MC3R links nutritional state to childhood growth and the timing of puberty.

The state of somatic energy stores in metazoans is communicated to the brain, which regulates key aspects of behaviour, growth, nutrient partitioning and development1. The central melanocortin system acts through melanocortin 4 receptor (MC4R) to control appetite, food intake and energy expenditure2. Here we present evidence that MC3R regulates the timing of sexual maturation, the rate of linear growth and the accrual of lean mass, which are all energy-sensitive processes. We found that humans who carry loss-of-function mutations in MC3R, including a rare homozygote individual, have a later onset of puberty. Consistent with previous findings in mice, they also had reduced linear growth, lean mass and circulating levels of IGF1. Mice lacking Mc3r had delayed sexual maturation and an insensitivity of reproductive cycle length to nutritional perturbation. The expression of Mc3r is enriched in hypothalamic neurons that control reproduction and growth, and expression increases during postnatal development in a manner that is consistent with a role in the regulation of sexual maturation. These findings suggest a bifurcating model of nutrient sensing by the central melanocortin pathway with signalling through MC4R controlling the acquisition and retention of calories, whereas signalling through MC3R primarily regulates the disposition of calories into growth, lean mass and the timing of sexual maturation.

Bottom-up Histone Post-translational Modification Analysis using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Tandem Mass Spectrometry.

The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K0) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me1-3) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

Conformational Characterization of Peptides and Proteins by 193 nm Ultraviolet Photodissociation in the Collision Cell of a Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometer.

Ultraviolet photodissociation (UVPD) has been shown to be a versatile ion activation strategy for the characterization of peptides and intact proteins among other classes of biological molecules. Combining the high-performance mass spectrometry (MS/MS) capabilities of UVPD with the high-resolution separation of trapped ion mobility spectrometry (TIMS) presents an opportunity for enhanced structural elucidation of biological molecules. In the present work, we integrate a 193 nm excimer laser in a TIMS-time-of-flight (TIMS-TOF) mass spectrometer for UVPD in the collision cell and use it for the analysis of several mass-mobility-selected species of ubiquitin and myoglobin. The resultant data displayed differences in fragmentation that could be correlated with changes in protein conformation. Additionally, this mobility-resolved UVPD strategy was applied to collision-induced unfolded ions of ubiquitin to follow changes in fragmentation patterns relating to the extent of protein unfolding. This platform and methodology offer new opportunities for exploring how conformational variations are manifested in the fragmentation patterns of gas-phase ions.

Ameloblastic carcinoma: A systematic review.

BACKGROUND: Ameloblastic carcinoma (AC) is the most common odontogenic malignancy, constituting approximately 30% of cases in this category. Literature is sparse on malignant odontogenic neoplasms, with a large proportion of current knowledge derived from case reports or small case series. METHODS: A systematic review of case series/case reports of AC was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) Statement guidelines. Demographic and clinical information, including duration of the lesion, location, clinical presentation and radiologic features, were analysed. Additionally, the origin of the lesion (primary/secondary), Ki-67 proliferation index, treatment performed, metastasis, tumour recurrence and prognosis were collected for analysis. RESULTS: A total of 126 studies, including 285 individual cases of AC, were included in this review. Patients presented with a near-equal distribution of painless and painful swellings. ACs presented at a median age of 45 years, with a male-to-female ratio of 1:2. The mandible was most frequently involved, with rare cases extending to involve more than one region, including crossing the midline. Although most lesions presented with poorly-demarcated borders (52.6%), unilocular lesions with well-demarcated borders (47.4%) comprised a substantial number in the sample. The proliferation index was only reported in 27 cases, with a mean score of 42% and a wide range. The probability of tumour recurrence increased, and the survival probability decreased with prolonged follow-up duration. CONCLUSION: This study provides more comprehensive, up-to-date descriptive data on these rare odontogenic malignancies, aiding clinicians and Pathologists with the diagnosis and surgeons in their management of cases.

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

Comparative Molecular Genetics of Odontogenic Keratocysts in Sporadic and Syndromic Patients.

