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Neurotrophic receptor tyrosine kinases (NTRKs) belong to the receptor tyrosine kinase (RTK) family. NTRKs are responsible for the activation of multiple downstream signaling pathways that regulate cell growth, proliferation, differentiation, and apoptosis. NTRK-associated mutations often result in oncogenesis and lead to aberrant activation of downstream signaling pathways including MAPK, JAK/STAT, and PLCγ1. This study characterizes the NACC2-NTRK2 oncogenic fusion protein that leads to pilocytic astrocytoma and pediatric glioblastoma. This fusion joins the BTB domain (Broad-complex, Tramtrack, and Bric-a-brac) domain of NACC2 (Nucleus Accumbens-associated protein 2) with the transmembrane helix and tyrosine kinase domain of NTRK2. We focus on identifying critical domains for the biological activity of the fusion protein. Mutations were introduced in the charged pocket of the BTB domain or in the monomer core, based on a structural comparison of the NACC2 BTB domain with that of PLZF, another BTB-containing protein. Mutations were also introduced into the NTRK2-derived portion to allow comparison of two different breakpoints that have been clinically reported. We show that activation of the NTRK2 kinase domain relies on multimerization of the BTB domain in NACC2-NTRK2. Mutations which disrupt BTB-mediated multimerization significantly reduce kinase activity and downstream signaling. The ability of these mutations to abrogate biological activity suggests that BTB domain inhibition could be a potential treatment for NACC2-NTRK2-induced cancers. Removal of the transmembrane helix leads to enhanced stability of the fusion protein and increased activity of the NACC2-NTRK2 fusion, suggesting a mechanism for the oncogenicity of a distinct NACC2-NTRK2 isoform observed in pediatric glioblastoma.

Toxoplasma gondii is an intracellular parasite that establishes a long-term infection in the brain of many warm-blooded hosts, including humans and rodents. Like all obligate intracellular microbes, Toxoplasma uses many effector proteins to manipulate the host cell to ensure parasite survival. While some of these effector proteins are universal to all Toxoplasma strains, some are polymorphic between Toxoplasma strains. One such polymorphic effector is GRA15. The gra15 allele carried by type II strains activates host NF-κB signaling, leading to the release of cytokines such as IL-12, TNF, and IL-1β from immune cells infected with type II parasites. Prior work also suggested that GRA15 promotes early host control of parasites in vivo, but the effect of GRA15 on parasite persistence in the brain and the peripheral immune response has not been well defined. For this reason, we sought to address this gap by generating a new IIΔgra15 strain and comparing outcomes at 3 weeks post infection between WT and IIΔgra15 infected mice. We found that the brain parasite burden and the number of macrophages/microglia and T cells in the brain did not differ between WT and IIΔgra15 infected mice. In addition, while IIΔgra15 infected mice had a lower number and frequency of splenic M1-like macrophages and frequency of PD-1+ CTLA-4+ CD4+ T cells and NK cells compared to WT infected mice, the IFN-γ+ CD4 and CD8 T cell populations were equivalent. In summary, our results suggest that in vivo GRA15 may have a subtle effect on the peripheral immune response, but this effect is not strong enough to alter brain parasite burden or parenchymal immune cell number at 3 weeks post infection.

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition.

Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. Here, we studied a molecular mechanism by which Med1 facilitates these processes, in particular, by focusing on TGFβ signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfβ1/β2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFβ1/β2) and diminishes activity of TGFβ signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFβ components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFβ ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFβ signaling pathway. Finally, we show that pharmacological inhibition of TGFβ signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration.

Chronic systemic sterile inflammation is implicated in the pathogenesis of cerebrovascular disease and white matter injury. Non-invasive blood markers for risk stratification and dissection of inflammatory molecular substrates in vivo are lacking. We sought to identify whether an interconnected network of inflammatory biomarkers centered on IL-18 and all previously associated with white matter lesions could detect overt and antecedent white matter changes in two populations at risk for cerebral small vessel disease. In a cohort of 167 older adults (mean age: 76, SD 7.1, 83 females) that completed a cognitive battery, physical examination, and blood draw in parallel with MR imaging including DTI, we measured cerebral white matter hyperintensities (WMH) and free water (FW). Concurrently, serum levels of a biologic network of inflammation molecules including MPO, GDF-15, RAGE, ST2, IL-18, and MCP-1 were measured. The ability of a log-transformed population mean-adjusted inflammatory composite score (ICS) to associate with MR variables was demonstrated in an age and total intracranial volume adjusted model. In this cohort, ICS was significantly associated with WMH (β = 0.222, p = 0.013), FW (β = 0.3, p = 0.01), and with the number of vascular risk factor diagnoses (r = 0.36, p

Migration of cells from both local and systemic sources is essential for the inflammatory and regenerative processes that occur during normal wound healing. CXCL12 is considered a critical regulator of CXCR4-positive cell migration during tissue regeneration. In this study, we investigated the expression of Cxcl12 and Cxcr4 during healing of a murine full thickness ear wound. We also investigated the expression of angiopoietin-like 4, which has been shown to participate in wound angiogenesis and reepithelialization. At time points up to 48hrs, complete blood counts were performed using automated hematology analysis, and the numbers of circulating stem and progenitor cells quantified using flow cytometry. Expression of both Cxcr4 and Angptl4 was significantly elevated within 3 days of wounding, and both were strongly expressed in cells of the epidermis. ANGPTL4 protein expression remained elevated in the epithelium through day 14. Cxcl12 expression was increased significantly at day 3, and remained elevated through day 21. Faint Cxcl12 staining was detectable in the epithelium at day 1, and thereafter staining was faint and more generalized. There were significantly fewer circulating total white blood cells and lymphocytes 1hr following ear punching. Similarly, there was a significant early (1hr) reduction in the number of circulating endothelial progenitor cells. Further studies are warranted to investigate whether ANGPTL4 and CXCL12/CXCR4 interact or synergize to facilitate cell recruitment and migration, and to potentiate reepithelialization and wound healing.

Because little is known about the role of corticotropin-releasing factor (CRF) agonists in regulating responses in pancreatitis, we evaluated the effects of urocortin 2 (UCN2) and stressin1 in caerulein-induced acute pancreatitis (AP) model in rats. Male rats were pretreated with UCN2 or stressin1 for 30 min followed by induction of AP with supraphysiologic doses of caerulein. Serum amylase and lipase activity, pancreatic tissue necrosis, immune cell infiltrate, nuclear factor (NF)-κB activity, trypsin levels, and intracellular Ca2+ ([Ca2+]i) were ascertained. UCN2, but not stressin1 attenuated the severity of AP in rats. UCN2, but not stressin1, reduced serum amylase and lipase activity, cell necrosis and inflammatory cell infiltration in AP. NF-κB activity in pancreatic nuclear extracts increased in AP and UCN2 treatment reduced caerulein-induced increases in NF-κB activity by 42%. UCN2 treatment prevented caerulein-induced degradation of IκB-α in the cytosolic fraction as well as increased levels of p65 subunit of NF-κB in the cytosolic fraction. Pancreatic UCN2 levels decreased in AP compared with saline. UCN2 evoked [Ca2+]i responses in primary acinar cells and abolished caerulein-evoked [Ca2+]i responses at 0.1nM, and decreased by ~50% at 1.0nM caerulein. UCN2 stimulation resulted in redistribution of a portion of F-actin from the apical to the basolateral pole. UCN2 prevented the massive redistribution of F-actin observed with supraphysiologic doses of caerulein. UCN2, but not stressin1 attenuated severity of an experimental pancreatitis model. The protective effects of UCN2, including anti-inflammatory and anti-necrotic effects involve activation of the CRF2 receptor, [Ca2+]i signaling, and inhibition of NF-κB activity.

