Fluoroquinolone Antibiotics Exhibit Low Antiviral Activity against SARS-CoV-2 and MERS-CoV

Repurposing FDA-approved drugs that treat respiratory infections caused by coronaviruses, such as SARS-CoV-2 and MERS-CoV, could quickly provide much needed antiviral therapies. In the current study, the potency and cellular toxicity of four fluoroquinolones (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) were assessed in Vero cells and A549 cells engineered to overexpress ACE2, the SARS-CoV-2 entry receptor. All four fluoroquinolones suppressed SARS-CoV-2 replication at high micromolar concentrations in both cell types, with enoxacin demonstrating the lowest effective concentration 50 value (EC₅₀) of 126.4 μM in Vero cells. Enoxacin also suppressed the replication of MERS-CoV-2 in Vero cells at high micromolar concentrations. Cellular toxicity of levofloxacin was not found in either cell type. In Vero cells, minimal toxicity was observed following treatment with ≥37.5 μM enoxacin and 600 μM ciprofloxacin. Toxicity in both cell types was detected after moxifloxacin treatment of ≥300 μM. In summary, these results suggest that the ability of fluoroquinolones to suppress SARS-CoV-2 and MERS-CoV replication in cultured cells is limited.     

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates the electrogenic Na/HCO3 cotransporter NBCe1-A expressed in Xenopus oocytes

Bicarbonate transporters are regulated by signaling molecules/ions such as protein kinases, ATP, and Ca²⁺. While phospholipids such as PIP₂ can stimulate Na-H exchanger activity, little is known about phospholipid regulation of bicarbonate transporters. We used the patch-clamp technique to study the function and regulation of heterologously expressed rat NBCe1-A in excised macropatches from Xenopus laevis oocytes. Exposing the cytosolic side of inside-out macropatches to a 5% CO₂/33 mM HCO₃⁻ solution elicited a mean inward current of 14 pA in 74% of macropatches attached to pipettes (−V_p = −60 mV) containing a low-Na⁺, nominally HCO₃⁻-free solution. The current was 80–90% smaller in the absence of Na⁺, approximately 75% smaller in the presence of 200 μM DIDS, and absent in macropatches from H₂O-injected oocytes. NBCe1-A currents exhibited time-dependent rundown that was inhibited by removing Mg²⁺ in the presence or absence of vanadate and F⁻ to reduce general phosphatase activity. Applying 5 or 10 μM PIP₂ (diC8) in the presence of HCO₃⁻ induced an inward current in 54% of macropatches from NBC-expressing, but not H₂O-injected oocytes. PIP₂-induced currents were HCO₃⁻-dependent and somewhat larger following more NBCe1-A rundown, 62% smaller in the absence of Na⁺, and 90% smaller in the presence of 200 μM DIDS. The polycation neomycin (250–500 μM) reduced the PIP₂-induced inward current by 69%; spermine (100 μM) reduced the current by 97%. Spermine, poly-D-lysine, and neomycin all reduced the baseline HCO₃⁻-induced inward currents by as much as 85%. In summary, PIP₂ stimulates NBCe1-A activity, and phosphoinositides are regulators of bicarbonate transporters.     

Effect of Fabrication Technique on the Microgap of CAD/CAM Cobalt–Chrome and Zirconia Abutments on a Conical Connection Implant: An In Vitro Study

The aim of this in vitro study was to investigate the microgaps at the implant–abutment interface when zirconia (Zr) and CAD/CAM or cast Co–Cr abutments were used. Methods: Sixty-four conical connection implants and their abutments were divided into four groups (Co–Cr (milled, laser-sintered and castable) and Zirconia (milled)). After chewing simulation (300,000 cycles, under 200 N loads at 2 Hz at a 30° angle) and thermocycling (10,000 cycles, 5 to 50 °C, dwelling time 55 s), the implant–abutment microgap was measured 14 times at each of the four anatomical aspects on each specimen by using a scanning electron microscope (SEM). Kruskal–Wallis and pair-wise comparison were used to analyze the data (α = 0.05). Results: The SEM analysis revealed smaller microgaps with Co–Cr milled abutments (0.69–8.39 μm) followed by Zr abutments (0.12–6.57 μm), Co–Cr sintered (7.31–25.7 μm) and cast Co–Cr (1.68–85.97 μm). Statistically significant differences were found between milled and cast Co–Cr, milled and laser-sintered Co–Cr, and between Zr and cast and laser-sintered Co–Cr (p < 0.05). Conclusions: The material and the abutment fabrication technique affected the implant–abutment microgap magnitude. The Zr and the milled Co–Cr presented smaller microgaps. Although the CAD/CAM abutments presented the most favorable values, all tested groups had microgaps within a range of 10 to 150 μm.     

