area in Iberic .Additional file 3: Figure S3. Elemental distribution maps of Iberia #25 (1948) obtained by MA-XRF: Ba Kα, Ba Lα, and composite Ba Lα (blue) and Ti Kα (red). The Ba Kα map shows scattering from the stretcher. The painting is also shown at bottom right for comparison.Additional file 4: Figure S4. Left, representative Raman spectra of some of the pigments identified in cross sections S3, S6, and S8 from Iberia #25 (1948), including a carbon-based black, b Prussian blue, and c–f cadmium yellow from yellow, orange, brown, and red areas, respectively. Right, EDS spectrum with intense Ca and P peaks, indicating bone or ivory black.Additional file 5: Figure S5. Reticulation pattern of a coating layer visible on top of the black field of Iberic (1949) near site where cross section S4 was removed.Additional file 6: Figure S6. Left, polarized light and UV light microphotographs of cross section S5 from Iberic (1949), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Cd Lα, Se Kα, S Kα, Ca Kα, P Kα, Mg Kα, Si Kα, and Ti Kα.Additional file 7: Figure S7. Left, polarized light and UV light microphotographs of cross section S4 from Iberic (1949), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Cd Lα, S Kα, Fe Kα, Ca Kα, Al Kα, P Kα, Mg Kα, Si Kα, and Ti Kα.Additional file 8: Figure S8. Left, polarized light and UV light microphotographs of cross section S6 from Iberic (1949), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Cd Lα, Se Kα, S Kα, Ca Kα, Ba Lα, P Kα, Mg Kα, Si Kα, and Ti Kα.Additional file 9: Figure S9. UV light microphotographs of cross sections S5, S6, S7, S9, and S10 from Iberic (1949), displaying particles with a bright greenish-yellow fluorescence.Additional file 10: Figure S10. Left, polarized light and UV light microphotographs of cross section S2 from Flights of Colors #16 (1949), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Cd Lα, S Kα, Fe Kα, Zn Kα, Al Kα, P Kα, Ca Kα, Si Kα, and Cr Kα.Additional file 11: Figure S11. Left, polarized light and UV light microphotographs of cross section S4 from Flights of Colors #16 (1949), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Cd Lα, Se Kα, S Kα, Ca Kα, Al Kα, P Kα, Ba Lα, Fe Kα, and Cr Kα.Additional file 12: Figure S12. Left, polarized light and UV light microphotographs of cross section S6 from Flights of Colors #16 (1949), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Cd Lα, Cr Kα, S Kα, Ca Kα, P Kα, Zn Kα, Co Kα, Al Kα, Si Kα, and Fe Kα.Additional file 13: Figure S13. Left, polarized light and UV light microphotographs of cross section S2 from Early Dynasty (1953), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Fe Kα, Co Kα, S Kα, Ca Kα, Al Kα, Mg Kα, Si Kα, and Ti Kα.Additional file 14: Figure S14. Left, polarized light and UV light microphotographs of cross section S9 from Early Dynasty (1953), with BSE image of a portion of the sample indicated by a yellow rectangle. Right, EDS elemental maps of Cd Lα, Se Kα, S Kα, Ca Kα, Al Kα, P Kα, Zn Kα, Si Kα, and Ti Kα.


Even at this level, however, glycopeptide analysis poses challenges: finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra. Here, we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, e.g., at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, e.g., ± 0.01 Da. Also, new is glycan “wildcard” search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally, the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.Glycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass,   •Filtering mass spectra by key peaks facilitates glycoproteomics search.•Glycan wildcard search finds unanticipated N-linked glycans.•User-defined diagnostic peaks for chemical biology.•Low-abundance O-linked glycosylation on neuropeptides derived from ProSAAS. New features have been added to popular commercial software. The features are demonstrated on several publicly available data sets and are used to discover sulfated N-glycans on Fc gamma receptor IIIa, O-linked glycosylation on rat neuropeptides, and prenylated cysteines in human cells. Site-specific glycosylation analysis  typicallvia in silico digestion from a protein database and combining them with all glycan compositions from one or more glycan composition databases. Byonic then predicts a theoretical glycopeptide spectrum and compares it with each observed spectrum that has a precursor mass within a user-defined precursor mass tolerance. This approach is the logical extension of the approach taken by the most popular proteomics search tools such as Sequest Inclusion List. Controls for limiting attention to tandem mass spectra (denoted MS2 or MS/MS) within a user-defined elution time and m/z range;2)MS2 Peak Filtering. Controls for limiting attention to tandem mass spectra containing user-defined or menu-selected m/z peaks;3)Custom Peaks. User-defined scoring and labels for m/z peaks associated with posttranslational modifications;4)Glycan Wildcard Search. Wildcard or “blind” modifications applied on top of glycan modifications.The database-search approach to glycopeptide identification is quite successful in practice, especially for samples with overall glycosylation already characterized from glycomics experiments, scientific literature, or biological knowledge. Database search, however, cannot identify a glycopeptide with a glycan not in the glycan database. In extreme cases, a search might return no glycopeptide matches at all because of an inaccurate glycan database. Searching with a very large glycan database to minimize missed glycopeptides incurs another problem: false or partially false matches, e.g., biopharmaceutical formulations.Feature (1), Inclusion List, enables the user to run searches targeting MS2 spectra from specific precursors; for example, time slices where glycopeptides have been previously found or where co-eluting “stripes” appear in a heatmap view of a chromatogram. This feature is especially helpful for thorough iterative analysis of simple samples, m/z’s such as 138.055, 204.087, and/or 274.092. As shown previously by Medzihradszky et al. (Feature (2), MS2 Peak Filtering, similar to MS-Filter in Protein Prospector can be used to focus attention on glycopeptide spectra by limiting the search to spectra containing glycan oxonium ions at y et al. , ordinary et al. . Filteriy et al. . MS2 Peay et al.  and phosy et al. , as wellThe Byonic Viewer, which is free software for viewing Byonic search results, already labels many diagnostic peaks for PTMs, including the peaks for the modifications just named, but new feature (3), Custom Peaks, gives the user a way to score and add labels to peaks not included in Byonic’s list of known diagnostic peaks. This feature should be especially useful for unusual samples such as bacterial glycopeptides and unnatural samples such as those produced by chemical biology.Finally, glycan wildcard search offers a partial solution to the problem of choosing a glycan database. With a glycan wildcard with mass ranging from −300 to +300 Da, Byonic will be able to make assignments of the form GLTT[+HexNAc(1)Hex(1)NeuAc(2) + 291.092]PR, if the glycan database contains only HexNAc(1)Hex(1)NeuAc(2) and not HexNAc(1)Hex(1)NeuAc(3). The wildcard glycan is readily correctible to HexNAc(1)Hex(1)NeuAc(3), based on close agreement of the 291.092 wildcard mass with the true mass of 291.095 for NeuAc. Glycan wildcard search is quite different from allowing a large-mass wildcard, say one with range up to +1300 Da, on any serine or threonine. Depending upon the size of the glycan database and the wildcard mass range, glycan wildcard search may be several times faster, especially for N-linked glycosylation. And, more importantly, it allows Byonic to score spectra more accurately, because Byonic will predict peaks for oxonium ions  and neutral losses of NeuAc, NeuAc(1)Hex(1), etc., for a modification of +HexNAc(1)Hex(1)NeuAc(2) + 291.092, but not for an “ordinary” wildcard modification of +1238.415, which is modeled as a nonfragmenting mass delta. Notice that a neutral loss of NeuAc(1)Hex(1) does not make sense for all possible topologies of HexNAc(1)Hex(1)NeuAc(2), but predicting a few extraneous peaks does not have a large impact on Byonic’s score, whereas not predicting highly reliable peaks such as 204 and 274 changes the score dramatically, because the lack of these peaks incurs large score penalties.In the computational experiments described below, we aimed to demonstrate and test search speedup, as well as discovery of unsuspected glycopeptides in ordinary proteomics samples, from MS2 Peak Filtering; improved scoring and spectrum annotation from Custom Peaks; and unanticipated glycan matching, which leads to glycan database improvement or customization, from Glycan Wildcard Search.Features (1), (2), and (4) appear in the June, 2020 release of Byonic (version 3.9.4). Feature (3), Custom Peaks, will appear in the September, 2020 release.1.Rattus rattus (black rat), but the journal article names Sprague-Dawley, which is a Rattus norvegicus (brown rat) strain.Rat brain peptidomics data , Proteom2.ftp://massive.ucsd.edu/MSV000080106. These “Wisconsin data” are Q-Exactive Orbitrap spectra from a study comparing neuropeptides in fed and unfed Sprague-Dawley rats. We used the files 041213_HT_A2.raw, 041213_HT_A3.raw, 041213_HT_A5.raw, 041213_HT_A6.raw, 041213_HT_B4.raw, and 041213_HT_B5.raw_20200705_Byonic.Rat brain peptidomics data , ftp://m3.Human T cells cultured with isotope-targeted glycoproteomics labels (IsoTaG), producing heavy and light O-linked N-azidoacetylGlcNAc in place of O-linked GlcNAc, ProteomeXchange PXD004559 . IsoTaG 4.Human endothelial cells (EA.hy92619) with prenylation probes YnF or YnGG  captured with three different azide reagents . The mas5.Purified CD16a/FcγRIIIa protein  from human NK cells , Proteom6.https://www.hupo.org/HPP-News/6272119Human plasma enriched for glycoproteins from the HUPO HGI study. We used B_glycopepnew_HCDEThcDiTCIDpeptide.raw. The data are available from We used the following publicly available proteomics data sets to test the software improvements:m/z limits apply to the monoisotopic m/z, which may be assigned either by the vendor software or by Byonic’s own precursor-calling code, depending upon the “Precursor and charge assignments” settings on the Advanced tab.  