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).

The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naïve T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.

Neointimal hyperplasia, stimulated by injury and certain vascular diseases, promotes artery obstruction and tissue ischemia. In vascular smooth muscle cell (VSMCs), multiple modulators of protein handling machinery regulate intimal hyperplasia. These include elements of the VSMC unfolded protein response to endoplasmic reticulum stress (UPRER), and transglutaminase 2 (TG2), which catalyzes post-translational protein modification. Previous results for deficiency of UPRER-specific mediator XBP1, and of TG2, have been significant, but in multiple instances contradictory, for effects on cultured VSMC function, and, using multiple models, for neointimal hyperplasia in vivo. Here, we engineered VSMC-specific deficiency of XBP1, and studied cultured VSMCs, and neointimal hyperplasia in response to carotid artery ligation in vivo. Intimal area almost doubled in Xbp1fl/fl SM22α-CRE+ mice 21 days post-ligation. Cultured murine Xbp1 deficient VSMCs migrated more in response to platelet derived growth factor (PDGF) than control VSMCs, and had an increased level of inositol-requiring enzyme 1α (Ire1α), a PDGF receptor-binding UPRER transmembrane endonuclease whose substrates include XBP1. Cultured XBP1-deficient VSMCs demonstrated decreased levels of TG2 protein, in association with increased TG2 polyubiquitination, but with increased TG transamidation catalytic activity. Moreover, IRE1α, and TG2-specific transamidation cross-links were increased in carotid artery neointima in Xbp1fl/fl SM22α-CRE+ mice. Cultured TG2-deficient VSMCs had decreased XBP1 associated with increased IRE1α, and increased migration in response to PDGF. Neointimal hyperplasia also was significantly increased in Tgm2fl/fl SM22α-CRE+ mice at 21 days after carotid ligation. In conclusion, a VSMC regulatory circuit between XBP1 and TG2 limits neointimal hyperplasia in response to carotid ligation.

Inflammation plays a critical role in initiation of adaptive immunity, pathogen clearance and tissue repair. Interleukin (IL)-1β is a potent pro-inflammatory cytokine and therefore its production is tightly regulated: its secretion requires the assembly of a macromolecular protein complex, termed the inflammasome. Aberrant activation of the inflammasome has been linked to debilitating human diseases including chronic inflammatory and autoimmune diseases. Thus, there is a great interest in understanding how inflammasomes are regulated. Here we show that Dicer, an enzyme necessary for the production of mature micro-RNAs (miRNAs), is required for optimal activation of NLRP3 inflammasomes in bone marrow macrophages. Our data indicate that miRNAs may play an important role in promoting inflammasome activation.

Dysregulation of the seven-transmembrane (7TM) receptor Smoothened (SMO) and other components of the Hedgehog (Hh) signaling pathway contributes to the development of cancers including basal cell carcinoma (BCC) and medulloblastoma (MB). However, SMO-specific antagonists produced mixed results in clinical trials, marked by limited efficacy and high rate of acquired resistance in tumors. Here we discovered that Nilotinib, an approved inhibitor of several kinases, possesses an anti-Hh activity, at clinically achievable concentrations, due to direct binding to SMO and inhibition of SMO signaling. Nilotinib was more efficacious than the SMO-specific antagonist Vismodegib in inhibiting growth of two Hh-dependent MB cell lines. It also reduced tumor growth in subcutaneous MB mouse xenograft model. These results indicate that in addition to its known activity against several tyrosine-kinase-mediated proliferative pathways, Nilotinib is a direct inhibitor of the Hh pathway. The newly discovered extension of Nilotinib's target profile holds promise for the treatment of Hh-dependent cancers.

A growing body of literature has demonstrated the potential for ketamine in the treatment of major depression. Sub-anesthetic doses produce rapid and sustained changes in depressive behavior, both in patients and rodent models, associated with reorganization of glutamatergic synapses in the prefrontal cortex (PFC). While ketamine is known to regulate N-methyl-D-aspartate (NMDA) -type glutamate receptors (NMDARs), the full complement of downstream cellular consequences for ketamine administration are not well understood. Here, we combine electrophysiology with 2-photon imaging and glutamate uncaging in acute slices of mouse PFC to further examine how ketamine alters glutamatergic synaptic transmission. We find that four hours after ketamine treatment, glutamatergic synapses themselves are not significantly affected. However, levels of the neuromodulatory Regulator of G-protein Signaling (RGS4) are dramatically reduced. This loss of RGS4 activity is associated with disruption of the normal compartmentalization of synaptic neuromodulation. Thus, under control conditions, α2 adrenergic receptors and type B γ-aminobutyric acid (GABAB) receptors selectively inhibit α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) -type glutamate receptors (AMPARs) and NMDARs, respectively. After ketamine administration and reduction in RGS4 activity, this selectivity is lost, with both modulatory systems broadly inhibiting glutamatergic transmission. These results suggest a novel mechanism by which ketamine may influence synaptic signaling and provide new avenues for the exploration of therapeutics directed at treating neuropsychiatric disorders, such as depression.

Preterm birth (PTB) is leading contributor to infant death in the United States and globally, yet the underlying mechanistic causes are not well understood. Histopathological studies of preterm birth suggest advanced villous maturity may have a role in idiopathic spontaneous preterm birth (isPTB). To better understand pathological and molecular basis of isPTB, we compared placental villous transcriptomes from carefully phenotyped cohorts of PTB due to infection or isPTB between 28-36 weeks gestation and healthy term placentas. Transcriptomic analyses revealed a unique expression signature for isPTB distinct from the age-matched controls that were delivered prematurely due to infection. This signature included the upregulation of three IGF binding proteins (IGFBP1, IGFBP2, and IGFBP6), supporting a role for aberrant IGF signaling in isPTB. However, within the isPTB expression signature, we detected secondary signature of inflammatory markers including TNC, C3, CFH, and C1R, which have been associated with placental maturity. In contrast, the expression signature of the gestational age-matched infected samples included upregulation of proliferative genes along with cell cycling and mitosis pathways. Together, these data suggest an isPTB molecular signature of placental hypermaturity, likely contributing to the premature activation of inflammatory pathways associated with birth and providing a molecular basis for idiopathic spontaneous birth.

