Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of collybistin are ...expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3−). The CBSH3+ mRNAs are predominantly expressed over CBSH3−. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3− protein might not be expressed in the brain. The expression or lack of expression of CBSH3− protein is an important issue because CBSH3− has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA‐A receptors (GABAARs). Moreover CBSH3− is constitutively active; therefore lower expression of CBSH3− protein might play a relatively stronger functional role than the more abundant but self‐inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3− protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3− protein than other neurons; (c) CBSH3− is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3− in hippocampal neurons decreases the synaptic clustering of gephyrin and GABAARs. The results show that CBSH3− protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse.
ARHGEF9 encodes several collybistin splice variants which can be grouped into ones that possess the auto‐inhibitory SH3 domain (CBSH3+) and ones that do not (CBSH3−), the latter being constitutively active. CBSH3+ and CBSH3− bind to gephyrin forming the GABAergic postsynaptic scaffold. The predominant CBSH3+ isoform has been extensively studied but not CBSH3−, whose function has been largely overlooked. We are now showing that CBSH3− variants play a significant role in regulating the size of the postsynaptic GABAergic scaffold.
Objectives The two currently available types of pegylated interferon (peg-IFN) used to treat hepatitis C have different pharmacokinetic properties. It is unclear how these differences affect response ...to therapy. We compared the effectiveness and safety of peg-IFN-α2a and peg-IFN-α2b, both with ribavirin, against chronic hepatitis C virus (HCV) infection in HIV-infected patients. Methods From the GESIDA HIV/HCV cohort, we analysed patients treated with peg-IFN-α2a (n = 315) or peg-IFN-α2b (n = 242). The primary endpoint was a sustained virological response (SVR). Results Both groups were well matched in baseline characteristics except for a higher frequency of injection drug users in the peg-IFN-α2b group than in the peg-IFN-α2a group (85% versus 76%; P = 0.01) and a higher frequency of bridging fibrosis and cirrhosis (F3–F4) in the peg-IFN-α2b group than in the peg-IFN-α2a group (42% versus 33%; P = 0.04). End-of-treatment response was significantly lower among patients treated with peg-IFN-α2b 40% versus 52%; odds ratio (OR), 1.63; 95% confidence interval (95% CI), 1.16–2.29; P < 0.01. However, no significant differences were found in SVR between patients treated with peg-IFN-α2b and those treated with peg-IFN-α2a (31% versus 33%; OR, 1.09; 95% CI, 0.75–1.59; P = 0.655). Therapy was interrupted due to adverse events in 33 (14%) patients treated with peg-IFN-α2b and 47 (15%) patients treated with peg-IFN-α2a. Conclusions No differences in effectiveness and safety were found between peg-IFN-α2b and peg-IFN-α2a for the treatment of chronic HCV infection in HIV-infected patients.
We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino ...acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is γ-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with γ-aminobutyric acid, type A, receptor (GABAAR) clusters than with α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABAAR clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.
It has been proposed that the combinatorial expression of γ‐protocadherins (Pcdh‐γs) and other clustered protocadherins (Pcdhs) provides a code of molecular identity and individuality to neurons, ...which plays a major role in the establishment of specific synaptic connectivity and formation of neuronal circuits. Particular attention has been directed to the Pcdh‐γ family, for which experimental evidence derived from Pcdh‐γ‐deficient mice shows that they are involved in dendrite self‐avoidance, synapse development, dendritic arborization, spine maturation, and prevention of apoptosis of some neurons. Moreover, a triple‐mutant mouse deficient in the three C‐type members of the Pcdh‐γ family (Pcdh‐γC3, Pcdh‐γC4, and Pcdh‐γC5) shows a phenotype similar to the mouse deficient in whole Pcdh‐γ family, indicating that the latter is largely due to the absence of C‐type Pcdh‐γs. The role of each individual C‐type Pcdh‐γ is not known. We have developed a specific antibody to Pcdh‐γC4 to reveal the expression of this protein in the rat brain. The results show that although Pcdh‐γC4 is expressed at higher levels in the embryo and earlier postnatal weeks, it is also expressed in the adult rat brain. Pcdh‐γC4 is expressed in both neurons and astrocytes. In the adult brain, the regional distribution of Pcdh‐γC4 immunoreactivity is similar to that of Pcdh‐γC4 mRNA, being highest in the olfactory bulb, dentate gyrus, and cerebellum. Pcdh‐γC4 forms puncta that are frequently apposed to glutamatergic and GABAergic synapses. They are also frequently associated with neuron‐astrocyte contacts. The results provide new insights into the cell recognition function of Pcdh‐γC4 in neurons and astrocytes.
