Early Expression Research Articles

Modulation of early gene expression responses to water deprivation stress by the E3 ubiquitin ligase ATL80: implications for retrograde signaling interplay

BackgroundPrimary response genes play a pivotal role in translating short-lived stress signals into sustained adaptive responses. In this study, we investigated the involvement of ATL80, an E3 ubiquitin ligase, in the dynamics of gene expression following water deprivation stress. We observed that ATL80 is rapidly activated within minutes of water deprivation stress perception, reaching peak expression around 60 min before gradually declining. ATL80, despite its post-translational regulation role, emerged as a key player in modulating early gene expression responses to water deprivation stress.ResultsThe impact of ATL80 on gene expression was assessed using a time-course microarray analysis (0, 15, 30, 60, and 120 min), revealing a burst of differentially expressed genes, many of which were associated with various stress responses. In addition, the diversity of early modulation of gene expression in response to water deprivation stress was significantly abolished in the atl80 mutant compared to wild-type plants. A subset of 73 genes that exhibited a similar expression pattern to ATL80 was identified. Among them, several are linked to stress responses, including ERF/AP2 and WRKY transcription factors, calcium signaling genes, MAP kinases, and signaling peptides. Promoter analysis predicts enrichment of binding sites for CAMTA1 and CAMTA5, which are known regulators of rapid stress responses. Furthermore, we have identified a group of differentially expressed ERF/AP2 transcription factors, proteins associated with folding and refolding, as well as pinpointed core module genes which are known to play roles in retrograde signaling pathways that cross-referenced with the early ATL80 transcriptome.ConclusionsBased on these findings, we propose that ATL80 may target one or more components within the retrograde signaling pathways for degradation. In essence, ATL80 serves as a bridge connecting these signaling pathways and effectively functions as an alarm signal.

Antiviral responses induced by Tdap-IPV vaccination are associated with persistent humoral immunity to Bordetella pertussis

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

Evaluation of the Effects of Whey Protein and Moringa Oleifera Leaves Extract Mixture on Osseointegration in Rabbits.

Osteogenic, antioxidant and anti-inflammatory effects of Whey protein and M. oleifera gel prompted us to evaluate their role alone or in combination on osseointegration in rabbits. In this study, 24 titanium implants were inserted in the femurs of six rabbits. One implant was placed without treatment, and another one was coated with a mixture of whey protein and M. oleifera gel for each side. The animals were divided into two groups of 2- and 6-week intervals and evaluated using histopathological and immunohistochemical techniques. Histological evaluation revealed a significant difference between the experimental and the control groups after two weeks in osteoblast and osteocyte counts. The experimental group had mature bone development after six weeks of implantation, while the control group had a woven bone. Immunohistochemical results showed that the experimental group, compared to the control group, exhibited early positive expression of osteoblast cells at two weeks after the experiment. Based on histopathological observations, the experimental group showed a tiny area of collagenous fiber in 6th week after the implantation. A mixture of whey protein and M. oleifera could accelerate osseointegration and healing processes.

The relationship between extreme inter-individual variation in macrophage gene expression and genetic susceptibility to inflammatory bowel disease

The differentiation of resident intestinal macrophages from blood monocytes depends upon signals from the macrophage colony-stimulating factor receptor (CSF1R). Analysis of genome-wide association studies (GWAS) indicates that dysregulation of macrophage differentiation and response to microorganisms contributes to susceptibility to chronic inflammatory bowel disease (IBD). Here, we analyzed transcriptomic variation in monocyte-derived macrophages (MDM) from affected and unaffected sib pairs/trios from 22 IBD families and 6 healthy controls. Transcriptional network analysis of the data revealed no overall or inter-sib distinction between affected and unaffected individuals in basal gene expression or the temporal response to lipopolysaccharide (LPS). However, the basal or LPS-inducible expression of individual genes varied independently by as much as 100-fold between subjects. Extreme independent variation in the expression of pairs of HLA-associated transcripts (HLA-B/C, HLA-A/F and HLA-DRB1/DRB5) in macrophages was associated with HLA genotype. Correlation analysis indicated the downstream impacts of variation in the immediate early response to LPS. For example, variation in early expression of IL1B was significantly associated with local SNV genotype and with subsequent peak expression of target genes including IL23A, CXCL1, CXCL3, CXCL8 and NLRP3. Similarly, variation in early IFNB1 expression was correlated with subsequent expression of IFN target genes. Our results support the view that gene-specific dysregulation in macrophage adaptation to the intestinal milieu is associated with genetic susceptibility to IBD.

