Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Time series transcriptome analysis implicates the circadian clock in the Drosophila melanogaster female's response to sex peptide.

Sex peptide (SP), a seminal fluid protein of Drosophila melanogaster males, has been described as driving a virgin-to-mated switch in females, through eliciting an array of responses including increased egg laying, activity, and food intake and a decreased remating rate. While it is known that SP achieves this, at least in part, by altering neuronal signaling in females, the genetic architecture and temporal dynamics of the female's response to SP remain elusive. We used a high-resolution time series RNA-sequencing dataset of female heads at 10 time points within the first 24 h after mating to learn about the genetic architecture, at the gene and exon levels, of the female's response to SP. We find that SP is not essential to trigger early aspects of a virgin-to-mated transcriptional switch, which includes changes in a metabolic gene regulatory network. However, SP is needed to maintain and diversify metabolic changes and to trigger changes in a neuronal gene regulatory network. We further find that SP alters rhythmic gene expression in females and suggests that SP's disruption of the female's circadian rhythm might be key to its widespread effects.

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

BACKGROUND: Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. RESULTS: We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. CONCLUSIONS: We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

Temporal Assessment of Protein Stability in Dried Blood Spots.

The use of protein biomarkers in blood for clinical settings is limited by the cost and accessibility of traditional venipuncture sampling. The dried blood spot (DBS) technique offers a less invasive and more accessible alternative. However, protein stability in DBS has not been well evaluated. Herein, we deployed a quantitative LC-MS/MS system to construct proteomic atlases of whole blood, DBSs, plasma, and blood cells. Approximately 4% of detected proteins' abundance was significantly altered during blood drying into blood spots, with overwhelming disturbances in cytoplasmic fraction. We also reported a novel finding suggesting a decrease in the level of membrane/cytoskeletal proteins (SLC4A1, RHAG, DSC1, DSP, and JUP) and an increase in the level of proteins (ATG3, SEC14L4, and NRBP1) related to intracellular trafficking. Furthermore, we identified 19 temporally dynamic proteins in DBS samples stored at room temperature for up to 6 months. There were three declined cytoskeleton-related proteins (RDX, SH3BGRL3, and MYH9) and four elevated proteins (XPO7, RAN, SLC2A1, and SLC29A1) involved in cytoplasmic transport as representatives. The instability was governed predominantly by hydrophilic proteins and enhanced significantly with an increasing storage time. Our analyses provide comprehensive knowledge of both short- and long-term storage stability of DBS proteins, forming the foundation for the widespread use of DBS in clinical proteomics and other analytical applications.

Dynamics of Liver Stiffness Measurement and Clinical Course of Primary Biliary Cholangitis.

BACKGROUND & AIMS: In primary biliary cholangitis (PBC), static liver stiffness measurement (LSM) has proven prognostic value. However, the added prognostic value of LSM time course in this disease remains uncertain. METHODS: We conducted an international retrospective cohort study among patients with PBC treated with ursodeoxycholic acid and followed by vibration-controlled transient elastography between 2003 and 2022. Using joint modeling, the association of LSM trajectory and the incidence of serious clinical events (SCE), defined as cirrhosis complications, liver transplantation, or death, was quantified using the hazard ratio and its confidence interval. RESULTS: A total of 6362 LSMs were performed in 3078 patients (2007 on ursodeoxycholic acid alone; 13% with cirrhosis), in whom 316 SCE occurred over 14,445 person-years (median follow-up, 4.2 years; incidence rate, 21.9 per 1000 person-years). LSM progressed in 59% of patients (mean, 0.39 kPa/year). After adjusting for prognostic factors at baseline, including LSM, any relative change in LSM was associated with a significant variation in SCE risk (P < .001). For example, the adjusted hazard ratios (95% confidence interval) associated with a 20% annual variation in LSM were 2.13 (1.89-2.45) for the increase and 0.40 (0.33-0.46) for the decrease. The association between LSM trajectory and SCE risk persisted regardless of treatment response or duration, when patients with cirrhosis were excluded, and when only death or liver transplantation was considered. CONCLUSIONS: Tracking longitudinal changes in LSM using vibration-controlled transient elastography provides valuable insights into PBC prognosis, offering a robust predictive measure for the risk of SCE. LSM could be used as a clinically relevant surrogate end point in PBC clinical trials.

