Refinement of paramagnetic bead-based digestion protocol for automatic sample preparation using an artificial neural network.

Despite technological advances in the proteomics field, sample preparation still represents the main bottleneck in mass spectrometry (MS) analysis. Bead-based protein aggregation techniques have recently emerged as an efficient, reproducible, and high-throughput alternative for protein extraction and digestion. Here, a refined paramagnetic bead-based digestion protocol is described for Opentrons® OT-2 platform (OT-2) as a versatile, reproducible, and affordable alternative for the automatic sample preparation for MS analysis. For this purpose, an artificial neural network (ANN) was applied to maximize the number of peptides without missed cleavages identified in HeLa extract by combining factors such as the quantity (µg) of trypsin/Lys-C and beads (MagReSyn® Amine), % (w/v) SDS, % (v/v) acetonitrile, and time of digestion (h). ANN model predicted the optimal conditions for the digestion of 50 µg of HeLa extract, pointing to the use of 2.5% (w/v) SDS and 300 µg of beads for sample preparation and long-term digestion (16h) with 0.15 µg Lys-C and 2.5 µg trypsin (≈1:17 ratio). Based on the results of the ANN model, the manual protocol was automated in OT-2. The performance of the automatic protocol was evaluated with different sample types, including human plasma, Arabidopsis thaliana leaves, Escherichia coli cells, and mouse tissue cortex, showing great reproducibility and low sample-to-sample variability in all cases. In addition, we tested the performance of this method in the preparation of a challenging biological fluid such as rat bile, a proximal fluid that is rich in bile salts, bilirubin, cholesterol, and fatty acids, among other MS interferents. Compared to other protocols described in the literature for the extraction and digestion of bile proteins, the method described here allowed identify 385 unique proteins, thus contributing to improving the coverage of the bile proteome.

Cryogels and Monoliths: Promising Tools for Chromatographic Purification of Nucleic Acids.

The increasing demand for highly pure biopharmaceuticals has put significant pressure on the biotechnological industry to innovate in production and purification processes. Nucleic acid purification, crucial for gene therapy and vaccine production, presents challenges due to the unique physical and chemical properties of these molecules. Meeting regulatory standards necessitates large quantities of biotherapeutic agents of high purity. While conventional chromatography offers versatility and efficiency, it suffers from drawbacks like low flow rates and binding capacity, as well as high mass transfer resistance. Recent advancements in continuous beds, including monoliths and cryogel-based systems, have emerged as promising solutions to overcome these limitations. This review explores and evaluates the latest progress in chromatography utilizing monolithic and cryogenic supports for nucleic acid purification.

Serum and Vitreous Levels of Placenta Growth Factor in Diabetic Retinopathy Patients: Correlation With Disease Severity and Optical Coherence Tomographic Parameters.

Purpose The primary objective of this study was to compare placenta growth factor (PlGF) levels in the serum and vitreous of diabetic retinopathy (DR) patients to non-diabetic controls. Additionally, the study aimed to establish associations between serum and vitreous PlGF concentrations and to examine the correlation between vitreous PlGF in DR patients and morphological parameters. Methods This study included serum and vitreous samples from 38 patients, including 21 patients with DR and 17 non-diabetic controls. The control group included non-diabetic patients with rhegmatogenous retinal detachment with retinal tears secondary to posterior vitreous detachment or trauma. PlGF levels were quantified in vitreous and serum samples using an enzyme-linked immunosorbent assay (ELISA). Optical coherence tomography (OCT) scans from DR patients were evaluated to measure the central retinal thickness (CRT) and macular volume (MV). Results DR patients had significantly higher mean vitreous PlGF levels compared to non-DR patients (70.0±39.2 vs. 46.47±9.7 pg/mL, p-value=0.004). However, no significant increase in mean serum PlGF levels was observed in DR patients (p-value=0.232). Within the DR group, proliferative DR (PDR) patients presented significantly higher vitreous PlGF levels than non-PDR (NPDR) patients (76.5±41.0 vs. 42.5±5.0 pg/mL, p-value=0.009). There was no association between serum and vitreous PlGF levels. The correlation between vitreous PlGF levels and morphological parameters was rsp=0.175, p-value=0.488 for CRT, and rsp=0.288, p-value=0.262 for MV. Conclusion This study emphasizes the important role of PlGF in neovascularization, specifically highlighting its overexpression exclusively in vitreous from PDR patients. The observed increase in PlGF levels may be indicative of disease severity. The lack of correlation between vitreous and serum PlGF levels suggests a potential dissociation between intravitreal and systemic PlGF synthesis. Consequently, targeting PlGF in therapeutic approaches may offer an additional strategy for ocular pathologies with a neovascular component.