Odontogenic keratocysts (OKCs) are common cysts of odontogenic origin that usually occur as a single nonsyndromic cyst in isolation (sporadic) or as syndromic multiple cysts as a manifestation of naevoid basal cell carcinoma syndrome. Alterations involving the PTCH gene are the most commonly identified factor associated with up to 85% and 84% of naevoid basal cell carcinoma syndrome and sporadic cases, respectively. Other Hedgehog pathway and non-Hedgehog pathway-associated genes have been implicated in the pathogenesis of OKCs. This pilot study used the Affymetrix OncoScan molecular assay to perform a comparative genomic analysis between 4 sporadic and 3 syndromic cases of OKC to identify molecular drivers that may be common and/or distinct in these 2 groups. The majority of alterations detected in both groups were copy number neutral loss of heterozygosity. Despite distinct molecular signatures observed in both groups, copy number neutral loss of heterozygosity alterations involving chromosome 9q affecting not only PTCH but also the NOTCH1 gene were detected in all syndromic and 3 sporadic cases. Loss of heterozygosity alterations involving 16p11.2 affecting genes not previously described in OKCs were also detected in all syndromic and 3 sporadic cases. Furthermore, alterations on 22q11.23 and 10q22.1 were also detected in both groups. Of note, alterations on 1p13.3, 2q22.1, and 6p21.33 detected in sporadic cases were absent in all syndromic cases. This study demonstrates that a more common group of genes may be affected in both groups of OKCs, whereas other alterations may be useful in distinguishing sporadic from syndromic cysts. These findings should be validated in larger OKC cohorts to improve molecular diagnosis and subsequent patient management.

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.

Effect of 2-methoxyestradiol treatment on early- and late-stage breast cancer progression in a mouse model.

The prevalence of breast cancer (BC) continues to increase and is the leading cause of cancer deaths in many countries. Numerous in vitro and in vivo studies have demonstrated that 2-methoxyestradiol (2-ME) has antiproliferative and antiangiogenic effects in BC, thereby inhibiting tumour growth and metastasis. We compared the effect of 2-ME in early- and late-stage BC using a transgenic mouse model-FVB/N-Tg(MMTV-PyVT)-of spontaneously development of aggressive mammary carcinoma with lung metastasis. Mice received 100 mg/kg 2-ME treatment immediately when palpable mammary tumours were identified (early-stage BC; Experimental group 1) and 28 days after palpable mammary tumours were detected (late-stage BC; Experimental group 2). 2-ME was administered via oral gavage three times a week for 28 days after initiation of treatment, whereas control mice received the vehicle containing 10% dimethyl sulfoxide and 90% sunflower oil for the same duration as the treatment group. Mammary tumours were measured weekly over the 28 days and at termination, blood, mammary and lung tissue were collected for analysis. Mice with a tumour volume threshold of 4000 mm3 were killed before the treatment regime was completed. 2-ME treatment of early-stage BC led to lower levels of mammary tumour necrosis, whereas tumour mass and volume were increased. Additionally, necrotic lesions and anti-inflammatory CD163-expressing cells were more frequent in pulmonary metastatic tumours in this group. In contrast, 2-ME treatment of late-stage BC inhibited tumour growth over the 28-day period and resulted in increased CD3+ cell number and tumour necrosis. Furthermore, 2-ME treatment slowed down pulmonary metastasis but did not increase survival of late-stage BC mice. Besides late-stage tumour necrosis, none of the other results were statistically significant. This study demonstrates that 2-ME treatment has an antitumour effect on late-stage BC, however, with no increase in survival rate, whereas the treatment failed to demonstrate any benefit in early-stage BC.

Trapped Ion Mobility Spectrometry and Parallel Accumulation-Serial Fragmentation in Proteomics.