Temporal profiling of physiological, histological, and transcriptomic dissection during auxin-induced adventitious root formation in tetraploid Robinia pseudoacacia micro-cuttings.

MAIN CONCLUSION: Optimal levels of indole-3-butyric acid (IBA) applied at the stem base promote adventitious root (AR) initiation and primordia formation, thus promoting the rooting of leafy micro-cuttings of tetraploid Robinia pseudoacacia. Tetraploid Robinia pseudoacacia L. is a widely cultivated tree in most regions of China that has a hard-rooting capability, propagated by stem cuttings. This study utilizes histological, physiological, and transcriptomic approaches to explore how root primordia are induced after indole butyric acid (IBA) treatment of micro-cuttings. IBA application promoted cell divisions in some cells within the vasculature, showing subcellular features associated with adventitious root (AR) founder cells. The anatomical structure explicitly showed that AR initiated from the cambium layer and instigate the inducible development of AR primordia. Meanwhile, the hormone data showed that similar to that of indole-3-acetic acid, the contents of trans-zeatin and abscisic acid peaked at early stages of AR formation and increased gradually in primordia formation across the subsequent stages, suggesting their indispensable roles in AR induction. On the contrary, 24-epibrassinolide roughly maintained at extremely high levels during primordium initiation thoroughly, indicating its presence was involved in cell-specific reorganization during AR development. Furthermore, antioxidant activities transiently increased in the basal region of micro-cuttings and may serve as biochemical indicators for distinct rooting phases, potentially aiding in AR formation. Transcriptomic analysis during the early stages of root formation shows significant downregulation of the abscisic acid and jasmonate signaling pathways, while ethylene and cytokinin signaling seems upregulated. Network analysis of genes involved in carbon metabolism and photosynthesis indicates that the basal region of the micro-cuttings undergoes rapid reprogramming, which results in the breakdown of sugars into pyruvate. This pyruvate is then utilized to fuel the tricarboxylic acid cycle, thereby sustaining growth through aerobic respiration. Collectively, our findings provide a time-course morphophysiological dissection and also suggest the regulatory role of a conserved auxin module in AR development in these species.

dynDeepDRIM: a dynamic deep learning model to infer direct regulatory interactions using time-course single-cell gene expression data.

Time-course single-cell RNA sequencing (scRNA-seq) data have been widely used to explore dynamic changes in gene expression of transcription factors (TFs) and their target genes. This information is useful to reconstruct cell-type-specific gene regulatory networks (GRNs). However, the existing tools are commonly designed to analyze either time-course bulk gene expression data or static scRNA-seq data via pseudo-time cell ordering. A few methods successfully utilize the information from multiple time points while also considering the characteristics of scRNA-seq data. We proposed dynDeepDRIM, a novel deep learning model to reconstruct GRNs using time-course scRNA-seq data. It represents the joint expression of a gene pair as an image and utilizes the image of the target TF-gene pair and the ones of the potential neighbors to reconstruct GRNs from time-course scRNA-seq data. dynDeepDRIM can effectively remove the transitive TF-gene interactions by considering neighborhood context and model the gene expression dynamics using high-dimensional tensors. We compared dynDeepDRIM with six GRN reconstruction methods on both simulation and four real time-course scRNA-seq data. dynDeepDRIM achieved substantially better performance than the other methods in inferring TF-gene interactions and eliminated the false positives effectively. We also applied dynDeepDRIM to annotate gene functions and found it achieved evidently better performance than the other tools due to considering the neighbor genes.

Protein turnover models for LC-MS data of heavy water metabolic labeling.

Protein turnover is vital for cellular functioning and is often associated with the pathophysiology of a variety of diseases. Metabolic labeling with heavy water followed by liquid chromatography coupled to mass spectrometry is a powerful tool to study in vivo protein turnover in high throughput and large scale. Heavy water is a cost-effective and easy to use labeling agent. It labels all nonessential amino acids. Due to its toxicity in high concentrations (20% or higher), small enrichments (8% or smaller) of heavy water are used with most organisms. The low concentration results in incomplete labeling of peptides/proteins. Therefore, the data processing is more challenging and requires accurate quantification of labeled and unlabeled forms of a peptide from overlapping mass isotopomer distributions. The work describes the bioinformatics aspects of the analysis of heavy water labeled mass spectral data, available software tools and current challenges and opportunities.

