Highly accurate measurement of the relative abundance of oral pathogenic bacteria using colony-forming unit-based qPCR.

PURPOSE: Quantitative polymerase chain reaction (qPCR) has recently been employed to measure the number of bacterial cells by quantifying their DNA fragments. However, this method can yield inaccurate bacterial cell counts because the number of DNA fragments varies among different bacterial species. To resolve this issue, we developed a novel optimized qPCR method to quantify bacterial colony-forming units (CFUs), thereby ensuring a highly accurate count of bacterial cells. METHODS: To establish a new qPCR method for quantifying 6 oral bacteria namely, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia, Fusobacterium nucleatum, and Streptococcus mutans, the most appropriate primer-probe sets were selected based on sensitivity and specificity. To optimize the qPCR for predicting bacterial CFUs, standard curves were produced by plotting bacterial CFU against Ct values. To validate the accuracy of the predicted CFU values, a spiking study was conducted to calculate the recovery rates of the predicted CFUs to the true CFUs. To evaluate the reliability of the predicted CFU values, the consistency between the optimized qPCR method and shotgun metagenome sequencing (SMS) was assessed by comparing the relative abundance of the bacterial composition. RESULTS: For each bacterium, the selected primer-probe set amplified serial-diluted standard templates indicative of bacterial CFUs. The resultant Ct values and the corresponding bacterial CFU values were used to construct a standard curve, the linearity of which was determined by a coefficient of determination (r²) >0.99. The accuracy of the predicted CFU values was validated by recovery rates ranging from 95.1% to 106.8%. The reliability of the predicted CFUs was reflected by the consistency between the optimized qPCR and SMS, as demonstrated by a Spearman rank correlation coefficient (ρ) value of 1 for all 6 bacteria. CONCLUSIONS: The CFU-based qPCR quantification method provides highly accurate and reliable quantitation of oral pathogenic bacteria.

Autoantibodies Neutralizing GM-CSF in HIV-Negative Colombian Patients Infected with Cryptococcus gattii and C. neoformans.

BACKGROUND: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. METHODS: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). RESULTS: We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. CONCLUSIONS: We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.

G-CSF promotes the development of hepatocellular carcinoma by activating the PI3K/AKT/mTOR pathway in TAM.

OBJECTIVE: This investigation seeks to elucidate the role of the Granulocyte Colony-Stimulating Factor (G-CSF) in the progression of hepatocellular carcinoma (HCC), as well as the impact of the substance on related signaling pathways within the disease matrix. METHODS: Nude mouse tumor-bearing assay was used to detect tumor progression. Levels of Mannose/CD68 and CD34/Mannose within these samples and the concentrations of Mannose and inducible Nitric Oxide Synthase (iNOS) in macrophages were quantified using immunofluorescence techniques. The angiogenic capability was assessed via tube formation assays, and protein expressions of G-CSF, Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-beta (TGF-ß), Matrix Metalloproteinases 2 and 9 (MMP2/9), SH2-containing protein tyrosine phosphatase-2 (SHP-2), phosphorylated PI3K/total PI3K (P-PI3K/t-PI3K), phosphorylated AKT/total AKT (P-AKT/t-AKT), and phosphorylated mTOR/total mTOR (P-mTOR/t-mTOR) were measured through Western Blot analysis in both tumor tissues and macrophages. RESULTS: Administration of G-CSF resulted in a marked augmentation of tumor volume. Macrophage Mannose expression was significantly elevated upon G-CSF treatment, while iNOS levels were conspicuously diminished. G-CSF substantially enhanced the secretion of VEGF, TGF-ß, and MMPs in tumor tissues. Macrophage parameters, following incubation in G-CSF pre-treated conditioned medium, indicated enhanced tube-forming capabilities relative to the control, an effect mitigated by the introduction of specific inhibitors. Furthermore, the G-CSF group exhibited a notable reduction in SHP-2 expression, alongside a substantial elevation in the phosphorylation levels of the PI3K/AKT/mTOR pathway proteins across all tumor-bearing paradigms. CONCLUSION: G-CSF ostensibly facilitates the advancement of hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling cascade within Tumor-Associated Macrophages (TAM).

Modelling the colony growth dynamics of Listeria monocytogenes single cells after exposure to peracetic acid and acidic conditions.