A Novel Porcine Graft for Regeneration of Bone Defects

Bone regeneration procedures require alternative graft biomaterials to those for autogenous bone. Therefore, we developed a novel porcine graft using particle sizes of 250–500 μm and 500–1000 μm in rabbit calvarial bone defects and compared the graft properties with those of commercial hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) over eight weeks. Surgery was performed in 20 adult male New Zealand white rabbits. During a standardized surgical procedure, four calvarial critical-size defects of 5 mm diameter and 3 mm depth were prepared. The defects were filled with HA/β-TCP, 250–500 μm or 500–1000 μm porcine graft, and control defects were not filled. The animals were grouped for sacrifice at 1, 2, 4, and 8 weeks post-surgery. Subsequently, sample blocks were prepared for micro-computed tomography (micro-CT) scanning and histological sectioning. Similar bone formations were observed in all three treatment groups, although the 250–500 μm porcine graft performed slightly better. Rabbit calvarial bone tissue positively responded to porcine grafts and commercial HA/β-TCP, structural analyses showed similar crystallinity and porosity of the porcine and HA/β-TCP grafts, which facilitated bone formation through osteoconduction. These porcine grafts can be considered as graft substitutes, although further development is required for clinical applications.     

Effect of Bonding Protocols on the Performance of Luting Agents Applied to CAD–CAM Composites

This in vitro study aimed to evaluate the effect of different bonding strategies on the micro-shear bond strength (μSBS) of luting agents to CAD–CAM composites. Surface scanning electron microscopy (SEM) and spectroscopy by energy-dispersive X-ray spectroscopy (EDS) were performed to analyze the surfaces of the composite before and after bonding treatment. Three CAD–CAM composites were evaluated: Lava Ultimate restorative (LU), Brava Blocks (BR), and Vita Enamic (VE). The LU and BR surfaces were sandblasted using aluminum oxide, while the VE surfaces were etched using a 5% hydrofluoric acid gel according to the manufacturers’ recommendations. All surfaces were subjected to the following bonding strategies (n = 15): adhesive with silane and MDP (ScotchBond Universal, 3M Oral Care, St Paul, MI, USA); adhesive with MDP (Ambar Universal, FGM, Joinville, Brazil); adhesive without silane or MDP (Prime&Bond Elect, Dentsply Sirona, Charlotte, NC, USA), pure silane without MDP (Angelus, Londrina, Brazil), and pure silane with MDP (Monobond N, Ivoclar Vivadent, Schaan, Liechtenstei). Afterwards, tygons were filled with RelyX Ultimate (3M Oral Care), AllCem (FGM), or Enforce (Dentsply Sirona), which were light-cured and subjected to the μSBS test. Data were analyzed using two-way ANOVA and Bonferroni’s post hoc test (α = 0.05). Additional blocks (n = 15) were subjected to scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) before and after the surface treatment. The μSBS values on VE surfaces were higher than those observed on LU and BR surfaces (p < 0.001). Silane without MDP (Allcem) promoted the highest μSBS values, while silane with MDP (RelyX Ultimate) provided the highest values among all bonding strategies (p < 0.001). Enforce promoted no significant difference in μSBS values. SEM and EDS analyses detected noticeable changes to the surface morphology and composition after the surface treatment. The effectiveness of the bonding strategy may vary according not only to the CAD–CAM composite but also to resin cement/bonding agent/silane used.     

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

Arabidopsis μ-adaptin subunit AP1M of adaptor protein complex 1 mediates late secretory and vacuolar traffic and is required for growth

Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, μ-adaptin, that selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their μ subunits in plants. Because of uncertain homology, the μ-adaptins of Arabidopsis have been designated muA through muD [Happel et al. (2004) Plant J 37(5):678–693]. Furthermore, only muD has been assigned to a specific AP complex, AP-3, involved in Golgi-vacuolar trafficking [Niihama et al. (2009) Plant Cell Physiol 50(12):2057–2068, Zwiewka et al. (2011) Cell Res 21(12):1711–1722, and Wolfenstetter et al. (2012) Plant Cell 24(1):215–232]. In contrast, the μ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 μ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans-Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans-Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.     