In any case, it is best to use an m/z window that is at least 2 Thomsons wide to account for monoisotopic m/z errors.We added two new tabs to Byonic’s input UI, containing the controls for Inclusion List and MS2 Peak Filtering. The Inclusion List UI allows any number of elution time and m/z boxes, defined by “m/z begin”, “m/z end”, “Elution time begin”, and “Elution time end”; these can be set individually or imported from a spreadsheet (.csv file). The m/z value. The user can also specify an m/z matching tolerance—the default value is 0.02 Thomsons—and a “required number” of peaks to match. For example, the user can require any one of three filtering peaks, or any two of four, and so forth. By default, MS2 Peak Filtering ignores peaks smaller than the 50th most intense peak in the spectrum, i.e., the 51st peak does not count as a match; this “Rank cutoff” value is changeable on the “MS/MS Filtering” tab.As shown in i.e., a search with a tight precursor mass tolerance and automatic filtering to low FDR, oxonium ion filters will provide a speed-up. If run with a loose precursor mass tolerance and no FDR control , filtered search can be used to find likely glycopeptide spectra  that can be targeted for identification in subsequent searches. Less obviously, MS2 Peak Filtering can also be used to find all glycopeptides with the same peptide part by filtering for peaks for predicted or observed Y-ions such as Y0 (bare peptide) and Y1 (bare peptide plus core HexNAc) or predicted or observed peptide fragments such as a2, b2, or y-ions at likely cleavage points.MS2 Peak Filtering can be used to find glycopeptides by filtering for common oxonium ion peaks such as 186.076 and 204.087. If run with a “normal” search, Inclusion List and MS2 Peak Filtering apply a binary filter, include/exclude, to MS2 spectra before they are searched against the protein database; these two new features do not affect Byonic scoring or annotation of included spectra. The third new feature, Custom Peaks, not yet released at the time of writing this article, gives the user a way to change scoring and annotation. The Custom Peaks feature uses a new command added to Byonic’s “fine control format”. For example, this modification ruleWoo/+346.1458@S,T | common2 | CustomPeaks{IsoTaG:347.1531, IsoTaG-36:311.1320, IsoTaG-90:255.0956}via the “CustomPeaks” command. If the top-scoring peptiform does indeed contain a “Woo” modification, then the Byonic Viewer will annotate peaks within the annotation m/z tolerance of 347.1531, 311.1320, and 255.0956 as “IsoTaG”, “IsoTaG-36”, and “IsoTaG-90”. Byonic scoring and Byonic Viewer annotation are two different software modules; one obvious difference is that annotation can be adjusted  after the Byonic search is complete.directs the software to look for peaks at 347.1531, 311.1320, 255.0956 for candidate “peptiforms” (specific modification states of peptides) containing a “Woo” modification on S or T. If the spectrum does contain the peaks within the fragment mass tolerance, the score will increase; if the peaks are not found, the score will decrease. Byonic’s scoring uses both observed and predicted peak intensities in its scoring, and for reliable peaks such as a y-ion on the N-terminal side of proline or an oxonium ion at 274.092 for NeuAc with water loss, Byonic predicts high intensity. Byonic uses a medium predicted peak intensity for new peaks added z = 3+ precursor with monoisotopic m/z 891.352 . If this candidate peptiform outscores all other candidate peptiforms, then Byonic will report a match of EEQYN[+1460.529]{+22.001}STYR, where 1460.529 specifies HexNAc(4)Hex(4). (Notice the curly braces for the wildcard mass delta.) The user might then decide that 22.001 is most likely sodiation, which has a theoretical mass of 21.982.Introduced in Byonic v3.9.4  is the fourth new feature, Glycan Wildcard Search. Byonic has always included “wildcard” modification search. Wildcard search is a version of “error-tolerant”, “blind”, or “open” search—names and details vary—that allows a modification of any mass within a user-specified mass range. When the search scores a potential PSM, the mass of the wildcard is set by the difference between the spectrum precursor mass and the mass of the (possibly modified) peptide without the wildcard. Byonic’s UI for wildcard search on the Modifications tab includes a box labeled “Restrict to residues”. Capital letters input to this box refer to the 20 standard amino acids, and “n” and “c” specify peptide N- and C-terminus. The innovation is that “g” in this box now specifies a wildcard “on top” of a glycan. For example, the glycopeptide EEQYN∗STYR, with N∗ corresponding to asparagine with a glycan of composition HexNAc(4)Hex(4) (sometimes denoted G1) has neutral mass 2649.034 Da, so a wildcard of mass 22.001 would be added to the glycan before scoring this candidate peptiform against an MS2 spectrum with Glycan wildcard search differs from standard wildcard search because Byonic predicts oxonium ions and glycan loss ions such as Y0 (bare peptide), Y1 (bare peptide + HexNAc), etc. from collisional dissociation for a peptide with a glycan and glycan wildcard but not for a peptide with a nonglycan wildcard. Thus, a spectrum must have unusually good peptide backbone fragmentation to match EEQYN{+1482.530}STYR, a nonglycan wildcard, but an MS2 spectrum from collisional dissociation may match EEQYN[+1460.529]{+22.001}STYR based solely on oxonium and glycan loss ions.et al. to test the speed gain from MS2 Peak Filtering for a large glycoproteomics search and to demonstrate that ordinary proteomics samples may contain unsuspected glycopeptides. We used the Uniprot proteome UP000002494 (Rattus norvegicus). We set Byonic for a nonspecific search, 6 ppm precursor tolerance, 20 ppm fragment tolerance, “Total common max” and “Total rare max” set to 1, and variable modificationsWe used the rat brain peptidomics data from Secher Oxidation/+15.994915 @ M | common1Amidated/-0.984016 @ CTerm | common1Gln->pyro-Glu/-17.026549 @ NTerm Q | rare1Glu->pyro-Glu/-18.010565 @ NTerm E | rare1Acetyl/+42.010565 @ NTerm | rare1Deamidated/+0.984016 @ N | common1Phospho/+79.966331 @ S, T | common1et al.  and three different capture reagents . We ran Byonic with and without “CustomPeaks” for the most prominent fragment ions to test the advantage of custom peak prediction. AzRB and AzRTB give the same mass deltas so the following rules suffice:AzRB-YnF/+459.2974 @ C | common2 | CustomPeaks{AzRB-YnF:460.3047,AzRB-YnF+H2S:494.2913}AzRB-YnGG/+527.3600 @ C | common2 | CustomPeaks{AzRB-YnGG:528.3673}Az3MRB + YnF/+629.4393 @ C | common2 | CustomPeaks{Az3MRB-YnF:630.4466}Az3MRB + YnF/+697.5019 @ C | common2 | CustomPeaks{Az3MRB-YnGG:698.5092}e.g., 120.080 and 129.103, but we did not include these as custom peaks, because they are at the low end of the m/z range and have low intensity in the spectra from data set 4.There are also low-mass peaks from geranylgeranylation fragments , e.g., 1We used CD16a/FcγRIIIa protein data to test if Glycan Wildcard Search could discover glycans even in a one-protein data set that had been thoroughly analyzed by experts. Finally, we ran Glycan Wildcard Search on the many-protein HUPO HGI plasma data to evaluate interpretability of spectrum assignments that include wildcard masses.For wildcard searches, both glycan and “ordinary”, as well as “open search” (wide precursor mass tolerance ), we usu7H8NO2 (a fragmentation product of HexNAc), HexNAc minus water, HexNAc, NeuAc minus water, and HexNAc(1)Hex(1), respectively. Every one of the 22 glycopeptide spectrum assignments with posterior error probability (PEP 2D) at most 0.001 found by the unfiltered search was also found by the filtered search, meaning that no confidently assigned glycopeptide spectrum was lost because of filtering. The reverse is automatically