Studies of classical microbiology rely on the average behaviour of large cell populations without considering that clonal bacterial populations may bifurcate into phenotypic distinct sub-populations by random switching mechanisms.Listeria monocytogenes exposure to sublethal stresses may induce different physiological states that co-exist (i.e., sublethal injury or dormancy) and present variable resuscitation capacity. Exposures to peracetic acid (PAA; 10-30 ppm; for 3 h), acetic acid and hydrochloric acid (AA and HCl; pH 3.0-2.5; for 5 h) at 20 °C were used to induce different physiological states in L. monocytogenes, Scott A strain. After stress exposure, colony growth of single cells was monitored, on Tryptic Soy Agar supplemented with 0.6 % Yeast Extract, using time-lapse microscopy, at 37 °C. Images were acquired every 5 min and were analyzed using BaSCA framework. Most of the obtained growth curves of the colonies were fitted to the model of Baranyi and Roberts for the estimation of lag time (λ) and maximum specific growth rate (µmax), except the ones obtained after exposure to AA pH 2.7 and 2.5 that were fitted to the Trilinear model. The data of λ and µmax that followed a multivariate normal distribution were used to predict growth variability using Monte Carlo simulations. Outgrowth kinetics after treatment with AA (pH 2.7 and 2.5; for 5 h at 20 °C), PAA (30 ppm; for 3 h at 20 °C) revealed that these stress conditions increase the skewness of the variability distributions to the right, meaning that the variability in lag times increases in favour of longer outgrowth. Exposures to AA pH 2.5 and 30 ppm PAA resulted in two distinct subpopulations per generation with different growth dynamics. This switching mechanism may have evolved as a survival strategy for L. monocytogenes cells, maximizing the chances of survival. Simulation of microbial growth showed that heterogeneity in growth dynamics is increased when cells are recovering from exposure to sublethal stresses (i.e. PAA and acidic conditions) that may induce injury or dormancy.

The Efficiency of Introducing Intrauterine Infusion of Autologous Platelet-Rich Plasma versus Granulocyte Colony-Stimulating Factor in Repeated Implantation Failure Patients: An Unblinded Randomised Clinical Trial.

BACKGROUND: Repeated implantation failure (RIF) refers to the condition where high quality embryos are unable to successfully implant after multiple cycles of in vitro fertilization (IVF) treatment. The aim of this study is to investigate the impact of intrauterine granulocyte colony-stimulating factor (G-CSF) and platelet-rich plasma (PRP) on pregnancy rate in patients with RIF. MATERIALS AND METHODS: The present randomised clinical trial study was conducted at the IVF Centre of Mehr Medical Institute in Rasht, Iran, from 2020 to 2022. The research consisted of 200 individuals who had experienced multiple failed cycles. These patients were randomised into two groups: intrauterine infusion of 1 ml of G-CSF and intrauterine infusion of 1 ml autologous PRP at least 48 hours before embryo transfer (ET). The groups were compared in terms of implantation rate, and chemical, clinical, and ongoing pregnancy. RESULTS: The implantation rate was significantly higher in patients who received PRP (P=0.016). Chemical pregnancy in the PRP group was significantly higher than G-CSF group (P=0.003). Both clinical pregnancy and ongoing pregnancy rates were significantly higher in the PRP group (P=0.001) compared to the G-CSF group (P=0.02). CONCLUSION: The utilisation of PRP via intrauterine infusion is considerably more successful than G-CSF in enhancing pregnancy and live birth rates among patients with RIF.

Sulfated Polyhydroxysteroid Glycosides from the Sea of Okhotsk Starfish Henricia leviuscula spiculifera and Potential Mechanisms for Their Observed Anti-Cancer Activity against Several Types of Human Cancer Cells.

Three new monosulfated polyhydroxysteroid glycosides, spiculiferosides A (1), B (2), and C (3), along with new related unsulfated monoglycoside, spiculiferoside D (4), were isolated from an ethanolic extract of the starfish Henricia leviuscula spiculifera collected in the Sea of Okhotsk. Compounds 1-3 contain two carbohydrate moieties, one of which is attached to C-3 of the steroid tetracyclic core, whereas another is located at C-24 of the side chain of aglycon. Two glycosides (2, 3) are biosides, and one glycoside (1), unlike them, includes three monosaccharide residues. Such type triosides are a rare group of polar steroids of sea stars. In addition, the 5-substituted 3-OSO3-α-L-Araf unit was found in steroid glycosides from starfish for the first time. Cell viability analysis showed that 1-3 (at concentrations up to 100 µM) had negligible cytotoxicity against human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer MDA-MB-231, and colorectal carcinoma HCT 116 cells. These compounds significantly inhibited proliferation and colony formation in HCT 116 cells at non-toxic concentrations, with compound 3 having the greatest effect. Compound 3 exerted anti-proliferative effects on HCT 116 cells through the induction of dose-dependent cell cycle arrest at the G2/M phase, regulation of expression of cell cycle proteins CDK2, CDK4, cyclin D1, p21, and inhibition of phosphorylation of protein kinases c-Raf, MEK1/2, ERK1/2 of the MAPK/ERK1/2 pathway.

