Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage ...through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 μm i.d. in-house-packed nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ∼20 nL/min flow rate provided by the LC separation. Ultrasensitive LC–MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 μm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.
Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago ...truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.
Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze ...low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 μm i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.
Hydration at low temperatures, commonly referred to as cold stratification, is widely used for releasing dormancy and triggering germination in a wide range of species including wheat. However, the ...molecular mechanism that underlies its effect on germination has largely remained unknown. Our previous studies showed that methyl-jasmonate, a derivative of jasmonic acid (JA), promotes dormancy release in wheat. In this study, we found that cold-stimulated germination of dormant grains correlated with a transient increase in JA content and expression of JA biosynthesis genes in the dormant embryos after transfer to 20 °C. The induction of JA production was dependent on the extent of cold imbibition and precedes germination. Blocking JA biosynthesis with acetylsalicylic acid (ASA) inhibited the cold-stimulated germination in a dose-dependent manner. In addition, we have explored the relationship between JA and abscisic acid (ABA), a well-known dormancy promoter, in cold regulation of dormancy. We found an inverse relationship between JA and ABA content in dormant wheat embryos following stratification. ABA content decreased rapidly in response to stratification, and the decrease was reversed by addition of ASA. Our results indicate that the action of JA on cold-stratified grains is mediated by suppression of two key ABA biosynthesis genes, TaNCED1 and TaNCED2.
Objectives
To determine whether hearing aid (HA) use affects social perceptions of general public adults and age‐matched peers and if so, determine if effects are modulated by lack of societal ...representation of pediatric HAs.
Methods
A 10‐year‐old boy was presented in six photographic conditions with and without HAs and eyeglasses (a worn sensory aid with wider societal representation). HAs were presented in neutral skin tone and bright blue colors. Photographic conditions were embedded into web‐based surveys with visual analog scales to capture social perceptions data and sourced to 206 adults (age 18–65) and 202 peers (age 10) with demographic characteristics representative of the general US population. Mean differences in scores for each condition compared to control images were computed using two‐tailed t‐tests.
Results
In both adult and child respondents, HAs were associated with decreased athleticism, confidence, health, leadership, and popularity. Glasses were associated with decreased athleticism and popularity but increased intelligence, overall success, and in the child respondents, friendliness. When worn in combination, the beneficial effects of glasses were mitigated by brightly colored but not neutrally colored HAs.
Conclusion
Negative effects of pediatric HAs on social perceptions may be influenced by poor societal representation of HAs. These results suggest that greater representation of pediatric HAs is necessary to make society more inclusive for children with hearing loss.
Level of Evidence
4 Laryngoscope, 131:E2387–E2392, 2021
Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using ...an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two domains encoded by Medicago truncatula CEP1 (MtCEP1). Novel arabinosylated and hydroxylated peptides were identified in root cultures overexpressing MtCEP1. The five most abundant CEP peptides were hydroxylated and these species were detected also in low amounts in vector control samples. Synthetic peptides with different hydroxylation patterns differentially affected root development. Notably, the domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5 mM KNO₃ and stimulated the highest increase in nodule number when grown with 0 mM KNO₃. Inhibition of lateral root emergence by D1:HyP4,11 was not alleviated by removing peptide exposure. In contrast, the domain 2 peptide hydroxylated at Pro11 (D2:HyP11) increased stage III–IV lateral root primordium numbers by 6-fold (P < 0.001) which failed to emerge. Auxin addition at levels which stimulated lateral root formation in wild-type plants had little or no ameliorating effect on CEP peptide-mediated inhibition of lateral root formation or emergence. Both peptides increased and altered the root staining pattern of the auxin-responsive reporter GH3:GUS suggesting CEPs alter auxin sensitivity or distribution. The results showed that CEP primary sequence and post-translational modifications influence peptide activities and the improved isolation procedure effectively and reproducibly identifies and characterises CEPs.
Plants can respond to insects that feed with stylet mouthparts using various processes that are initiated via the salicylic acid metabolic pathway. In Australia, scale insects of the genus
can cause ...economic damage to grapevines as they feed on the vines and produce honeydew as a waste by-product, which supports the growth of black sooty mould on fruit and leaves, potentially affecting the plant growth and yield. Using rootlings of Sauvignon Blanc (SB, resistant) and Chardonnay (Char, susceptible), the growth and production of volatile organic compounds (VOCs) following exposure to scale insect infestations were measured under controlled greenhouse conditions. At harvest, the numbers of scale insects per five leaves were higher on plants infested at the start of the study compared with the control plants. Infested SB had increased dry root and shoot mass compared with the SB control, which was also the case with Char (control and infested). Leaf volatiles differed between cultivars in response to scale infestation. Benzyl alcohol decreased among infested SB plants compared with the other treatments. A change in the salicylic acid pathway as indicated by the change in benzyl alcohol may cause the increased growth in SB associated with the increased scale insect infestation.