Thyroid hormone (TH) is essential for brain development. While disruption of TH signaling by environmental chemicals has been discussed as a mechanism of developmental neurotoxicity (DNT) for more than a decade, there remains a paucity of information linking specific TH disrupting chemicals to adverse neurodevelopmental outcomes. This data gap reflects, in part, the fact that the molecular machinery of TH signaling is complex and varies according to cell type and developmental time. Thus, establishing a baseline of the ontogenetic profile of expression of TH signaling molecules in relevant cell types is critical for developing in vitro and alternative systems-based models for screening TH disrupting chemicals for DNT. Here, we characterize the transcriptomic profile of molecules critical to TH signaling across three species-human, rat, and zebrafish-in vitro and in vivo across different stages of neurodevelopment. Our data indicate that while cultured human and rat neural progenitor cells, primary cultures of rat cortical cells, and larval zebrafish all express a fairly comprehensive transcriptome of TH signaling molecules, the spatiotemporal expression profiles as well as the responses to TH vary across species and developmental stages. The data presented here provides a roadmap for identifying appropriate in vitro and in simpler systems-based models for mechanistic studies and screening of chemicals that alter neurodevelopment via interference with TH action.

Bile acids are critical contributors to the regulation of whole body glucose homeostasis; however, the mechanisms remain incompletely defined. While the hydrophilic bile acid subtype, ursodeoxycholic acid, has been shown to attenuate hepatic endoplasmic reticulum (ER) stress and thereby improve glucose regulation in mice, the effect of hydrophobic bile acid subtypes on ER stress and glucose regulation in vivo is unknown. Therefore, we investigated the effect of the hydrophobic bile acid subtype, deoxycholic acid (DCA), on ER stress and glucose regulation. Eight week old C57BL/6J mice were fed a high fat diet supplemented with or without DCA. Glucose regulation was assessed by oral glucose tolerance and insulin tolerance testing. In addition, circulating bile acid profile and hepatic insulin and ER stress signaling were measured. DCA supplementation did not alter body weight or food intake, but did impair glucose regulation. Consistent with the impairment in glucose regulation, DCA increased the hydrophobicity of the circulating bile acid profile, decreased hepatic insulin signaling and increased hepatic ER stress signaling. Together, these data suggest that dietary supplementation of DCA impairs whole body glucose regulation by disrupting hepatic ER homeostasis in mice.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

Corticotropin-releasing hormone (CRH) signaling in the hippocampus has been established to be important for mediating the effects of stress on learning and memory. Given our laboratory's recent characterization of a subset of hippocampal CRH neurons as a novel class of GABAergic interneurons, we hypothesized that these local GABAergic hippocampal CRH neurons may influence hippocampal function. Here we applied an array of molecular tools to selectively label and manipulate hippocampal CRH neurons in mice, in order to assess this interneuron population's impact on hippocampus-dependent behaviors and hippocampal network excitability. Genetically-targeted ablation of hippocampal CRH neurons in vivo impaired object recognition memory and substantially enhanced the severity of kainic acid-induced seizures. Conversely, selective activation of CRH neurons in vitro suppressed the excitability of the mossy fiber-CA3 pathway. Additional experiments are needed to reconcile the functions of GABA and CRH signaling of hippocampal CRH neurons on hippocampal function. However, our results indicate that this interneuron population plays an important role in maintaining adaptive network excitability, and provide a specific circuit-level mechanism for this role.

The lungs are highly susceptible to injury, including ischemia/reperfusion (I/R) injury. Pulmonary I/R injury can occur when correcting conditions such as primary pulmonary hypertension, and is also relatively common after lung transplantation or other cardiothoracic surgery. Methods to reduce pulmonary I/R injury are urgently needed to improve outcomes following procedures such as lung transplantation. Remote liver ischemic preconditioning (RLIPC) is an effective cardioprotective measure, reducing damage caused by subsequent cardiac I/R injury, but little is known about its potential role in pulmonary protection. Here, we analyzed the efficacy and mechanistic basis of RLIPC in a rat model of pulmonary I/R injury. RLIPC reduced lung I/R injury, lessening structural damage, inflammatory cytokine production and apoptosis. In addition, RLIPC preserved pulmonary function compared to controls following lung I/R injury. RLIPC stimulated phosphorylation of pulmonary STAT3, a component of the SAFE signaling pathway, but not phosphorylation of RISK pathway signaling proteins. Accordingly, STAT3 inhibition using AG490 eliminated the pulmonary protection afforded by RLIPC. Our data demonstrate for the first time that RLIPC protects against pulmonary I/R injury, via a signaling pathway requiring STAT3 phosphorylation.

Mutations at position K171 in the kinase activation loop of Inhibitor of κB kinase beta (IKKβ) occur in multiple myeloma, spleen marginal zone lymphoma and mantle cell lymphoma. Previously, we demonstrated that these result in constitutive kinase activation and stimulate Signal Transducer and Activator of Transcription 3 (STAT3). This work also identified K147 as a site of K63-linked regulatory ubiquitination required for activation of signaling pathways. We now present a more detailed analysis of ubiquitination sites together with a comprehensive examination of the signaling pathways activated by IKKβ K171E mutants. Downstream activation of STAT3 is dependent upon the activity of: UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; TAK1 (MAP3K7), or TGFβ Activated Kinase, which forms a complex required for NFκB activation; JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; and gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKβ K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKKβ K171E mutants trigger an autocrine loop in which IL-6 is secreted and binds to the IL-6 receptor complex gp130, resulting in JAK activation. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKβ K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKKβ are likely to also exhibit activated STAT3 and p44/42 MAPK (Erk1/2), this suggests the possibility of using MAPK (Erk1/2) and JAK inhibitors, or specific ubiquitination inhibitors. K63-linked ubiquitination occurs in other kinases at sites homologous to K147 in IKKβ, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis.

