CFTR involvement in nasal potential differences in mice and pigs studied using a thiazolidinone CFTR inhibitor

Nasal potential difference (PD) measurements have been used to demonstrate defective CFTR function in cystic fibrosis (CF) and to evaluate potential CF therapies. We used the selective thiazolidinone CFTR inhibitor CFTR(inh)-172 to define the involvement of CFTR in nasal PD changes in mice and pigs. In normal mice infused intranasally with a physiological saline solution containing amiloride, nasal PD was -4.7 +/- 0.7 mV, hyperpolarizing by 15 +/- 1 mV after a low-Cl- solution, and a further 3.9 +/- 0.5 mV after forskolin. CFTR(inh)-172 produced 1.1 +/- 0.9- and 4.3 +/- 0.7-mV depolarizations when added after low Cl- and forskolin, respectively. Systemically administered CFTR(inh)-172 reduced the forskolin-induced hyperpolarization from 4.7 +/- 0.4 to 0.9 +/- 0.1 mV but did not reduce the low Cl(-)-induced hyperpolarization. Nasal PD was -12 +/- 1 mV in CF mice after amiloride, changing by <0.5 mV after low Cl- or forskolin. In pigs, nasal PD was -14 +/- 3 mV after amiloride, hyperpolarizing by 13 +/- 2 mV after low Cl- and a further 9 +/- 1 mV after forskolin. CFTR(inh)-172 and glibenclamide did not affect nasal PD in pigs. Our results suggest that cAMP-dependent nasal PDs in mice primarily involve CFTR-mediated Cl- conductance, whereas cAMP-independent PDs are produced by a different, but CFTR-dependent, Cl- channel. In pigs, CFTR may not be responsible for Cl- channel-dependent nasal PDs. These results have important implications for interpreting nasal PDs in terms of CFTR function in animal models of CFTR activation and inhibition.     

Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

Cyclic AMP-activated intestinal Cl⁻ secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl⁻ secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl⁻ secretion in human intestinal epithelial (T84) cells with IC₅₀ of ∼20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl⁻ current showed that diclofenac reversibly inhibited CFTR Cl⁻ channel activity (IC₅₀∼10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na⁺-K⁺ ATPases and Na⁺-K⁺-Cl⁻ cotransporters, but inhibited cAMP-activated basolateral K⁺ channels with IC₅₀ of ∼3 µM. In addition, diclofenac suppressed Ca²⁺-activated Cl⁻ channels, inwardly rectifying Cl⁻ channels, and Ca²⁺-activated basolateral K⁺ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl⁻ secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca²⁺-activated Cl⁻ secretion by inhibiting both apical Cl⁻ channels and basolateral K⁺ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal hypersecretion of Cl⁻.     

Construction of high-density display of CD147 ectodomain on VCSM13 phage via gpVIII: effects of temperature, IPTG, and helper phage infection-period

Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfully increased when growing the transformed TG1 at 25 degrees C with IPTG-stimulation. In addition to temperature and IPTG-stimulation, the VCSM13 helper phage infection-period particularly affected the insertion of CD147Ex into phage progeny. By sandwich ELISA, incorporation of the CD147Ex into phage particle was confirmed. The correct size of the CD147Ex-gpVIII fusion protein at 28kDa was demonstrated by Western immunoblotting. Multivalent display of CD147Ex on phage particles will be valuable in discovering its ligand partner.     

Baculovirus display of single chain antibody (scFv) using a novel signal peptide

Background  

Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.     

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.     

Hydroxyxanthone as an inhibitor of cAMP-activated apical chloride channel in human intestinal epithelial cell