Recent advances in efficiency and ease of implementation have rekindled interest in ion mobility spectrometry, a technique that separates gas phase ions by their size and shape and that can be hybridized with conventional LC and MS. Here, we review the recent development of trapped ion mobility spectrometry (TIMS) coupled to TOF mass analysis. In particular, the parallel accumulation-serial fragmentation (PASEF) operation mode offers unique advantages in terms of sequencing speed and sensitivity. Its defining feature is that it synchronizes the release of ions from the TIMS device with the downstream selection of precursors for fragmentation in a TIMS quadrupole TOF configuration. As ions are compressed into narrow ion mobility peaks, the number of peptide fragment ion spectra obtained in data-dependent or targeted analyses can be increased by an order of magnitude without compromising sensitivity. Taking advantage of the correlation between ion mobility and mass, the PASEF principle also multiplies the efficiency of data-independent acquisition. This makes the technology well suited for rapid proteome profiling, an increasingly important attribute in clinical proteomics, as well as for ultrasensitive measurements down to single cells. The speed and accuracy of TIMS and PASEF also enable precise measurements of collisional cross section values at the scale of more than a million data points and the development of neural networks capable of predicting them based only on peptide sequences. Peptide collisional cross section values can differ for isobaric sequences or positional isomers of post-translational modifications. This additional information may be leveraged in real time to direct data acquisition or in postprocessing to increase confidence in peptide identifications. These developments make TIMS quadrupole TOF PASEF a powerful and expandable platform for proteomics and beyond.

Top-"Double-Down" Mass Spectrometry of Histone H4 Proteoforms: Tandem Ultraviolet-Photon and Mobility/Mass-Selected Electron Capture Dissociations.

Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approach─ultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)─is illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (∼75%) with significant faster data acquisition in the ToF MS/MS platform (∼3 vs ∼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (∼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.

Tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS): a promising analytical method for investigating heterogenous samples.

Ion mobility spectrometry/mass spectrometry (IMS/MS) is widely used to study various levels of protein structure. Here, we review the current state of affairs in tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS). Two different tTIMS/MS instruments are discussed in detail: the first tTIMS/MS instrument, constructed from coaxially aligning two TIMS devices; and an orthogonal tTIMS/MS configuration that comprises an ion trap for irradiation of ions with UV photons. We discuss the various workflows the two tTIMS/MS setups offer and how these can be used to study primary, tertiary, and quaternary structures of protein systems. We also discuss, from a more fundamental perspective, the processes that lead to denaturation of protein systems in tTIMS/MS and how to soften the measurement so that biologically meaningful structures can be characterised with tTIMS/MS. We emphasize the concepts underlying tTIMS/MS to underscore the opportunities tandem-ion mobility spectrometry methods offer for investigating heterogeneous samples.

Reservoir sedimentation and spatiotemporal land use changes in their watersheds: the case of two sub-catchments of the White Volta Basin.

Reservoir storage is compromised by sedimentation for which reason it has become an important matter in reservoir operation and management. While many studies have investigated sediment deposition rate in reservoirs, few have analyzed reservoir sedimentation from their catchment's land use change perspective. Based on bathymetric survey conducted on two reservoirs in the White Volta Basin in 2020 and analysis of four Landsat satellite imagery (1986, 1996, 2006, and 2020) within their watersheds, this study assessed the land cover change within the watersheds to draw inferences on the rate of sedimentation of the reservoirs located downstream of their catchments. The results revealed rapid sedimentation in the small-sized reservoir (Vea), with an annual sedimentation rate of 0.304% and a nominal sedimentation rate of 0.17% for the mid-sized reservoir (Tono). Furthermore, the savannah forest within the Vea catchment declined drastically from 29.4% (1985) to 9.9% (2020) influenced by the rapid expansion of farmlands from 18.7% to 47.9% within the same period, respectively. On the other hand, the savannah forest within the Tono catchment declined from 34.7% (1985) to 21.6% (2020) due to farmland expansion from 19.2% to 39% within the same period, respectively. The higher sedimentation rate observed in the small-sized reservoir was observed to be worsened by extensive tree cover removal in its catchment. Therefore, land cover characteristics within a watershed have a significant bearing on the rate of sedimentation in the reservoirs located downstream of their catchment. Hence, adopting a multi-sectorial approach to dealing with land use management is necessary to sustain reservoirs' storage.

Surface-Induced Dissociation of Protein Complexes Selected by Trapped Ion Mobility Spectrometry.