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium.

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.

RNA-Seq time-course analysis of neural precursor cell transcriptome in response to herpes simplex Virus-1 infection.

The neurogenic niches within the central nervous system serve as essential reservoirs for neural precursor cells (NPCs), playing a crucial role in neurogenesis. However, these NPCs are particularly vulnerable to infection by the herpes simplex virus 1 (HSV-1). In the present study, we investigated the changes in the transcriptome of NPCs in response to HSV-1 infection using bulk RNA-Seq, compared to those of uninfected samples, at different time points post infection and in the presence or absence of antivirals. The results showed that NPCs upon HSV-1 infection undergo a significant dysregulation of genes playing a crucial role in aspects of neurogenesis, including genes affecting NPC proliferation, migration, and differentiation. Our analysis revealed that the CREB signaling, which plays a crucial role in the regulation of neurogenesis and memory consolidation, was the most consistantly downregulated pathway, even in the presence of antivirals. Additionally, cholesterol biosynthesis was significantly downregulated in HSV-1-infected NPCs. The findings from this study, for the first time, offer insights into the intricate molecular mechanisms that underlie the neurogenesis impairment associated with HSV-1 infection.

Excessive psychological stress preceding the onset of idiopathic cervical dystonia.

Idiopathic cervical dystonia (ICD) is the largest subgroup of dystonia. Psychological stress as a triggering factor has long been discussed, but detailed descriptions are lacking. We report on a group of 13 patients with ICD and preceding excessive psychological stress (age at ICD onset 39.0 ± 13.9 years, 7 females, 6 males). The observation period was 7.8 ± 5.0 years. Excessive psychological stress included partner conflicts (divorce and separation, domestic violence), special familial burdens, legal disputes and migration. It started 8.3 ± 3.9 months before ICD onset. In 85% of our patients (typical cases), ICD developed within 5.8 ± 4.4 weeks, then lasted 18.5 ± 8.3 months, before it started to remit 2.7 ± 0.8 years after its onset to 54.5 ± 35.3% of its maximal severity. Idiopathic dystonia is thought to be based upon a genetic predisposition triggered by epigenetic factors. Our study suggests that excessive psychological stress could be one of them. Pathophysiologic elements are only vaguely identified, but could include the endoplasmic reticulum stress response, cerebellar 5HT-2A receptors and the metabolism of heat shock proteins. Whilst the clinical presentation of ICD preceded by excessive psychological stress is typical, its course is atypical with rapid onset and fast and substantial remission.

Persistent high sepsis-induced coagulopathy and sequential organ failure assessment scores can predict the 28-day mortality of patients with sepsis: A prospective study.

BACKGROUND: The performance of the sepsis-induced coagulopathy (SIC) and sequential organ failure assessment (SOFA) scores in predicting the prognoses of patients with sepsis has been validated. This study aimed to investigate the time course of SIC and SOFA scores and their association with outcomes in patients with sepsis. METHODS: This prospective study enrolled 209 patients with sepsis admitted to the emergency department. The SIC and SOFA scores of the patients were assessed on days 1, 2, and 4. Patients were categorized into survivor or non-survivor groups based on their 28-day survival. We conducted a generalized estimating equation analysis to evaluate the time course of SIC and SOFA scores and the corresponding differences between the two groups. The predictive value of SIC and SOFA scores at different time points for sepsis prognosis was evaluated. RESULTS: In the non-survivor group, SIC and SOFA scores gradually increased during the first 4 days (P < 0.05). In the survivor group, the SIC and SOFA scores on day 2 were significantly higher than those on day 1 (P < 0.05); however, they decreased on day 4, dropping below the levels observed on day 1 (P < 0.05). The non-survivors showed higher SIC scores on days 2 (P < 0.05) and 4 (P < 0.001) than the survivors, whereas no significant differences were found between the two groups on day 1 (P > 0.05). The performance of SIC scores on day 4 for predicting mortality was more accurate than that on day 2, with areas under the curve of 0.749 (95% confidence interval [CI]: 0.674-0.823), and 0.601 (95% CI: 0.524-0.679), respectively. The SIC scores demonstrated comparable predictive accuracy for 28-day mortality to the SOFA scores on days 2 and 4. Cox proportional hazards models indicated that SIC on day 4 (hazard ratio [HR] = 3.736; 95% CI: 2.025-6.891) was an independent risk factor for 28-day mortality. CONCLUSIONS: The time course of SIC and SOFA scores differed between surviving and non-surviving patients with sepsis, and persistent high SIC and SOFA scores can predict 28-day mortality.

