Aging-associated weakening of the action potential in fast-spiking interneurons in the human neocortex.

Aging is associated with the slowdown of neuronal processing and cognitive performance in the brain; however, the exact cellular mechanisms behind this deterioration in humans are poorly elucidated. Recordings in human acute brain slices prepared from tissue resected during brain surgery enable the investigation of neuronal changes with age. Although neocortical fast-spiking cells are widely implicated in neuronal network activities underlying cognitive processes, they are vulnerable to neurodegeneration. Herein, we analyzed the electrical properties of 147 fast-spiking interneurons in neocortex samples resected in brain surgery from 106 patients aged 11-84 years. By studying the electrophysiological features of action potentials and passive membrane properties, we report that action potential overshoot significantly decreases and spike half-width increases with age. Moreover, the action potential maximum-rise speed (but not the repolarization speed or the afterhyperpolarization amplitude) significantly changed with age, suggesting a particular weakening of the sodium channel current generated in the soma. Cell passive membrane properties measured as the input resistance, membrane time constant, and cell capacitance remained unaffected by senescence. Thus, we conclude that the action potential in fast-spiking interneurons shows a significant weakening in the human neocortex with age. This may contribute to the deterioration of cortical functions by aging.

Ventilator Weaning in Prolonged Mechanical Ventilation-A Narrative Review.

Patients requiring mechanical ventilation (MV) beyond 21 days, usually referred to as prolonged MV, represent a unique group with significant medical needs and a generally poor prognosis. Research suggests that approximately 10% of all MV patients will need prolonged ventilatory care, and that number will continue to rise. Although we have extensive knowledge of MV in the acute care setting, less is known about care in the post-ICU setting. More than 50% of patients who were deemed unweanable in the ICU will be liberated from MV in the post-acute setting. Prolonged MV also presents a challenge in care for medically complex, elderly, socioeconomically disadvantaged and marginalized individuals, usually at the end of their life. Patients and their families often rely on ventilator weaning facilities and skilled nursing homes for the continuation of care, but home ventilation is becoming more common. The focus of this review is to discuss recent advances in the weaning strategies in prolonged MV, present their outcomes and provide insight into the complexity of care.

Corrigendum: The mycoremediation potential of the armillarioids: a comparative genomics analysis.

[This corrects the article DOI: 10.3389/fbioe.2023.1189640.].

Valproate treatment induces age- and sex-dependent neuronal activity changes according to a patch clamp study.

Autism spectrum disorder is a heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restricted, and stereotyped behaviors. The valproic acid model is one of the most recognized and broadly used models in rats to induce core symptoms of this disorder. Comorbidity of epilepsy and autism occurs frequently, due to similar background mechanisms that include the imbalance of excitation and inhibition. In this series of experiments, treatment was performed on rat dams with a single 500 mg/kg dose i.p. valproate injection on embryonic day 12.5. Intracellular whole-cell patch clamp recordings were performed on brain slices prepared from adolescent and adult offspring of both sexes on pyramidal neurons of the medial prefrontal cortex and entorhinal cortex. Current clamp stimulation utilizing conventional current step protocols and dynamic clamp stimulation were applied to assess neuronal excitability. Membrane properties and spiking characteristics of layer II-III pyramidal cells were analyzed in both cortical regions. Significant sex-dependent and age-dependent differences were found in several parameters in the control groups. Considering membrane resistance, rheobase, voltage sag slope, and afterdepolarization slope, we observed notable changes mainly in the female groups. Valproate treatment seemed to enhance these differences and increase network excitability. However, it is possible that compensatory mechanisms took place during the maturation of the network while reaching the age-group of 3 months. Based on the results, the expression of the hyperpolarization-activated cyclic nucleotide-gated channels may be appreciably affected by the valproate treatment, which influences fundamental electrophysiological properties of the neurons such as the voltage sag. Remarkable changes appeared in the prefrontal cortex; however, also the entorhinal cortex shows similar tendencies.

The mycoremediation potential of the armillarioids: a comparative genomics analysis.

