Comparison of Dual-Dispatch Protocols on Return of Spontaneous Circulation in Patients with Out-of-Hospital Cardiac Arrest: A Nationwide Observational Study.

Objective: In South Korea, the National Fire Agency (NFA) conducted a pilot project on the advanced life support (ALS) protocol, including epinephrine administration, to improve the survival rate of out-of hospital cardiac arrest (OHCA). Therefore, this study aimed to evaluate the effect of the ALS protocol of NFA on prehospital return of spontaneous circulation (PROSC) in patients with OHCA. Methods: This study was conducted on patients with adult-presumed cardiac arrest between January and December 2020. The main factor of interest was ambulance type according to the ALS protocol, which was divided into dedicated ALS(DA), smartphone-based ALS(SALS), and non-dedicated ALS(Non-DA), and the main analysis factor was PROSC. Multivariate logistic regression analysis was performed. Results: During the study period, a total of 18,031 adult patients with OHCA were treated by the emergency medical service (EMS), including 7,520 (41.71 %) DA, 2,622 (14.54 %) SALS, and 7,889 (43.75 %) Non-DA. The prehospital ROSC ratio was 13.19% for the DA, 11.17% for the SALS, and 7.91% for the Non-DA ambulance (P < 0.01). Compared with that of the DA group, the odds ratio (95% confidence interval [CI]) for PROSC ratio in the SALS and Non-DA groups were 0.97 (0.82-1.15) and 0.57 (0.50-0.65), respectively. It was shown that the PROSC ratio of the DA group was higher than that of the Non-DA group and was not lower than that of the SALS group. Conclusion: ALS protocol intervention was associated with difference in PROSC rates. Therefore, continuous efforts on the systemic implementation of the ALS protocol to improve OHCA outcomes are necessary.

Immune-Stimulating Potential of Lacticaseibacillus rhamnosus LM1019 in RAW 264.7 Cells and Immunosuppressed Mice Induced by Cyclophosphamide.

Probiotics, including Lacticaseibacillus rhamnosus (L. rhamnosus), have gained recognition for their potential health benefits, such as enhancing immune function, maintaining gut health, and improving nutrient absorption. This study investigated the effectiveness of L. rhamnosus LM1019 (LM1019) in enhancing immune function. In RAW 264.7 cells, LM1019 demonstrated dose-dependent immune stimulation by increasing nitric oxide production, gene expression of proinflammatory cytokines, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). These effects were mediated through the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) translocation without inducing cytotoxicity. Furthermore, orally administered LM1019 was evaluated in immunosuppressed mice induced by cyclophosphamide (CTX). High-dose administration of LM1019 significantly increased the subpopulations of lymphocytes, specifically helper T cells (CD4+), as well as two subtypes of natural killer (NK) cells, namely, IFN-γ+ and granzyme B+ NK cells. Additionally, LM1019 at a high dose led to elevated levels of proinflammatory cytokines, including IFN-γ and IL-12, compared to CTX-treated mice. These findings highlight the potential of LM1019 in enhancing the immune system. The study contributes to the growing body of research on the beneficial effects of probiotics on immune function.

Multi-Species Probiotic Strain Mixture Enhances Intestinal Barrier Function by Regulating Inflammation and Tight Junctions in Lipopolysaccharides Stimulated Caco-2 Cells.

Although leaky gut syndrome is not recognized as an official diagnosis for human diseases, it is now believed that dysfunction of the cell barrier causes increased permeability of intestinal epithelial cells leading to this condition. Probiotics have been widely used to improve gut health, and studies have investigated the relevance of protecting the intestinal barrier by taking probiotic strains in vitro and in vivo. However, most studies have restricted the use of single or several probiotic strains and do not consider commercially available probiotic products composed of multi-species. In this study, we provide experimental evidence that a multi-species probiotic mixture composed of eight different strains and a heat-treated probiotic strain is effective in preventing leaky gut conditions. We employed an in vitro co-culture model system utilizing two different differentiated cell lines to mimic human intestinal tissue. The integrity of epithelial barrier function was protected by the preserving the occludin protein level and activating the AMPK signaling pathway, associated with tight junctions (TJs), through treatment with the probiotic strain mixture in Caco-2 cells. Moreover, we confirmed that application of the multi-species probiotic mixture reduced the expression of proinflammatory cytokine genes by inhibiting NFκB signaling pathway when artificial inflammation was induced in an in vitro co-culture model system. Finally, we proved that the epithelial permeability measured by trans-epithelial electrical resistance (TEER) was significantly decreased in the probiotic mixture treated cells, indicating that the integrity of the epithelial barrier function was not compromised. The multi-species probiotic strain mixture exhibited the protective effect on the integrity of intestinal barrier function via enhancing TJ complexes and reducing inflammatory responses in the human intestinal cells.