Multigenerational exposure of Ag materials (nano and salt) in soil - environmental hazards in Enchytraeus crypticus (Oligochaeta).

Because of its properties, silver is among the most used metals both as salt and as nanomaterials (NMs), hence reaching the environment. Multigenerational (MG) exposure testing is scarce, and especially so for NMs and soil invertebrates. In this study the MG effects of Ag NMs (Ag NM300K) and Ag salt (AgNO3) were assessed, using Enchytraeus crypticus in LUFA 2.2 soil. Survival, reproduction and internal Ag concentration in the animals were measured throughout 7 generations (5 generations (F0-F4) in spiked soil plus 2 (F5-F6) in clean soil) exposed to sublethal concentrations corresponding to the reproduction EC10 and EC50 obtained in standard toxicity tests (45 and 60 mg Ag per kg soil DW for AgNO3; 20 and 60 mg Ag per kg soil DW for Ag NM300K). MG exposure caused a dose-related decrease in reproduction for both Ag forms. Ag uptake peaked in the F1 (64 days) for AgNO3 and F2 (96 days) for Ag NM300K, after which it decreased. In agreement with toxicokinetic studies, a maximum body Ag concentration was reached (20 mg Ag per kg body DW (AgNO3) and 70 mg Ag per kg body DW (Ag NM300K)) and after which detoxification mechanisms seem to be activated with elimination of Ag accompanied by a decrease in reproduction. Transfer to clean soil allowed Ag to be (fully) eliminated from the animals. This MG study confirmed the effects determined in standard reproduction toxicity tests but further allowed to monitor the dynamics between exposure and effects of the Ag materials, and how the animals seem to cope with Ag for 7 generations by compensating between detoxification and reproductive output.

Molecular basis of progressive familial intrahepatic cholestasis 3. A proteomics study.

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe rare liver disease that affects between 1/50,000 and 1/100,000 children. In physiological conditions, bile is produced by the liver and stored in the gallbladder, and then it flows to the small intestine to play its role in fat digestion. To prevent tissue damage, bile acids (BAs) are kept in phospholipid micelles. Mutations in phosphatidyl choline transporter ABCB4 (MDR3) lead to intrahepatic accumulation of free BAs that result in liver damage. PFIC3 onset usually occurs at early ages, progresses rapidly, and the prognosis is poor. Currently, besides the palliative use of ursodeoxycholate, the only available treatment for this disease is liver transplantation, which is really challenging for short-aged patients. To gain insight into the pathogenesis of PFIC3 we have performed an integrated proteomics and phosphoproteomics study in human liver samples to then validate the emerging functional hypotheses in a PFIC3 murine model. We identified 6246 protein groups, 324 proteins among them showing differential expression between control and PFIC3. The phosphoproteomic analysis allowed the identification of 5090 phosphopeptides, from which 215 corresponding to 157 protein groups, were differentially phosphorylated in PFIC3, including MDR3. Regulation of essential cellular processes and structures, such as inflammation, metabolic reprogramming, cytoskeleton and extracellular matrix remodeling, and cell proliferation, were identified as the main drivers of the disease. Our results provide a strong molecular background that significantly contributes to a better understanding of PFIC3 and provides new concepts that might prove useful in the clinical management of patients.

Proteomic Serum Profiling of Holstein Friesian Cows with Different Pathological Forms of Bovine Paratuberculosis Reveals Changes in the Acute-Phase Response and Lipid Metabolism.