Insights into the P-to-Q conversion in the catalytic cycle of methane monooxygenase from a synthetic model system

For the catalytic cycle of soluble methane monooxygenase (sMMO), it has been proposed that cleavage of the O–O bond in the (μ-peroxo)diiron(III) intermediate P gives rise to the diiron(IV) intermediate Q with an Fe₂(μ–O)₂ diamond core, which oxidizes methane to methanol. As a model for this conversion, (μ–oxo) diiron(III) complex 1 ([Fe^III₂(μ–O)(μ–O₂H₃)(L)₂]³⁺, L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) has been treated consecutively with one eq of H₂O₂ and one eq of HClO₄ to form 3 ([Fe^IV₂(μ–O)₂(L)₂]⁴⁺). In the course of this reaction a new species, 2, can be observed before the protonation step; 2 gives rise to a cationic peak cluster by ESI-MS at m/z 1,399, corresponding to the {[Fe₂O₃L₂H](OTf)₂}⁺ ion in which 1 oxygen atom derives from 1 and the other two originate from H₂O₂. Mössbauer studies of 2 reveal the presence of two distinct, exchange coupled iron(IV) centers, and EXAFS fits indicate a short Fe–O bond at 1.66 Å and an Fe–Fe distance of 3.32 Å. Taken together, the spectroscopic data point to an HO-Fe^IV-O-Fe^IV = O core for 2. Protonation of 2 results in the loss of H₂O and the formation of 3. Isotope labeling experiments show that the [Fe^IV₂(μ–O)₂] core of 3 can incorporate both oxygen atoms from H₂O₂. The reactions described here serve as the only biomimetic precedent for the conversion of intermediates P to Q in the sMMO reaction cycle and shed light on how a peroxodiiron(III) unit can transform into an [Fe^IV₂(μ–O)₂] core.     

Process–Structure–Property Relationships of Copper Parts Manufactured by Laser Powder Bed Fusion

The process–structure–property relationships of copper laser powder bed fusion (L-PBF)-produced parts made of high purity copper powder (99.9 wt %) are examined in this work. A nominal laser beam diameter of 100 μm with a continuous wavelength of 1080 nm was employed. A wide range of process parameters was considered in this study, including five levels of laser power in the range of 200 to 370 W, nine levels of scanning speed from 200 to 700 mm/s, six levels of hatch spacing from 50 to 150 μm, and two layer thickness values of 30 μm and 40 μm. The influence of preheating was also investigated. A maximum relative density of 96% was obtained at a laser power of 370 W, scanning speed of 500 mm/s, and hatch spacing of 100 μm. The results illustrated the significant influence of some parameters such as laser power and hatch spacing on the part quality. In addition, surface integrity was evaluated by surface roughness measurements, where the optimum Ra was measured at 8 μm ± 0.5 μm. X-ray photoelectron spectroscopy (XPS) and energy-dispersive X-ray spectroscopy (EDX) were performed on the as-built samples to assess the impact of impurities on the L-PBF part characteristics. The highest electrical conductivity recorded for the optimum density-low contaminated coils was 81% IACS.     

A study of Chitosan and c‐di‐GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine

Please cite this paper as: Svindland et al. (2012) A study of Chitosan and c‐di‐GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses 10.1111/irv.12056000(000), 000–000. Background  Highly pathogenic avian influenza A/H5N1 virus remains a potential pandemic threat, and it is essential to continue vaccine development against this subtype. A local mucosal immune response in the upper respiratory tract may stop influenza transmission. It is therefore important to develop effective intranasal pandemic influenza vaccines that induce mucosal immunity at the site of viral entry. Objectives  We evaluated the humoral and cellular immune responses of two promising mucosal adjuvants (Chitosan and c‐di‐GMP) for intranasal influenza H5N1 vaccine in a murine model. Furthermore, we evaluated the concept of co‐adjuvanting an experimental adjuvant (c‐di‐GMP) with chitosan. Methods  BALB/c mice were intranasally immunised with two doses of subunit NIBRG‐14 (H5N1) vaccine (7·5, 1·5 or 0·3 μg haemagglutinin (HA) adjuvanted with chitosan (CSN), c‐di‐GMP or both adjuvants. Results  All adjuvant formulations improved the serum and local antibody responses, with the highest responses observed in the 7·5 μg HA CSN and c‐di‐GMP‐adjuvanted groups. The c‐di‐GMP provided dose sparing with protective single radial haemolysis (SRH), and haemagglutination inhibition (HI) antibody responses found in the 0·3 μg HA group. CSN elicited a Th2 response, whereas c‐di‐GMP induced higher frequencies of virus‐specific CD4⁺ T cells producing one or more Th1 cytokines (IFN‐γ⁺, IL‐2⁺, TNF‐α⁺). A combination of the two adjuvants demonstrated effectiveness at 7·5 μg HA and triggered a more balanced Th cytokine profile. Conclusion  These data show that combining adjuvants can modulate the Th response and in combination with ongoing studies of adjuvanted intranasal vaccines will dictate the way forward for optimal mucosal influenza vaccines.     