Genes involved in mycoremediation were identified by comparative genomics analysis in 10 armillarioid species and selected groups of white-rot Basidiomycota (14) and soft-rot Ascomycota (12) species to confine the distinctive bioremediation capabilities of the armillarioids. The genomes were explored using phylogenetic principal component analysis (pPCA), searching for genes already documented in a biocatalysis/biodegradation database. The results underlined a distinct, increased potential of aromatics-degrading genes/enzymes in armillarioids, with particular emphasis on a high copy number and diverse spectrum of benzoate 4-monooxygenase [EC:1.14.14.92] homologs. In addition, other enzymes involved in the degradation of various monocyclic aromatics were more abundant in the armillarioids than in the other white-rot basidiomycetes, and enzymes involved in the degradation of polycyclic aromatic hydrocarbons (PAHs) were more prevailing in armillarioids and other white-rot species than in soft-rot Ascomycetes. Transcriptome profiling of A. ostoyae and A. borealis isolates confirmed that several genes involved in the degradation of benzoates and other monocyclic aromatics were distinctively expressed in the wood-invading fungal mycelia. Data were consistent with armillarioid species offering a more powerful potential in degrading aromatics. Our results provide a reliable, practical solution for screening the likely fungal candidates for their full biodegradation potential, applicability, and possible specialization based on their genomics data.

Recommendations for wider adoption of clinical pharmacy in Central and Eastern Europe in order to optimise pharmacotherapy and improve patient outcomes.

Clinical pharmacy as an area of practice, education and research started developing around the 1960s when pharmacists across the globe gradually identified the need to focus more on ensuring the appropriate use of medicines to improve patient outcomes rather than being engaged in manufacturing and supply. Since that time numerous studies have shown the positive impact of clinical pharmacy services (CPS). The need for wider adoption of CPS worldwide becomes urgent, as the global population ages, and the prevalence of polypharmacy as well as shortage of healthcare professionals is rising. At the same time, there is great pressure to provide both high-quality and cost-effective health services. All these challenges urgently require the adoption of a new paradigm of healthcare system architecture. One of the most appropriate answers to these challenges is to increase the utilization of the potential of highly educated and skilled professionals widely available in these countries, i.e., pharmacists, who are well positioned to prevent and manage drug-related problems together with ensuring safe and effective use of medications with further care relating to medication adherence. Unfortunately, CPS are still underdeveloped and underutilized in some parts of Europe, namely, in most of the Central and Eastern European (CEE) countries. This paper reviews current situation of CPS development in CEE countries and the prospects for the future of CPS in that region.

Dual RNA-Seq Profiling Unveils Mycoparasitic Activities of Trichoderma atroviride against Haploid Armillaria ostoyae in Antagonistic Interaction Assays.

Armillaria ostoyae, a species among the destructive forest pathogens from the genus Armillaria, causes root rot disease on woody plants worldwide. Efficient control measures to limit the growth and impact of this severe underground pathogen are under investigation. In a previous study, a new soilborne fungal isolate, Trichoderma atroviride SZMC 24276 (TA), exhibited high antagonistic efficacy, which suggested that it could be utilized as a biocontrol agent. The dual culture assay results indicated that the haploid A. ostoyae-derivative SZMC 23085 (AO) (C18/9) is highly susceptible to the mycelial invasion of TA. In the present study, we analyzed the transcriptome of AO and that of TA in in vitro dual culture assays to test the molecular arsenal of Trichoderma antagonism and the defense mechanisms of Armillaria. We conducted time-course analysis and functional annotation and analyzed enriched pathways and differentially expressed genes including biocontrol-related candidate genes from TA and defense-related candidate genes from AO. The results indicated that TA deployed several biocontrol mechanisms when confronted with AO. In response, AO initiated multiple defense mechanisms to protect against the fungal attack. To our knowledge, the present study offers the first transcriptome analysis of a biocontrol fungus attacking AO. Overall, this study provides insights that aid the further exploration of plant pathogen-biocontrol agent interaction mechanisms. IMPORTANCE Armillaria species can survive for decades in the soil on dead woody debris, develop rapidly under favorable conditions, and harmfully infect newly planted forests. Our previous study found Trichoderma atroviride to be highly effective in controlling Armillaria growth; therefore, our current work explored the molecular mechanisms that might play a key role in Trichoderma-Armillaria interactions. Direct confrontation assays combined with time course-based dual transcriptome analysis provided a reliable system for uncovering the interactive molecular dynamics between the fungal plant pathogen and its mycoparasitic partner. Furthermore, using a haploid Armillaria isolate allowed us to survey the deadly prey-invading activities of the mycoparasite and the ultimate defensive strategies of its prey. Our current study provides detailed insights into the essential genes and mechanisms involved in Armillaria defense against Trichoderma and the genes potentially involved in the efficiency of Trichoderma to control Armillaria. In addition, using a sensitive haploid Armillaria strain (C18/9), with its complete genome data already available, also offers the opportunity to test possible variable molecular responses of Armillaria ostoyae toward diverse Trichoderma isolates with various biocontrol abilities. Initial molecular tests of the dual interactions may soon help to develop a targeted biocontrol intervention with mycoparasites against plant pathogens.