Upregulation of MMPs in placentas of patients with gestational diabetes mellitus: Involvement of the PI3K/Akt pathway.

In recent years, there has been a notable rise in the incidence of pregnancies complicated by gestational diabetes mellitus (GDM), characterized by glucose intolerance first identified during pregnancy. Analysis of placental tissue has revealed that placentas from women with GDM tend to be larger and heavier compared to control placentas, indicating potential changes in trophoblast proliferation, differentiation, and apoptosis. In this study, transcriptome sequencing was conducted on placentas obtained from both normal pregnancies and pregnancies with GDM to investigate the molecular mechanisms underlying this condition. The original sequencing data were subjected to sequencing analysis, resulting in the identification of 935 upregulated genes and 256 downregulated genes. The KEGG and GO analysis techniques on differential genes uncovered evidence suggesting that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway may contribute to the pathogenesis of GDM. Subsequent analysis indicated that the expression levels of matrix metalloproteinases (MMP) 11, MMP12, MMP14, and MMP15, which are regulated by the PI3K/Akt pathway, were upregulated in the placentas of patients with GDM when compared to those of individuals with normal placental function. Additionally, our investigation into alternative splicing patterns revealed an increase in exon skipping alternative splicing of CSF3R in the placenta of patients with GDM compared to that in the control group. The CSF3R-PI3K-MMP pathway is speculated to regulate the pathogenesis of GDM.

A clustering mining method for sports behavior characteristics of athletes based on the ant colony optimization.

This study aims to enhance the precision of analyzing athlete behavior characteristics, thereby optimizing sports training and competitive strategies. This study introduces an innovative Ant Colony Optimization (ACO) clustering model designed to address the high-dimensional clustering issues in athlete behavior data by simulating the path selection mechanism of ants searching for food. The development process of this model includes fine-tuning ACO parameters, optimizing for features specific to sports data, and comparing it with traditional clustering algorithms, and similar research models based on the neural network, support vector machines, and deep learning. The results indicate that the ACO model significantly outperforms the comparison algorithms in terms of silhouette coefficient (0.72) and Davies-Bouldin index (1.05), demonstrating higher clustering effectiveness and model stability. Particularly noteworthy is the recall rate (0.82), a key performance indicator, where the ACO model accurately captures different behavioral characteristics of athletes, validating its effectiveness and reliability in athlete behavior analysis. The innovation lies not only in the application of the ACO algorithm to address practical issues in the field of sports but also in showcasing the advantages of the ACO algorithm in handling complex, high-dimensional sports data. However, its generality and efficiency on a larger scale or different types of sports data still need further validation. In conclusion, through the introduction and optimization of the ACO clustering model, this study provides a novel and effective approach for a deeper understanding and analysis of athlete behavior characteristics. This study holds significant importance in advancing sports science research and practical applications.

A novel clustered-based detection method for shilling attack in private environments.

The topic of privacy-preserving collaborative filtering is gaining more and more attention. Nevertheless, privacy-preserving collaborative filtering techniques are vulnerable to shilling or profile injection assaults. Hence, it is crucial to identify counterfeit profiles in order to achieve total success. Various techniques have been devised to identify and prevent intrusion patterns from infiltrating the system. Nevertheless, these strategies are specifically designed for collaborative filtering algorithms that do not prioritize privacy. There is a scarcity of research on identifying shilling attacks in recommender systems that prioritize privacy. This work presents a novel technique for identifying shilling assaults in privacy-preserving collaborative filtering systems. We employ an ant colony clustering detection method to effectively identify and eliminate fake profiles that are created by six widely recognized shilling attacks on compromised data. The objective of the study is to categorize the fraudulent profiles into a specific cluster and separate this cluster from the system. Empirical experiments are conducted with actual data. The empirical findings demonstrate that the strategy derived from the study effectively eliminates fraudulent profiles in privacy-preserving collaborative filtering.