true: any peptide-spectrum match (PSM) made by the filtered search will also be made by the larger, unfiltered search and will give the same Byonic score. PEPs, however, may change, because the filtered search has fewer “true” and “false” PSMs for postsearch machine learning (The search described above on the Copenhagen data took 23 h and 30 min using six cores of an 8-core laptop (Intel Xeon CPU running at 2.8 GHz) on a single file (20100805_Velos3_AnSe_Batch12faste_hypot_S3.raw) with 30,882 scans (both MS1 and MS2). The time improved to 9 h and 16 min, about 2.5× speedup, with MS2 Peak Filtering requiring any three of the following five peaks: 138.055, 186.076, 204.087, 274.092, and 366.139, which are Clearning , 34. IndThe 2.5× speedup is surprisingly small. Only 229 out of 20,943 spectra pass the Peak Filtering test, so one might expect a speedup around 90×. The actual speedup is much less because of reduced parallelism for small sets of spectra. Byonic breaks a search into “chunks”, each containing 500 to 2000 spectra, so the filtered search used only a single CPU core. Reducing the minimum size of a chunk from 500 to 50 did not improve the speed, because with such a small number of spectra, most of the computing time is spent generating billions of candidate glycopeptides rather than scoring the relatively small number of spectra. On other searches, the speedup from MS2 Peak Filtering varied from 1×  to more than 20×.A–B are known neuroptides  which was not observed unmodified, probably because of the data acquisition method, which avoided acquiring MS2 scans of singly charged peptides. See the table in roptides , but the2(c), which was not observed in the Wisconsin data. A much longer, unmodified peptide with sequence DGPTGPDVEDAADETPDVDPELLRYLLGRILTGSSEPEAAPAPRRL was observed, which tends to confirm the interpretation that shorter unmodified peptides such as DGPTGPDVED were not observed in the Copenhagen data because of the data acquisition method. Figure 2A but identified from the hypothalamus samples in the Wisconsin data set and compared across six rats. Figure 2B, compared across rats. With so few experimental animals (4 unfed and 2 fed rats), it is impossible to say whether there is any difference in glycosylation between the unfed and fed conditions.The Wisconsin data set confirmed all the proSAAS glycosylation sites from On the epithelial cell data set, the search with Custom Peaks gave 37,391 PSMs and 8847 unique peptides, and an otherwise identical search without Custom Peaks gave 37,307 PSMs and 8820 unique peptides, with “false discovery rate (FDR) 2D” at most 0.01, that is, with estimated percentage of false positives 1%.  These results again appear to be essentially identical, but this time, there is an important difference. The search without Custom Peaks gave 120 PSMs and 41 unique peptides with prenylation, which is a rare but important group of posttranslational modifications, and the search with Custom Peaks gave 148 PSMs and 56 unique peptides with prenylation, an improvement of 37% at the peptide level. Here, we counted the same prenylated peptide captured with AzRB/AzRTB and Az3MRB as unique peptides because the “clicked” modifications are distinct. Expert curation revealed that the FDR on prenylated peptides, which tend to be short peptides with poor fragmentation, is clearly higher than 1%, but we still consider Custom Peaks a success because the search yielded more candidates for curation and the peak annotations made curation faster and more accurate. The peaks for AzRB/AzRTB or Az3MRB clicked to YnGG or YnFF are generally intense peaks, so manual curation discarded all PSMs without the expected peak. e.g., the same peptide with HexNAc(2)Hex(4) and with HexNAc(2)Hex(3) + 162.13 wildcard. To reduce duplication, the wildcard range should be narrowed to roughly the size of the gaps between the masses of glycans in the glycan database. For completeness, we mention one more option related to Glycan Wildcard Search: an open search allowing a wide precursor mass tolerance, e.g., ±250 Da. An open search is at least as fast as a glycan wildcard search with similar mass range on the peptides with Nx{S/T} motif, but the results may require more time and effort to interpret, as we discuss below.Glycan Wildcard Search does not give a speedup for O-linked glycan search. With a glycan wildcard, each S/T will be considered with a glycan or a glycan and a wildcard, and with an ordinary wildcard restricted to S/T, each S/T will be considered with a glycan or a wildcard but not both; the ordinary wildcard search is actually smaller, and an open search is probably still smaller (depending upon the number of S/T’s in the peptides). Also note that, as with Byonic’s original wildcard modification, there is a limit of one glycan wildcard per candidate peptiform.Glycan Wildcard Search (or open search) pairs well with MS2 Peak Filtering to find all the MS2 spectra of glycopeptides with the same peptide part. As a demonstration, we searched for all glycopeptides with peptide YLGNATAIFFLPDEGK from alpha-1-antitrypsin, by filtering for spectra containing three of six peaks: 204.087 (HexNAc oxonium ion), 545.257 (y5), 658.341 (y6), 942.478 (y8), 1755.896 (Y0), and 1958.974 (Y1). We allowed a wildcard of mass from −50 to 700 Da. The search on the HCD spectra of B_glycopepnew_HCDEThcDiTCIDpeptide.raw using a “focused” protein database containing 246 target and decoy proteins took less than 1 min, and every returned PSM had peptide part YLGNATAIFFLPDEGK. (Notice that ETD spectra would require different filtering peaks). We note that in data from glyco-enriched samples such as B_glycopepnew_HCDEThcDiTCIDpeptide.raw, many MS2 spectra, even those of ordinary peptides, contain small peaks at 204.087, 274.092, and 366.139 from background glycopeptides. A “Rank cutoff” of 20 or 30 on filtering peaks should eliminate most ordinary peptide spectra with acceptably low loss of glycopeptides.Finally, we report on the interpretability of N-linked glycan wildcard PSMs. Glycan wildcard search, like open search, generally requires expert interpretation to map PSMs with wildcards or large precursor mass errors to likely explanations such as adducts, nonspecific termini, known but unanticipated PTMs, and—on rare occasion—previously unknown PTMs. Glycan wildcard search differs from open search in that only peptides containing the consensus sequence motif will be allowed large precursor mass errors, thus reducing the time for expert curation.We examined the top-scoring PSMs from a search of B_glycopepnew_HCDEThcDiTCIDpeptide.raw using a glycan wildcard with mass from −30 to +205 Da and the 246-protein focused database. Out of 547 PSMs with error probability (PEP 2D) at most 0.01, the most common glycan wildcard masses, to the closest integer, were 201 to 205 Da (55 occurrences), 161 to 164 (47 occurrences), −1 to 1 (44 occurrences), 62 to 64 (37 occurrences), 37 to 39 (27 occurrences), and 21 to 22 (14 occurrences), which we interpret as HexNAc, Hex, precursor mass out of tolerance, copper(?), calcium or potassium, and sodium, respectively. The work reported here suggests some subjects for future study in both glycobiology and glycoproteomics software. On the biological side, the work reported here found O-linked glycosylation on neuropeptides, similar to corresponding recent discoveries of O-linked glycosylation on human and mouse insulin  and O-liCD16a/FcγRIIIa glycosylation plays a key role in antibody-dependent cellular cytotoxicity. Glycosylation on N-162 enhances binding to antibodies lacking core fucosylation  and hencOn the software side, the work reported here continues our effort to support and improve glycopeptide search within a general-purpose proteomics search program. There are several advantages to building an integrated proteomics tool, rather than a number of special purpose tools, especially for a commercial product: single user interface (UI), code reuse, larger market size, and wider education and training. The finding of unsuspected glyconeuropeptides in unenriched samples from two different laboratories, along with a previous finding on MHC Class I bound glycopeptides , demonstAn obvious next task for software development is to improve the speedup provided by MS2 Peak Filtering. One possibility is to preprocess the spectra for the predefined filtering peaks such as 204.087, 274.092, etc., rather than to check peaks anew for each search. Another, similar, possibility is to cache the peak filtering results of the first search, which would allow user-defined as well as predefined peaks to be included in the speed-up. Glycan Wildcard Search would also benefit from speed improvement. This new feature is currently quite usable for searching for glycopeptides with only N-linked glycans, but it is slower than ordinary wildcard or open search for unanticipated O-linked glycans.Planned improvements include specialized FDR control for glycopeptides and incorporation of retention time information as in . CurrentAuthors declare no competing interests.