Native mass spectrometry (nMS), particularly in conjunction with gas-phase ion mobility spectrometry measurements, has proven useful as a structural biology tool for evaluating the stoichiometry, conformation, and topology of protein complexes. Here, we demonstrate the combination of trapped ion mobility spectrometry (TIMS) and surface-induced dissociation (SID) on a Bruker SolariX XR 15 T FT-ICR mass spectrometer for the structural analysis of protein complexes. We successfully performed SID on mobility-selected protein complexes, including the streptavidin tetramer and cholera toxin B with bound ligands. Additionally, TIMS-SID was employed on a mixture of the peptides desArg1 and desArg9 bradykinin to mobility-separate and identify the individual peptides. Importantly, results show that native-like conformations can be maintained throughout the TIMS analysis. The TIMS-SID spectra are analogous to SID spectra acquired using quadrupole mass selection, indicating little measurable, if any, structural rearrangement during mobility selection. Mobility parking was used on the ion or mobility of interest and 50-200 SID mass spectra were averaged. High-quality TIMS-SID spectra were acquired over a period of 2-10 min, comparable to or slightly longer than SID coupled with ion mobility on various instrument platforms in our laboratory. The ultrahigh resolving power of the 15 T FT-ICR allowed for the identification and relative quantification of overlapping SID fragments with the same nominal m/z based on isotope patterns, and it shows promise as a platform to probe small mass differences, such as protein/ligand binding or post-translational modifications. These results represent the potential of TIMS-SID-MS for the analysis of both protein complexes and peptides.

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies.

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K0 = 0.185-1.84 cm2·V-1·s-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCSN2) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6-73), Tuning Mix oligomers (n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1-4), carbonic anhydrase, ß clamp (n = 1-4), topoisomerase IB, bovine serum albumin (n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1-2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 Å2 with <0.6% relative standard deviation (RSD)).

Photo-Detachable Self-Cleaning Surfaces Inspired by Gecko Toepads.

Strong, reversible, and self-cleaning adhesion in the toe pads of geckos allow the lizards to climb on a variety of vertical and inverted surfaces, regardless of the surface conditions, whether hydrophobic or hydrophilic, smooth or tough, wet or dry, clean or dirty. Development of synthetic gecko-inspired surfaces has drawn a great attention over the past two decades. Despite many external-stimuli responsive mechanisms (i.e., thermal, electrical, magnetic) have been successfully demonstrated, smart adhesives controlled by light signals still substantially lag behind. Here, in this report, we integrate tetramethylpiperidinyloxyl (TEMPO)-doped polydopamine (PDA), namely, TDPDA, with PDMS micropillars using a template-assisted casting method, to achieve both improved adhesion and self-cleaning performances. To the best of our knowledge, this is the first report on PDA being used as a doping nanoparticle in bioinspired adhesive surfaces to achieve highly efficient self-cleaning controllable by light signals. Notably, the adhesion of the 5% TDPDA-PDMS sample is ∼688.75% higher than that of the pure PDMS at the individual pillar level, which helps to explain the highly efficient self-cleaning mechanism. The sample surfaces (named TDPDA-PDMS) can efficiently absorb 808 nm wavelength of light and heat up from 25 °C to 80.9 °C in 3 min with NIR irradiation. The temperature rise causes significant reduction of adhesion, which results in outstanding self-cleaning rate of up to 55.8% within five steps. The exploration of the photoenabled switching mechanism with outstanding sensitivity may bring the biomimetic smart surfaces into a new dimension, rendering varied applications, e.g., in miniaturized climbing robot, artificial intelligence programmable manipulation/assembly/filtration, active self-cleaning solar panels, including high output sensors and devices in many engineering and biomedical frontiers.

Tandem-trapped ion mobility spectrometry/mass spectrometry coupled with ultraviolet photodissociation.