Time-course and dose-effect of omalizumab in treating chronic idiopathic urticaria/chronic spontaneous urticaria.

PURPOSE: Several studies have shown that subcutaneous injections of omalizumab can treat chronic idiopathic/spontaneous urticaria (CIU/CSU) patients by only assessing the efficacy on specific endpoints. This study aimed to quantitatively analyze different doses of omalizumab in CIU/CSU and compare it with ligelizumab. METHODS: Literature searches were performed in PubMed, Embase, and Web of Science databases. A model-based meta-analysis (MBMA) was utilized to develop a model incorporating time since the initiation of treatment and dose for omalizumab, with the change from baseline in Urticaria Activity Score (CFB-UAS7) as the primary efficacy endpoint. The time-course and dose-effect relationship throughout the omalizumab treatment period was analyzed, and the findings were compared with those of the investigational ligelizumab. RESULTS: The model equation for the CFB-UAS7 was established as E = -Emax × time/(ET50 + time) × (b0 + b1 × dose). The estimated values of the model parameters E max , ET 50 , b 0 , and b 1 were -1.16, 1.26 weeks, -9.90, and -0.0361 mg-1, respectively. At week 12 after the first dose, the model-predicted CFB-UAS7 for 150 mg and 300 mg of omalizumab were -16.0 (95% CI, -17.2 to -14.8) and -21.7 (95% CI, -22.9 to -20.5), respectively. In the PEARL-1 trial, the CFB-UAS7 for 72 mg and 120 mg of ligelizumab were -19.4 (95% CI, -20.7 to -18.1) and -19.3 (95% CI, -20.6 to -18.0), respectively. In the PEARL-2 trial, these values were -19.2 (95% CI, -20.5 to -17.9) and -20.3 (95% CI, -21.6 to -19.0), respectively. CONCLUSION: Omalizumab showed a significant dose-dependent effect in the treatment of CSU. Both 72 mg and 120 mg ligelizumab might have the potential to outperform 150 mg (but not 300 mg) omalizumab.

The time course of spontaneous closure of idiopathic full-thickness macular holes.

PURPOSE: Spontaneous closure of idiopathic full-thickness macular holes (iFTMH) has been reported regularly. However, little is known about its probability and timeline. METHODS: In this retrospective study all consecutive patients who presented between August 2008 and August 2019 were screened for the presence of a macular hole and only iFTMHs were included. The primary outcome measure was the spontaneous closure of the iFTMH. RESULTS: Of 1256 eyes with macular holes, 338 fulfilled the inclusion criteria. Spontaneous closure of the iFTMH was detected in 31 eyes (9.2%) with a median time of 44 days after diagnosis. Eyes exhibiting spontaneous closure demonstrated a higher baseline best-corrected visual-acuity (BCVA) and smaller iFTMH diameter (p < 0.0001 and p < 0.0001, respectively). The mean BCVA improved from 0.4 logMAR (SD ± 0.21) to 0.29 logMAR (SD ± 0.20) after spontaneous closure (p = 0.031). The iFTMH diameter was positively correlated with the time to spontaneous closure (Pearson-r = 0.37, p = 0.0377). Spontaneously closed iFTMHs reopened in 16% (n = 5) of cases, with a median of 136 days after closure. A logistic regression model showed the hole diameter was associated with spontaneous closure (odds-Ratio 0.97, 95%CI [0.96, 0.98]). The Kaplan-Meier-Curve revealed that approximately 25% of small-iFTMH (n = 124) and 55% of iFTMH with a diameter < 150µm (n = 48) closed spontaneously within two months. CONCLUSION: The established gold-standard for the treatment of iFTMHs is macular surgery. However, the potential for spontaneous closure of small iFTMHs must be acknowledged. Therefore, if surgical treatment is delayed in individual cases, close observation is recommended.

Temporal dynamics of gait function in acute cervical spinal cord injury.