Patients with Polycystic ovary syndrome (PCOS) have chronic low-grade ovarian inflammation. Inflammation can cause telomere dysfunction, and telomere and telomerase complex are also involved in regulating inflammation. However, the specific mechanisms of inflammatory signaling feedback and telomere-telomerase mutual regulation remain to be discovered. This study elucidates the role of Nuclear factor kappa-B (NF-κB)-Telomerase reverse transcriptase (TERT) feedback in PCOS granulosa cell apoptosis. Using letrozole and a high-fat diet, a PCOS rat model was established, along with a Lipopolysaccharide (LPS) -treated KGN cell inflammation model was established. NF-κB and TERT inhibitors (BAY 11-7082 and BIBR1532) were then administered to LPS-induced KGN cells. PCOS rats displayed disrupted estrous cycles, increased weight, elevated serum testosterone, cystic follicles, granulosa cell layer thinning, and reduced corpora lutea count (P are all less than 0.05). In PCOS rat ovaries, NF-κB, Interleukin-6 (IL-6), Tumor Necrosis Factor α (TNF-α), TERT, Bax, and Caspase-3 exhibited notable upregulation, while Bcl-2 decreased, with telomere elongation (P are all less than 0.05). There were significant correlations among NF-κB-related inflammatory factors, TERT and apoptotic factors, and they were positively correlated with Bax and Caspase-3, and negatively correlated with Bcl-2 (P are all less than 0.05). LPS-treated KGN cells demonstrated increased expression of inflammatory and pro-apoptotic factors, later restored post-treatment with NF-κB and TERT inhibitors (P are all less than 0.05). In conclusion, TERT may induce granulosa cell apoptosis by participating in the regulation of the NF-κB signaling pathway, thereby mediating the chronic inflammatory response of PCOS through downstream inflammatory factors IL-6 and TNF-α., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Xue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study delved into the interplay between CD244 and Src Homology 2 Domain Containing Phosphatase-2 (SHP2) in chronic obstructive pulmonary disease (COPD) pathogenesis, focusing on apoptosis and inflammation in cigarette smoke extract (CSE)-treated human bronchial epithelial (HBE) cells. Analysis of the GSE100153 dataset identified 290 up-regulated and 344 down-regulated differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) highlighted the turquoise module had the highest correlation with COPD samples. Functional enrichment analysis linked these DEGs to critical COPD processes and pathways like neutrophil degranulation, protein kinase B activity, and diabetic cardiomyopathy. Observations on CD244 expression revealed its upregulation with increasing CSE concentrations, suggesting a dose-dependent relationship with inflammatory cytokines (IL-6, IL-8, TNF-α). CD244 knockdown mitigated CSE-induced apoptosis and inflammation, while overexpression exacerbated these responses. Co-immunoprecipitation (Co-IP) confirmed the physical interaction between CD244 and SHP2, emphasizing their regulatory connection. Analysis of Concurrently, the Nuclear Factor-kappa B (NF-κB) and Mitogen-activated protein kinase (MAPK) signaling pathways showed that modulating CD244 expression impacted key pathway components (p-JNK, p-IKKβ, p-ERK, p-P38, p-lkBα, p-P65), an effect reversed upon SHP2 knockdown. These findings underscore the pivotal role of the CD244/SHP2 axis in regulating inflammatory and apoptotic responses in CSE-exposed HBE cells, suggesting its potential as a therapeutic target in COPD treatment strategies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The imbalance between T helper 17 (Th17) and T regulatory (Treg) cells plays a key role in the progression of multiple myeloma (MM)., Methods: The gene expression profiles of MM were acquired and examined from the Gene Expression Omnibus (GEO) database (GSE72213). Our research involved experimental investigations conducted using the MOPC-MM mouse model. Dysregulation of Treg and Th17 cells was evaluated through flow cytometry, while the levels of inflammatory factors were measured using the enzyme-linked immunosorbent assay. Cell proliferation was gauged using the Cell Counting Kit-8 assay, and cell apoptosis was quantified via flow cytometry. Cell metastasis capabilities were determined by conducting transwell assays. To confirm the relationship between miR-27a and PI3K, a dual-luciferase reporter assay was employed. Finally, proteins associated with the PI3K/AKT/mTOR signaling pathway were assessed using western blotting., Results: MiR-27a exhibited reduced expression levels in MM. Moreover, it exerted control over the equilibrium of Th17 and Treg cells while reducing the expression of inflammatory mediators such as TGF-β1 and IL-10 in an in vivo setting. Elevated miR-27a levels led to the inhibition of cell viability, colony formation capacity, migratory and invasive traits in an in vitro context. The PI3K/AKT/mTOR signaling pathway was identified as a direct target of miR-27a and could reverse the effects induced by miR-27a in MM cells. Notably, PI3K was directly targeted by miR-27a., Conclusions: Our study revealed that miR-27a inhibited MM evolution by regulating the Th17/Treg balance. Inhibition of the PI3K/AKT/mTOR signaling pathway by miR-27a may play a potential mechanistic role., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Patients with chronic kidney disease (CKD) have a high incidence of dyslipidemia comprising high triglyceride (TG) and low high-density lipoprotein (HDL)-cholesterol levels. An abnormal increase of TGs within cells can lead to intracellular lipid accumulation. In addition to dyslipidemia, hyperglycemia in diabetes may elicit ectopic lipid deposition in non-adipose tissues. Hyperglycemia increases intracellular levels of methylglyoxal (MG) leading to cellular dysfunction. A deficit of glyoxalase I (GLO1) contributes to dicarbonyl stress. Whether dicarbonyl stress induced by MG causes renal lipotoxicity through alteration of lipid metabolism signaling is still unknown. In this study, mice with high fat diet-induced diabetes were used to investigate the renal pathology induced by MG. NRK52E cells treated with MG were further used in vitro to delineate the involvement of lipogenic signaling. After treatment with MG for 12 weeks, plasma TG levels, renal fatty changes, and tubular injuries were aggravated in diabetic mice. In NRK52E cells, MG activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and sterol regulatory element-binding protein 1 (SREBP1), resulting in stimulation of fatty acid synthase. The intracellular accumulation of lipid droplets was mainly contributed by TGs, which increased the oxidative stress accompanied by high Nrf2 expression. In addition, MG time-dependently activated cyclin D, cyclin-dependent kinase 4 (CDK4), and cleaved caspase-3, evidencing that G0/G1 arrest was associated with apoptosis of NRK52E cells. In conclusion, our studies revealed the mechanism of lipotoxicity caused by MG. The target of such dicarbonyl stress may become a promising therapy for diabetic CKD., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Peng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pulmonary fibrosis is characterized by excessive extracellular matrix (ECM) accumulation caused by detrimental stimuli. The progressive impairment in lung functions is chronic and highly fatal, presenting itself as a global health challenge. Because of the lack of efficacious treatments, the underlying mechanism should be investigated. The progression of fibrosis involves transforming growth factor-beta 1 (TGF-β1), which accelerates ECM production via epithelial-mesenchymal transition and cell invasion. As microRNAs (miRNAs) serve as regulators of disease development and progression, this study aimed to investigate the interaction of miRNAs and target genes that could contribute to pulmonary fibrosis when exposed to TGF-β1. Differentially expressed mRNA and miRNA were identified in respiratory epithelial cells via transcriptome analysis by using the constructed TGF-β1-induced fibrosis model. Our results revealed a significant increase in the expression of thrombospondin 1 (THBS1), which participates in TGF-β1 activation, where THBS1 was identified as a core gene in protein interactions analyzed through bioinformatics. The expression of miR-335-3p, which targets 3'-UTR of THBS1, substantially decreased upon TGF-β1 treatment. The TGF-β1 downstream signal was suppressed by inhibiting the interaction between TGF-β1 and THBS1, consequently alleviating fibrosis. When the miR-335-3p mimic was transfected in TGF-β1-treated respiratory epithelial cells, THBS1 and fibrosis markers were downregulated, while the introduction of miR-335-3p inhibitor exhibited a reverse phenomenon. Our findings demonstrated that TGF-β1 exposure to respiratory epithelial cells led to a decrease in miR-335-3p expression, resulting in the upregulation of THBS1 and ultimately exacerbating fibrosis. This study provides insights into TGF-β1-induced pulmonary fibrosis, suggesting new therapeutic targets and mechanisms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Armando et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Regulation of visual system function demands precise gene regulation. Dysregulation of miRNAs, as key regulators of gene expression in retinal cells, contributes to different eye disorders such as diabetic retinopathy (DR), macular edema, and glaucoma. MIR-96, a member of the MIR-183 cluster family, is widely expressed in the retina, and its alteration is associated with neovascular eye diseases. MIR-96 regulates protein cascades in inflammatory and insulin signaling pathways, but further investigation is required to understand its potential effects on related genes. For this purpose, we identified a series of key target genes for MIR-96 based on gene and protein interaction networks and utilized text-mining resources. To examine the MIR-96 impact on candidate gene expression, we overexpressed MIR-96 via adeno-associated virus (AAV)-based plasmids in human retinal pigment epithelial (RPE) cells. Based on Real-Time PCR results, the relative expression of the selected genes responded differently to overexpressed MIR-96. While the expression levels of IRS2, FOXO1, and ERK2 (MAPK1) were significantly decreased, the SERPINF1 gene exhibited high expression simultaneously. pAAV-delivered MIR-96 had no adverse effect on the viability of human RPE cells. The data showed that changes in insulin receptor substrate-2 (IRS2) expression play a role in disrupted retinal insulin signaling and contribute to the development of diabetic complications. Considered collectively, our findings suggest that altered MIR-96 and its impact on IRS/PI3K/AKT/VEGF axis regulation contribute to DR progression. Therefore, further investigation of the IRS/PI3K/AKT/VEGF axis is recommended as a potential target for DR treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hosseinpoor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: This study aims to systematically analyze the potential active components of Eucommiae cortex in the treatment of post- myocardial infarction heart failure through network analysis and molecular docking methods. In vitro experiments were conducted to verify that medioresinol, a component of Eucommiae cortex, improves oxygen-glucose deprivation-induced cell failure through its anti-inflammatory and antioxidant capacities., Methods: Potential active components of Eucommiae cortex were screened using specific data. The targets of these components were predicted using Swiss Institute of Bioinformatics database and TargetNet, and key targets were identified by intersecting with the disease targets of myocardial infarction and heart failure. Protein-Protein Interaction analysis was performed on the key targets to screen for core targets. Genomics Institute of the Novartis Research Foundation and Human Protein Atlas were used to identify myocardial highly expressed targets. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses were conducted using the Database for Annotation, Visualization, and Integrated Discovery. Molecular docking was performed for the final components and target proteins. In vitro experiments were carried out using H9c2 cells subjected to oxygen and glucose deprivation conditions to validate the effects of the screened potential active components., Results: Network analysis revealed that Eucommiae cortex might exert its effects through the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR), hypoxia-inducible factor 1, and Janus kinase/signal transducer and activator of transcription pathways, which are crucial for myocardial contraction, vascular tone regulation, inflammatory response, and oxidative stress. Molecular docking indicated stable binding of the selected compounds to PI3K, AKT, and mTOR. Medioresinol was selected for further study and shown to significantly improve oxidative stress and inflammatory response in myocardial ischemia-hypoxia model cells by activating the PI3K/AKT/mTOR pathway., Conclusion: This study confirms the role of the PI3K/AKT/mTOR pathway in the cardiovascular protective effects of Eucommiae cortex and provides evidence at the cellular level. Medioresinol demonstrated potential therapeutic effects on myocardial infarction induced heart failure by reducing oxidative stress and inflammatory responses. These findings offer a theoretical basis for the application of Eucommiae cortex in the treatment of heart failure and support the development of new therapeutic drugs for cardiovascular diseases. Future research should further validate these effects in animal models and explore the overall efficacy of Eucommiae cortex., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Qin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Renal fibrosis is a major cause of renal dysfunction and is a common pathological event in almost all forms of chronic kidney disease (CKD). Currently, the pathomechanisms of renal fibrosis are not well understood. However, researchers have demonstrated that aerobic exercise can improve renal fibrosis. Klotho is considered to be a negative regulator of renal fibrosis. In this study, we aimed to investigate the role and mechanism of Klotho in the improvement of renal fibrosis through aerobic exercise. We performed a 12-week aerobic exercise intervention in 19-month-old male C57BL/6J mice. Physiological and biochemical indexes were performed to assess renal function and renal fibrosis. The roles of Klotho were further confirmed through knockdown of Klotho by small interfering RNA (siRNA) in C57BL/6J mice.Q-PCR and Western blot were performed to quantify determine the expression of relevant genes and proteins in the kidney. Results: Aging decreased Klotho expression via activated the upstream TGF-β1/p53/miR34a signaling pathway and affected its downstream signaling pathways, ultimately leading to renal fibrosis. Exposure to aerobic exercise for 12 weeks significantly improved renal fibrosis and alleviated the intrarenal genetic alterations induced by aging. Conclusion: Our results showed that aerobic exercise increased Klotho expression by inhibiting the TGF-β1/p53/miR34a signaling pathway and further inhibited its downstream TGF-β1/smad3 and β-linker protein signaling pathways. These results provide a theoretical basis supporting the feasibility of exercise in the prevention and treatment of CKD., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE) in pigs, horses and wide range of mammals. Little is known about the role of innate immune response during L. intracellularis infection. In this study, we investigated the nuclear factor-κB (NF-κB)-regulated immune response against infection of a clinical strain Dkp23 and a live-attenuated Enterisol vaccine strain in PK-15 cells. We found that expression of NF-κB target genes TNF-α, IFN-γ, IL-6 and IL-8 were modulated during the course of infection. At 5 dpi, there was a significant increase in p65 NF-κB activation, including protein nuclear translocation and phosphorylation, synchronous with the induction of IL-6, IFN-γ and IL-8 expression in