Using double‐ and triple‐label immunofluorescence, the authors reveal the expression of protocadherin‐γC4 in neurons and astrocytes in the rat brain.
We have used RNA interference (RNAi) to knock down the expression of the gamma2 subunit of the GABA(A) receptors (GABA(A)Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small ...interference RNAs (shRNAs) for the gamma2 subunit, one targeting the coding region and the other one the 3'-untranslated region (UTR) of the gamma2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA(A) receptor gamma2 subunit and the clustering of other GABA(A)R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the gamma2 shRNAs had no effect on the clustering of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A gamma2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3'-UTR targeted by gamma2 RNAi, rescued both the postsynaptic clustering of GABA(A)Rs and the GABAergic innervation. Decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the gamma2 shRNAs. The results indicate that the postsynaptic clustering of GABA(A)Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.
Although the use of mycophenolate mofetil (MMF) has reduced the incidence of acute rejection in heart and kidney allograft recipients, its role in lung transplantation remains controversial. ...Therefore, we conducted a randomized, prospective, open-label, multicenter study in lung transplant recipients to determine whether MMF decreases episodes of acute allograft rejection when compared with azathioprine (AZA).
Between March of 1997 and January of 1999, 81 consecutive lung transplant recipients from two centers were prospectively randomized to receive cyclosporine, corticosteroids, and either 2 mg/kg per day of AZA or 1 g twice daily of MMF. The primary study endpoint was biopsy-proven acute allograft rejection over the first 6 months posttransplant. Secondary endpoints included clinical rejection, cytomegalovirus (CMV) infection, adverse events, and survival. Surveillance bronchoscopies were performed at 1, 3, and 6 months, or if clinically indicated. Pathologists interpreting the biopsy results were blinded to the randomization. Results were analyzed according to intention-to-treat. Between group comparisons of means and proportions were made by using two sample t tests and Fisher's exact tests, respectively. Six-month survival was calculated by the Kaplan-Meier method and compared by the log rank test.
Thirty-eight patients were prospectively randomized to receive AZA, and 43 MMF. The incidence of biopsy proven grade II or greater acute allograft rejection at 6 months was 58% in the AZA group and 63% in the MMF group (P=0.82). The 6-month survival rates in the MMF and AZA groups were 86% and 82%, respectively (P=0.57). Rates of CMV infection and adverse events were not significantly different between the two groups.
Acute rejection rates and overall survival at 6 months are similar in lung transplant recipients treated with either MMF- or AZA-based immunosuppression.
The dysfunction of TAR DNA-binding protein 43 (TDP-43) is implicated in various neurodegenerative diseases, though the specific contributions of its toxic gain-of-function versus loss-of-function ...effects remain unclear. This study investigates the impact of TARDBP loss on cellular metabolism and viability using human-induced pluripotent stem cell-derived motor neurons and HeLa cells. TARDBP silencing led to reduced metabolic activity and cell growth, accompanied by neurite degeneration and decreased oxygen consumption rates in both cell types. Notably, TARDBP depletion induced a metabolic shift, impairing ATP production, increasing metabolic inflexibility, and elevating free radical production, indicating a critical role for TDP-43 in maintaining cellular bioenergetics. Furthermore, TARDBP loss triggered non-apoptotic cell death, increased ACSL4 expression, and reprogrammed lipid metabolism towards lipid droplet accumulation, while paradoxically enhancing resilience to ferroptosis inducers. Overall, our findings highlight those essential cellular traits such as ATP production, metabolic activity, oxygen consumption, and cell survival are highly dependent on TARDBP function.
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We have found that the brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the beta subunits of the gamma-aminobutyric acid type-A receptor (GABA(A)R). BIG2 is a Sec7 ...domain-containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C-terminal fragment of BIG2 and the large intracellular loop of the GABA(A)R beta subunits was revealed with a yeast two-hybrid assay. The native BIG2 and GABA(A)Rs interact in the brain since both coprecipitated from detergent extracts with either anti-GABA(A)R or anti-BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABA(A)Rs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans-Golgi network. BIG2 is also present in vesicle-like structures in the dendritic cytoplasm, sometimes colocalizing with GABA(A)Rs. BIG2 is present in both inhibitory GABAergic synapses that contain GABA(A)Rs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABA(A)R beta subunits plays a role in the exocytosis and trafficking of assembled GABA(A)R to the cell surface.