Error-Prone Replication through UV Lesions by DNA Polymerase θ Protects against Skin Cancers.

Cancers from sun-exposed skin accumulate "driver" mutations, causally implicated in oncogenesis. Because errors incorporated during translesion synthesis (TLS) opposite UV lesions would generate these mutations, TLS mechanisms are presumed to underlie cancer development. To address the role of TLS in skin cancer formation, we determined which DNA polymerase is responsible for generating UV mutations, analyzed the relative contributions of error-free TLS by Polη and error-prone TLS by Polθ to the replication of UV-damaged DNA and to genome stability, and examined the incidence of UV-induced skin cancers in Polθ-/-, Polη-/-, and Polθ-/- Polη-/- mice. Our findings that the incidence of skin cancers rises in Polθ-/- mice and is further exacerbated in Polθ-/- Polη-/- mice compared with Polη-/- mice support the conclusion that error-prone TLS by Polθ provides a safeguard against tumorigenesis and suggest that cancer formation can ensue in the absence of somatic point mutations.

Genetic control of predominantly error-free replication through an acrolein-derived minor-groove DNA adduct.

Acrolein, an α,ß-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1-N2-propano-2' deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Polι in inserting a nucleotide (nt) opposite the adduct and for Pols η and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r) γ-HOPdG, Polι incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Polι-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.

Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine.

N3-Methyladenine (3-MeA) is formed in DNA by reaction with S-adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro, we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells.

Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ.

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

Identification of two functional PCNA-binding domains in human DNA polymerase κ.

Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA-binding motifs, PIP1 and PIP2, and that a C-terminal deletion of Polη that lacks the ubiquitin-binding UBZ domain and the PIP2 domain but retains the PIP1 domain promotes normal levels of translesion synthesis (TLS) opposite a cis-syn TT dimer in human cells. Here, we identify two PIP domains in Polκ and show that TLS occurs normally in human fibroblast cells in which the pip1 or pip2 mutant Polκ is expressed, but mutational inactivation of both PIP domains renders Polκ nonfunctional in TLS opposite the thymine glycol lesion. Thus, the two PIP domains of Polκ function redundantly in TLS opposite this DNA lesion in human cells. However, and surprisingly, whereas mutational inactivation of the PIP1 domain completely inhibits the stimulation of DNA synthesis by Polκ in the presence of proliferating cell nuclear antigen (PCNA), replication factor C, and replication protein A, mutations in PIP2 have no adverse effect on PCNA-dependent DNA synthesis. This raises the possibility that activation of Polκ PIP2 as a PCNA-binding domain occurs during TLS in human cells and that protein-protein interactions and post-transcriptional modifications are involved in such activation.

A role for DNA polymerase θ in promoting replication through oxidative DNA lesion, thymine glycol, in human cells.

The biological functions of human DNA polymerase (pol) θ, an A family polymerase, have remained poorly defined. Here we identify a role of polθ in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (TG) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, polymerases κ and ζ function together and mediate error-free TLS, whereas in the other, polθ functions in an error-prone manner. Human polθ is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite TG in human cells. In contrast to TLS mediated by polκ and polζ, in which polζ would elongate the chain from the TG:A base pair formed by polκ action, the ability of polθ alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5' template base, suggests that the polθ active site can accommodate highly distorting DNA lesions.

CCK2 receptor expression transforms non-tumorigenic human NCM356 colonic epithelial cells into tumor forming cells.

Expression of gastrin and cholecystokinin 2 (CCK(2)) receptor splice variants (CCK(2)R and CCK(2i4sv)R) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK(2) receptor variant expression on colonic epithelial cell growth in vitro and in vivo, we employed the non-tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK(2i4sv)R or CCK(2)R. RT-PCR and intracellular Ca(2+) ([Ca(2+)](i)) imaging of RIE/CCK(2)R cells treated with conditioned media (CM) from NCM356 revealed that NCM356 cells express gastrin mRNA and secrete endogenous, biologically active peptide. NCM356 cells expressing either CCK(2)R or CCK(2i4sv)R (71 and 81 fmol/mg, respectively) grew faster in vitro, and exhibited an increase in basal levels of phosphorylated ERK (pERK), compared with vector. CCK(2) receptor selective antagonist, YM022, partially inhibited the growth of both receptor-expressing NCM356 cells, but not the control cells. Inhibitors of mitogen activated protein kinase pathway (MEK/ERK) or protein kinase C (PKC) isozymes partially inhibited the elevated levels of basal pERK and in vitro growth of receptor-expressing cells. Vector-NCM356 cells did not form tumors in nude mice, whereas, either CCK(2) receptor-expressing cells formed large tumors. Autocrine activation CCK(2) receptor variants are sufficient to increase in vitro growth and tumorigenicity of non-transformed NCM356 colon epithelial cells through a pathway involving PKC and the MEK/ERK axis. These findings support the hypothesis that expression of gastrin and its receptors in human colonic adenomas contributes to tumor growth and progression.