Lithium-loaded GelMA-Phosphate glass fibre constructs: Implications for astrocyte response.

Combinations of different biomaterials with their own advantages as well as functionalization with other components have long been implemented in tissue engineering to improve the performance of the overall material. Biomaterials, particularly hydrogel platforms, have shown great potential for delivering compounds such as drugs, growth factors, and neurotrophic factors, as well as cells, in neural tissue engineering applications. In central the nervous system, astrocyte reactivity and glial scar formation are significant and complex challenges to tackle for neural and functional recovery. GelMA hydrogel-based tissue constructs have been developed in this study and combined with two different formulations of phosphate glass fibers (PGFs) (with Fe3+ or Ti2+ oxide) to impose physical and mechanical cues for modulating astrocyte cell behavior. This study was also aimed at investigating the effects of lithium-loaded GelMA-PGFs hydrogels in alleviating astrocyte reactivity and glial scar formation offering novel perspectives for neural tissue engineering applications. The rationale behind introducing lithium is driven by its long-proven therapeutic benefits in mental disorders, and neuroprotective and pronounced anti-inflammatory properties. The optimal concentrations of lithium and LPS were determined in vitro on primary rat astrocytes. Furthermore, qPCR was conducted for gene expression analysis of GFAP and IL-6 markers on primary astrocytes cultured 3D into GelMA and GelMA-PGFs hydrogels with and without lithium and in vitro stimulated with LPS for astrocyte reactivity. The results suggest that the combination of bioactive phosphate-based glass fibers and lithium loading into GelMA structures may impact GFAP expression and early IL-6 expression. Furthermore, GelMA-PGFs (Fe) constructs have shown improved performance in modulating glial scarring over GFAP regulation.

Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila.

Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, we identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. We find that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. We also find that Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.

Delta-dependent Notch activation closes the early neuroblast temporal program to promote lineage progression and neurogenesis termination in Drosophila

Neuroblasts in Drosophila divide asymmetrically, sequentially expressing a series of intrinsic factors to generate a diversity of neuron types. These intrinsic factors known as temporal factors dictate timing of neuroblast transitions in response to steroid hormone signaling and specify early versus late temporal fates in neuroblast neuron progeny. After completing their temporal programs, neuroblasts differentiate or die, finalizing both neuron number and type within each neuroblast lineage. From a screen aimed at identifying genes required to terminate neuroblast divisions, we identified Notch and Notch pathway components. When Notch is knocked down, neuroblasts maintain early temporal factor expression longer, delay late temporal factor expression, and continue dividing into adulthood. We find that Delta, expressed in cortex glia, neuroblasts, and after division, their GMC progeny, regulates neuroblast Notch activity. We also find that Delta in neuroblasts is expressed high early, low late, and is controlled by the intrinsic temporal program: early factor Imp promotes Delta, late factors Syp/E93 reduce Delta. Thus, in addition to systemic steroid hormone cues, forward lineage progression is controlled by local cell-cell signaling between neuroblasts and their cortex glia/GMC neighbors: Delta transactivates Notch in neuroblasts bringing the early temporal program and early temporal factor expression to a close.

Early expression of GluN2A-containing NMDA receptors in a model of fragile X syndrome.

NMDA-type glutamate receptors (NMDARs) play a crucial role in synaptogenesis, circuit development, and synaptic plasticity, serving as fundamental components in cellular models of learning and memory. Their dysregulation has been implicated in several neurological disorders and synaptopathies. NMDARs are heterotetrameric complexes composed of two GluN1 and two GluN2 subunits. The composition of GluN2 subunits determines the main biophysical properties of the channel, such as calcium permeability and gating kinetics, and influences the ability of the receptor to interact with postsynaptic proteins involved in normal synaptic physiology and plasticity, including scaffolding proteins and signaling molecules. During early development, NMDARs in the forebrain contain solely the GluN2B subunit, a necessary subunit for proper synaptogenesis and synaptic plasticity. As the animal matures, the expression of the GluN2A subunit increases, leading to a partial replacement of GluN2B-containing synaptic NMDARs with GluN2A-containing receptors. The switch in the synaptic GluN2A-to-GluN2B ratio has a significant impact on the kinetics of excitatory postsynaptic currents and diminishes the synaptic plasticity capacity. In this study, we present findings indicating that GluN2A expression occurs earlier in a mouse model of fragile X syndrome (FXS). This altered timing of GluN2A expression affects various important parameters of NMDAR-mediated excitatory postsynaptic currents, including maximal current amplitude, decay time, and response to consecutive stimuli delivered in close temporal proximity. These observations suggest that the early expression of GluN2A during a critical period when synapses and circuits are developing could be an underlying factor contributing to the formation of pathological circuits in the FXS mouse model.NEW & NOTEWORTHY NMDA receptors (NMDARs) play important roles in synaptic transmission and are involved in multiple neurological disorders. During development, GluN2A in the forebrain becomes incorporated into previously GluN2B-dominated NMDARs, leading to the "GluN2A/GluN2B ratio switch." This is a crucial step for normal brain development. Here we present findings indicating that GluN2A expression occurs earlier in the fragile X mouse and this could be an underlying factor contributing to the pathology found in the fragile X model.

Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses.

We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors. Using virological assays, we show that these bisbenzimide compounds inhibit VACV spread, plaque formation, and the production of infectious progeny VACV with relatively low cell toxicity. Further analysis of the VACV lifecycle indicated that the effective bisbenzimide compounds had little impact on VACV early gene expression but inhibited VACV late gene expression and truncated the formation of VACV replication sites. Additionally, we found that bisbenzimide compounds, including H42, can inhibit both monkeypox and a VACV mutant resistant to the widely used anti-poxvirus drug TPOXX (Tecovirimat). Therefore, the tested bisbenzimide compounds were inhibitors of both prototypic and pandemic potential poxviruses and could be developed for use in situations where anti-poxvirus drug resistance may occur. Additionally, these data suggest that bisbenzimide compounds may serve as broad-activity antiviral compounds, targeting diverse DNA viruses such as poxviruses and betaherpesviruses.IMPORTANCEThe 2022 mpox (monkeypox) outbreak served as a stark reminder that due to the cessation of smallpox vaccination over 40 years ago, most of the human population remains susceptible to poxvirus infection. With only two antivirals approved for the treatment of smallpox infection in humans, the need for additional anti-poxvirus compounds is evident. Having shown that the bisbenzimide H33342 is a potent inhibitor of poxvirus gene expression and DNA replication, here we extend these findings to include a set of novel bisbenzimide compounds that show anti-viral activity against mpox and a drug-resistant prototype poxvirus mutant. These results suggest that further development of bisbenzimides for the treatment of pandemic potential poxviruses is warranted.

In vitro SELEX and application of an African swine fever virus (ASFV) p30 protein specific aptamer

The African swine fever virus (ASFV) has caused severe economic losses in the pig industry. To monitor ASFV spread, the p30 protein has been identified as an ideal infection marker due to its early and long-term expression during the ASFV infection period. Timely monitoring of ASFV p30 enables the detection of ASFV infection and assessment of disease progression. Aptamers are an outstanding substitute for antibodies to develop an efficient tool for ASFV p30 protein detection. In this study, a series of aptamer candidates were screened by in vitro magnetic bead-based systematic evolution of ligands by exponential enrichment (MB-SELEX). An aptamer (Atc-20) finally showed high specificity and affinity (Kd = 140 ± 10 pM) against ASFV p30 protein after truncation and affinity assessment. Furthermore, an aptamer/antibody heterogeneous sandwich detection assay was designed based on Atc20, achieving a linear detection of ASFV p30 ranging from 8 to 125 ng/ml and a detection limit (LOD) of 0.61 ng/ml. This assay showed good analytical performances and effectively detected p30 protein in diluted serum samples, presenting promising potential for the development of ASFV biosensors.

Signal peptide peptidase-like 2b modulates the amyloidogenic pathway and exhibits an Aβ-dependent expression in Alzheimer's disease

Alzheimer's disease (AD) is a multifactorial disorder driven by abnormal amyloid β-peptide (Aβ) levels. In this study, we investigated the role of presenilin-like signal peptide peptidase-like 2b (SPPL2b) in AD pathophysiology and its potential as a druggable target within the Aβ cascade. Exogenous Aβ42 influenced SPPL2b expression in human cell lines and acute mouse brain slices. SPPL2b and its AD-related substrate BRI2 were evaluated in the brains of AppNL-G-F knock-in AD mice and human postmortem AD brains. An early high cortical expression of SPPL2b was observed, followed by a downregulation in late AD pathology in AppNL-G-F mice, correlating with synaptic loss. To understand the consequences of pathophysiological SPPL2b dysregulation, we found that SPPL2b overexpression significantly increased APP cleavage, while genetic deletion reduced APP cleavage and Aβ production. Notably, postmortem AD brains showed higher levels of SPPL2b's BRI2 substrate compared to healthy control samples. These results strongly support the involvement of SPPL2b in AD pathology. The early Aβ-induced upregulation of SPPL2b may enhance Aβ production in a vicious cycle, further aggravating Aβ pathology. Therefore, SPPL2b emerges as a potential anti-Aβ drug target.