BackgroundImpaired functionality, cognitive decline, comorbidity and polypharmacy in nonagenarians increase mortality risks associated with age. Polypharmacy (>4 chronic drugs) in elderly people is ...related to an increase in drug-related problems (DRP) and worse health outcomes due to potentially inappropriate prescriptions (PIP). To optimise medical care for chronic patients, our healthcare system stratifies chronic patients according to their grade of chronicity in chronic complex patients (CCP) or CCP with advance chronic disease (CCP-ACD).PurposeTo evaluate the differences related to functionality, cognition, polypharmacy and pharmacist interventions due to DRP (PI-DRP) regarding the grade of chronicity.Material and methodsWe included ≥90 years-old patients with polypharmacy discharged between January and June of 2017 from an Acute Geriatric Unit (81 beds) of a Geriatric Healthcare Centre. Registered variables: age, sex, grade of chronicity, Barthel Index and Pfeiffer Test before admission. Number of chronic drug/patient, number of PIP/patient and chronic benzodiazepines use before admission, and PI-DRP. Data are presented as median (Q1–Q3). We use Fisher’s exact test for qualitative and the Mann–Whitney U test and the Wilcoxon signed-rank test for quantitative data. Statistical analysis was performed with Stata 13.ResultsOne hundred and eighteen patients included: 83 CCP and 35 CCP-ACD. Differences between CCP and CCP-ACD: age 92 (90–94) vs 94 (91–95), p=0.029. Females 58 (69.9%) vs 20 (57.1%), p=0.205. Data at admission: Barthel Index 55 (40–80) vs 40 (20–60), p=0.010; Pfeiffer Test three (1–6) vs four (2–8), p=0.432. Chronic drugs/patient 10 (8–12) vs 10 (7–14), p=0.972. Average of PIP/patient 1.2 (±0.88) vs 0.86 (±0.85), p=0.049; chronic benzodiazepines use 30 (36.1%) vs 6 (17.1%), p=0.050. PI-DRP: indication 10 (12%) vs 2 (5.7%) patients, p=0.506; effectivity 11 (13.3%) vs five (14.3), p=0.881; safety one (1.2%) vs four (11.4%), p=0.027; advice to nursing three (3.6%) vs two (5.7%), p=0.632; others 11 (13.3%) vs five (14.3%), p=0.881.ConclusionCCP–ACD group are older than CCP, and have worse results in functional status without differences in cognitive function.Although the number of chronic drugs prescribed between the two groups are similar, CCP–ACD have significantly less PIP and use less chronic benzodiazepines than CCP.The major pharmaceutical interventions have been those of safety in the CCP–ACD group.References and/or AcknowledgementsAll medical and nursing staff of Geriatric-Healthcare Centre.No conflict of interest
Background. We analyzed the effect of exposure to nonnucleoside reverse‐transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) on the progression of liver fibrosis in patients with human ...immunodeficiency virus (HIV) and hepatitis C virus coinfection. Methods. We analyzed data and liver biopsy findings for 201 coinfected patients. Fibrosis was scored following the French METAVIR Cooperative Study Group. We used multinomial logistic regression analysis and the fibrosis progression rate to assess the association between cumulative exposure to antiretroviral drugs and stage of fibrosis. Results. The adjusted odds ratio (AOR) and 95% confidence interval (CI) of having a fibrosis stage score of 0 or 1, compared with 3 or 4, increased with each additional year of exposure to HAART (AOR, 1.32; 95% CI, 1.04–1,67), to NNRTIs as a class (AOR, 1.64; 95% CI, 1.18–2.27), to efavirenz (AOR, 1.54; 95% CI, 1.03–2.30), and to nevirapine (AOR, 1.72; 95% CI, 1.15–2.78). This effect was not found with PIs as a class. The AOR (95% CI) of having a fibrosis stage score of 2 versus 3 or 4 increased with each additional year of exposure to NNRTIs (AOR, 1.51; 95% CI, 1.08–2.10) and nevirapine (AOR, 1.58; 95% CI, 1.06–2.37). This effect was not found with highly active antiretroviral therapy, PIs, or efavirenz. The AOR (95% CI) of having a fibrosis progression rate ⩽0.1 versus >0.1 increased with each additional year of exposure to highly active antiretroviral therapy (AOR, 1.31; 95% CI, 1.07–1.60), to NNRTIs (AOR, 1.33; 95% CI, 1.03–1.70), and to nevirapine (AOR, 1.44; 95% CI, 1.07–1.95). This effect was not found with PIs or with efavirenz. Conclusions. In contrast with previous studies, we found that exposure to NNRTIs was clearly associated with a reduction in fibrosis progression, whereas exposure to PIs was not. Of note, exposure to nevirapine was more consistently associated with a reduction in fibrosis progression than was exposure to efavirenz. Prospective work is needed in this area.
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