Recent studies have established that dietary protein restriction improves metabolic health and glucose homeostasis. SLC6A19 (B⁰AT1) is the major neutral amino acid transporter in the intestine and ...carries out the bulk of amino acid absorption from the diet. Mice lacking SLC6A19 show signs of protein restriction, have improved glucose tolerance, and are protected from diet-induced obesity. Pharmacological blockage of this transporter could be used to induce protein restriction and to treat metabolic diseases such as type 2 diabetes. A few novel inhibitors of SLC6A19 have recently been identified using in vitro compound screening, but it remains unclear whether these compounds block the transporter in vivo. To evaluate the efficacy of SLC6A19 inhibitors biomarkers are required that can reliably detect successful inhibition of the transporter in mice. A gas chromatography mass spectrometry (GC-MS)-based untargeted metabolomics approach was used to discriminate global metabolite profiles in plasma, urine and faecal samples from SLC6A19ko and wt mice. Due to inefficient absorption in the intestine and lack of reabsorption in the kidney, significantly elevated amino acids levels were observed in urine and faecal samples. By contrast, a few neutral amino acids were reduced in the plasma of male SLC6A19ko mice as compared to other biological samples. Metabolites of bacterial protein fermentation such as p-cresol glucuronide and 3-indole-propionic acid were more abundant in SLC6A19ko mice, indicating protein malabsorption of dietary amino acids. Consistently, plasma appearance rates of
C-labelled neutral amino acids were delayed in SLC6A19ko mice as compared to wt after intra-gastric administration of a mixture of amino acids. Receiver operating characteristic (ROC) curve analysis was used to validate the potential use of these metabolites as biomarkers. These findings provide putative metabolite biomarkers that can be used to detect protein malabsorption and the inhibition of this transporter in intestine and kidney.
The study used the mixed probiotics of Bacillus amyloliquefaciens 54A and B. pumilus 47B isolated from striped catfish (Pangasianodon hypophthalmus) intestine aiming to stimulate growth performance, ...innate immunity, stress tolerance of striped catfish. The average weight gain (AWG), specific growth rate (SGR), and feed conversion ratio (FCR) were analyzed after fish were fed the mixture of probiotics (B. amyloliquefaciens 54A and B. pumilus 47B) at concentrations of 1 × 108, 3 × 108, and 5 × 108 CFU g−1 feed for 90 days. Immunity parameters, survival rate of fish challenged with Edwardsiella ictaluri and ammonia tolerance were also investigated. The amounts of B. amyloliquefaciens and B. pumilus were counted and identified by specific primer pairs of Ba1-F/Ba1-R, and 16-F/Bpu-R to confirm the presence of probiotics in fish intestine. The AWG (476.6 ± 7.81 g fish−1) of fish fed probiotics at 5 × 108 CFU g−1 was significant higher than the control (390 ± 25.7 g fish−1) after 90 days of feeding, but there was no significant (P > 0.05) effect of probiotics on FCR and SGR. Fish fed diet containing probiotics at 5 × 108 CFU g−1 also expressed resistance to E. ictaluri infection and higher immune parameters such as phagocytic activity, respiratory bursts, and lysozyme activity than the control. Stress response with ammonia showed significantly lower mortality rate (25%, 20% and 27%) of fish fed probiotics at all three levels of 1, 3 and 5 × 108 CFU g−1 than the fish fed control diet (75%). The study also demonstrated that the probiotics survived in the intestine of striped catfish after 90 days of feeding. Therefore, the dietary supplementation of a mixture of B. amyloliquefaciens and B. pumilus at 5 × 108 CFU g−1 can be used to improve the health and growth rate of striped catfish.
•The spores of B. amyloliquefaciens 54A and B. pumilus 47B were first applied as probiotics in striped catfish.•Dietary supplementation with B. amyloliquefaciens 54A and B. pumilus 47B for 90 days enhanced fish growth.•Innate immunity of striped catfish were stimulated after feeding B. amyloliquefaciens 54A and B. pumilus 47B for 90 days.•The mixture of B. amyloliquefaciens 54A and B. pumilus 47B in the diet enhanced disease resistance and stress tolerance.•Colonization of B. amyloliquefaciens 54A and B. pumilus 47B in the intestine of striped catfish was observed.
Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel ...D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.
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