L. intracellularis infected cells, especially for Enterisol vaccine strain-infected cells. This result suggests that NF-κB signalling level is induced when L. intracellularis bacterial load peaks at 5 dpi. The induction of pro-inflammatory cytokines expression is consistent with the decreased viability of L. intracellularis-infected cells especially that of the vaccine strain. There were no significant changes in NF-κB signalling between vaccine and Dkp23 infection in PK-15 cells, except for moderate levels of differences in NF-κB target genes expression which might be a reflection of differences in intracellular bacterial load. Overall, the data presented here indicate a correlation between the induction of NF-κB signalling and the L. intracellularis bacterial load in PK-15 cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Heme and iron metabolic pathways are highly intertwined, both compounds being essential for key biological processes, yet becoming toxic if overabundant. Their concentrations are exquisitely regulated, including via dedicated two-component systems (TCSs) that sense signals and regulate adaptive responses. HemKR is a TCS present in both saprophytic and pathogenic Leptospira species, involved in the control of heme metabolism. However, the molecular means by which HemKR is switched on/off in a signal-dependent way, are still unknown. Moreover, a comprehensive list of HemKR-regulated genes, potentially overlapped with iron-responsive targets, is also missing. Using the saprophytic species Leptospira biflexa as a model, we now show that 5-aminolevulinic acid (ALA) triggers the shutdown of the HemKR pathway in live cells, and does so by stimulating the phosphatase activity of HemK towards phosphorylated HemR. Phospho~HemR dephosphorylation leads to differential expression of multiple genes, including of heme metabolism and transport systems. Besides the heme-biosynthetic genes hemA and the catabolic hmuO, which we had previously reported as phospho~HemR targets, we now extend the regulon identifying additional genes. Finally, we discover that HemR inactivation brings about an iron-deficit tolerant phenotype, synergistically with iron-responsive signaling systems. Future studies with pathogenic Leptospira will be able to confirm whether such tolerance to iron deprivation is conserved among Leptospira spp., in which case HemKR could play a vital role during infection where available iron is scarce. In sum, HemKR responds to abundance of porphyrin metabolites by shutting down and controlling heme homeostasis, while also contributing to integrate the regulation of heme and iron metabolism in the L. biflexa spirochete model., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Imelio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: To explore whether miR-199a-5p regulated BBB integrity through PI3K/Akt pathway after ischemia stroke., Methods: Adult male Sprague-Dawley rats with permanent middle cerebral artery occlusion(MCAO) were used in experiment. The Ludmila Belayev 12-point scoring was used to measure the neurological function of MCAO rats. The Evans Blue Stain, immunofluorescence staining, western-blotting and RT-PCR were performed to evaluate the effects of miR-199a-5p mimic on BBB integrity in rats following MCAO., Results: The result suggested that miR-199a-5p mimic treatment possessed the potential to boost proprioception and motor activity of MCAO rats. MiR-199a-5p decreased the expression of PIK3R2 after MCAO, activated Akt signaling pathway, and increased the expression of Claudin-5 and VEGF in the ischemic penumbra. Furthermore, miR-199a-5p alleviated inflammation after cerebral ischemia. BBB leakage and neurocyte apoptosis were cut down in MCAO rats treated with miR-199a-5p mimic., Conclusions: MiR-199a-5p mimic decreased the expression of PIK3R2 and activated Akt signaling pathway after ischemia stroke, reduced the expression of inflammatory cytokines, and attenuated BBB disruption after ischemic stroke., Competing Interests: All authors declare that they have no conflicts of interest to report., (Copyright: © 2024 Ni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Francisella tularensis is a causative agent of the zoonotic disease tularemia, and is highly pathogenic to humans. The pathogenicity of this bacterium is largely attributed to intracellular growth in host cells. Although several bacterial factors important for the intracellular growth have been elucidated, including the type VI secretion system, the host factors involved in the intracellular growth of F. tularensis are largely unknown. To identify the host factors important for F. tularensis infection, 368 compounds were screened for the negative regulation of F. tularensis subsp. novicida (F. novicida) infection. Consequently, 56 inhibitors were isolated that decreased F. novicida infection. Among those inhibitors, we focused on cucurbitacin I, an inhibitor of the JAK2/ STAT3 pathway. Cucurbitacin I and another JAK2/STAT3 inhibitor, Stattic, decreased the intracellular bacterial number of F. novicida. However, these inhibitors failed to affect the cell attachment or the intrasaccular proliferation of F. novicida. In addition, treatment with these inhibitors destabilized actin filaments. These results suggest that the JAK2/STAT3 pathway plays an important role in internalization of F. novicida into host cells through mechanisms involving actin dynamics, such as phagocytosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Matsumoto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Myocardial fibrosis can trigger heart failure in diabetic cardiomyopathy (DCM), and irisin, an exercise-induced myokine, may have a beneficial effect on cardiac function. However, the specific molecular mechanism between exercise and irisin in the diabetic heart remains not fully explored. This study aimed to investigate how miR-34a mediates exercise-induced irisin to ameliorate myocardial fibrosis and its underlying mechanisms. Type 2 diabetes mellitus (T2DM) with DCM was induced in adult male rats with high-fat diet and streptozotocin injection. The DCM rats were subjected to swimming (60 min/d) and recombinant irisin (r-irisin, 500 μg/kg/d) interventions for 8 weeks, respectively. Cardiac function, cardiomyocyte structure, myocardial fibrosis and its correlated gene and protein expression were analyzed. Swimming intervention alleviated insulin resistance, myocardial fibrosis, and myocardial hypertrophy, and promoted blood glucose homeostasis in T2DM model rats. This improvement was associated with irisin upregulation and miR-34a downregulation in the myocardium, thus enhancing cardiac function. Similar efficacy was observed via intraperitoneal injection of exogenous recombinant irisin. Inhibition of miR-34a in vivo exhibited an anti-myocardial fibrotic effect by promoting irisin secretion through activating sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α)/fibronectin type III domain-containing protein 5 (FNDC5) signal pathway and downregulating myocardial fibrosis markers (collagen I, collagen III, and transforming growth factor-β1). Therefore, swimming-induced irisin has the potential therapeutic effect on diabetic myocardial fibrosis through activating the miR-34a-mediated SIRT1/PGC-1α/FNDC5 signal pathway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Micro and nanoscale patterning of surface features and biochemical cues have emerged as tools to precisely direct neurite growth into close proximity with next generation neural prosthesis electrodes. Biophysical cues can exert greater influence on neurite pathfinding compared to the more well studied biochemical cues; yet the signaling events underlying the ability of growth cones to respond to these microfeatures remain obscure. Intracellular Ca2+ signaling plays a critical role in how a growth cone senses and grows in response to various cues (biophysical features, repulsive peptides, chemo-attractive gradients). Here, we investigate the role of inositol triphosphate (IP3) and ryanodine-sensitive receptor (RyR) signaling as sensory neurons (spiral ganglion neurons, SGNs, and dorsal root ganglion neurons, DRGNs) pathfind in response to micropatterned substrates of varied geometries. We find that IP3 and RyR signaling act in the growth cone as they navigate biophysical cues and enable proper guidance to biophysical, chemo-permissive, and chemo-repulsive micropatterns. In response to complex micropatterned geometries, RyR signaling appears to halt growth in response to both topographical features and chemo-repulsive cues. IP3 signaling appears to play a more complex role, as growth cones appear to sense the microfeatures in the presence of xestospongin C but are unable to coordinate turning in response to them. Overall, key Ca2+ signaling elements, IP3 and RyR, are found to be essential for SGNs to pathfind in response to engineered biophysical and biochemical cues. These findings inform efforts to precisely guide neurite regeneration for improved neural prosthesis function, including cochlear implants., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vecchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: This study aims to explore the impact of Nesfatin-1 on type 2 diabetic erectile dysfunction (T2DMED) and its underlying mechanism in regulating the phenotypic switching of corpus cavernosum smooth muscle cells (CCSMCs)., Methods: Twenty-four 4-week-old male C57 wild-type mice were randomly assigned to the control group, model group, and Nesfatin-1 treatment group. Monitoring included body weight, blood glucose levels, and penile cavernous pressure (ICP). Histochemistry and Western blot analyses were conducted to assess the expressions of α-SMA, OPN, and factors related to the PI3K/AKT/mTOR signaling pathway. CCSMCs were categorized into the control group, high glucose and high oleic acid group (GO group), Nesfatin-1 treatment group (GO+N group), sildenafil positive control group (GO+S group), and PI3K inhibitor group (GO+N+E group). Changes in phenotypic markers, cell morphology, and the PI3K/AKT/mTOR signaling pathway were observed in each group., Results: (1) Nesfatin-1 significantly ameliorated the body size, body weight, blood glucose, glucose tolerance, and insulin resistance in T2DMED mice. (2) Following Nesfatin-1 treatment, the ICP/MSBP ratio and the peak of the ICP curve demonstrated a significant increase. (3) Nesfatin-1 significantly enhanced smooth muscle and reduced collagen fibers in the corpus cavernosum. (4) Nesfatin-1 notably increased α-SMA expression and decreased OPN expression in CCSMCs. (5) Nesfatin-1 elevated PI3K, p-AKT/AKT, and p-mTOR/mTOR levels in penile cavernous tissue., Conclusions: Nesfatin-1 not only effectively improves body weight and blood glucose levels in diabetic mice but also enhances erectile function and regulates the phenotypic switching of corpus cavernosum smooth muscle. The potential mechanism involves Nesfatin-1 activating the PI3K/AKT/mTOR signaling pathway to induce the conversion of CCSMCs to a contractile phenotype., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Obesity is a growing concern. 42.3% of people in the U.S were considered obese between 2017-2018. Much is still unknown about the genetic components that contribute to weight gain. In humans, the hormone glucagon is a major contributor to the body's energy regulation as it signals for the breakdown of lipids. Treatments targeting the glucagon pathway have helped patients with both weight loss and appetite suppression. Understanding the genetic modifiers of glucagon signaling and its downstream pathways could enable the development of a wider variety of effective therapeutics. In this study, we blocked the glucagon pathway in Drosophila melanogaster by reducing the expression of the fly ortholog of the glucagon receptor (AKHR). We then crossed our model to the Drosophila Genetic Reference Panel (DGRP) and looked for natural variation in fat content. We used variation in larval density to identify candidate modifier genes through a genome-wide association study. We then tested these modifier genes by increasing or decreasing their expression in the AKHR model. We screened these candidates initially with the same density assay used in the original study to narrow down to four candidate genes that substantially impacted the density of the larvae: THADA, AmyD, GluRIIC, and CG9826. We further characterized these candidates using biochemical assays to analyze stored metabolites such as triglycerides, glucose, glycogen, and protein under control, high sugar, and high fat conditions to see if the larvae are resistant to environmental changes. Our results indicate consistency between the results of the density assay and direct measurement of metabolite levels. In particular, THADA and AmyD are highlighted as interesting genes for additional study. We hope to improve our understanding of the glucagon signaling pathway, obesity, and lipid metabolism. We also aim to provide candidate genes that can be regarded as future therapeutic targets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nicol et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: This study comprises an investigation of the role of meteorin-like (Metrnl) in an experimental model of diabetic kidney disease (DKD)., Methods: Twenty-four db/db mice were randomly assigned to one of the following groups: DKD, DKD + Metrnl-/-, and DKD + Metrnl+/+. Plasma Metrnl concentrations were measured using ELISA. Kidney tissues were examined via western blotting, qRT-PCR, and immunohistochemistry to determine the expression levels of inflammatory factors. Electron microscopy was employed to observe stained kidney sections., Results: Compared with the NC group, FBG, BW, and UACR were elevated in the DKD and Metrnl-/- groups, with severe renal pathological injury, decreased serum Metrnl concentration, decreased renal Metrnl expression, and increased expression levels of TNF-α, TGF-β1, TGF-R1, pSmad2, pSmad3, and α-SMA. In contrast, the Metrnl+/+ group showed decreased FBG and UACR, BUN, TC and TG, increased HDL-C and serum Metrnl concentration, increased renal Metrnl expression, and decreased expression of TNF-α, TGF-β1, TGF-R1, pSmad2, pSmad3, and α-SMA, compared to the DKD and Metrnl-/- groups. A Pearson bivariate correlation analysis revealed a negative correlation between UACR and Metrnl, and a positive correlation between UACR and TGF-β1., Conclusion: Upregulation of renal Metrnl expression can improve renal injury by downregulating the expression of molecules in the TGF-β1/Smads signaling pathway in the renal tissues of type 2 diabetic mice; and by reducing the production of fibrotic molecules such as α-SMA., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neuronal loss is a hallmark of stroke and other neurodegenerative diseases, and as such, neuronal loss caused by microglia has been thought to be a contributing factor to disease progression. Here, we show that microglia indeed contribute significantly to neuronal loss in a mouse model of stroke, but this microglial-dependent process of neuronal clearance specifically targets stressed and degenerating neurons in the ischemic cortical region and not healthy non-ischemic neurons. Nonspecific stimulation of microglia decreased the density of neurons in the ischemic cortical region, whereas specific inhibition of MFG-E8 signaling, which is required for microglial phagocytosis of neurons, had the opposite effect. In both scenarios, the effects were microglia specific, as the same treatments had no effect in mice whose microglia were depleted prior to stroke. Finally, even though the inhibition of MFG-E8 signaling increased neuronal density in the ischemic brain region, it substantially exacerbated the development of cortical infarction. In conclusion, microglia through MFG-E8 signaling contribute to the loss of ischemic neurons and, in doing so, minimize the development of cortical infarction after stroke., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinformatic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac β-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species.