Unsupervised analysis of whole transcriptome data from human pluripotent stem cells cardiac differentiation

The main objective of the present work was to highlight differences and similarities in gene expression patterns between different pluripotent stem cell cardiac differentiation protocols, using a workflow based on unsupervised machine learning algorithms to analyse the transcriptome of cells cultured as a 2D monolayer or as 3D aggregates. This unsupervised approach effectively allowed to portray the transcriptomic changes that occurred throughout the differentiation processes, with a visual representation of the entire transcriptome. The results allowed to corroborate previously reported data and also to unveil new gene expression patterns. In particular, it was possible to identify a correlation between low cardiomyocyte differentiation efficiencies and the early expression of a set of non-mesodermal genes, which can be further explored as predictive markers of differentiation efficiency. The workflow here developed can also be applied to analyse other stem cell differentiation transcriptomic datasets, envisaging future clinical implementation of cellular therapies.

Spatio-temporal transcriptome and storage compound profiles of developing faba bean (Vicia faba) seed tissues.

Faba bean (Vicia faba) is a legume grown in diverse climate zones with a high potential for increased cultivation and use in food due to its nutritional seeds. In this study, we characterized seed tissue development in faba bean to identify key developmental processes; from embryo expansion at the expense of the endosperm to the maturing storage stages of the bean seed. A spatio-temporal transcriptome profiling analysis, combined with chemical nutrient analysis of protein, starch, and lipid, of endosperm and embryo tissues at different developmental stages, revealed gene expression patterns, transcriptional networks, and biochemical pathways in faba bean. We identified key players in the LAFL (LEC1, ABI3, FUS3, and LEC2) transcription factor network as well as their major repressors VAL1 and ASIL1. Our results showed that proteins accumulated not only in the embryo but also in the endosperm. Starch accumulated throughout seed development and oil content increased during seed development but at very low levels. The patterns of differentially expressed transcripts encoding proteins with functions in the corresponding metabolic pathways for the synthesis of these storage compounds, to a high extent, aligned with these findings. However, the early expression of transcripts encoding WRI1 combined with the late expression of oil body proteins indicated a not manifested high potential for lipid biosynthesis and oil storage. Altogether, this study contributes to increased knowledge regarding seed developmental processes applicable to future breeding methods and seed quality improvement for faba bean.

Quality Initiative Program to Improve the Percentage of Very Preterm Neonates Receiving Expressed Breast Milk Within 24 Hours of Birth in a Tertiary Care (Level III-B) NICU

Background There is often a delay in the initiation of feeding in preterm babies and more dependence on PDHM milk due to the non-availability of the mother’s own milk. This study aimed to increase the percentage of very preterm neonates receiving expressed breast milk (EBM) within 24 hours of birth. Methods A quality improvement (QI) initiative study was conducted from May 2022 to August 2022 in a tertiary care (Level III-B) NICU. A QI support team was formed. The reasons for the delay in the initiation of expression of breastmilk were evaluated. We included preterm neonates (<32 weeks gestation) born within the study period. Antenatal and postnatal counseling regarding the expression of breast milk, using leaflets and video, frequent visits, telephonic reminders, and emphasizing Kangaroo Mother Care through various Plan-Do-Study-Act cycles were done. Results A total of 17 very preterm babies were studied over a period of 3 months. The percentage of preterm receiving EBM within 24 hours of birth, increased from 20% (observation phase) to 83.3% (initiation phase) and maintained to 90% in the continuation phase. The time of receiving the first EBM reduced from a median of 32.5 hours (1.35 days) in the retrospective phase to 17 hours (0.7 days) during the initiation phase and it was sustained later on. Mothers were able to express as early as within 4.5 hours. Conclusion A simple QI intervention showed early expression and sustenance of breast milk in mothers of preterm neonates.

