Activation of pancreatic stellate cells attenuates intracellular Ca2+ signals due to downregulation of TRPA1 and protects against cell death induced by alcohol metabolites

Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca²⁺ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca²⁺ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-β-activated PSCs, compare the expression of Ca²⁺ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca²⁺ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca²⁺-handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca²⁺ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca²⁺ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis.Subject terms: Cell death, Ion channel signalling, Gastrointestinal diseases, Preclinical research, Physiology     

FLASH radiotherapy: an emerging approach in radiation therapy

FLASH radiotherapy (RT) is a technique involving the delivery of ultra-high dose rate radiation to the target. FLASH-RT has been shown to reduce radiation-induced toxicity in healthy tissues without compromising the anti-cancer effects of treatment compared to conventional radiation therapy. In the present article, we review the published data on FLASH-RT and discuss the current state of knowledge of this novel approach. We also highlight the technological constraints and complexity of FLASH-RT and describe the physics underlying this modality, particularly how technology supports energy transfer by ionising radiation (e.g., beam on/off sequence, pulse-energy load, intervals). We emphasise that current preclinical experience is mostly based on FLASH electrons and that clinical application of FLASH-RT is very limited. The incorporation of FLASH-RT into routine clinical radiotherapy will require the development of devices capable of producing FLASH photon beams.     

New Potentially Probiotic Strains Isolated from Humans – Comparison of Properties with Strains from Probiotic Products and ATCC Collection

Lactic acid bacteria are used in various types of probiotic products. Due to the constantly growing probiotics market, new strains with pro-health properties are sought. The present study compared 39 strains of Lactobacillus, Lacticaseibacillus, and Lactiplantibacillus, isolated from probiotic products and healthy people. The current research aimed to search for new, potentially probiotic strains. For this purpose the relationship between Lactobacillaceae strains was carried out; moreover, the basic properties of probiotic microorganisms, such as survival at low pH and bile salt environment, antibiotic susceptibility, aggregation and antagonism were estimated. The properties of these isolates were also compared with the properties of probiotic strains from the ATCC collection. In comparing the genetic relationship (PFGE method) between the tested isolates, it was observed that some of them show a high degree of similarity. All tested strains tolerated an environment with a pH value of 3.0, and the addition of 0.3% bile salt; showed auto-aggregation properties and displayed antagonism against pathogenic microorganisms. In the present study, the bacteria were susceptible to tetracycline, chloramphenicol and ampicillin; the resistance to vancomycindepended on the bacteria type. All the properties were strain-depended. Most of the tested strains had properties comparable to the reference strains. Three L. acidophilus strains isolated from cervical swabs seem to be promising candidates for probiotic strains. Key words: lactobacilli, PFGE, antimicrobial susceptibility, antagonism     

Susceptibility of Clostridium Sporogenes Spores to Selected Reference Substances and Disinfectants

Research on the susceptibility of the spores of anaerobic bacteria such as Clostridium sporogenes or Clostridioides difficile is vital for assessing the sporicidal activity of disinfectants. The diverse susceptibility of anaerobic bacteria spores may lead to different disinfection parameters being determined by laboratories that prepare spore suspensions to test sporicidal effectiveness. The tests were performed using the suspension method according to PN-EN 13704:2018-09. In order to assess the susceptibility of the C. sporogenes spores, the criterion established for the C. difficile ribotype 027 spores was used in accordance with PN‑EN 17126:2019-01. The susceptibility of the C. sporogenes spores to glutardialdehyde corresponded to the susceptibility ranges established for the C. difficile ribotype 027 spores. The C. sporogenes spore suspension was susceptible to low concentrations of peracetic acid (0.01%). A disinfectant containing peracetic acid as the active substance showed high sporicidal activity at a low concentration (1%), a short contact time (15 minutes), and a high organic load (3.0 g/l bovine albumin + 3.0 ml/l sheep erythrocytes), as compared to a disinfectant with glutardialdehyde, which was sporicidal at a higher concentration (2.5%), at a longer contact time(60 minutes) and lower organic conditions (3.0 g/l bovine albumin). There is a need to define the minimum susceptibility criteria for the C. sporogenes spores to the reference substances most often found in disinfectants with sporicidal activity. Excessive susceptibility of the C. sporogenes spores to reference substances may result in low-performance parameters of disinfection products with sporicidal activity and lead to ineffective disinfection in practice. Open in a separate window     

Sex and age-specific differences in wing pointedness and wing length in blackcaps Sylvia atricapilla migrating through the southern Baltic coast

The blackcap Sylvia atricapilla shows a complex migratory pattern and is a suitable species for the studies of morphological migratory syndrome, including adaptations of wing shape to different migratory performance. Obligate migrants of this species that breed in northern, central, and Eastern Europe differ by migration distance and some cover shorter distance to the wintering grounds in the southern part of Europe/North Africa or the British Isles, although others migrate to sub-Saharan Africa. Based on ˃40 years of ringing data on blackcaps captured during autumn migration in the Southern Baltic region, we studied age- and sex-related correlations in wing pointedness and wing length of obligate blackcap migrants to understand the differences in migratory behavior of this species. Even though the recoveries of blackcaps were scarce, we reported some evidence that individuals which differ in migration distance differed also in wing length. We found that wing pointedness significantly increased with an increasing wing length of migrating birds, and adults had longer and more pointed wings than juvenile birds. This indicates stronger antipredator adaptation in juvenile blackcaps than selection on flight efficiency, which is particularly important during migration. Moreover, we documented more pronounced differences in wing length between adult and juvenile males and females. Such differences in wing length may enhance a faster speed of adult male blackcaps along the spring migration route and may be adaptive when taking into account climatic effects, which favor earlier arrival from migration to the breeding grounds.     