Multiple small-dose infusions of G-CSF-mobilized haploidentical lymphocytes after autologous haematopoietic stem cell transplantation for acute myeloid leukaemia.

This retrospective study analysed 106 acute myeloid leukaemia (AML) patients undergoing autologous haematopoietic stem cell transplantation (ASCT) to assess the impact of multiple small-dose infusions of granulocyte-colony-stimulating factor (G-CSF)-mobilized haploidentical lymphocytes as post-ASCT maintenance therapy. Among them, 50 patients received lymphocyte maintenance therapy, 21 received alternative maintenance therapy, and 35 received no maintenance therapy. Patients receiving lymphocyte maintenance therapy demonstrated significantly higher overall survival (OS) and disease-free survival (DFS) compared to those without maintenance therapy, with 4-year OS and DFS rates notably elevated. While there were no significant differences in recurrence rates among the three groups, lymphocyte maintenance therapy showcased particular benefits for intermediate-risk AML patients, yielding significantly higher OS and DFS rates and lower relapse rates compared to alternative maintenance therapy and no maintenance therapy. The study suggests that multiple small-dose infusions of G-CSF-mobilized haploidentical lymphocytes may offer promising outcomes for AML patients after ASCT, particularly for those classified as intermediate-risk. These findings underscore the potential efficacy of lymphocyte maintenance therapy in reducing disease relapse and improving long-term prognosis in this patient population.

Enhancement of cyber security in IoT based on ant colony optimized artificial neural adaptive Tensor flow.

The Internet of Things (IoT) is a network that connects various hardware, software, data storage, and applications. These interconnected devices provide services to businesses and can potentially serve as entry points for cyber-attacks. The privacy of IoT devices is increasingly vulnerable, particularly to threats like viruses and illegal software distribution lead to the theft of critical information. Ant Colony-Optimized Artificial Neural-Adaptive Tensorflow (ACO-ANT) technique is proposed to detect malicious software illicitly disseminated through the IoT. To emphasize the significance of each token in source duplicate data, the noise data undergoes processing using tokenization and weighted attribute techniques. Deep learning (DL) methods are then employed to identify source code duplication. Also the Multi-Objective Recurrent Neural Network (M-RNN) is used to identify suspicious activities within an IoT environment. The performance of proposed technique is examined using Loss, accuracy, F measure, precision to identify its efficiency. The experimental outcomes demonstrate that the proposed method ACO-ANT on Malimg dataset provides 12.35%, 14.75%, 11.84% higher precision and 10.95%, 15.78%, 13.89% higher f-measure compared to the existing methods. Further, leveraging block chain for malware detection is a promising direction for future research the fact that could enhance the security of IoT and identify malware threats.

A systematic literature review on meta-heuristic based feature selection techniques for text classification.

Feature selection (FS) is a critical step in many data science-based applications, especially in text classification, as it includes selecting relevant and important features from an original feature set. This process can improve learning accuracy, streamline learning duration, and simplify outcomes. In text classification, there are often many excessive and unrelated features that impact performance of the applied classifiers, and various techniques have been suggested to tackle this problem, categorized as traditional techniques and meta-heuristic (MH) techniques. In order to discover the optimal subset of features, FS processes require a search strategy, and MH techniques use various strategies to strike a balance between exploration and exploitation. The goal of this research article is to systematically analyze the MH techniques used for FS between 2015 and 2022, focusing on 108 primary studies from three different databases such as Scopus, Science Direct, and Google Scholar to identify the techniques used, as well as their strengths and weaknesses. The findings indicate that MH techniques are efficient and outperform traditional techniques, with the potential for further exploration of MH techniques such as Ringed Seal Search (RSS) to improve FS in several applications.

Correlation between the Colony Phenotype and Amino Acid Sequence of the Variable Vaa Antigen in Clinical Isolates of Mycoplasma hominis.