All-trans-retinoic acid (RA) inhibits adipogenesis in established preadipocyte cell lines. Dosing pharmacological amounts of RA reduces weight gain in mice fed a high-fat diet, i.e. counteracts diet-induced obesity (DIO). The aldehyde dehydrogenase Raldh1 (Aldh1a1) functions as one of three enzymes that converts the retinol metabolite retinal into RA, and one of many proteins that contribute to RA homeostasis. Female Raldh1-ablated mice resist DIO. This phenotype contrasts with ablations of other enzymes and binding-proteins that maintain RA homeostasis, which gain adiposity. The phenotype observed prompted the conclusion that loss of Raldh1 causes an increase in adipose tissue retinal, and therefore, retinal functions independently of RA to prevent DIO. A second deduction proposed that low nM concentrations of RA stimulate adipogenesis, in contrast to higher concentrations. Using peer-reviewed LC/MS/MS assays developed and validated for quantifying tissue RA and retinal, we show that endogenous retinal and RA concentrations in adipose tissues from Raldh1-null mice do not correlate with the phenotype. Moreover, male Raldh1-null mice resist weight gain regardless of dietary fat content. Resistance to weight gain occurs during adolescence in both sexes. We show that RA concentrations as low as 1 nM, i.e. in the sub-physiological range, impair adipogenesis of embryonic fibroblasts from wild-type mice. Embryonic fibroblasts from Raldh1-null mice resist differentiating into adipocytes, but retain ability to generate RA. These fibroblasts remain sensitive to an RA receptor pan-agonist, and are not affected by an RA receptor pan-antagonist. Thus, the data do not support the hypothesis that retinal itself represses weight gain and adipogenesis independently of RA. Instead, the data indicate that Raldh1 functions as a retinal and atRA-independent promoter of adiposity during adolescence, and enhances adiposity through pre-adipocyte cell autonomous actions.