Vitis. However, genes responsible for sexual differentiation and its mechanism in V. amurensis remain unknown. Here, we identify a sex determination candidate gene coding adenine phosphoribosyl transferase 3 (VaAPRT3) in V. amurensis. Cloning and sequencing of the VaAPRT3 gene allowed us to develop a molecular marker able to discriminate female individuals from males or hermaphrodites based on a 22-bp InDel. Gene expression and endogenous cytokinin content analysis revealed that the VaAPRT3 gene is involved in sex determination or, to be precise, in female organ differentiation, through regulating cytokinin metabolism in V. amurensis. This study enlarged the understanding of sex determination mechanism in the genus Vitis, and the sex marker could be used as a helpful tool for sexual identification in breeding programs as well as in investigation and collection of V. amurensis germplasms.In the past decade, progress has been made in sex determination mechanism in  Vitis L.) is an important temperate fruit crop widely used for fresh fruit supply, raisin production, and juice and wine making. In China, the area for grapevine cultivation was nearly 0.75 million ha with a harvest of 14.37 million tons  as a putative gene for the control of sexual traits in grapevine. V. vinifera physical map, and developed a marker able to discriminate female plants from hermaphroditic/male ones by analyzing a candidate gene APRT in detail. V. vinifera physical map with three populations, further supporting the previously identified location of grapevine flower sex (V. vinifera plants. Based on the two genes identified by Vitis rotundifolia). Recently, V. vinifera physical map, which included the 143 kb region defined by INP1. Based on the above studies, several genes might be involved in sex determination in grapevines. Among these genes, the APRT3 gene may play an important role. Firstly, APRT gene has been successfully used in development of sex markers in V. vinifera  of 117 individuals was generated by crossing “Zuoshanyi” and “043”, and was planted at the Institute of Pomology, Jilin Academy of Agricultural Science . Another two cross populations were obtained by the cross “Zuoshanyi” × “75042” (named “Z7”) and “Zuoshanyi”× “Shuangqing” (named “ZS”), and they are both growing in the vineyard of Shenyang Agricultural University .Vitis L.)” . The common female parent “Zuoshanyi” possesses two female alleles, and each of the two male parents carries both a male and a female allele and the hermaphroditic parent “Shuangqing” carries both a hermaphroditic and a female allele. Individuals of the populations were scored for sex during the flowering period by visual inspection using the “Descriptors and data standard for grape (tis L.)” . For allV. amurensis germplasms distributed in different areas of the Northeast three provinces in China was also used for marker verification, and they were collected in the 1970s and 1980s and now conserved in the National Field Gene Bank for Amur Grapevine . Information of all the germplasms used in this study is shown in The “Z0” population was used for linkage group construction, and the other two populations consisting of 126 and 93 individuals, respectively, were used for marker verification. A small group of dioecious Young leaves were collected and immediately frozen in liquid nitrogen and stored at −80°C until DNA extraction. The genomic DNA was extracted using an improved cetyl trimethylammonium bromide (CTAB) method . DNA wasVitis vinifera grapevine reference genome (12× version). InDel and SNP calling was performed using the Variant Filtration parameter in the Genome Analysis Toolkit  was used for paired-end sequencing of the two bulks and the two parents. The raw sequence data have been submitted to the NCBI’s Sequence Read Archive (SRA) under the BioProject accession number PRJNA719652. Raw reads were processed to get high-quality clean reads according to the following filtering standards: (1) removing reads with the number of N bases accounting for over 10%; (2) removing reads with the number of low-quality bases  accounting for over 50%; and (3) removing adaptor-polluted reads (reads containing more than five adaptor-polluted bases). The Burrows-Wheeler Aligner software program  was used Toolkit  with the Toolkit .Initial location of sex locus was done by BSA + NGS approach. SNP index was calculated for all variants. The SNP index was the ratio of the number of reads harboring a mutant SNP to that of reads corresponding to the SNP. When all the reads are identical to the reference genome, the index equals 0, whereas when all the reads are different from the reference genome, the index equals 1 . ΔSNP in2+ plus), 0.15 mmol/L of each dNTPs, 5 pmol of each primer, and 1 U Taq DNA polymerase, with the following amplification protocol: 4 min at 94°C followed by 25 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C followed by a final extension of 7 min at 72°C. Amplification products were separated using 10% non-denaturing polyacrylamide gel electrophoresis (PAGE). SNP marker development and genotyping was done using Kompetitive Allele Specific PCR (KASP) technology  and complementary DNAs (cDNA) of “Zuoshanyi”, “043”, “75042”, and “Shuangqing”. The gene sequence was firstly downloaded from Genonscope 8× genome version (https://www.genoscope.cns.fr/cgi-bin/ggb/vitis/geneView?src=vitis&name=GSVIVT00007310001). The full length of the DNA sequence of the VaAPRT3 gene was amplified using Max Master Mix DNA polymerase  with the following primer pair: forward, 5′-CTTTCCCTCTCTCTTATCGA-3′, and reverse, 5′-GGCCAGAGGAATTCATACT-3’. The cDNA from small inflorescence , were calculated using the 2−ΔΔCt method with three biological replicates. Standard PCR was performed for semi-quantitative analysis, with 5-min denaturation at 95°C followed by 30 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s. The PCR products were analyzed on a 1% agarose gel electrophoresis.Using Baggiolini’s description as a reference , flowersPlant samples were the same as described in 2.6. The measurement of endogenous cytokinins was performed as described with minor modifications . In brieχ2 value of 0.14, 0.21, and 0.03 for “Z0”, “Z7”, and “ZS” population, respectively, being less than χ20.05,1 = 3.84. It can be concluded from the segregation pattern of the three populations that the sex genotypes for all the female, male, and hermaphroditic individuals, including the four cross parents, were FF, MF, and HF respectively.Sexual phenotype segregated in all three cross populations used in this study. The segregation of sex fit the single major gene model proposed by V. vinifera reference genome. The average sequencing depths ranged from 17.78 to 33.80 (The Illumina HiSeq X Ten platform generated nearly 49.4 Gb of raw sequencing data (SRA accession: PRJNA719652). A total of 55,848,330, 66,428,654, 100,942,254, and 106,175,722 raw reads were obtained from “Zuoshanyi”, “043”, and the F- and M-bulk, respectively. After removing low-quality, adaptor polluted, and N reads, nearly 312 million of clean reads were produced and 81.63%–89.10% clean reads were mapped on the to 33.80 .V. vinifera reference sequence |. Initial mapping generated a peak of variants in a specific region on chromosome 2 . Accordisequence .V. vinifera reference sequence (12× version) and the position estimation of two genes that were absent from the 12× reference sequence version , and each of “043” and “75042” only expressed another allele , though the DNA sequence of the VaAPRT3 gene in “043” and “75042” was heterozygous. The hermaphroditic “Shuangqing” expressed both the female and the male alleles at this stage. Sequence alignment displayed several SNPs among these alleles, and three of them were non-synonymous, causing amino acid changes at position 61, 124, and 163 in the protein sequences  adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ) in three sexes -N3-phenylurea (CPPU) at a time point just near stage C described in this present study (VaAPRT3 gene at that certain stage. The later increase of cytokinin (iP and cZ) content might be needed for floral organ development.To investigate the affinity of certain ee sexes . The matnt study . So, takV. amurensis intraspecific cross populations. Although the dominance relationship between M and H cannot be illustrated here due to the lack of an HH × MF or an HF × MF crossing pattern as described by V. amurensis revealed a 2:1:1 segregating ratio of male, hermaphroditic, and female progenies from the crossing pattern HF × MF, proving the dominance of M over H  was 18.78 Mb. So, in our linkage group, comparatively, the small