A new Mycoplasma hominis phenotype forming mini-colonies (MC) on agar and distinct from the phenotype forming typical colonies (TC) not only in size, but also in morphology, growth rate, and resistance to adverse factors, has been previously identified. In this study, the phenotype of colonies was determined and a comparative analysis of the amino acid sequence of the main variable antigen Vaa of the laboratory strain N-34 and seven clinical isolates of M. hominis was performed. It is demonstrated that the amino acid sequence of Vaa in clinical isolates forming TC (similar to the laboratory strain N-34) is entirely analogous to that of laboratory strain. Clinical isolates forming MC carry amino acid substitutions in the variable C-terminal region of Vaa, which can contribute to adhesion to eukaryotic cells and immune evasion. The connection between colony phenotype and amino acid sequence of Vaa is established.

Pegylated recombinant human granulocyte colony-stimulating factor for primary prophylaxis of neutropenia in patients with cervical cancer receiving concurrent chemoradiotherapy: a prospective study.

BACKGROUND: This study aimed to investigate the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) for primary prophylaxis of neutropenia in patients with cervical cancer receiving concurrent chemoradiotherapy. METHODS: In this prospective, single-center, single-arm study, we enrolled patients (18-70 years) with 2018 International Federation of Gynecology and Obstetrics (FIGO) stage IIIC1r-IVA and IVB (distant metastasis only with inguinal lymph node metastasis) cervical cancer. Eligible patients should have normal function of the bone marrow (absolute neutrophil count (ANC) ≥ 2.0 × 109/L) and adequate hepatic and renal functions. Key exclusion criteria included: previous chemotherapy and/or radiotherapy; a history of bone marrow dysplasia or other hematopoietic abnormalities. All patients underwent radical radiotherapy (pelvic radiotherapy or extended-field irradiation) plus brachytherapy. The chemotherapy regimen included four cycles of 3-weekly paclitaxel and cisplatin. PEG-rhG-CSF was administered 48-72 h after each treatment cycle. Salvage granulocyte colony-stimulating factor (G-CSF) was only permitted in certain circumstances. The primary endpoint was the incidence of grade 3-4 neutropenia. The secondary endpoints included frequency of febrile neutropenia (FN), chemotherapy completion rate in cycles 2-4, time to complete radiotherapy, and safety. RESULTS: Overall, 52 patients were enrolled in this study from July 2019 to October 2020. The incidence of grade 3-4 neutropenia was 28.8%, with an average duration of grade 3-4 neutropenia persistence of 3.85 days (1-7 days). The incidence rate of FN was 3.8%. The chemotherapy completion rate was 94.2%, 82.7%, and 75.0% for cycles 2-4, respectively. The incidences of grade 3-4 neutropenia for cycles 1-4 were 9.6% (5/52), 8.2% (4/49), 14.0% (6/43), and 2.6% (1/39), respectively. All patients completed radiotherapy within 8 weeks (median, 48 days; range: 41-56 days), except one patient who withdrew consent and did not receive radiotherapy. Severe non-hematologic toxicity was not observed in any patient. CONCLUSION: PEG-rhG-CSF is an effective and safe prophylactic treatment for neutropenia in patients with cervical cancer undergoing concurrent chemoradiotherapy. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1900024494. Date of Registration:13/July/2019.

Inhibition of fusarium graminearum growth and spore germination by a streptomyces amritsarensis strain capable of killing and growing on microcystis scum.

AIMS: Developing energy-saving and ecofriendly strategies for treating harvested Microcystis biomass. METHODS AND RESULTS: Streptomyces amritsarensis HG-16 was first reported to effectively kill various morphotypes of natural Microcystis colonies at very high cell densities. Concurrently, HG-16 grown on lysed Microcystis maintained its antagonistic activity against plant pathogenic fungus Fusarium graminearum. It could completely inhibit spore germination and destroy mycelial structure of F. graminearum. Transcriptomic analysis revealed that HG-16 attacked F. graminearum in a comprehensive way: interfering with replication, transcription, and translation processes, inhibiting primary metabolisms, hindering energy production and simultaneously destroying stress-resistant systems of F. graminearum. CONCLUSIONS: The findings of this study provide a sustainable and economical option for resource reclamation from Microcystis biomass: utilizing Microcystis slurry to propagate HG-16, which can subsequently be employed as a biocontrol agent for managing F. graminearum.

Stay or go? Changing breeding conditions affect sexual difference in colony attendance strategies of Atlantic puffins Fratercula arctica.