BACKGROUND: Following spinal cord injury (SCI), gait function reaches a post-recovery plateau that depends on the paralysis severity. However, the plateau dynamics during the recovery period are not known. This study aimed to examine the gait function temporal dynamics after traumatic cervical SCI (CSCI) based on paralysis severity. METHODS: This retrospective cohort study included 122 patients with traumatic CSCI admitted to a single specialized facility within 2 weeks after injury. The Walking Index for Spinal Cord Injury II (WISCI II) was estimated at 2 weeks and 2, 4, 6, and 8 months postinjury for each American Spinal Injury Association Impairment Scale (AIS) grade, as determined 2 weeks postinjury. Statistical analysis was performed at 2 weeks to 2 months, 2-4 months, 4-6 months, and 6-8 months, and the time at which no significant difference was observed was considered the time at which the gait function reached a plateau. RESULTS: In the AIS grade A and B groups, no significant differences were observed at any time point, while in the AIS grade C group, the mean WISCI II values continued to significantly increase up to 6 months. In the AIS grade D group, the improvement in gait function was significant during the entire observation period. CONCLUSIONS: The plateau in gait function recovery was reached at 2 weeks postinjury in the AIS grade A and B groups and at 6 months in the AIS grade C group.

Time-Course Analysis and Transcriptomic Identification of a Group III ERF CmTINY2 Involved in Waterlogging Tolerance in Chrysanthemums × morifolium Ramat.

'Hangju' is a variety of Chrysanthemum × morifolium Ramat. with both edible and medicinal value, cultivated as a traditional Chinese medicine for four centuries. The cultivation of 'Hangju' is currently at risk due to waterlogging, yet there is a lack of comprehensive understanding regarding its response to waterlogging stress. This study compared the waterlogging-tolerant 'Hangju' variety Enhanced Waterlogging Tolerance (EWT) with the waterlogging-sensitive variety CK ('zaoxiaoyangju'). EWT exhibited a more developed aeration tissue structure and demonstrated rapid growth regarding the adventitious roots following waterlogging. The time-course transcriptome analysis indicated that EWT could swiftly adjust the expression of the genes involved in the energy metabolism signaling pathways to acclimate to the waterlogged environment. Through WGCNA analysis, we identified Integrase-Type DNA-Binding Protein (CmTINY2) as a key factor in regulating the waterlogging tolerance in EWT. CmTINY2, a transcription factor belonging to the ethylene-responsive factor (ERF) subfamily III, operated within the nucleus and activated downstream gene expression. Its role in enhancing the waterlogging tolerance might be linked to the control of the stomatal aperture via the Ethylene-Responsive Element (ERE) gene. In summary, our research elucidated that the waterlogging tolerance displayed by EWT is a result of a combination of the morphological structure and molecular regulatory mechanisms. Furthermore, the study of the functions of CmTINY2 from ERF subfamily III also broadened our knowledge of the role of the ERF genes in the waterlogging signaling pathways.

Time-course cross-species transcriptomics reveals conserved hepatotoxicity pathways induced by repeated administration of cyclosporine A.

Cyclosporine A (CsA) has shown efficacy against immunity-related diseases despite its toxicity in various organs, including the liver, emphasizing the need to elucidate its underlying hepatotoxicity mechanism. This study aimed to capture the alterations in genome-wide expression over time and the subsequent perturbations of corresponding pathways across species. Six data from humans, mice, and rats, including animal liver tissue, human liver microtissues, and two liver cell lines exposed to CsA toxic dose, were used. The microtissue exposed to CsA for 10 d was analyzed to obtain dynamically differentially expressed genes (DEGs). Single-time points data at 1, 3, 5, 7, and 28 d of different species were used to provide additional evidence. Using liver microtissue-based longitudinal design, DEGs that were consistently up- or down-regulated over time were captured, and the well-known mechanism involved in CsA toxicity was elucidated. Thirty DEGs that consistently changed in longitudinal data were also altered in 28-d rat in-house data with concordant expression. Some genes (e.g. TUBB2A, PLIN2, APOB) showed good concordance with identified DEGs in 1-d and 7-d mouse data. Pathway analysis revealed up-regulations of protein processing, asparagine N-linked glycosylation, and cargo concentration in the endoplasmic reticulum. Furthermore, the down-regulations of pathways related to biological oxidations and metabolite and lipid metabolism were elucidated. These pathways were also enriched in single-time-point data and conserved across species, implying their biological significance and generalizability. Overall, the human organoids-based longitudinal design coupled with cross-species validation provides temporal molecular change tracking, aiding mechanistic elucidation and biologically relevant biomarker discovery.