The amyloid precursor protein intracellular domain induces sleep disruptions and its nuclear localization fluctuates in circadian pacemaker neurons in Drosophila and mice

The most prominent symptom of Alzheimer's disease (AD) is cognitive decline; however, sleep and other circadian disruptions are also common in AD patients. Sleep disruptions have been connected with memory problems and therefore the changes in sleep patterns observed in AD patients may also actively contribute to cognitive decline. However, the underlying molecular mechanisms that connect sleep disruptions and AD are unclear. A characteristic feature of AD is the formation of plaques consisting of Amyloid-β (Aβ) peptides generated by cleavage of the Amyloid Precursor Protein (APP). Besides Aβ, APP cleavage generates several other fragments, including the APP intracellular domain (AICD) that has been linked to transcriptional regulation and neuronal homeostasis. Here we show that overexpression of the AICD reduces the early evening expression of two core clock genes and disrupts the sleep pattern in flies. Analyzing the subcellular localization of the AICD in pacemaker neurons, we found that the AICD levels in the nucleus are low during daytime but increase at night. While this pattern of nuclear AICD persisted with age, the nighttime levels were higher in aged flies. Increasing the cleavage of the fly APP protein also disrupted AICD nuclear localization. Lastly, we show that the day/nighttime nuclear pattern of the AICD is also detectable in neurons in the suprachiasmatic nucleus of mice and that it also changes with age. Together, these data suggest that AD-associated changes in APP processing and the subsequent changes in AICD levels may cause sleep disruptions in AD.

Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.

Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.

Silencing ANGPTL8 reduces mouse preadipocyte differentiation and insulin signaling

ANGPTL8, expressed mainly in the liver and adipose tissue, regulates the activity of lipoprotein lipase (LPL) present in the extracellular space and triglyceride (TG) metabolism through its interaction with ANGPTL3 and ANGPTL4. Whether intracellular ANGPTL8 can also exert effects in tissues where it is expressed is uncertain. ANGPTL8 expression was low in preadipocytes and much increased during differentiation. To better understand the role of intracellular ANGPTL8 in adipocytes and assess whether it may play a role in adipocyte differentiation, we knocked down its expression in normal mouse subcutaneous preadipocytes. ANGPTL8 knockdown reduced adipocyte differentiation, cellular TG accumulation and also isoproterenol-stimulated lipolysis at day 7 of differentiation. RNA-Seq analysis of ANGPTL8 siRNA or control siRNA transfected SC preadipocytes on days 0, 2, 4 and 7 of differentiation showed that ANGPTL8 knockdown impeded the early (day 2) expression of adipogenic and insulin signaling genes, PPARγ, as well as genes related to extracellular matrix and NF-κB signaling. Insulin mediated Akt phosphorylation was reduced at an early stage during adipocyte differentiation. This study based on normal primary cells shows that ANGPTL8 has intracellular actions in addition to effects in the extracellular space, like modulating LPL activity. Preadipocyte ANGPTL8 expression modulates their differentiation possibly via changes in insulin signaling gene expression.

Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells. Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes. The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified. This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

Cystic Fibrosis in Adults: A Paradigm of Frailty Syndrome? An Observational Study.

This study aimed to assess the main clinical and anamnestic characteristics of adult Cystic Fibrosis (CF) patients and to evaluate the association of frailty with the CF genotyping classification. In an observational cross-sectional study, all ambulatory CF patients over 18 years old who received a diagnosis at the Regional Cystic Fibrosis Center for adults were enrolled and assessed by spirometry for respiratory function, by ADL and IADL for functional status, and by the Study of Osteoporotic Fractures (SOF) Index for frailty. The study population consisted of 139 CF patients (mean age 32.89 ± 10.94 years old, 46% women). Most of the subjects were robust (60.4%). The pre-frail/frail group was more frequently females (p = 0.020), had a lower BMI (p = 0.001), worse respiratory function, a higher number of pulmonary exacerbations/years, cycles of antibiotic therapy, and hospitalization (all p < 0.001) with respect to robust patients. The pre-frail/frail subjects used more drugs and were affected by more CF-related diseases (all p < 0.001). In relation to logistic regression, the best predictor of the pre-frail/frail status was a low FEV1 level. The CF patients show similarities to older pre-frail/frail subjects, suggesting that CF might be considered an early expression of this geriatric syndrome. This finding could help to better define the possible progression of CF, but overall, it could also suggest the usefulness employing of some tools used in the management and therapy of frailty subjects to identify the more severe CF subjects.