Male and female birds have different roles in reproduction and, thereby in their reproductive investment, which in turn may increase negative effects of poorer breeding conditions caused by e.g., climate change or ecosystem regime shifts. By using a 33-year time series of resightings of Atlantic puffins Fratercula arctica individually colour-ringed as breeders in previous years, we showed that the difference in colony attendance of male and female birds depended on the environmental conditions for raising young, proxied by the average duration of the chick period and size of the herring Clupea harengus fed to the chicks in the colony each year. The longer the chick period, the more was the sex ratio of adults sitting visibly in the colony biased in favour of males. An increase in herring size, indicating better feeding conditions for raising chicks, led to more observations of both sexes. Additionally, we found that birds were observed less with age and females more so than males. We discuss the results in relation to general life-history theory on sexual differences in trade-offs between individual investment in breeding and own survival. Our results suggest that females are increasingly more willing than males to invest in provisioning for the chick the more and longer the chick needs such care.

Protein phosphatase 6 promotes stemness of colorectal cancer cells.

Colorectal cancer (CRC) remains a significant global health concern, demanding a more profound comprehension of its molecular foundations for the development of improved therapeutic strategies. This study aimed to elucidate the role of protein phosphatase 6 (PP6), a member of the type 2A protein phosphatase family, in CRC. Protein phosphatase 6 functions as a heterotrimer with a catalytic subunit (PP6c), regulatory subunits (PP6Rs; PP6R1, PP6R2, and PP6R3), and scaffold subunits (ANKRD28, ANKRD44, and ANKRD52). Elevated PP6c expression has been identified in CRC tissues compared to normal mucosa, aligning with its potential involvement in CRC pathogenesis. PP6c knockdown resulted in decreased colony-forming ability and in vivo proliferation of various CRC cell lines. Transcriptome analysis revealed that PP6c knockdown resulted in altered expression of genes associated with cancer stemness. Notably, the PP6c-PP6R3 complex is a key player in regulating cancer stem cell (CSC) markers. Additionally, increased PP6c expression was observed in CSC-like cells induced by sphere formation, implicating the role of PP6c in CSC maintenance. This study highlights the role of PP6c in CRC and suggests that it is a potential therapeutic target disrupting a pathway critical for CRC progression and stem cell maintenance.

Phenotypic and genomic characterization of a small colony variant of Klebsiella pneumoniae isolated from urine of a prostate cancer case.

Small colony variants (SCVs) in Klebsiella pneumoniae are rare and understudied. We report an SCV of Klebsiella pneumoniae isolated from the urine of a prostate cancer patient undergoing prolonged radiotherapy. The strain was non-lactose fermenting, non-mucoid, slow-growing, multi-drug resistant, and showed atypical biochemical reactions and biofilm formation. On whole genome sequencing, it showed low-level virulence, sequence type 231 and gene CTX-M-15. Three major porins OmpK35, OmpK36 and OmpK37 were found. SCVs pose challenges like difficulties in identification, altered metabolism, and increased biofilm formation, which contribute to persistent infections. Radiotherapy and chemotherapy may have led to the formation of the SCV phenotype.

An optimal promoter regulating cytokine transgene expression is crucial for safe and effective oncolytic virus immunotherapy.

In general, ensuring safety is the top priority of a new modality. Although oncolytic virus armed with an immune stimulatory transgene (OVI) showed some promise, the strategic concept of simultaneously achieving maximum effectiveness and minimizing side effects has not been fully explored. We generated a variety of survivin-responsive "conditionally replicating adenoviruses that can target and treat cancer cells with multiple factors (m-CRAs)" (Surv.m-CRAs) armed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene downstream of various promoters using our m-CRA platform technology. We carefully analyzed both therapeutic and adverse effects of them in the in vivo syngeneic Syrian hamster cancer models. Surprisingly, an intratumor injection of a conventional OVI, which expresses the GM-CSF gene under the constitutively and strongly active "cytomegalovirus enhancer and ß-actin promoter", provoked systemic and lethal GM-CSF circulation and shortened overall survival (OS). In contrast, a new conceptual type of OVI, which expressed GM-CSF under the cancer-predominant and mildly active E2F promoter or the moderately active "Rous sarcoma virus long terminal repeat", not only abolished lethal adverse events but also prolonged OS and systemic anti-cancer immunity. Our study revealed a novel concept that optimal expression levels of an immune stimulatory transgene regulated by a suitable upstream promoter is crucial for achieving high safety and maximal therapeutic effects simultaneously in OVI therapy. These results pave the way for successful development of the next-generation OVI and alert researchers about possible problems with ongoing clinical trials.