physical distance corresponded to a very large genetic distance. What is more, there exists discrepancy in the ratio of physical to genetic distance in our linkage group. For example, between the two adjacent markers InDel1 and InDel2, the ratio was 31.6 kb/cM, but the ratio was only 0.68 kb/cM between InDel2 and InDel3. This phenomenon was also observed in other plants, such as barley, where long genetic distances translated into short physical distances, and vice versa, and that was thought to be related to different frequencies of recombination events at different chromosome regions (Vitis species (In our study, the total genetic distance of the linkage group was 52.8 cM, and the linkage group was constructed based on molecular markers within a 1.43-Mb physical distance, whereas in the  86.4 cM , and the regions . However species .Vitis species almost corresponded to a same small region in the genome, including a 143-kb region in V. riparia or V. cinerea (V. v. sylvestris (V. v. sylvestris (V. amurensis by us, and even a 450-kb region in the subgenus Muscadinia (Vitis species could be explained by the estimated age of the sex-determining region (Vitis and Muscadinia subgenera was only thought to have occurred 18 million years ago (Vitis species. So, the formation of the small sex region can be a very ancient event in grapevine. The small size of the region could result from rare recombination between M and F haplotypes, preventing its evolution to a full chromosome in Vitis (APRT3 gene, based on which a sex marker was developed to distinguish females from males/hermaphrodites, might be a candidate contributing to sex determination in Vitis (VviFSEX gene was considered as a candidate responsible for male sterility, while a VviAPRT3 gene was proved functional in the arrest of carpel development (V. vinifera individuals when used simultaneously (VviINP1 gene containing an 8-bp deletion interrupted male function, making VviINP1 a plausible male-sterility candidate, and another M-linked transcription factor VviYABBY3 was postulated to be associated with female sterility (VaAPRT3 gene was hypothesized to be associated with female organ initiation in V. amurensis. In all, multiple genes may be involved in different sexual organ formation in grapevine, illustrating a complexity of sex determination mechanism in Vitis.The sex locus defined in  cinerea , a 151.8lvestris , a 111-klvestris , a 257-klvestris , the 130scadinia . The colg region . In grapg region . Howeverears ago . That mein Vitis . Althougin Vitis . A VviFSelopment . They alaneously . In a reterility . In thisVaAPRT3 was highly expressed in female and hermaphroditic flowers at the key developmental stage (stage C in V. vinifera subs sylvestris, the VviAPRT3 gene showed a significant high expression in male flowers compared with female or hermaphroditic flowers at a similar developmental stage (APRT3 gene in female organ development. Interestingly, in Arabidopsis thaliana, the APRT-deficient mutants have been reported to be male sterile due to the failure of microspore mitotic division (APRT gene may play different roles in different plant species, or they have different function mechanisms though for the same genus but different species.In our study, the candidate sex-determining gene al stage . These tdivision . So baseVaAPRT3 gene and endogenous cytokinin contents between different sexes. As for the function of the amino acid changes in the protein sequence of the VaAPRT3 gene, we do not have experimental evidence to prove their role in sex determination; thus, this is worth researching in the next step. Recently, an interesting result was obtained by a phylogenic analysis tending to divide the sex-determining region of grapevine into two parts, where the first 8-kb part was M-linked potentially responsible for female sterility, and the F-linked part started from 40 kb downstream, suggesting a possible role in male sterility (VaAPRT3 gene identified in this study is only available to explain female organ differentiation. In the future, with the help of recently published genome of V. amurensis (V. amurensis, and to uncover the sex determination mechanism comprehensively through various experimental methods, such as gene editing and transgenic verification (The sex determination mechanism hypothesized in this study was based on the combination of differential expressions of the terility . This mamurensis , more infication .V. amurensis (V. amurensis depending on the polymorphism inside the candidate gene VaAPRT3 among different sexes. No recombinant individual was found in any of the three cross populations or the natural V. amurensis germplasms used in this study, suggesting the co-segregant character of the marker, at least based on the population size of this study. This marker could be further used in V. amurensis breeding programs, to remove fruitless male individuals in an FF × MF cross population and to retain self-fruitful ones in FF × HF or HF × HF cross populations. As the allelic bands of M and H are identical, the marker can also help distinguish hermaphrodites from males in an HH × MF cross population. The marker can also facilitate the work of investigation and collection of V. amurensis germplasm resources, which dates back to the middle of the 19th century (V. amurensis resources and to keep sex balance of this precious species, the work of investigation and collection is still needed. The marker developed in this study will be very helpful in sex identification when the germplasm resources were at the plantlet stage.The perennial woody grapevine has a large plant size and a long juvenile period, which prolong the breeding process. Molecular markers, especially the ones co-segregating with the target trait, can be very useful for selecting desired individuals at the young seedling stage, thus reducing the cost of time and labor as well as economic input. Chinese researchers developed molecular markers related to sex in murensis , but the century . The invVitis amurensis, we developed a molecular marker for sex identification based on a 22-bp InDel within the sex candidate gene VaAPRT3. This marker is able to distinguish female individuals from males or hermaphrodites in V. amurensis. By analyzing gene polymorphism and expression level as well as endogenous cytokinin content, we hypothesize that the VaAPRT3 gene was involved in sex determination through regulating cytokinin metabolism in V. amurensis. Our findings enriched the theory of sex determination in the genus Vitis.In


Kleg)/vertical stiffness (Kvert) in recreationally trained runners. Thirty-one male runners were recruited and underwent three separate tests. In the first session, the body composition, Kleg, and Kvert at running speeds of 12 and 14 km⋅h–1 were measured. In the second session, isokinetic knee and ankle joint PT at 60°⋅s–1 were tested. The force-time characteristics of the IMTP were evaluated in the final session. Pearson’s product-moment correlations, with the Benjamini–Hochberg correction procedure, showed that the knee flexor concentric and eccentric and extensor concentric PT  were moderate to largely correlated with Kleg and Kvert at 12 and 14 km⋅h–1. The knee extensor eccentric PT  was moderately correlated with the 14 km⋅h–1Kvert. The ankle plantar flexor concentric and dorsiflexor eccentric PT  were largely correlated with Kleg at 12 km⋅h–1. The ankle plantar flexor concentric and eccentric and dorsiflexor eccentric PT  were moderate to largely correlated with Kvert at 12 and 14 km⋅h–1. For IMTP testing, high correlation was only found between the IMPT peak force (PF) and Kvert at 14 km⋅h–1 . Thus, superior leg-spring stiffness in recreational runners may be related to increased knee and ankle joint strength, eccentric muscular capacity, and maximal force production.Neuromuscular characteristics, such as lower-limb joint strength and the ability to rapidly generate force, may play an important role in leg-spring stiffness regulation. This study aimed to investigate the relationship between isokinetic knee and ankle joint peak torque (PT), the force-time characteristics of isometric mid-thigh pull (IMTP), and leg stiffness ( Kleg) and vertical stiffness (Kvert), are typically used to characterize the mechanical properties of a “leg-spring,” representing the lower limbs during running  and the maximum compression in the lower-limb length   . Greaterss (COM) . Previouss (COM)  and thatss (COM) . Thus, eKleg and Kvert after training  are mostly due to neuromuscular adaptation (Neuromuscular strength is an important factor in determining leg-spring stiffness. Improvements in aptation . It has