AimsPrevious investigation showed that polyphenols abundantly found in many plants could inhibit Cl^? secretion. The present study was aimed to investigate the effect of phenol containing xanthone derivatives on cAMP-activated intestinal Cl^? secretion and evaluate potential benefits of these compounds in the treatment of cholera.Main methodsFour hydroxy xanthones were synthesized via oxidative coupling reaction of the corresponding ortho-hydroxybenzoic acids and hydroxyphenols. Short-circuit current and apical Cl^? current measurements across monolayers of human intestinal epithelial (T84) cell and Fisher rat thyroid cells transfected with human CFTR (FRT-hCFTR cell) were performed to determine the effect of hydroxyxanthones on cAMP-activated Cl^? secretion. Intracellular cAMP was measured by immunoassay methods. Anti-diarrheal efficacy was evaluated using closed loop model of cholera.Key findingsAmong the tested xanthones, 1,3,6-trihydroxyxanthone (THX-001) was found to be the most potent derivative in the inhibition of cAMP-activated Cl^? secretion across T84 cell monolayers (IC₅₀ ~ 100 μM). Electrophysiological analysis of T84 cells and FRT-hCFTR cells revealed that THX-001 targeted two distinct cAMP-activated Cl^? channels in the apical membrane of T84 cells, namely, CFTR and inward rectifying Cl^? channel (IRC). In contrast, THX-001 had no effect on intracellular cAMP levels in these cells. Importantly, THX-001 completely abolished cholera toxin-induced Cl^? secretion across T84 cell monolayers and significantly inhibited cholera toxin-induced intestinal fluid secretion in mouse closed loop models.SignificanceThis study revealed that hydroxyxanthone represents another chemical class of polyphenolic compounds that may hold promise as anti-secretory therapy for cholera.     

Context-dependent tree species effects on soil nitrogen transformations and related microbial functional genes

Although it is generally accepted that tree species can influence nutrient cycling processes in soils, effects are not consistently found, nor are the mechanisms behind tree species effects well understood. Our objectives were to gain insights into the mechanism(s) underlying the effects of tree species on soil nitrogen cycling processes, and to determine the consistency of tree species effects across sites. We compared N cycling in soils beneath six tree species (ash, sycamore maple, lime, beech, pedunculate oak, Norway spruce) in common garden experiments planted 42 years earlier at three sites in Denmark with distinct land-use histories (forest and agriculture). We measured: (1) net and gross rates of N transformations using the ¹⁵N isotope pool-dilution method, (2) soil microbial community composition through qPCR of fungal ITS, bacterial and archaeal 16S, and (3) abundance of functional genes associated with N cycling processes—for nitrification the archaeal and bacterial ammonia-monooxygenase genes (amoA AOA and amoA AOB, respectively) and for denitrification, the nitrate reductase genes nirK and nirS. Carbon concentrations were higher in soils under spruce than under broadleaves, so N transformation rates were standardized per g soil C. Soil NH₄⁺ parameters (gross ammonification, gross NH₄⁺ consumption, net ammonification (net immobilization in this case), and NH₄⁺ concentrations, per g C) were all lowest in soils under spruce. Soils under spruce also had the lowest gene abundance of bacteria, bacterial:fungal ratio, denitrifying microorganisms, ammonia-oxidizing archaea and ammonia-oxidizing bacteria. Differences in N-cycling processes and organisms among the five broadleaf species were smaller. The ‘spruce effect’ on soil microbes and N transformations appeared to be driven by its acidifying effect on soil and tighter N cycling, which occurred at the previously forested sites but not at the previously agricultural site. We conclude that existing characteristics of soils, including those resulting from previous land use, mediate the effects of tree species on the soil microbial communities and activities that determine rates of N-cycling processes.     

The Occluded Epitope Residing in Spike Receptor-Binding Motif Is Essential for Cross-Neutralization of SARS-CoV-2 Delta Variant

Concerns over vaccine efficacy after the emergence of the SARS-CoV-2 Delta variant prompted revisiting the vaccine design concepts. Monoclonal antibodies (mAbs) have been developed to identify the neutralizing epitopes on spike protein. It has been confirmed that the key amino acid residues in epitopes that induce the formation of neutralizing antibodies do not have to be on the receptor-binding domain (RBD)- angiotensin-converting enzyme 2 (ACE2) contact surface, and may be conformationally hidden. In addition, this epitope is tolerant to amino acid mutations of the Delta variant. The antibody titers against RBD in health care workers in Thailand receiving two doses of CoronaVac, followed by a booster dose of BNT162b2, were significantly increased. The neutralizing antibodies against the Delta variant suggest that the overall neutralizing antibody level against the Wuhan strain, using the NeutraLISA, was consistent with the levels of anti-RBD antibodies. However, individuals with moderate anti-RBD antibody responses have different levels of a unique antibody population competing with a cross-neutralizing mAb clone, 40591-MM43, determined by in-house competitive ELISA. Since 40591-MM43 mAb indicates cross-neutralizing activity against the Delta variant, this evidence implies that the efficiency of the vaccination regimen should be improved to facilitate cross-protective antibodies against Delta variant infections. The RBD epitope recognized by 40591-MM43 mAb is hidden in the close state.