Alzheimer’s-specific effects of soluble β-amyloid on protein kinase C-α and -γ degradation in human fibroblasts

Alzheimer’s disease (AD) is a multifactorial disease in which β-amyloid peptide (βAP) plays a critical role. We report here that the soluble fraction 1–40 of βAP differentially degrades protein kinase C-α and -γ (PKCα and PKCγ) isoenzymes in normal (age-matched controls, AC) and AD fibroblasts most likely through proteolytic cascades. Treatment with nanomolar concentrations of βAP(1–40) induced a 75% decrease in PKCα, but not PKCγ, immunoreactivity in AC fibroblasts. In the AD fibroblasts, a 70% reduction of the PKCγ, but not PKCα, immunoreactivity was observed after βAP treatment. Preincubation of AC or AD fibroblasts with 50 μM lactacystine, a selective proteasome inhibitor, prevented β-AP(1–40)-mediated degradation of PKCα in the AC cells, and PKCγ in the AD fibroblasts. The effects of βAP(1–40) on PKCα in AC fibroblasts were prevented by inhibition of protein synthesis and reversed by PKC activation. A 3-hr treatment with 100 nM phorbol 12-myristate 13-acetate restored the PKCα signal in treated AC cells but it did not reverse the effects of βAP(1–40) on PKCγ in the AD fibroblasts. Pretreatment with the protein synthesis inhibitor, cycloheximide (CHX, 100 μM), inhibited the effects of βAP(1–40) on PKCα and blocked the rescue effect of phorbol 12-myristate 13-acetate in AC fibroblasts but did not modify PKCγ immunoreactivity in AD cells. These results suggest that βAP(1–40) differentially affects PKC regulation in AC and AD cells via proteolytic degradation and that PKC activation exerts a protective role via de novo protein synthesis in normal but not AD cells.     

Enhancement of Bentonite Materials with Cement for Gamma-Ray Shielding Capability

The gamma-ray shielding ability of various Bentonite–Cement mixed materials from northeast Egypt have been examined by determining their theoretical and experimental mass attenuation coefficients, μ_m (cm²g⁻¹), at photon energies of 59.6, 121.78, 344.28, 661.66, 964.13, 1173.23, 1332.5 and 1408.01 keV emitted from ²⁴¹Am, ¹³⁷Cs, ¹⁵²Eu and ⁶⁰Co point sources. The μ_m was theoretically calculated using the chemical compositions obtained by Energy Dispersive X-ray Analysis (EDX), while a NaI (Tl) scintillation detector was used to experimentally determine the μ_m (cm²g⁻¹) of the mixed samples. The theoretical values are in acceptable agreement with the experimental calculations of the XCom software. The linear attenuation coefficient (μ), mean free path (MFP), half-value layer (HVL) and the exposure buildup factor (EBF) were also calculated by knowing the μ_m values of the examined samples. The gamma-radiation shielding ability of the selected Bentonite–Cement mixed samples have been studied against other puplished shielding materials. Knowledge of various factors such as thermo-chemical stability, availability and water holding capacity of the bentonite–cement mixed samples can be analyzed to determine the effectiveness of the materials to shield gamma rays.     