Development of a Multicomponent Microbiological Soil Inoculant and Its Performance in Sweet Potato Cultivation.

The cultivation and consumption of sweet potato (Ipomoea batatas) are increasing globally. As the usage of chemical fertilizers and pest control agents during its cultivation may lead to soil, water and air pollution, there is an emerging need for environment-friendly, biological solutions enabling increased amounts of healthy crop and efficient disease management. Microbiological agents for agricultural purposes gained increasing importance in the past few decades. Our goal was to develop an agricultural soil inoculant from multiple microorganisms and test its application potential in sweet potato cultivation. Two Trichoderma strains were selected: Trichoderma ghanense strain SZMC 25217 based on its extracellular enzyme activities for the biodegradation of plant residues, and Trichoderma afroharzianum strain SZMC 25231 for biocontrol purposes against fungal plant pathogens. The Bacillus velezensis strain SZMC 24986 proved to be the best growth inhibitor of most of the nine tested strains of fungal species known as plant pathogens, therefore it was also selected for biocontrol purposes against fungal plant pathogens. Arthrobacter globiformis strain SZMC 25081, showing the fastest growth on nitrogen-free medium, was selected as a component with possible nitrogen-fixing potential. A Pseudomonas resinovorans strain, SZMC 25872, was selected for its ability to produce indole-3-acetic acid, which is among the important traits of potential plant growth-promoting rhizobacteria (PGPR). A series of experiments were performed to test the selected strains for their tolerance to abiotic stress factors such as pH, temperature, water activity and fungicides, influencing the survivability in agricultural environments. The selected strains were used to treat sweet potato in two separate field experiments. Yield increase was observed for the plants treated with the selected microbial consortium (synthetic community) in comparison with the control group in both cases. Our results suggest that the developed microbial inoculant has the potential to be used in sweet potato plantations. To the best of our knowledge, this is the first report about the successful application of a fungal-bacterial consortium in sweet potato cultivation.

HCN channels at the cell soma ensure the rapid electrical reactivity of fast-spiking interneurons in human neocortex.

Accumulating evidence indicates that there are substantial species differences in the properties of mammalian neurons, yet theories on circuit activity and information processing in the human brain are based heavily on results obtained from rodents and other experimental animals. This knowledge gap may be particularly important for understanding the neocortex, the brain area responsible for the most complex neuronal operations and showing the greatest evolutionary divergence. Here, we examined differences in the electrophysiological properties of human and mouse fast-spiking GABAergic basket cells, among the most abundant inhibitory interneurons in cortex. Analyses of membrane potential responses to current input, pharmacologically isolated somatic leak currents, isolated soma outside-out patch recordings, and immunohistochemical staining revealed that human neocortical basket cells abundantly express hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel isoforms HCN1 and HCN2 at the cell soma membrane, whereas these channels are sparse at the rodent basket cell soma membrane. Antagonist experiments showed that HCN channels in human neurons contribute to the resting membrane potential and cell excitability at the cell soma, accelerate somatic membrane potential kinetics, and shorten the lag between excitatory postsynaptic potentials and action potential generation. These effects are important because the soma of human fast-spiking neurons without HCN channels exhibit low persistent ion leak and slow membrane potential kinetics, compared with mouse fast-spiking neurons. HCN channels speed up human cell membrane potential kinetics and help attain an input-output rate close to that of rodent cells. Computational modeling demonstrated that HCN channel activity at the human fast-spiking cell soma membrane is sufficient to accelerate the input-output function as observed in cell recordings. Thus, human and mouse fast-spiking neurons exhibit functionally significant differences in ion channel composition at the cell soma membrane to set the speed and fidelity of their input-output function. These HCN channels ensure fast electrical reactivity of fast-spiking cells in human neocortex.