Wnt3a is a major regulator of bone metabolism however, very few of its target genes are known in bone. Wnt3a preferentially signals through transmembrane receptors Frizzled and co-receptors Lrp5/6 to activate the canonical signaling pathway. Previous studies have shown that the canonical Wnt co-receptors Lrp5 and Lrp6 also play an essential role in normal postnatal bone homeostasis, yet, very little is known about specific contributions by these co-receptors in Wnt3a-dependent signaling. We used high-throughput sequencing technology to identify target genes regulated by Wnt3a in osteoblasts and to elucidate the role of Lrp5 and Lrp6 in mediating Wnt3a signaling. Our study identified 782 genes regulated by Wnt3a in primary calvarial osteoblasts. Wnt3a up-regulated the expression of several key regulators of osteoblast proliferation/ early stages of differentiation while inhibiting genes expressed in later stages of osteoblastogenesis. We also found that Lrp6 is the key mediator of Wnt3a signaling in osteoblasts and Lrp5 played a less significant role in mediating Wnt3a signaling.

We have reported that integrins crosstalk with growth factors through direct binding to growth factors (e.g., fibroblast growth factor-1, insulin-like growth factor 1 (IGF1), neuregulin-1, fractalkine) and subsequent ternary complex formation with cognate receptor [e.g., integrin/IGF1/IGF1 receptor (IGF1R)]. IGF1 and IGF2 are overexpressed in cancer and major therapeutic targets. We previously reported that IGF1 binds to integrins ανβ3 and α6β4, and the R36E/R37E mutant in the C-domain of IGF1 is defective integrin binding and signaling functions of IGF1, and acts as an antagonist of IGF1R. We studied if integrins play a role in the signaling functions of IGF2, another member of the IGF family. Here we describe that IGF2 specifically binds to integrins ανβ3 and α6β4, and induced proliferation of CHO cells (IGF1R+) that express ανβ3 or α6β4 (β3- or α6β4-CHO cells). Arg residues to Glu at positions 24, 34, 37 and/or 38 in or close to the C-domain of IGF2 play a critical role in binding to integrins and signaling functions. The R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E mutants were defective in integrin binding and IGF2 signaling. These mutants suppressed proliferation induced by WT IGF2, suggesting that they are dominant-negative antagonists of IGF1R. These results suggest that IGF2 also requires integrin binding for signaling functions, and the IGF2 mutants that cannot bind to integrins act as antagonists of IGF1R. The present study defines the role of the C-domain in integrin binding and signaling.