RATIONALE: Tandem-ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem-ion mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes. METHODS: Here, we describe the coupling of the separation capabilities of tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis. RESULTS: We establish the feasibility of dissociating intact proteins by UV irradiation at 213 nm between the two TIMS devices in tTIMS/MS and at pressure conditions compatible with ion mobility spectrometry (2-3 mbar). We validate that the fragments produced by UVPD under these conditions result from a radical-based mechanism in accordance with prior literature on UVPD. The data suggest stabilization of fragment ions produced from UVPD by collisional cooling due to the elevated pressures used here ("UVnoD2"), which otherwise do not survive to detection. The data account for a sequence coverage for the protein ubiquitin comparable to recent reports, demonstrating the analytical utility of our instrument in mobility-separating fragment ions produced from UVPD. CONCLUSIONS: The data demonstrate that UVPD carried out at elevated pressures of 2-3 mbar yields extensive fragment ions rich in information about the protein and that their exhaustive analysis requires IMS separation post-UVPD. Therefore, because UVPD and tTIMS/MS each have been shown to be valuable techniques on their own merit in proteomics, our contribution here underscores the potential of combining tTIMS/MS with UVPD for structural proteomics.

Factors associated with North-South research collaboration focusing on HIV/AIDS: lessons from ClinicalTrials.gov.

BACKGROUND: A North-South (N-S) research collaboration is one way through which research capacity of developing countries can be strengthened. Whereas N-S collaboration in HIV/AIDS area may result in research capacity strengthening of Southern partners, it is not clear what factors are associated with this type of collaboration. The study aims to characterize N-S research collaboration focusing on HIV/AIDS and to determine factors associated with such N-S research collaborations. METHODS: Clinical trial data on HIV/AIDS-related studies conducted between 2000 and 2019 were obtained from ClinicalTrials.gov. Using these data, we characterized N-S collaborative studies focusing on HIV/AIDS and summarized them using frequencies and percentages. To determine factors associated with these studies, we used logistic regression and reported results as adjusted odds ratios with Wald 95% confidence intervals. RESULTS AND DISCUSSION: Of the 4,832 HIV/AIDS-related studies retrieved from the registry, less than one-quarter (n = 1133, 23%) involved a Southern institution, with 77% of these studies classified as N-S collaborations. Majority of these studies have single PI (50%), are conducted at single location (39%); have large sample sizes (41%); are federally-funded (32%) or receive funding from other sources (32%); are intervention studies (64%); and involve a mixture of male and female participants (58%) and adult participants (54%). Single PIs (as opposed to multiple PIs) were more likely to be from the North than South institution (odds ratio = 5.59, 95%CI: 4.16 - 11.57). Trend analyses showed that N-S research collaborations produced HIV/AIDS-related studies at a faster rate than S-S research collaborations. N-S collaborations involving female or children produced HIV/AIDS-related studies between 2000 and 2019 at a significantly faster rate than S-S collaborations involving females and children during the same period. Holding other factors constant, N-S collaborative research focusing on HIV/AIDS are associated with: multiple PIs as opposed to single PI, multiple institutions as opposed to a single institution, multiple locations as opposed to a single location, large number of participants as opposed to small sample sizes, and public funding as opposed to industry funding. Almost half of these studies had a Northern PI only, about one-third had a Southern PI only, and much fewer had PIs from both North and South. However, these studies were less likely to receive funding from other sources than industry funding. CONCLUSIONS: HIV/AIDS-related research is increasingly becoming a more collaborative global research involving more N-S collaborations than S-S collaborations. Factors associated with N-S collaborative studies focusing on HIV/AIDS include multiple PIs, institutions, and locations; large sample sizes; publicly funded; and involve vulnerable populations such as women and children. Whereas almost half of these studies have a Northern PI only, about one-third have a Southern PI only, and much fewer have PIs from both North and South. Our results inform future design and implementation of N-S research collaborations in this area. Suggestions for improvement of ClinicalTrials.gov registry are provided.

Barnyard grass [Echinochloa crus-galli (L.) Beauv] leaves extract against tomato pests.