PHYLOGENETIC POSITION OF KOLIELLA (CHLOROPHYTA) AS INFERRED FROM NUCLEAR AND CHLOROPLAST SMALL SUBUNIT rDNA

The phylogenetic position of Koliella , a chlorophyte characterized by Klebsormidium type cell division, was inferred from analyses of partial 18S rDNA and partial 16S rDNA. Parsimony and distance analyses of separate and combined data sets indicated that the members of Koliella belonged to Trebouxiophyceae, and high decay indices and bootstrap values supported this affinity. However, the genus appeared to be polyphyletic. Koliella spiculiformis , the nomenclatural type of the genus, was allied with Nannochloris eucaryota and the "true" chlorellas ( Chlorella vulgaris , C. lobophora , C. sorokiniana , and C. kessleri ). The close relatives of Koliella longiseta (≡ Raphidonema longiseta ) and Koliella sempervirens appeared to be Stichococcus bacillaris and some species traditionally classified in Chlorella that were characterized by the production of secondary carotenoids under nitrogen-deficient conditions. This clade was also supported by the presence of a relatively phylogenetically stable group I intron (1506) in the 18S rRNA gene. Because of the presence of Klebsormidium type cell division, some authors regarded the members of Koliella as closely related to charophytes. Molecular analyses, however, did not confirm this affinity and suggested that a Klebsormidium type cell division is homoplastic in green plants.     

VULNERABILITY OF NOSTOC MUSCORUM AGARDH (CYANOPHYCEAE) MOTILE HORMOGONIA TO CILIATE GRAZING1

Experiments were carried out to investigate if the stage of life cycle of Nostoc muscorum Agardh alters vulnerability to grazing by Pseudomicrothorax dubius Maupas. When the percentage of motile hormogonia of all counted trichomes exceeded 10%, most of the grazers (80%–100%) became satiated within 2 h. In most cases (90%) grazers successfully attacked motile hormogonia. Attacks on nonmotile trichomes were much rarer (8%) and mainly unsuccessful. Direct observations revealed that hormogonia could be ingested by the ciliates as long as they remained motile. Hormogonia already adhered to the bottom were still recognized by ciliates as potential food but were not ingested. We did not observe attacks on old vegetative colonies. This is apparently the first report on the motile stage of Nostoc being susceptible to ciliate grazing. Experiments with other grazers, Nassula tumida Maskell and two different clones of Furgassonia blochmanni Faure‐Fremiet, showed that only one clone of F. blochmanni was able to feed on motile hormogonia, whereas the second clone and N. tumida showed no interest in them.     

Regulatory T cell vaccination without autoantigen protects against experimental autoimmune encephalomyelitis

Regulatory T (T(reg)) cells show promise for treating autoimmune diseases, but their induction to elevated potency has been problematic when the most optimally derived cells are from diseased animals. To circumvent reliance on autoantigen-reactive T(reg) cells, stimulation to myelin-independent Ags may offer a viable alternative while maintaining potency to treat experimental autoimmune encephalomyelitis (EAE). The experimental Salmonella vaccine expressing colonization factor Ag I possesses anti-inflammatory properties and, when applied therapeutically, reduces further development of EAE in SJL mice. To ascertain T(reg) cell dependency, a kinetic analysis was performed showing increased levels of FoxP3(+)CD25(+)CD4(+) T cells. Inactivation of these T(reg) cells resulted in loss of protection. Adoptive transfer of the vaccine-induced T(reg) cells protected mice against EAE with greater potency than naive or Salmonella vector-induced T(reg) cells, and cytokine analysis revealed enhanced production of TGF-beta, not IL-10. The development of these T(reg) cells in conjunction with immune deviation by Th2 cells optimally induced protective T(reg) cells when compared those induced in the absence of Th2 cells. These data show that T(reg) cells can be induced to high potency to non-disease-inducing Ags using a bacterial vaccine.     

Protein-induced local DNA bends regulate global topology of recombination products

The tyrosine family of recombinases produces two smaller DNA circles when acting on circular DNA harboring two recombination sites in head-to-tail orientation. If the substrate is supercoiled, these circles can be unlinked or form multiply linked catenanes. The topological complexity of the products varies strongly even for similar recombination systems. This dependence has been solved here. Our computer simulation of the synapsis showed that the bend angles, phi, created in isolated recombination sites by protein binding before assembly of the full complex, determine the product topology. To verify the validity of this theoretical finding we measured the values of phi for Cre/loxP and Flp/FRT systems. The measurement was based on cyclization of the protein-bound short DNA fragments in solution. Despite the striking similarity of the synapses for these recombinases, action of Cre on head-to-tail target sites produces mainly unlinked circles, while that of Flp yields multiply linked catenanes. In full agreement with theoretical expectations we found that the values of phi for these systems are very different, close to 35 degrees and 80 degrees, respectively. Our findings have general implications in how small protein machines acting locally on large DNA molecules exploit statistical properties of their substrates to bring about directed global changes in topology.