aptation . For exaaptation . The plaaptation  and are aptation . Meanwhiaptation . The kneaptation  and helpaptation . Therefor = 0.50) in older adults, whereas Kleg/Kvert during practical running.However, the relationship between knee and ankle strength and leg-spring stiffness is a topic of debate in scientific and sports communities. As current literature commonly used the ultrasonography technique to measure tendon stiffness or tendinous tissues stiffness, which cannot directly reflect leg-spring stiffness during running. For example, Kleg at 12 km⋅h–1. Notably, the time required to make contact with the ground and propel the body forward is >200 ms at sub-maximal speed (12 km⋅h–1) (The runners’ ability to generate force and power within a transitory period during landing could effectively regulate leg-spring stiffness . The iso km⋅h–1) . It is, Kleg and Kvert) in recreational male runners.Thus, in the present study, we aimed to evaluate the relationship between isokinetic lower-limb joint peak torque (PT) (such as knee and ankle joint concentric and eccentric PT) and IMTP force-time characteristics , and leg-spring stiffness  from a collegiate running club volunteered to participate in this study. The basic information and physical characteristics of the subjects are presented in Kleg, and Kvert at running speeds of 12 and 14 km⋅h–1 were measured. In the second session, PT of concentric and eccentric action at 60°⋅s–1 velocity of isokinetic knee flexor and extensor muscles, and that of ankle plantar flexor and dorsiflexor muscles, was conducted. Force-time characteristics  of the IMTP were evaluated in the final session.This was a cross-sectional study involving recreational runners to investigate the association between leg-spring stiffness and isokinetic lower-limb joint strength, and isometric time force characteristics. Each subject performed three separate test sessions in the laboratory with a minimum rest interval of 48 h. Before each session, participants were asked to refrain from strenuous exercise for 24 h and acquire more than 8 h of adequate sleep; it was also ensured that they did not experience any muscle soreness or fatigue. The researcher examined the subjects’ commitment to the instructions with the help of the questionnaire when the subjects came to the laboratory for testing. In the first session, the body composition, Body composition was measured prior to the leg-spring stiffness test. Subjects were required to achieve a rested and hydrated state on the day before the body composition test. On the measurement day, they were required to avoid drink and food intake 2 h before testing. Standing height was measured with a wall-mounted measuring device , and the measurement accuracy was within 0.1 cm. Bodyweight, fat mass, and fat-free were measured using a bioimpedance analyzer . Body mass index (BMI) was calculated by dividing weight in kilograms by height in square meters.–1, the subjects performed running at speeds of 12 and 14 km⋅h–1 for 4 min, during which time data were recorded for analysis. An 8-camera, 3-dimensional (3D) motion capture system  was used to record the position of 36 reflective markers (100 Hz) to accurately obtain the vertical displacement of the COM [calculated by the body segmental analysis technique , using inverse dynamics. The vGRF was filtered with a cut-off frequency of 200 Hz, and the marker trajectories were filtered with a cut-off frequency of 7 Hz using a fourth-order Butterworth low-pass filter . Based oKleg) and vertical stiffness (Kvert) were calculated according to Morin’s method  between 0.86 and 0.99. Kleg (kN⋅m–1) is defined as the ratio between the maximal vGRF and the maximum compression in the lower-limb length , Δy is the maximal displacement of the COM during ground contact, v is the running velocity (m⋅s–1), and tc is the ground contact time (s) (a threshold of 50 N vGRF was set when the foot contacted the force plate).In this study, Kvert (kN⋅m–1) is the ratio of the maximum vGRF to the vertical displacement when the COM reaches its lowest point , eccentric (Kflex–ecc), knee extensor concentric (Kex–con), and eccentric (Kex–ecc) PT at 60°⋅s–1.In the knee joint isokinetic strength test, the subjects sat on the testing table with the knee at 90° flexion. The shaft of the dynamometer was visually aligned with the lateral condyle of the femur and remained constant during the concentric and eccentric contractions . The shoAdors–con), eccentric (Adors–ecc), plantar flexor concentric (Aplan–con), and eccentric (Aplan–ecc) PT were measured at 60°⋅s–1.In the ankle joint isokinetic strength test, subjects laid supine on the dynamometer seat with their hips and knees fully extended, and the right foot was placed on the foot plate. The shaft of the dynamometer was visually aligned with the lateral malleolus of the foot and kept constant during the concentric and eccentric contractions . The rigThe IMTP test was performed using a portable test rack and two force platforms at a sampling frequency of 1,000 Hz . This test has consistently proven to be highly reliable for ICCs ranging from 0.80 to 0.97 . Before F50,F100,F150,F200,F250,F300, and F350) from the initiation of the pull were collected for every trial. The calculation formula for the RFD is:Force-time curves were collected at rest for 2 s and at full pull for 5 s during the IMTP test, with the highest force generated reported as the absolute PF. In addition, the time-specific forces at 50–350 ms  (The RFD was applied to pre-determined time bands: 0–50 to 0–350 ms (RFD–350ms) . These t–350ms) .Kleg and Kvert at 12 and 14 km⋅h–1) were analyzed using Pearson’s product-moment correlations. Magnitudes of correlation (r) were defined as “small” when r = 0.1–0.3, “moderate” when r = 0.3–0.5, “large” when r = 0.5–0.7, “very large” when r = 0.7–0.9, and “extremely large” when r = 0.9–1.0  and the statistical programming language R.1The Shapiro–Wilk test was used to test data normality. Values are expressed as mean ± SD. The correlations between isokinetic strength (knee and ankle joint concentric and eccentric PT), IMTP force-time characteristics , and leg-spring stiffness ( 0.9–1.0 . All reprocedure . StatistKleg and Kvert at 12 and 14 km⋅h–1) are shown in All isokinetic strength, IMTP time force characteristics, and leg-spring stiffness values are listed in Kflex–con , Kflex–ecc , and Kex–con  was moderately to largely correlated with Kleg at 12 and 14 km⋅h–1. The PT of Aplan–con  and Adors–ecc  was largely correlated with 12 km⋅h–1Kleg after Benjamini–Hochberg adjustment , Kflex–ecc , Aplan–con , Aplan–ecc , and Adors–ecc  was moderately to largely correlated with Kvert at 12 and 14 km⋅h–1. The PT of Kex–con  and Kex–ecc  was moderately to largely correlated with the 14 km⋅h–1Kvert  and Kvert at 14 km⋅h–1, after Benjamini–Hochberg correction  is an important parameter regulating running mechanics because it reflects the amount of energy storage and recoil for a given compression. However, scientific literature on the relationship of neuromuscular strength with leg-spring stiffness remains unclear. This study aimed to investigate the relationship between isokinetic lower-limb joint strength, isometric force-time characteristics, and leg-spring stiffness in recreationally trained male runners. Our primary finding was that the maximum torque of knee and ankle production was strongly correlated with Kleg and Kvert. Furthermore, only the IMTP PF was correlated with Kvert at 14 km⋅h–1. No correlations were found between leg-spring stiffness and specific time force at 50–350 ms or RFD at 0–50 to 0–350 ms. The potential mechanisms of how these neuromuscular characteristics regulate leg-spring stiffness are shown in Leg-spring stiffness  and Kvert values (26.89–29.26 kN⋅m–1) reported are similar to those of previous investigations at similar running speeds , and those of previous studies  between IMTP PF and eccentric strength . At a higher speed (14 km⋅h–1), the muscle activity during the stance phase may reinforce the driving force in the forward direction  was relatively low (–1) and did not require explosive contraction, which may have resulted in a limited correlation with leg-spring stiffness. By contrast, the PF of IMPT was largely correlated with vertical stiffness, as Kvert was calculated using Fmax. Although no statistical correlation was found, the ability to quickly generate a higher force is essential for runners. Theoretically, it enables runners to rapidly push off the ground, thereby decreasing ground contact and resulting in a quicker transition from the braking to the propulsive phase of the running gait . In addition, a large correlation does not imply a cause-effect relationship. Therefore, studies with a longitudinal study design should be performed in the future to confirm the effects of isokinetic joint strength, isometric force-time characteristics, and their manipulation through intervention on leg-spring stiffness.