Optogenetic stimulation of the adrenergic C1 neurons produces cardiorespiratory activation, and selective depletion of these cells attenuates breathing responses induced by hypoxia. The preBötzinger complex (preBötC) is a group of neurons located in the intermediate aspect of the ventrolateral medulla, critical for respiratory rhythmogenesis, and is modulated by glutamate and catecholamines. Our hypothesis is that selective activation of C1 neurons leads to breathing responses by excitatory connections with the preBötC neurons. Anatomical connection between C1 cells and preBötC was evaluated using retrograde (Cholera Toxin b; preBötC) and anterograde (LVV-PRSx8-ChR2-eYFP; C1 region) tracers. LVV-PRSx8-ChR2-eYFP (viral vector that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter (PRSx8) was also injected into the C1 region of male Wistar rats for the functional experiments. Anatomical results demonstrated that preBötC neurons receive projections from C1 cells, and these projections express tyrosine hydroxylase and vesicular glutamate transporter 2. Functional connection between C1 cells and preBötC was evaluated by photostimulation of ChR2-transduced C1 neurons before and after unilateral injection of the ionotropic glutamate antagonist, kynurenic acid (kyn), or cocktail of adrenergic antagonists in the preBötC. Kyn injection into preBötC blocked the increase in Dia frequency without changing the MAP increase elicited by photostimulation of C1 neurons, while the injection of adrenergic antagonists into the preBötC did not change Dia frequency and MAP increase induced by photostimulation of C1 cells. Our results suggest that the increase in breathing produced by photostimulation of C1 neurons can be caused by a direct glutamatergic activation of preBötC neurons.
Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.
Phosphatidylinositol-phosphate kinases (PIPKins) belong to a family of lipid kinase enzymes that generate lipid messengers derivate from inositol, including the second messenger phosphatidylinositol-4,5-biphosphate [PI(4,5)P2], which participates in several cell regulation processes including gene expression. Previous studies developed in our laboratory suggested that the enzyme phosphatidylinositol-4-phosphate kinase-II-alpha (PIP4KIIα) may be related with the expression of the globin genes. Thus, the aim of this work was to evaluate the expression levels of the α, β and γ globin genes after disruption and overexpression of the PIP4KIIα gene (PIP4KIIA) in KU812 erythroleukemic cell line. The PIP4KIIα gene disruption was performed by using CRISPR/Cas9 and CRISPR/dCas9 techniques: KU812 cells were electro-transfected (Lonza 4D Nucleofector) with CRISPR/Cas9 lentiviral plasmid construct containing 3 gRNA targets to exon 8 of PIP4KIIA to knockout the gene; RNA samples from the pool of cells were collected and quantified by qPCR after 9 (t1) and 14 days (t2) of transfection. To overexpress PIP4K2A, CRISPR/dCas9 system (five lentiviral plasmids - 3 gRNAs targeting 214 nt upstream the transcriptional start site of PIP4K2A and 2 SAM accessorial factors) was transfected (Lipofectamine 3000) and RNA quantified by qPCR 24 (h1), 48 (h2) and 72 (h3) hours after the procedure. β-ACTIN and GAPDH genes were used as endogenous controls for qPCR. Our results with transfected cells without selection revealed, as expected, a reduction of PIP4KIIA compared to the control (no transfected cells - t1 and t2). The α globin genes presented a variation of 42% (increase in t1) and a 60% reduction in t2 (RQ = 1.42; RQ = 0.40, respectively), while β globin genes presented a 123% increase in t1 and remained similar to the control at t2 (RQ = 2.23; RQ = 1.01). The γ globin genes presented variation of 18% and 59% reduction in t1 and t2, respectively (RQ = 0.82; RQ = 0.41). The overexpression results suggested that PIP4KIIA leads to a higher γ globin expression in h2 and h3 (RQ = 3.26; RQ = 2.01) as well as a lower β globin expression in h1, h2 and h3 (RQ = 0.54; RQ = 0.002; RQ = 0.18), while a globin remained similar to the control at all analysed times (RQ variation of 1.26; 0.82 and 1.25, respectively). These results, although preliminary, suggest a possible relationship between PIP4KIIα and the regulation of expression of the globin genes. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp. Disclosures No relevant conflicts of interest to declare.
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