Warburg-like metabolic transformation underlies neuronal degeneration in sporadic Alzheimer's disease.

The drivers of sporadic Alzheimer's disease (AD) remain incompletely understood. Utilizing directly converted induced neurons (iNs) from AD-patient-derived fibroblasts, we identified a metabolic switch to aerobic glycolysis in AD iNs. Pathological isoform switching of the glycolytic enzyme pyruvate kinase M (PKM) toward the cancer-associated PKM2 isoform conferred metabolic and transcriptional changes in AD iNs. These alterations occurred via PKM2's lack of metabolic activity and via nuclear translocation and association with STAT3 and HIF1α to promote neuronal fate loss and vulnerability. Chemical modulation of PKM2 prevented nuclear translocation, restored a mature neuronal metabolism, reversed AD-specific gene expression changes, and re-activated neuronal resilience against cell death.

Response of the mushroom pathogen Cladobotryum mycophilum to prochloraz and metrafenone fungicides and Streptomyces flavovirens actinobacteria.

After an outbreak of cobweb disease of cultivated button mushroom in Serbia in 2003, the isolated fungal pathogen was initially identified as Cladobotryum dendroides (teleomorph Hypomyces rosellus) based on morpho-physiological traits. Molecular analysis indicated re-classification of two strains (isolated in 2004 and 2007) as Cladobotryum mycophilum (teleomorph Hypomyces odoratus). However, subsequent analysis of further five strains (isolated over the period 2003-2010) within the frames of the present study, also confirmed their identification as the exclusive cobweb causal agent C. mycophilum. After artificial inoculation, the symptoms observed on harvested and growing mushrooms were consistent with the appearance of cobweb disease. Pathogen sensitivity to fungicides was estimated by probit analyses. Fungicide susceptibility tests showed that C. mycophilum strains were highly sensitive both to prochloraz (ED50<0.087 µg mL-1) and the newly introduced metrafenone (ED50<0.15 µg mL-1). Furthermore, the growth of all examined strains of C. mycophilum was significantly inhibited by the indigenous actinobacterial strain Streptomyces flavovirens A06. A dual culture assay showed after 72 h that the percentage of radial growth inhibition of the pathogen ranged from 22.38 to 55.73%. Our findings suggest that the antagonistic S. flavovirens A06 might be a potential candidate for controlling the cobweb disease of cultivated button mushroom.

Epileptiform Events and Kainate Receptor Activity Changes Considerably during Entorhinal Cortex Development: An ex vivo Study.

Epilepsy is a commonly diagnosed neurological disease, which often develops already in childhood. The prominent feature of this dysfunction is the strong, unprovoked hypersynchronous neuronal activity of the brain, especially in the cortex, which appears in recurrent seizures. Previous studies indicated a potential modulatory role of kainate types of glutamate receptors in this mechanism. In our experiments, we used combined hippocampal-entorhinal rat brain slices of different ages. Developing (2-, 3-, and 4-week-old), adolescent (6-week-old), and adult (3-month-old) groups were investigated. During the experiments, first, we provoked convulsions with magnesium-free perfusion solution; then, to investigate the role of kainate receptors, seizure-like events (SLEs) were suppressed by applying a specific GluK1/2 antagonist (UBP-296). Neuronal network activity was recorded by a multi-electrode array chip, and temporal features of field potentials and single-cell activity were analyzed in the different age-groups. The frequency, duration of spontaneous events, the overall seizure characteristics, and spike activities were compared. Spontaneous events were categorized into interictal epileptiform discharges (IEDs) and SLEs on the basis of the temporal structure of activities. In 3- and 4-week-old animals, IEDs were observable, which entirely disappeared after the 4th week. The structure and the length of SLEs varied in the younger animals (3- and 4-week-old animals); however, after the 6th week, these events became more stabilized. In most groups, the count of detected spikes was significantly higher in layer II/III than in layer V. The neuronal networks started to behave like adult ones at 4 weeks of age. The length of events decreased in adult animals due to the maturation of the network, and the inhibition becomes stronger. The IEDs disappeared completely, and the SLEs became stable and stereotypic in 6-week-old animals. UBP-296 administration reduced the number of IEDs; however, this had no substantial effect on the SLEs.