–1 were significantly correlated with Kleg and Kvert. Furthermore, the IMTP PF was largely correlated with Kvert at 14 km⋅h–1. Therefore, leg-spring stiffness is associated with specific neuromuscular characteristics in recreational runners. Runners should focus on improving their knee and ankle joint strength, eccentric muscular capacity, and maximal force production. Although we did not find a correlation between specific time interval force or RFD and leg-spring stiffness, a previous study indicated that quicker force production facilitates leg-spring stiffness regulation . The participants provided written informed consent to participate in this study. Written informed consent was obtained from the participants for the publication of any potentially identifiable images or data included in this manuscript.SC and QZ conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents, materials, and analysis tools, prepared figures and/or tables, authored or reviewed drafts of the manuscript, and approved the final draft. DW conceived and designed the experiments, and authored or reviewed drafts of the manuscript. YS performed the experiments, and prepared figures and/or tables. HD guided the writing of the manuscript, critically revised the manuscript, and approved the final draft. FL conceived and designed the experiments, and approved the final draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to systematically review the efficacy and clinical safety of different courses and doses of tripterygium glycoside (TG) adjuvant methotrexate (MTX) therapy in the treatment of rheumatoid arthritis (RA).  Randomized controlled trials (RCTs) of TG adjuvant MTX therapy in patients with RA were retrieved from SinoMed, China Network Knowledge Infrastructure, WanFang Data, PubMed, Cochrane Library, and Embase from inception to September 30, 2021. The effects and clinical safety evaluations were conducted using RevMan 5.3 software. P = 0.005, P = 0.0001, and P = 0.004, respectively. Simultaneously, the efficacy of the TG adjuvant MTX therapy was improved at either 30 or 60 mg/day over a six-month course compared to MTX monotherapy (P < 0.0001). There was no statistical difference in the effects between the doses of 30 and 60 mg/day after three months (P = 0.82). TG adjuvant MTX also reduced the expression rate of the swollen joint count, tender joint count, erythrocyte sedimentation rate, rheumatoid factor, and C-reactive protein in subgroup analyses with different courses and doses. In terms of hepatic adverse effects (P = 0.28), leukopenia (P = 0.78), gastrointestinal adverse effects (P = 0.17), cutaneous adverse effects (P = 0.94), and irregular menstruation adverse effects (P = 0.29), there was no statistically significant difference with TG adjuvant MTX therapy and MTX monotherapy with different courses and doses.  A total of 9 RCTs and 892 patients with RA were included in this study. In the meta-analysis, a total of 463 and 429 patients were enrolled into the TG adjuvant MTX therapy group and MTX monotherapy group, respectively. In comparison with MTX monotherapy, the results of the analyzed effects showed that the TG adjuvant MTX therapy can achieve 20%, 50%, and 70% improvements in American College of Rheumatology (ACR) criteria ACR20, ACR50, and ACR70 at  TG adjuvant MTX therapy is more effective than MTX monotherapy and is a safe strategy for RA treatment in doses of 30 or 60 mg/day over a treatment course of six months. However, high-quality multicenter RCT studies with large sample sizes are still needed to confirm the effects and clinical safety of different courses and doses of TG adjuvant MTX therapy. Rheumatoid arthritis (RA) is a complex, inflammatory, and systemic autoimmune disease. It is associated with progressive disability, and it primarily affects the lining of the synovial joints , 2. PatiTripterygium wilfordii Hook F, have been widely used as anti-inflammatory drugs and immunosuppressants for treating RA. The effective parts of TGs mainly include diterpenoids, alkaloids, triterpenoids, and glycosides. Most of these active constituents of TGs are effective in anti-inflammation and immunosuppression [Traditional Chinese herbal medicine has achieved considerable progress in treating RA , 8. Trippression . In the pression –12.During RA treatment, adjuvant therapeutics, which involve several drugs that interact with multiple targets in the molecular networks of RA, may achieve better effects compared with monotherapy , 14. TG This study was investigated according to the preferred reporting items for systematic review and meta-analysis (PRISMA) 2020 checklist (Supplementary Information 1).This meta-analysis study and its protocol were registered in PROSPERO of the Centre for Reviews and Dissemination (NO. CRD42021224095).The eligibility criteria for the enrollment of studies into meta-analysis are as follows: (1) randomized controlled trials (RCTs) comparing TG adjuvant MTX therapy and MTX monotherapy, (2) patients with a diagnosis of RA based on the criteria revised by the American College of Rheumatology (ACR) in 1987 or the ACR/European Association of Anti-Rheumatology Annual in 2010 , (3) priThe exclusion criteria are as follows: (1) studies that focused only secondary outcomes or safety outcomes without primary outcomes, (2) patients in studies with additional treatment factors in TG adjuvant MTX therapy group and/or the MTX monotherapy group, and (3) incomplete or duplicate data.∗,” “Tripterygium hypoglaucum,” “Tripterygium hypoglaucums,” “Tripterygiums,” “hypoglaucums, Tripterygium,” “Tripterygium wilfordii,” “Tripterygium wilfordius,” “wilfordius, Tripterygium,” “Leigong Teng,” “Leigong Tengs,” “Teng, Leigong,” “Tengs, Leigong,” “Thundergod Vine,” “Thundergod Vines,” “Vine, Thundergod,” “Vines, Thundergod,” “Arthritis, Rheumatoid∗,” “Caplan Syndrome∗,” “Felty Syndrome∗,” “Rheumatoid Nodule∗,” “Rheumatoid Vasculitis∗,” “Sjogren's Syndrome∗,” “Still's Disease, Adult-Onset∗,” “Rheumatoid Arthritis,” “Caplan Syndromes,” “Caplan's Syndrome,” “Caplans Syndrome,” “Syndrome, Felty,” “Felty's Syndrome,” “Feltys Syndrome,” “Syndrome, Felty's,” “Familial Felty's Syndrome,” “Familial Feltys Syndrome,” “Felty's Syndrome, Familial,” “Syndrome, Familial Felty's,” “Rheumatoid Arthritis,” “Splenomegaly and Neutropenia,” “Familial Felty Syndrome,” “Felty Syndrome, Familial,” “Syndrome, Familial Felty,” “Nodule, Rheumatoid,” “Nodules, Rheumatoid,” “Rheumatoid Nodules,” “Rheumatoid Nodulosis,” “Rheumatoid Noduloses,” “Rheumatoid Vasculitides,” “Vasculitides, Rheumatoid,” “Vasculitis, Rheumatoid,” “Sjogrens Syndrome,” “Syndrome, Sjogren's,” “Sjogren Syndrome,” “Sicca Syndrome,” “Syndrome, Sicca,” “Still's Disease, Adult-Onset,” “Stills Disease, Adult-Onset,” “Adult-Onset Still's Disease,” “Adult-Onset Still's Disease,” “Adult-Onset Stills Disease,” “Still Disease, Adult-Onset,” “Still Disease, Adult-Onset,” “Adult-Onset Still Disease,” “Adult-Onset Still Disease,” “Methotrexate∗,” “Amethopterin,” “Methotrexate, (D)-Isomer,” “Methotrexate, (DL)-Isomer,” “Mexate,” “Methotrexate Sodium,” “Sodium, Methotrexate,” “Methotrexate, Sodium Salt,” “Methotrexate, Disodium Salt,” “Methotrexate Hydrate,” “Hydrate, Methotrexate,” “Methotrexate, Dicesium Salt,” and “Dicesium Salt Methotrexate.” Supplementary Information 2 presents the detailed search strategies employed in this study. Unpublished studies and clinical trial registries were also obtained from the databases of GreyNet International, Open Grey, Cochrane Library, and Chinese Clinical Trial Registry.An electronic search of databases, including SinoMed, China Network Knowledge Infrastructure (CNKI), WanFang Data, PubMed, Cochrane Library and Embase, was performed and completed on September 30, 2021. The references of all retrieved articles were also reviewed for potentially relevant studies. The search strategy involved the use of the following keywords: “TripterygiumThe titles and abstracts of the searched results were independently assessed by two investigators (Q. Geng and B. Liu). The full texts of the potentially eligible studies were then screened for final inclusion in the current study. Disagreements were resolved by a third opinion (C. Lu). The extracted data included the study characteristics , patient characteristics , intervention, control, and outcomes.The two investigators (Q. Geng and