Dauer juvenile recovery transcriptome of two contrasting EMS mutants of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode Heterorhabditis bacteriophora, symbiotically associated with enterobacteria of the genus Photorhabdus, is a biological control agent against many insect pests. Dauer Juveniles (DJ) of this nematode are produced in industrial-scale bioreactors up to 100 m3 in liquid culture processes lasting approximately 11 days. A high DJ yield (> 200,000 DJ·mL-1) determines the success of the process. To start the mass production, a DJ inoculum proceeding from a previous monoxenic culture is added to pre-cultured (24 h) Photorhabdus bacteria. Within minutes after contact with the bacteria, DJ are expected to perceive signals that trigger their further development (DJ recovery) to reproductive hermaphrodites. A rapid, synchronized, and high DJ recovery is a key factor for an efficient culture process. In case of low percentage of DJ recovery, the final DJ yield is drastically reduced, and the amount of non-desired stages (males and non-fertilized females) hinders the DJ harvest. In a preliminary work, a huge DJ recovery phenotypic variability in H. bacteriophora ethyl methanesulphonate (EMS) mutants was determined. In the present study, two EMS-mutant lines (M31 and M88) with high and low recovery phenotypes were analyzed concerning their differences in gene expression during the first hours of contact with Photorhabdus supernatant containing food signals triggering recovery. A snapshot (RNA-seq analysis) of their transcriptome was captured at 0.5, 1, 3 and 6 h after exposure. Transcripts (3060) with significant regulation changes were identified in the two lines. To analyze the RNA-seq data over time, we (1) divided the expression profiles into clusters of similar regulation, (2) identified over and under-represented gene ontology categories for each cluster, (3) identified Caenorhabditis elegans homologous genes with recovery-related function, and (4) combined the information with available single nucleotide polymorphism (SNP) data. We observed that the expression dynamics of the contrasting mutants (M31 and M88) differ the most within the first 3 h after Photorhabdus supernatant exposure, and during this time, genes related to changes in the DJ cuticle and molting are more active in the high-recovery line (M31). Comparing the gene expression of DJ exposed to the insect food signal in the haemolymph, genes related to host immunosuppressive factors were not found in DJ upon bacterial supernatant exposure. No link between the position of SNPs associated with high recovery and changes in gene expression was determined for genes with high differential expression. Concerning specific transcripts, nine H. bacteriophora gene models with differential expression are provided as candidate genes for further studies.

Toward a hypothesis-free understanding of how phosphorylation dynamically impacts protein turnover.

The turnover measurement of proteins and proteoforms has been largely facilitated by workflows coupling metabolic labeling with mass spectrometry (MS), including dynamic stable isotope labeling by amino acids in cell culture (dynamic SILAC) or pulsed SILAC (pSILAC). Very recent studies including ours have integrated themeasurement of post-translational modifications (PTMs) at the proteome level (i.e., phosphoproteomics) with pSILAC experiments in steady state systems, exploring the link between PTMs and turnover at the proteome-scale. An open question in the field is how to exactly interpret these complex datasets in a biological perspective. Here, we present a novel pSILAC phosphoproteomic dataset which was obtained during a dynamic process of cell starvation using data-independent acquisition MS (DIA-MS). To provide an unbiased "hypothesis-free" analysis framework, we developed a strategy to interrogate how phosphorylation dynamically impacts protein turnover across the time series data. With this strategy, we discovered a complex relationship between phosphorylation and protein turnover that was previously underexplored. Our results further revealed a link between phosphorylation stoichiometry with the turnover of phosphorylated peptidoforms. Moreover, our results suggested that phosphoproteomic turnover diversity cannot directly explain the abundance regulation of phosphorylation during cell starvation, underscoring the importance of future studies addressing PTM site-resolved protein turnover.

Transcriptomic and Proteomic Changes Driving Pulmonary Fibrosis Resolution in Young and Old Mice.

Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFß antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.