BACKGROUND: Tomato is one of the widely cultivated crops worldwide that is affected by several pests, such as fungi (Fusarium oxysoporum, Alternaria solani), bacteria (Pectobacterium carotovorum) and weeds (Cyperus iria L., Amaranthus spinosus). A growing interest has emerged for developing plant-derived pesticidal compounds to counteract these pests. One attractive alternative is to use barnyard grass (Echinochloa crus-galli), known to be widely resistant to synthetic herbicides, as a potential biopesticide compound source. RESULTS: Phytochemical screening of the crude extract showed that phenolic compounds were the most abundant component present in barnyard grass. The crude extract was evaluated for antifungal, antibacterial and herbicidal activities. Bioassays showed inhibition against F. oxysporum (10.73 ± 1.30%) and A. solani (20.47 ± 3.51%), the causative agent of Fusarium rot and early blight disease in tomato, respectively. Antibacterial activity against P. carotovorum gave a mean zone of inhibition (paper disc diffusion assay) of 17.00 ± 1.00 mm and an IC50 (dose-response assay) of 2.26 mg mL-1 was observed. Dose-responsive herbicidal activity on the lettuce seed germination bioassay produced an IC50 of 459.30 ppm. Selectivity studies showed inhibition towards C. iria and A. spinosus with no effect on tomato. Lastly, bioassay-guided fractionation coupled with untargeted metabolomics studies using ultra-performance liquid chromatography with diode array detection-tandem mass spectrometry mass analyses revealed loliolide and tricin as the putative metabolites present in barnyard grass. CONCLUSION: To date, this is the first reported study on using barnyard grass as a potential alternative biopesticide against tomato pests such as fungi, bacteria and weeds. © 2021 Society of Chemical Industry.

Structural Analysis of the Glycoprotein Complex Avidin by Tandem-Trapped Ion Mobility Spectrometry-Mass Spectrometry (Tandem-TIMS/MS).

Glycoproteins play a central role in many biological processes including disease mechanisms. Nevertheless, because glycoproteins are heterogeneous entities, it remains unclear how glycosylation modulates the protein structure and function. Here, we assess the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (tandem-TIMS/MS) to characterize the structure and sequence of the homotetrameric glycoprotein avidin. We show that (1) tandem-TIMS/MS retains native-like avidin tetramers with deeply buried solvent particles; (2) applying high activation voltages in the interface of tandem-TIMS results in collision-induced dissociation (CID) of avidin tetramers into compact monomers, dimers, and trimers with cross sections consistent with X-ray structures and reports from surface-induced dissociation (SID); (3) avidin oligomers are best described as heterogeneous ensembles with (essentially) random combinations of monomer glycoforms; (4) native top-down sequence analysis of the avidin tetramer is possible by CID in tandem-TIMS. Overall, our results demonstrate that tandem-TIMS/MS has the potential to correlate individual proteoforms to variations in protein structure.

Accurate Identification of Isomeric Glycans by Trapped Ion Mobility Spectrometry-Electronic Excitation Dissociation Tandem Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) has become a powerful tool for glycan structural characterization due to its ability to separate isomers and provide collision cross section (CCS) values that facilitate structural assignment. However, IM-based isomer analysis may be complicated by the presence of multiple gas-phase conformations of a single structure that not only increases difficulty in isomer separation but can also introduce the possibility for misinterpretation of conformers as isomers. Here, the ion mobility behavior of several sets of isomeric glycans, analyzed as their permethylated derivatives, in both nonreduced and reduced forms, was investigated by gated-trapped ion mobility spectrometry (G-TIMS). Notably, reducing-end reduction, commonly performed to remove anomerism-induced chromatographic peak splitting, did not eliminate the conformational heterogeneity of permethylated glycans in the gas phase. At a mobility resolving power of ∼100, 14 out of 22 structures showed more than one conformation. These results highlight the need to use IMS devices with high mobility resolving power for better separation of isomers and to acquire additional structural information that can differentiate isomers from conformers. Online electronic excitation dissociation (EED) MS/MS analysis of isomeric glycan mixtures following G-TIMS separation showed that EED can generate isomer-specific fragments while producing nearly identical tandem mass spectra for conformers, thus allowing confident identification of isomers with minimal evidence of any ambiguity resulting from the presence of conformers. G-TIMS EED MS/MS analysis of N-linked glycans released from ovalbumin revealed that several mobility features previously thought to arise from isomeric structures were conformers of a single structure. Finally, analysis of ovalbumin N-glycans from different sources showed that the G-TIMS EED MS/MS approach can accurately determine the batch-to-batch variations in glycosylation profiles at the isomer level, with confident assignment of each isomeric structure.