Targeting of a CCK(2) receptor splice variant with (111)In-labelled cholecystokinin-8 (CCK8) and (111)In-labelled minigastrin.

PURPOSE: Radiolabelled cholecystokinin (CCK) and gastrin-derived peptides potentially can be used for peptide receptor radionuclide therapy (PRRT). Recently, a splice variant version of the CCK2R has been identified, designated CCK2i4svR. Constitutive expression of this receptor has been demonstrated in human colorectal cancer and in pancreatic cancer, but not in normal tissue. So far, it has never been shown whether radiolabelled peptides can target the CCK2i4svR in vivo. In this paper, we investigated the potential of sulfated (111)In-labelled DOTA-CCK8 (sCCK8), a pan-CCKR-binding peptide, and [(111)In]DOTA-minigastrin (MG0), a CCK2R selective peptide, for the targeting of the CCK2i4svR. MATERIALS AND METHODS: The receptor binding affinity of [(111)In]DOTA-sCCK8 and [(111)In]DOTA-MG0 for the CCK2R and CCK2i4svR was determined using stably transfected HEK293 cell lines, expressing either CCK2R or CCK2i4svR. Tumour targeting was studied in HEK293-CCK2i4svR tumour-bearing athymic mice. RESULTS: [(111)In]DOTA-sCCK8 as well as [(111)In]DOTA-MG0 specifically bound both CCK2R and CCK2i4svR with affinities in the low nanomolar range. In vivo experiments revealed that accumulation of both peptides in CCK2i4svR-positive tumours was similar (3.21 +/- 0.77 and 3.01 +/- 0.67%ID/g, sCCK8 and MG0, respectively, 24 h p.i.). Kidney retention of [(111)In]DOTA-MG0 (32.4 +/- 7.5%ID/g, 24 h p.i.) was markedly higher than that of [(111)In]DOTA-sCCK8 (2.75 +/- 0.31%ID/g, 24 h p.i.). CONCLUSION: We demonstrated that the CCK2i4svR is a potential target for PRRT using a radiolabelled sulfated CCK8 peptide. As this receptor is expressed on colorectal and pancreatic tumours, but not in normal tissue, these tumours are potentially new targets for PRRT with CCK8 and gastrin analogs.

Epidermal growth factor potentiates cholecystokinin/gastrin receptor-mediated Ca2+ release by activation of mitogen-activated protein kinases.

Small differences in amplitude, duration, and temporal patterns of change in the concentration of free intracellular Ca2+ ([Ca2+](i)) can profoundly affect cell physiology, altering programs of gene expression, cell proliferation, secretory activity, and cell survival. We report a novel mechanism for amplitude modulation of [Ca2+](i) that involves mitogen-activated protein kinase (MAPK). We show that epidermal growth factor (EGF) potentiates gastrin-(1-17) (G17)-stimulated Ca2+ release from intracellular Ca2+ stores through a MAPK-dependent pathway. G17 activation of the cholecystokinin/gastrin receptor (CCK(2)R), a G protein-coupled receptor, stimulates release of Ca2+ from inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Pretreating rat intestinal epithelial cells expressing CCK(2)R with EGF increased the level of G17-stimulated Ca2+ release from intracellular stores. The stimulatory effect of EGF on CCK(2)R-mediated Ca2+ release requires activation of the MAPK kinase (MEK)1,2/extracellular signal-regulated kinase (ERK)1,2 pathway. Inhibition of the MEK1,2/ERK1,2 pathway by either serum starvation or treatment with selective MEK1,2 inhibitors PD98059 and U0126 or expression of a dominant-negative mutant form of MEK1 decreased the amplitude of the G17-stimulated Ca2+ release response. Activation of the MEK1,2/ERK1,2 pathway either by pretreating cells with EGF or by expression of constitutively active K-ras (K-rasV12G) or MEK1 (MEK1*) increased the amplitude of G17-stimulated Ca2+ release. Although EGF, MEK1*, and K-rasV12G activated the MEK1,2/ERK1,2 pathway, they did not increase [Ca2+](i) in the absence of G17. These data demonstrate that the activation state of the MEK1,2/ERK1,2 pathway can modulate the amplitude of the CCK(2)R-mediated Ca2+ release response and identify a novel mechanism for cross-talk between EGF receptor- and CCK(2)R-regulated signaling pathways.