The lack of sensitive diagnostic methods to detect Mycobacterium avium subsp. paratuberculosis (Map) subclinical infections has hindered the control of paratuberculosis (PTB). The serum proteomic profiles of naturally infected cows presenting focal and diffuse pathological forms of PTB and negative controls (n = 4 per group) were analyzed using TMT-6plex quantitative proteomics. Focal and diffuse are the most frequent pathological forms in subclinical and clinical stages of PTB, respectively. One (focal versus (vs.) control), eight (diffuse vs. control), and four (focal vs. diffuse) differentially abundant (DA) proteins (q-value < 0.05) were identified. Ingenuity pathway analysis of the DA proteins revealed changes in the acute-phase response and lipid metabolism. Six candidate biomarkers were selected for further validation by specific ELISA using serum from animals with focal, multifocal, and diffuse PTB-associated lesions (n = 108) and controls (n = 56). Overall, the trends of the serum expression levels of the selected proteins were consistent with the proteomic results. Alpha-1-acid glycoprotein (ORM1)-based ELISA, insulin-like growth factor-binding protein 2 (IGFBP2)-based ELISA, and the anti-Map ELISA had the best diagnostic performance for detection of animals with focal, multifocal, and diffuse lesions, respectively. Our findings identify potential biomarkers that improve diagnostic sensitivity of PTB and help to elucidate the mechanisms involved in PTB pathogenesis.

SARS-CoV-2 accessory proteins involvement in inflammatory and profibrotic processes through IL11 signaling.

SARS-CoV-2, the cause of the COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome. Their roles during infection are still not completely understood. In this study, transcriptomics analysis revealed that both WNT5A and IL11 were significantly up-regulated in A549 cells expressing individual accessory proteins ORF6, ORF8, ORF9b or ORF9c from SARS-CoV-2 (Wuhan-Hu-1 isolate). IL11 is a member of the IL6 family of cytokines. IL11 signaling-related genes were also differentially expressed. Bioinformatics analysis disclosed that both WNT5A and IL11 were involved in pulmonary fibrosis idiopathic disease and functional assays confirmed their association with profibrotic cell responses. Subsequently, data comparison with lung cell lines infected with SARS-CoV-2 or lung biopsies from patients with COVID-19, evidenced altered profibrotic gene expression that matched those obtained in this study. Our results show ORF6, ORF8, ORF9b and ORF9c involvement in inflammatory and profibrotic responses. Thus, these accessory proteins could be targeted by new therapies against COVID-19 disease.

Proteomic analysis of STEAP1 knockdown in human LNCaP prostate cancer cells.

Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. The six transmembrane epithelial antigen of the prostate 1 (STEAP1) protein is overexpressed in several types of human tumors, particularly in PCa. Our research group has demonstrated that STEAP1 overexpression is associated with PCa progression and aggressiveness. Therefore, understanding the cellular and molecular mechanisms triggered by STEAP1 overexpression will provide important insights to delineate new strategies for PCa treatment. In the present work, a proteomic strategy was used to characterize the intracellular signaling pathways and the molecular targets downstream of STEAP1 in PCa cells. A label-free approach was applied using an Orbitrap LC-MS/MS system to characterize the proteome of STEAP1-knockdown PCa cells. More than 6700 proteins were identified, of which a total of 526 proteins were found differentially expressed in scramble siRNA versus STEAP1 siRNA (234 proteins up-regulated and 292 proteins down-regulated). Bioinformatics analysis allowed us to explore the mechanism through which STEAP1 exerts influence on PCa, revealing that endocytosis, RNA transport, apoptosis, aminoacyl-tRNA biosynthesis, and metabolic pathways are the main biological processes where STEAP1 is involved. By immunoblotting, it was confirmed that STEAP1 silencing induced the up-regulation of cathepsin B, intersectin-1, and syntaxin 4, and the down-regulation of HRas, PIK3C2A, and DIS3. These findings suggested that blocking STEAP1 might be a suitable strategy to activate apoptosis and endocytosis, and diminish cellular metabolism and intercellular communication, leading to inhibition of PCa progression.

Assessment of Aptamer as a Potential Drug Targeted Delivery for Retinal Angiogenesis Inhibition.