Impact of Mass Azithromycin Distribution on Malaria Parasitemia during the Low-Transmission Season in Niger: A Cluster-Randomized Trial

We assessed the effect of mass azithromycin treatment on malaria parasitemia in a trachoma trial in Niger. Twenty-four study communities received treatment during the wet, high-transmission season. Twelve of the 24 communities were randomized to receive an additional treatment during the dry, low-transmission season. Outcome measurements were conducted at the community-level in children < 1–72 months of age in May–June 2011. Parasitemia was higher in the 12 once-treated communities (29.8%, 95% confidence interval [CI] = 21.5–40.0%) than in the 12 twice-treated communities (19.5%, 95% CI = 13.0–26.5%, P = 0.03). Parasite density was higher in once-treated communities (354 parasites/μL, 95% CI = 117–528 parasites/μL) than in twice-treated communities (74 parasites/μL, 95% CI = 41–202 parasites/μL, P = 0.03). Mass distribution of azithromycin reduced malaria parasitemia 4–5 months after the intervention. The results suggest that drugs with antimalaria activity can have long-lasting impacts on malaria during periods of low transmission.     

The barrier-to-autointegration protein is a host factor for HIV type 1 integration

In vivo, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display in vitro integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein–DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5–20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5–10 μM) human host factor HMG I(Y) was required. Similarly high levels (3–8 μM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein–DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.     

Learning-related postburst afterhyperpolarization reduction in CA1 pyramidal neurons is mediated by protein kinase A

Learning-related reductions of the postburst afterhyperpolarization (AHP) in hippocampal pyramidal neurons have been shown ex vivo, after trace eyeblink conditioning. The AHP is also reduced by many neuromodulators, such as norepinephrine, via activation of protein kinases. Trace eyeblink conditioning, like other hippocampus-dependent tasks, relies on protein synthesis for consolidating the learned memory. Protein kinase A (PKA) has been shown to be a key contributor for protein synthesis via the cAMP-response element-binding pathway. Here, we have explored a potential involvement of PKA and protein kinase C (PKC) in maintaining the learning-related postburst AHP reduction observed in CA1 pyramidal neurons. Bath application of isoproterenol (1 μM), a β-adrenergic agonist that activates PKA, significantly reduced the AHP in CA1 neurons from control animals, but not from rats that learned. This occlusion suggests that PKA activity is involved in maintaining the AHP reduction measured ex vivo after successful learning. In contrast, bath application of the PKC activator, (–) indolactam V (0.2 μM), significantly reduced the AHP in CA1 neurons from both control and trained rats, indicating that PKC activity is not involved in maintaining the AHP reduction at this point after learning.     

GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum

GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP⁺ cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP⁺ cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC₅₀ of 52.2 ± 11.8 μM for GABA. The evoked currents were inhibited by bicuculline (100 μM) and TPMPA (IC₅₀, 5.9 ± 0.6 μM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein–protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A–GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP⁺ cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 μm and a net distance traveled of 1–2 μm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP⁺ cells during early postnatal development.The role of GABAergic signaling is fundamental in the cerebellum, not only for influencing cell differentiation and neurotransmitter specification during early postnatal development, but also for controlling precise movements in the adult life (1–3). The expression of ionotropic GABA-A receptors with high affinity for the neurotransmitter is now well recognized, although the source of GABA involved in this process is controversial (4–6).GABA-A receptors mediating synaptic (phasic) or extrasynaptic (tonic) transmission are molecularly and functionally distinct. In contrast to neurons, the depolarizing effect of astrocytic GABA-A receptors persists through postnatal development, although the response may attenuate with age (7). In cerebellar astrocytes, the array of GABA-A subunits is heterogeneous, and modulation by benzodiazepines is different from that by neurons (8). Indeed, GABA responses of Bergmann cells and ependymal glial cells (EGCs) are insensitive to benzodiazepines or pentobarbital, owing to the assembly of receptors that include GABAδ or GABAρ subunits (9, 10).GABAρ subunits are part of the ionotropic GABA-A receptor family, which includes 19 identified genes that code for the same number of known proteins: α1–α6, β1–β3, γ1–γ3, δ, ε, Θ, π, and ρ1–ρ3 (11). GABA-A receptors are pentameric heterocomplexes composed of a combination of subunits, most commonly the α1β2γ2 combination, that gate a Cl⁻ channel on activation (11, 12). Other arrays may include δ, ρ, or ε subunits, which confer distinctive functional and pharmacologic properties. GABAρ subunits can combine in homopentameric arrangements that form receptors with high affinity for the neurotransmitter (GABA EC₅₀, 1–5 μM) and a low rate of desensitization, making them suitable for tonic transmission (11, 13, 14). GABAρ subunits are known to be expressed in the retina, where their presence in bipolar neurons controls the glutamatergic output (15, 16); they are present in other areas of the central nervous system as well, including striatum, hippocampus, and cerebellum, but their function there is not fully understood (17–19).The role of GABAρ in neuronal tonic (extrasynaptic) and phasic (synaptic) transmission has been demonstrated in the Purkinje neurons of the cerebellum (20); however, GABAρ subunits are also expressed in a large fraction of EGCs, specialized, ciliated GFAP⁺ cells that permit the flow of cerebrospinal fluid circulating in the fourth ventricle (10, 21). GABA-ionic currents in these cells are insensitive to pentobarbital and partially blocked by the GABA-A antagonist bicucculline as well as by (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), the first synthesized GABAρ antagonist (22). GABA-A receptors that include GABAρ subunits are also expressed in approximately 30% of the GFAP⁺ cells present in the striatum (19). Thus, it seems that cells of glial origin, such as EGCs and astrocytes, present a diverse array of ionotropic GABA receptors that include GABAρ subunits and whose functional role remains unidentified.In the course of recent work on assessing the presence of GABAρ subunits during early postnatal development of cerebellar EGCs, we corroborated their presence in this area but, unexpectedly, found that they are also widely distributed in a large proportion of GFAP⁺ cells of the granular layer (GL) that appear to be astrocytes. In this paper we report in detail the expression pattern of GABAρ subunits in GFAP⁺ cells of the cerebellum, the functional characterization of GABA responses of cerebellar astrocytes grown in vitro, and the participation of a GABAρ component in these responses. In addition, we provide evidence of the intracellular trafficking of GABAρ in astrocytes grown in vitro, and speculate about the possible synthesis of proteins in the processes of these cells.     

Recovery of HPA Axis Function After Successful Gonadotropin-Induced Pregnancy and Delivery in a Woman With Panhypopituitarism: Case Report and Review

Hypopituitarism is defined as the partial or complete defect of anterior pituitary hormone secretion. Patients with hypopituitarism usually need life-long hormone replacement therapy. However, in this case, we report a patient with panhypopituitarism whose hypothalamus–pituitary–adrenal (HPA) axis function was completely recovered after pregnancy and delivery.In this case study, we reported the case management and conducted a review of literature to identify the possible mechanism of pituitary function recovery.The patient who suffered from secondary amenorrhea was found a nonfunctioning pituitary macroadenoma, and the hormone test showed serum cortisol, FT3, FT4, thyrotropic hormone, and prolactin were at normal range. After surgical removal of the tumor which invasion in the sellar region, the patient had panhypopituitarism confirmed by the routine hormone test. Though spontaneous pregnancy is impossible in female patients with panhypopituitarism, the patient was restored fertility by the help of artificial reproductive techniques. After the confirmation of the pregnancy, levothyroixine was increased to 75 μg daily and readjusted to 150 μg daily before delivery according to the monthly measurement thyroid function. Hydrocortisone 10 mg daily replaced cortisone acetate; the dose was increased according to the symptoms of morning sickness. A single stress dose of hydrocortisone (200 mg) was used before elective cesarean delivery and was tapered to the dose of 10 mg per day in 1 week. Levothyroixine was reduced to 75 μg daily after delivery. During follow-up, her hypothalamus–pituitary–adrenal (HPA) axis function was completely recovered. The peak serum cotisol level could increase to 19.08 μg/dL by insulin-induced hypoglycemia. However, growth hormone remained unresponsive to the insulin-tolerance test, and thyroid hormone still needed exogenous supplementation.Hormone replacement therapy needed closely followed by endocrinologist and multidisciplinary cooperation during the pregnancy of patients with hypopituitarism. This case indicates that the pituitary function may partially recover after pregnancy in panhypopituitarism patients.     

Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway

AIM: To investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways.METHODS: The effect of carvedilol on angiogenesis was examined using a human umbilical vascular endothelial cell (HUVEC) model. The effect of carvedilol on cell viability was measured by CCK8 assay. Flow cytometry was used to assess the effect of carvedilol on cell cycle progression. Cell migration, transwell migration and tube formation assays were performed to analyze the effect of carvedilol on HUVEC function. Vascular endothelial growth factor (VEGF) induced activation of HUVECs, which were pretreated with different carvedilol concentrations or none. Western blot analysis detected the phosphorylation levels of three cell signaling pathway proteins, VEGFR-2, Src, and extracellular signal-regulated kinase (ERK). The specific Src inhibitor PP2 was used to assess the role of Src in the VEGF-induced angiogenic pathway.RESULTS: Carvedilol inhibited HUVEC proliferation in a dose-dependent manner (IC50 = 38.5 mmol/L). The distribution of cells in the S phase decreased from 43.6% to 37.2%, 35.6% and 17.8% by 1, 5 and 10 μmol/L carvedilol for 24 h, respectively. Carvedilol (10 μmol/L) reduced VEGF-induced HUVEC migration from 67.54 ± 7.83 to 37.11 ± 3.533 (P < 0.001). Carvedilol concentrations of 5 μmol/L and 10 μmol/L reduced cell invasion from 196.3% ± 18.76% to 114.0% ± 12.20% and 51.68% ± 8.28%, respectively. VEGF-induced tube formation was also reduced significantly by 5 μmol/L and 10 μmol/L carvedilol from 286.0 ± 36.72 to 135.7 ± 18.13 (P < 0.05) and 80.27 ± 11.16 (P < 0.01) respectively. We investigated several intracellular protein levels to determine the reason for these reductions. Treatment with 10 μmol/L carvedilol reduced VEGF-induced tyrosine phosphorylation of VEGFR-2 from 175.5% ± 8.54% to 52.67% ± 5.33% (P < 0.01). Additionally, 10 μmol/L carvedilol reduced VEGF-induced ERK 1/2 phosphorylation from 181.9% ± 18.61% to 56.45% ± 7.64% (P < 0.01). The VEGF-induced increase in Src kinase activity was alleviated by carvedilol [decreased from 141.8% ± 15.37% to 53.57 ± 7.18% (P < 0.01) and 47.04% ± 9.74% (P < 0.01) at concentrations of 5 and 10 μmol/L, respectively]. Pretreatment of HUVECs with Src kinase inhibitor almost completely prevented the VEGF-induced ERK upregulation [decreased from 213.2% ± 27.68% to 90.96% ± 17.16% (P < 0.01)].CONCLUSION: Carvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced Src-ERK signaling pathways.     

Comparison of opioid local anesthetic combination regimens using the number of self-administrated boluses in patient-controlled epidural analgesia after cesarean section: A retrospective single-center study

The aim of this study was to assess the efficacy of combined opioids by comparing four regimens of patient-controlled epidural analgesia (PCEA) after cesarean section.Parturient patients who underwent elective or emergent cesarean section under combined spinal and epidural anesthesia from April 2013 to March 2016 were retrospectively analyzed. Based on PCEA, they were assigned to one of 4 groups: local anesthetic alone (LA), epidural single morphine administration during surgery followed by local anesthetic alone (M), local anesthetic combined with fentanyl 10 μg/h (F10), or local anesthetic combined with fentanyl 20 μg/h (F20). The primary outcome was the number of PCEA boluses used. Secondary outcomes included the use of rescue analgesia, postoperative nausea and vomiting, and postoperative pruritus.A total of 250 parturients were analyzed. Whereas the number of PCEA boluses in the LA group was significantly higher than in the other combined opioid groups on the day of surgery and postoperative day 1 (LA: 3 [1–6] and 7 [4–9] vs M: 2 [0–4] and 4 [0–7] vs F10: 1 [0–4] and 3 [0–6] vs F20: 1 [0–3] and 2 [0–8], P = .012 and 0.010, respectively), within the combined opioid groups, the number was not significantly different. Significantly fewer patients in the F20 group required rescue analgesia on postoperative day 1 and 2 (25 and 55%) than those in the M (66 and 81%) and F10 (62 and 66%) groups (P < .001 and P = .007, respectively). Postoperative nausea and vomiting and pruritus were significantly higher in the M group (P < .008 and P = .024, respectively).The results of the present study suggest that local anesthetic alone after a single administration of morphine, or local anesthetic combined with fentanyl 10 μg/h would generally be adequate for PCEA, whereas local anesthetic combined with fentanyl 20 μg/h would be suitable for conventional epidural analgesia.