B. Liu) used the Cochrane Collaboration  “Risk ofI2 statistic. The I2 values ranged from 0 to 100% and were categorized as follows: I2 <40%, might not be important; 50% < I2 < 90%, moderate heterogeneity; 75% < I2 < 100%, considerable heterogeneity [I2 ≤ 50%,P ≥ 0.1. I2 > 50% and P < 0.1 indicated the possibility of statistical heterogeneity, and random-effects model was adopted. Potential sources of heterogeneity were explored using subgroup and sensitivity analyses. We conducted a subgroup analysis of different courses and doses . The results are presented as forest plots. Sensitivity analyses were also performed to test the stability of the results via the leave-one-out method. Funnel plots and Egger's test were used to assess for publication bias when there are at least 10 studies included in the meta-analysis [Statistical analyses were performed using RevMan 5.3 software from the Cochrane Collaboration. The data were summarized using risk ratio (RR) at 95% confidence intervals (CIs) for dichotomous data. For continuous variables, the mean difference (MD) was used if the outcome measurement units of each study were the same, however, standardized MD (SMD) was used if the measurement units and methods were different among the studies. Statistical heterogeneity was tested by examining both the chi-square test and the ogeneity . A fixedanalysis .The results of the meta-analysis were evaluated using the GRADE method , and degn = 258), CNKI (n = 330), WanFang Data (n = 352), PubMed (n = 19), Cochrane Library (n = 9), and Embase (n = 52). Duplicate checking was conducted using NoteExpress 3.5.0, and 485 papers were ultimately selected. Furthermore, there were 16 reviews and 91 irrelevant studies. After reading through the full articles, the following were excluded from this review: Chinese and English language papers (n = 7), duplicate non-TG studies (n = 201), studies with primary effect points not meeting ACR20, ACR50, or ACR70 (n = 101), experimental studies (n = 105), and non-RA studies (n = 7). Two records were identified by manual searching. A total of nine RCTs [A total of 1020 articles were identified by literature search: SinoMed  (P = 0.0001) (P = 0.004) . The forest plot (P = 0.46) or 60 mg/day  showed no statistically significant difference. The results showed that the difference in ACR50 improvement was not statistically significant  in a three-month course, and the results of the 60 mg dose were consistent with the above results . However, the difference was statistically significant when using a dose of 30 mg   was observed at doses of 30  and 60 mg/day   for doses of 30  and 60 mg/day . The results were all statistically significant in the overall ACR70 efficacies  of doses of 30  and 60 mg/day  (The forest plot (P ˂ 0.00001), TJC , ESR , CRP , and RF  , TJC (P ˂  0.00001), ESR (P ˂  0.00001), CRP (P = 0.009), and RF (P = 0.0002) were statistically significant in a three-month course of TG treatment. Subgroup analysis showed statistically significant differences regardless of whether the dose was 30 or 60 mg/day (Supplementary P = 0.004), TJC (P = 0.02), ESR (P = 0.0005), CRP (P = 0.005), and RF (P < 0.0001) significantly improved, and the difference was statistically significant , leukopenia , gastrointestinal adverse effects , cutaneous adverse effects , and irregular menstruation adverse effects . A heterogeneity test showed that the I2 of each index was less than 50. Hence, the fixed-effects model was adopted.The forest plot (Supplementary P = 0.36), leukopenia , gastrointestinal adverse effects , cutaneous adverse effects , 60 mg/day dose in terms of leukopenia , and gastrointestinal adverse effects  , leukopenia , gastrointestinal adverse effects , cutaneous adverse effects , and irregular menstruation adverse effects  between the six-month course and the 30 mg/day dose. Similar to the result for the 30 mg dose, the result for the 60 mg dose showed that there was no statistical difference in hepatic adverse effects , leukopenia , gastrointestinal adverse effects , cutaneous adverse effects , and irregular menstruation adverse effects  , CRP (I2 = 94%), ESR (I2 = 95%), and RF (I2 = 97%) heterogeneity were high. The robustness and variance of SJC, CRP, ESR, and RF were between 73% and 87%, 90% and 94%, 86% and 95%, and 97% and 98%, respectively. All results remained relatively stable according to the leave-one-out sensitivity analysis.Sensitivity analysis was performed to evaluate the results of different studies. The secondary outcomes of SJC (P = 0.513) and ACR50 (Egger's test: P = 0.539) and on the secondary outcomes of SJC (Egger's test: P = 0.555), TJC (Egger's test: P = 0.834), CRP (Egger's test: P = 0.217), ESR (Egger's test: P = 0.05), leukopenia (Egger's test: P = 0.250), and gastrointestinal adverse reaction (Egger's test: P = 0.844). The symmetrical funnel plot indicated that there was no significant publication bias in this study. These results suggested that there was no significant publication bias in this meta-analysis. In addition, the publication bias could not be assessed for other outcomes because of the small number of studies.Egger's tests were performed to evaluate the publication bias of the studies on the primary outcomes of ACR20 . The main reason for this result is that the study design is not rigorous, and the sample size is not sufficient.This study focused on 9 RCTs with 892 RA participants to evaluate the effects and clinical safety of different courses and doses of TG adjuvant MTX therapy in the treatment of RA in comparison with MTX monotherapy. The test results show that there were no significant differences in the baseline of patients and interventions in all evaluated studies. Most system baselines also showed no significant differences, thus conforming to the principle of meta-analysis. The results of this study showed that TG adjuvant MTX therapy is more effective than MTX monotherapy and is a safe strategy for RA treatment with different courses and doses. For effects, a six-month course of TG adjuvant MTX for RA increased the primary outcomes of ACR20, ACR50, and ACR70, which are the gold standard composite measures used in clinical trials of patients with RA . For secα), and promote the development of RA [α is involved in the pathogenesis of RA by activating endothelial cells and promoting the synthesis and release of inflammatory cytokines [α, however, the expressions of IL-6 and TNF-α decrease more significantly in the adjuvant therapy. It suggests that TG adjuvant MTX treatment can enhance the synergistic effect of the two drugs by inhibiting the activities of IL-6 and TNF-α and control the progression of RA [Currently, although the etiology and pathological mechanism of RA are not clear, a large number of studies have shown that the abnormality and interaction of cytokines play important roles in the pathogenesis of RA , 37. Intnt of RA . TNF-α iytokines . Severalon of RA . This fiThe heterogeneity of secondary outcomes was high. Although we performed subgroup analysis, we were unable to reduce the heterogeneity probably because of the fact that most secondary outcomes were laboratory measures with different reference ranges in different hospitals or probably because there was an uneven distribution among the subgroups in terms of the number of studies and cases.This meta-analysis has some limitations. Firstly, random allocation principle, allocation concealment, and blinding were not described in detail in some of the included documents. Secondly, given that the sample of raw data in this study was small and because all involved trials were conducted in China, the results of this review might have introduced potential selection bias. It may have caused measurement bias in the implementation and outcome evaluation. Thirdly, the high heterogeneity of individual literature may be because of the low quality of the included literature, the difference in sample size, the difference in disease activity of patients, and the difference in the course of treatment. Although these limitations may undermine the level of evidence of this meta-analysis, the selected trials are highly comparable, and the documents were selected in strict accordance with the inclusion criteria.According to the nine RCTs included, a six-month course of TG adjuvant MTX therapy at 30 or 60 mg/day is more effective than MTX monotherapy and is a safe strategy for treating RA. However, because of the low quality of GRADE evaluations and given the limitations of existing research, further high-quality multicenter RCT studies with large sample sizes are needed to confirm the clinical safety of TG combination therapy.