AT11-L0 is an aptamer derivative of AS1411 composed of G-rich sequences that can adopt a G-quadruplex (G4) structure and target nucleolin (NCL), a protein that acts as a co-receptor for several growth factors. Hence, this study aimed to characterize the AT11-L0 G4 structure and its interaction with several ligands for NCL targeting and to evaluate their capacity to inhibit angiogenesis using an in vitro model. The AT11-L0 aptamer was then used to functionalize drug-associated liposomes to increase the bioavailability of the aptamer-based drug in the formulation. Biophysical studies, such as nuclear magnetic resonance, circular dichroism, and fluorescence titrations, were performed to characterize the liposomes functionalized with the AT11-L0 aptamer. Finally, these liposome formulations with the encapsulated drugs were tested on the human umbilical vein endothelial cell (HUVEC) model to assess their antiangiogenic capacity. The results showed that the AT11-L0 aptamer-ligand complexes are highly stable, presenting melting temperatures from 45 °C to 60 °C, allowing for efficient targeting of NCL with a KD in the order of nM. The aptamer-functionalized liposomes loaded with ligands C8 and dexamethasone did not show cytotoxic effects in HUVEC cells compared with the free ligands and AT11-L0, as assessed by cell viability assays. AT11-L0 aptamer-functionalized liposomes encapsulating C8 and dexamethasone did not present a significant reduction in the angiogenic process when compared with the free ligands. In addition, AT11-L0 did not show anti-angiogenic effects at the concentrations tested. However, C8 shows potential as an angiogenesis inhibitor, which should be further developed and optimized in future experiments.

Proteomics profiling of vitreous humor reveals complement and coagulation components, adhesion factors, and neurodegeneration markers as discriminatory biomarkers of vitreoretinal eye diseases.

Introduction: Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and proliferative DR (PDR), no treatment options exist for the highly prevalent dry form of AMD. Methods: To unravel the biological processes underlying these pathologies and to find new potential biomarkers, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR (n=4), AMD (n=4) compared to idiopathic epiretinal membranes (ERM) (n=4). Results and discussion: Post-hoc tests revealed 96 proteins capable of differentiating among the different groups, whereas 118 proteins were found differentially regulated in PDR compared to ERM and 95 proteins in PDR compared to dry AMD. Pathway analysis indicates that mediators of complement, coagulation cascades and acute phase responses are enriched in PDR vitreous, whilst proteins highly correlated to the extracellular matrix (ECM) organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development were found underexpressed. According to these results, 35 proteins were selected and monitored by MRM (multiple reaction monitoring) in a larger cohort of patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and retinal detachment (n=13). Of these, 26 proteins could differentiate between these vitreoretinal diseases. Based on Partial least squares discriminant and multivariate exploratory receiver operating characteristic (ROC) analyses, a panel of 15 discriminatory biomarkers was defined, which includes complement and coagulation components (complement C2 and prothrombin), acute-phase mediators (alpha-1-antichymotrypsin), adhesion molecules (e.g., myocilin, galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2).

Toxicokinetics and toxicodynamics of Ag nanomaterials (NM300K) in the soil environment-impact on Enchytraeus crypticus (Oligochaeta).

Silver (Ag) is one of the most used elements in the nanomaterials (NMs) form, which upon release to the environment can be harmful to organisms. We compared the toxicokinetics (TK) and toxicodynamics (TD) of Ag from AgNO3 (0, 15, 45, 135, 405 mg Ag/kg soil) and AgNM300K (0, 75, 150, 300, 600, 1200 mg Ag/kg soil) in the model organism Enchytraeus crypticus. Organisms were exposed in LUFA 2.2 soil, and besides body Ag concentrations, survival and reproduction were determined, in a time series (for 21 days). In the soil, the available (CaCl2 extractable) Ag fraction from Ag NM300K increased from 0 to 21 days but did not consistently change for AgNO3. Internal concentrations reached equilibrium in most exposures to both Ag forms. The organisms were able to internalize and eliminate Ag, but less when exposed to Ag NM300K. The overall uptake rate constants for Ag from AgNO3 and Ag NM300K exposures were 0.05 and 0.06 kg soil/kg organism/day, respectively, the elimination rate constants 0.2 and 0.1 day-1, respectively. For AgNO3 the median lethal concentrations decreased steadily with time, while for Ag NM300K they remained constant during the first 10 days of exposure followed by a 2-fold decline in the last 7 days. The 21-d LC50s for both Ag forms were similar but the LC50inter (based on internal concentrations) were 63 and 121 mg Ag/kg body DW (Dry Weight) for AgNO3 and Ag NM300K, respectively, showing higher toxicity of AgNO3. These results show the importance of assessing time to toxicity, a relevant factor in toxicity assessment, especially for NMs.