Epidemiologic studies have reported an inverse relationship between childhood Helicobacter pylori infection and development of allergic asthma. Because lung epithelium plays an important role in allergic asthma pathogenesis, we hypothesized that H. pylori may directly influence airway epithelial cell innate immune function, particularly in early childhood. To test our hypothesis, we established an in vitro H. pylori infection model using primary tracheobronchial epithelial cell cultures derived from infant, juvenile and adult rhesus monkeys. Airway epithelial cell cultures were infected with wild-type or cag pathogenicity island mutant H. pylori strains, followed by evaluation of IL-8 and IL-6 protein synthesis. We found that H. pylori primarily increased IL-8 synthesis in a MOI and age-dependent fashion, with a greater than 4-fold induction in infant versus adult cultures. H. pylori-induced IL-8 synthesis in infant and juvenile cultures was significantly reduced by cag pathogenicity island mutants, indicating a requirement for the type IV secretion system. Although peptidoglycan recognition of nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NF-kappaB have been implicated as key cytokine signaling molecules for H. pylori infection in gastric epithelium, NOD1 (ML130) or NF-kappaB (JSH-23) inhibitors minimally affected IL-8 synthesis in airway epithelial cell cultures following H. pylori infection. In contrast, inhibition of the p38 MAP kinase pathway (SB203580) resulted in almost complete suppression of H. pylori-induced IL-8 synthesis. Collectively, these results indicate that H. pylori can preferentially elicit IL-8 synthesis in a model of pediatric airway epithelium using the type IV secretion system via p38 MAP kinase.

Background and objectivesResolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.MethodsVSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.ResultsTreatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.ConclusionsOur results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.

Following the primary mechanical impact, traumatic brain injury (TBI) induces the simultaneous production of a variety of pro- and anti-inflammatory molecular mediators. Given the variety of cell types and their requisite expression of cognate receptors this creates a highly complex inflammatory milieu. Increasingly in neurotrauma research there has been an effort to define injury-induced inflammatory responses within the context of in vitro defined macrophage polarization phenotypes, known as "M1" and "M2". Herein, we expand upon our previous work in a rodent model of TBI to show that the categorization of inflammatory response cannot be so easily delineated using this nomenclature. Specifically, we show that TBI elicited a wide spectrum of concurrent expression responses within both pro- and anti-inflammatory arms. Moreover, we show that the cells principally responsible for the production of these inflammatory mediators, microglia/macrophages, simultaneously express both "M1" and "M2" phenotypic markers. Overall, these data align with recent reports suggesting that microglia/macrophages cannot adequately switch to a polarized "M1-only" or "M2-only" phenotype, but display a mixed phenotype due to the complex signaling events surrounding them.

Renal endothelial cells (REc) are the first target of HIV-1 in the kidney. The integrity of REc is maintained at least partially by heparin binding growth factors that bind to heparan sulfate proteoglycans located on their cell surface. However, previous studies showed that the accumulation of two heparin-binding growth factors, Vascular Endothelial Cell Growth Factor-A (VEGF-A) and Fibroblast Growth Factor-2 (FGF-2), in combination with the viral protein Tat, can precipitate the progression of HIV-renal diseases. Nonetheless, very little is known about how these factors affect the behavior of REc in HIV+ children. We carried out this study to determine how VEGF-A, FGF-2, and HIV-Tat, modulate the cytoskeletal structure and permeability of cultured REc, identify key signaling pathways involved in this process, and develop a functional REc assay to detect HIV+ children affected by these changes. We found that VEGF-A and FGF-2, acting in synergy with HIV-Tat and heparin, affected the cytoskeletal structure and permeability of REc through changes in Rho-A, Src, and Rac-1 activity. Furthermore, urine samples from HIV+ children with renal diseases, showed high levels of VEGF-A and FGF-2, and induced similar changes in cultured REc and podocytes. These findings suggest that FGF-2, VEGF-A, and HIV-Tat, may affect the glomerular filtration barrier in HIV+ children through the induction of synergistic changes in Rho-A and Src activity. Further studies are needed to define the clinical value of the REc assay described in this study to identify HIV+ children exposed to circulating factors that may induce glomerular injury through similar mechanisms.