Alterations of the Hippocampal Networks in Valproic Acid-Induced Rat Autism Model.

Autism Spectrum Disorder (ASD) is one of the most frequently diagnosed neurodevelopmental disorders, characterized among others by impairments in social interactions and repetitive behavior. According to one of the leading hypotheses about its origin, ASD is caused by the imbalance of excitatory and inhibitory circuit activity. ASD-related morphological and functional changes can be observed in several brain regions i.e., in the prefrontal cortex and the hippocampus. It is well-established that prenatal valproic-acid (VPA) exposure of rats on day 12.5 leads to neurodevelopmental alterations with autism-like clinical and behavioral symptoms. The aim of this study was to investigate potential changes in the excitability of neuronal networks and individual neurons of the hippocampus elicited by prenatal VPA treatment. As there are marked sex differences in ASD, offspring of both sexes were systematically tested, using two different age groups, to elucidate eventual differences in neurodevelopment after VPA treatment. Excitatory connections and long-term synaptic plasticity as well as intrinsic excitability of CA1 pyramidal cells were examined. Pregnant female Wistar rats received saline or 500 mg/kg VPA i. p. on gestation day 12.5. Brain slices of 6-week-old and 3-month-old offspring were investigated using extra- and intracellular electrophysiological techniques. Field potential- and whole-cell patch clamp recordings were carried out to measure network excitability and single cell activity in the CA1 region hippocampus. Enhanced excitability of hippocampal networks was detected in the 6-week-old VPA-treated male rats; however, this change could not be observed in 3-month-old males. Intrinsic excitability of single neurons, however, was increased in 3-month-old males. In 6-week-old treated females, the most prominent effect of VPA was an increase in voltage sag, to a similar degree to the neurons of the older age group. In 3-month-old females, a network excitability increase could be demonstrated, in a lesser degree than in younger males. It can be concluded, that VPA treatment had diverse effects on hippocampal excitability depending on the sex and the age of the animals. We found that certain alterations manifested in 6-week-old rats were compensated later, on the other hand, other changes persisted until the age of 3 months.

Anatomical Assessment vs. Pullback REsting full-cycle rAtio (RFR) Measurement for Evaluation of Focal and Diffuse CoronarY Disease: Rationale and Design of the "READY Register".

Background: The morphology and functional severity of coronary stenosis show poor correlation. However, in clinical practice, the visual assessment of the invasive coronary angiography is still the most common means for evaluating coronary disease. The fractional flow reserve (FFR), the coronary flow reserve (CFR), and the resting full-cycle ratio (RFR) are established indices to determine the hemodynamic significance of a coronary stenosis. Design/Methods: The READY register (NCT04857762) is a prospective, multicentre register of patients who underwent invasive intracoronary FFR and RFR measurement. The main aim of the registry is to compare the visual estimate of coronary lesions and the functional severity of the stenosis assessed by FFR, as well as the RFR pullback. Characterizations of the coronary vessel for predominantly focal, diffuse, or mixed type disease according to visual vs. RFR pullback determination will be compared. The secondary endpoint of the study is a composite of major adverse cardiac events, including death, myocardial infarction, and repeat coronary revascularization at 1 year. These endpoints will be compared in patients with non-ischemic FFR in the subgroup of cases where the local pressure drop indicates a focal lesion according to the definition of ΔRFR > 0.05 (for <25 mm segment length) and in the subgroup without significant ΔRFR. In case of an FFR value above 0.80, an extended physiological analysis is planned to diagnose or exclude microvascular disease using the CFR/FFR index. This includes novel flow dynamic modeling for CFR calculation (CFRp-3D). Conclusion: The READY register will define the effect of RFR measurement on visual estimation-based clinical decision-making. It can identify a prognostic value of ΔRFR during RFR pullback, and it would also explore the frequency of microvascular disease in the patient population with FFR > 0.80. Clinical Trial Registration: ClinicalTrials.gov (NCT04857762).