Multi-omics analyses demonstrate a critical role for EHMT1 methyltransferase in transcriptional repression during oogenesis.

EHMT1 (also known as GLP) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 (also known as G9A). Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models (Ehmt2 cKO, Ehmt1 cKO, Ehmt1/2 cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 in Ehmt1 cKO and Ehmt1/2 cDKO oocytes recapitulated meiotic defects observed in the Ehmt2 cKO; however, there was a significant impairment in oocyte maturation and developmental competence in Ehmt1 cKO and Ehmt1/2 cDKO oocytes beyond that observed in the Ehmt2 cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)-based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome in Ehmt1/2 cDKO than Ehmt2 cKO oocytes, with transcriptional derepression leading to increased protein abundance and local changes in genic DNA methylation in Ehmt1/2 cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence.

Toxicokinetics and toxicodynamics of chromium in the soil invertebrate Enchytraeus crypticus (Oligochaeta).

Chromium emissions led to increased concentrations in soil, where it can affect soil organisms to relevant levels. With the aim of better understanding the effects of Cr throughout time, its toxicokinetics-toxicodynamics (TKTD) were evaluated in the soil model organism Enchytraeus crypticus to assess the development of internal concentrations and consequent toxic effects. To achieve this goal, organisms were exposed in LUFA 2.2 soil spiked with increasing CrCl3 concentrations. During the 21-day exposure period, survival, internal concentrations, and reproduction were evaluated at several time points up to 21 days. Uptake and elimination rate constants were 0.0044 kg soil/kg organism/day and 0.023 per day, respectively. Internal Cr concentrations increased with time, generally reaching equilibrium within 14 days with an estimated LC50inter (based on internal metal concentrations) of 57.7 mg Cr/kg body DW. Internal Cr concentrations were regulated by the organisms up to exposure to 360 mg Cr/kg soil DW, where the elimination rate was highest, but at 546 mg Cr/kg soil DW the animals were no longer able to eliminate Cr, and the internal concentrations were well above the estimated LC50inter. At day 21, exposure to 546 mg Cr/kg soil DW significantly reduced survival by 23 %, while reproduction EC50 was 344 mg Cr/kg soil DW. This study highlights the advantages of using a TKTD approach to understand the development of internal metal concentrations in time and relate it to the phenotypical effects observed. Toxicity is better understood when also taking into account time and not just exposure concentration alone.

Stable Cobalt-Mediated Monolithic Dye-Sensitized Solar Cells by Full Glass Encapsulation.

Dye-sensitized solar cells (DSSCs) emerged in the market as one of the most promising indoor photovoltaic technologies to address the need for wireless powering of low-consuming electronics and sensor nodes of the internet of things (IoT). The monolithic design structure of the cell (M-DSSCs) makes the devices simpler and cheaper, and it is straightforward for constructing in-series modules. The most efficient DSSCs reported so far are Co(III/II)-mediated liquid junction cells with acetonitrile electrolytes; however, they are mostly unstable. This study reports on highly stable cobalt-mediated M-DSSCs, passing thermal cycling tests up to 85 °C according to ISOS standard protocols. Under 1000 h of aging in the dark and under simulated solar and artificial light soaking, all tested cells improved or retained their initial power conversion efficiency. Advanced long-term stability was achieved by eliminating the extrinsic factors of degradation, such as the interaction of the cell components with the environment and electrolyte leakage. This was obtained by encapsulation of the devices using a glass-frit sealant, including the holes for filling up the liquid components of the cells. The hermeticity of the encapsulation complies with the MIL-STD-883 standard fine helium gas leakage test, and its hermeticity remained unchanged after humidity-freeze cycles according to IEC 61646. The elimination of extrinsic degradation factors allowed reliable assessment of inner factors accountable for aging. The impact of the ISOS-protocol test conditions on the intrinsic device stability and long-term photovoltaic history of the M-DSSCs is discussed.

Vitreous humor proteome: unraveling the molecular mechanisms underlying proliferative and neovascular vitreoretinal diseases.

Proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and neovascular age-related macular degeneration (nAMD) are among the leading causes of blindness. Due to the multifactorial nature of these vitreoretinal diseases, omics approaches are essential for a deeper understanding of the pathophysiologic processes underlying the evolution to a proliferative or neovascular etiology, in which patients suffer from an abrupt loss of vision. For many years, it was thought that the function of the vitreous was merely structural, supporting and protecting the surrounding ocular tissues. Proteomics studies proved that vitreous is more complex and biologically active than initially thought, and its changes reflect the physiological and pathological state of the eye. The vitreous is the scenario of a complex interplay between inflammation, fibrosis, oxidative stress, neurodegeneration, and extracellular matrix remodeling. Vitreous proteome not only reflects the pathological events that occur in the retina, but the changes in the vitreous itself play a central role in the onset and progression of vitreoretinal diseases. Therefore, this review offers an overview of the studies on the vitreous proteome that could help to elucidate some of the pathological mechanisms underlying proliferative and/or neovascular vitreoretinal diseases and to find new potential pharmaceutical targets.

Exploring the Applicability of Calorespirometry to Assess Seed Metabolic Stability Upon Temperature Stress Conditions-Pisum sativum L. Used as a Case Study.

The availability of phenotyping tools to assist breeding programs in the selection of high-quality crop seeds is of obvious interest with consequences for both seed producers and consumers. Seed germination involves the activation of several metabolic pathways, such as cellular respiration to provide the required ATP and reducing power. This work tested the applicability of calorespirometry, the simultaneous measurement of heat and CO2 rates, as a phenotyping tool to assess seed respiratory properties as a function of temperature. The effect of temperature on seed germination was evaluated after 16 h of seed imbibition by calorespirometric experiments performed in isothermal mode at 15, 20, 25, and 28°C on the seeds of three cultivars of peas (Pisum sativum L.) commonly used in conventional agriculture (cvs. 'Rondo', 'Torta de Quebrar', and 'Maravilha d'América'). Significant differences in metabolic heat rate and CO2 production rate (R CO2 ) as well as in the temperature responses of these parameters were found among the three cultivars. A seed germination trial was conducted during the 6 days of imbibition to evaluate the predictive power of the parameters derived from the calorespirometric measurements. The germination trial showed that the optimal germination temperature was 20°C and low germination rates were observed at extreme temperatures (15 or 28°C). The cv. 'Torta de Quebrar' showed significantly higher germination in comparison with the other two cultivars at all three temperatures. In comparison with the other two cultivars, 'Torta de Quebrar' has the lowest metabolic heat and CO2 rates and the smallest temperature dependence of these measured parameters. Additionally, 'Torta de Quebrar' has the lowest values of growth rate and carbon use efficiency calculated from the measured variables. These data suggest that calorespirometry is a useful tool for phenotyping physiologic efficiency at different temperatures during early germination stages, and can determine the seeds with the highest resilience to temperature variation, in this case 'Torta de Quebrar'.

Multi-omic rejuvenation of human cells by maturation phase transient reprogramming.

Ageing is the gradual decline in organismal fitness that occurs over time leading to tissue dysfunction and disease. At the cellular level, ageing is associated with reduced function, altered gene expression and a perturbed epigenome. Recent work has demonstrated that the epigenome is already rejuvenated by the maturation phase of somatic cell reprogramming, which suggests full reprogramming is not required to reverse ageing of somatic cells. Here we have developed the first "maturation phase transient reprogramming" (MPTR) method, where reprogramming factors are selectively expressed until this rejuvenation point then withdrawn. Applying MPTR to dermal fibroblasts from middle-aged donors, we found that cells temporarily lose and then reacquire their fibroblast identity, possibly as a result of epigenetic memory at enhancers and/or persistent expression of some fibroblast genes. Excitingly, our method substantially rejuvenated multiple cellular attributes including the transcriptome, which was rejuvenated by around 30 years as measured by a novel transcriptome clock. The epigenome was rejuvenated to a similar extent, including H3K9me3 levels and the DNA methylation ageing clock. The magnitude of rejuvenation instigated by MPTR appears substantially greater than that achieved in previous transient reprogramming protocols. In addition, MPTR fibroblasts produced youthful levels of collagen proteins, and showed partial functional rejuvenation of their migration speed. Finally, our work suggests that optimal time windows exist for rejuvenating the transcriptome and the epigenome. Overall, we demonstrate that it is possible to separate rejuvenation from complete pluripotency reprogramming, which should facilitate the discovery of novel anti-ageing genes and therapies.