Chaetomium and Chaetomium-like Species from European Indoor Environments Include Dichotomopilus finlandicus sp. nov.

The genus Chaetomium is a frequently occurring fungal taxon world-wide. Chaetomium and Chaetomium-like species occur in indoor environments, where they can degrade cellulose-based building materials, thereby causing structural damage. Furthermore, several species of this genus may also cause adverse effects on human health. The aims of this research were to identify Chaetomium and Chaetomium-like strains isolated from indoor environments in Hungary and Finland, two geographically distant regions of Europe with drier and wetter continental climates, respectively, and to study their morphological and physiological properties, as well as their extracellular enzyme activities, thereby comparing the Chaetomium and Chaetomium-like species isolated from these two different regions of Europe and their properties. Chaetomium and Chaetomium-like strains were isolated from flats and offices in Hungary, as well as from schools, flats, and offices in Finland. Fragments of the translation elongation factor 1α (tef1α), the second largest subunit of RNA polymerase II (rpb2) and ß-tubulin (tub2) genes, as well as the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster were sequenced, and phylogenetic analysis of the sequences performed. Morphological examinations were performed by stereomicroscopy and scanning electron microscopy. Thirty-one Chaetomium sp. strains (15 from Hungary and 16 from Finland) were examined during the study. The most abundant species was Ch. globosum in both countries. In Hungary, 13 strains were identified as Ch. globosum, 1 as Ch. cochliodes, and 1 as Ch. interruptum. In Finland, 10 strains were Ch. globosum, 2 strains were Ch. cochliodes, 2 were Ch. rectangulare, and 2 isolates (SZMC 26527, SZMC 26529) proved to be representatives of a yet undescribed phylogenetic species from the closely related genus Dichotomopilus, which we formally describe here as the new species Dichotomopilus finlandicus. Growth of the isolates was examined at different temperatures (4, 15, 20, 25, 30, 37, 35, 40, and 45 °C), while their extracellular enzyme production was determined spectrophotometrically.

Protein kinase D promotes activity-dependent AMPA receptor endocytosis in hippocampal neurons.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.

Conventional measures of intrinsic excitability are poor estimators of neuronal activity under realistic synaptic inputs.

Activity-dependent regulation of intrinsic excitability has been shown to greatly contribute to the overall plasticity of neuronal circuits. Such neuroadaptations are commonly investigated in patch clamp experiments using current step stimulation and the resulting input-output functions are analyzed to quantify alterations in intrinsic excitability. However, it is rarely addressed, how such changes translate to the function of neurons when they operate under natural synaptic inputs. Still, it is reasonable to expect that a strong correlation and near proportional relationship exist between static firing responses and those evoked by synaptic drive. We challenge this view by performing a high-yield electrophysiological analysis of cultured mouse hippocampal neurons using both standard protocols and simulated synaptic inputs via dynamic clamp. We find that under these conditions the neurons exhibit vastly different firing responses with surprisingly weak correlation between static and dynamic firing intensities. These contrasting responses are regulated by two intrinsic K-currents mediated by Kv1 and Kir channels, respectively. Pharmacological manipulation of the K-currents produces differential regulation of the firing output of neurons. Static firing responses are greatly increased in stuttering type neurons under blocking their Kv1 channels, while the synaptic responses of the same neurons are less affected. Pharmacological blocking of Kir-channels in delayed firing type neurons, on the other hand, exhibit the opposite effects. Our subsequent computational model simulations confirm the findings in the electrophysiological experiments and also show that adaptive changes in the kinetic properties of such currents can even produce paradoxical regulation of the firing output.

Mislocalization of CFTR expression in acute pancreatitis and the beneficial effect of VX-661 + VX-770 treatment on disease severity.

Cystic fibrosis transmembrane conductance regulator (CFTR) has an essential role in maintaining pancreatic ductal function. Impaired CFTR function can trigger acute pancreatitis (AP) and exacerbate disease severity. We aimed to investigate the localization and expression of CFTR during AP, and determined the effects of a CFTR corrector (VX-661) and potentiator (VX-770) on disease severity. AP was induced in FVB/n mice by 6-10 hourly intraperitoneal injections of 50 µg/kg cerulein. Some mice were pre-treated with five to six daily injections of 2 mg/kg VX-661 + VX-770. Control animals were administered physiological saline instead of cerulein and dimethyl sulfoxide instead of VX compounds. AP severity was determined by measuring laboratory and histological parameters; CFTR and CK19 expression was measured. Activity of ion transporters was followed by intracellular pH or fluid secretion measurement of isolated pancreatic intra-/interlobular ducts. Cerulein-induced AP severity was greatest between 12 and 24 h. CFTR mRNA expression was significantly increased 24 h after AP induction. Immunohistochemistry demonstrated disturbed staining morphology of CFTR and CK19 proteins in AP. Mislocalization of CFTR protein was observed from 6 h, while expression increased at 24 h compared to control. Ductal HCO3- transport activity was significantly increased 6 h after AP induction. AP mice pre-treatment with VX-661 + VX-770 significantly reduced the extent of tissue damage by about 20-30%, but other parameters were unchanged. Interestingly, VX-661 + VX-770 in vitro administration significantly increased the fluid secretion of ducts derived from AP animals. This study described the course of the CFTR expression and mislocalization in cerulein-induced AP. Our results suggest that the beneficial effects of CFTR correctors and potentiators should be further investigated in AP. KEY POINTS: Cystic fibrosis transmembrane conductance regulator (CFTR) is an important ion channel in epithelial cells. Its malfunction has several serious consequences, like developing or aggravating acute pancreatitis (AP). Here, the localization and expression of CFTR during cerulein-induced AP in mice were investigated and the effects of CFTR corrector (VX-661) and a potentiator (VX-770) on disease severity were determined. CFTR mRNA expression was significantly increased and mislocalization of CFTR protein was observed in AP compared to the control group. Interestingly, pre-treatment of AP mice with VX-661 + VX-770 significantly reduced the extent of pancreatic tissue damage by 20-30%. In vitro administration of VX-661 + VX-770 significantly increased the fluid secretion of ducts derived from AP animals. Based on these results, the utilization of CFTR correctors and potentiators should be further investigated in AP.

Effect of E2 and long control region polymorphisms on disease severity in human papillomavirus type 11 mediated mucosal disease: Protein modelling and functional analysis.

Interaction of the long control region (LCR) and the E2 protein of HPV11s was studied by in silico modelling and in vitro functional analysis. Genomes of HPV11s from fifteen (six known and nine novel) patients (two solitary papillomas, eleven respiratory papillomatoses of different severity, one condyloma acuminatum and one cervical atypia) were sequenced; E2 polymorphisms were analysed in silico by protein modelling. E2 and LCR variants were cloned into pcDNA3.1+ expression vector and into pALuc reporter vector, respectively, transfected to HEp2 cells alone or in different combinations and the luciferase activity was measured. In the E2, the ubiquitous polymorphism K308R caused stronger binding between the dimers but did not alter DNA binding; E2s with this polymorphism were significantly less efficient than the reference in promoting LCR activity. The unique polymorphism Q86K changed the negative surface charge of E2 (Q86) to positive (K86). The unique polymorphisms S245F and N247T in the hinge region disrupt a probable phosphorylation site in a RXXS motif targeted by protein kinase A and B, but do not affect directly the amino acids critical to nuclear transport. Both unique patterns partly restored the LCR activating potential disrupted by K308R. A unique E2/E4 ORF with a 58-bp deletion leading to a frameshift and an early stop codon resulted in a practically nonfunctional E2, and was associated with a papillomatosis with dysplasia. When testing existing LCR-E2 combinations, LCR with intrinsically lower enhancer capacity was only marginally activated by its E2 (R308 and the deletion mutant), and did not significantly exceed the activity of the reference LCR without E2. Combined with more potent LCRs associated with more severe disease, the activity was significantly higher, but still significantly lower than LCRs with reference E2. In summary, LCR-E2 interaction determined by their polymorphisms may explain, at least partly, differences in disease severity.