Molecular Biological Methods Research Articles

Identification of Novel Variations in Exon 1 of ABO Blood Group Gene

Serological and molecular biology methods were used to identify the blood type of a patient with forward and reverse ABO typing inconsistency, and to explore the genetic characteristics of this blood type. The ABO phenotype of the proband was identified by tube method, and the ABO blood group genotype of the proband and her parents was determined by fluorescent PCR. The 7 exons of the ABO gene were directly sequenced and analyzed. According to preliminary serological identification, the ABO phenotype of this patient was Bel subtype. Genotyping tests showed that the ABO genotype of the proband and her father was B/O1 , and her mother was O1/O1. Sequencing of exons revealed novel heterozygous variations in exon 1: c.16_17delinsTGTTGCA. The Novel variations in exon 1 led to Bel subtype in the ABO blood group of the proband, and these variations are heritable.

Overexpressing miR-222-3p in cultured <i>Mycobacterium Tuberculosis</i>-infected macrophages does not affect their bacteriostatic activity

Tuberculosis, a disease caused by the bacterium Mycobacterium tuberculosis, is a major public health concern. Innate and adaptive immunity provide robust defense against pathogens. However, M. tuberculosis, which co-evolved with humans, has acquired many mechanisms to evade the immune response and ensure its intracellular existence and long-term survival within the host. Moreover, emerging evidence suggests that this secretive bacterium can alter expression of regulatory noncoding RNAs (including microRNAs), leading to dysregulation of biological processes underlying tuberculosis pathogenesis. For example, miR-222-3p has been shown to regulate the functional reprogramming of macrophages and is involved in the regulation of host innate immunity. Previously, we demonstrated the important role of miR-222-3p as a biological marker of tuberculosis activity. To confirm their biological targets and understand their role in the pathogenesis of tuberculosis, many research groups are working to establish functional relationships between miRNA expression under different conditions and their actual biological action using molecular biology and bioinformatics methods. In the present study, we demonstrated the effect of miR-222-3p overexpression on several functions of human macrophages of monocytic origin activated with M. tuberculosis antigens in in vitro culture. Specifically, we found that miR-222-3p overexpression significantly decreased IL-6 and IFNγ expression and increased IL-1β and cxcl10 expression in cultures of uninfected macrophages. Infected macrophages overexpressing miR-222-3p were characterized by increased NF-κB and IL-6 expression, as were infected macrophages without transfection. Another important finding was that miR-222-3p overexpression caused a small but significant increase in reactive nitrogen species production by infected macrophages, but did not affect their bacteriostatic activity against M. tuberculosis. Elucidating the functions of different microRNAs in regulating different pathogenic pathways in TB may lead to discovering new therapeutic targets. The detailed study of microRNAs that regulate immune-associated pathways will be useful for the design of miRNA mimetic molecules, either as inhibitors or as activators. Immune effects induced by miRNA drugs are currently a major challenge for miRNA therapeutics.

Translocase of Outer Mitochondrial Membrane 40, as a Promising Biomarker for the Diagnosis of Polycystic Ovary Syndrome and Pan-Cancer

Polycystic ovary syndrome (PCOS) is a metabolic disease that affects the reproductive system, and its pathogenesis remains unresolved. Through the application of bioinformatics and molecular biology techniques, this study has identified a significant association between translocase of outer mitochondrial membrane 40 (TOMM40) and both PCOS and pan-cancers. The selection of PCOS biomarkers included TOMM40, which we found to be significantly decreased in the PCOS group both in vitro and in vivo, using molecular biology methods such as Western Blot as well as immunohistochemistry. Over-expression TOMM40 can rescue the effect on apoptosis rate and proliferation suppression induced by DHEA in KGN cells. TOMM40 as a biomarker for the diagnosis of PCOS. The pan-cancer analysis revealed an association between elevated TOMM40 expression in Uterine Corpus Endometrial Carcinoma and an unfavorable prognosis, while increased TOMM40 expression in six tumor types was linked to a favorable prognosis. Therefore, TOMM40 can be regarded as a promising biomarker for diagnosing both PCOS and pan-cancer.

Increase in congenital syphilis in a tertiary-care centre from Madrid, Spain: Back to the classics.

Globally, an increase in sexually transmitted diseases worldwide has been reported, including acquired Treponema pallidum infection. Congenital syphilis is the mother-to-child transmission syphilis infection, experiencing an increase in cases returning to incidence rates of years ago. We report three cases of symptomatic congenital syphilis occurring in a tertiary-care hospital in Madrid (Spain). Cases were diagnosed by serology and molecular biology methods. The use of molecular techniques in specimens such as skin lesion, subplacental exudate swabs, bronchoalveolar aspirate, or cerebrospinal fluid could favour the diagnosis of this clinical entity, especially in symptomatic newborns with systemic involvement.

Skin microbiome changes in patients with atopic dermatitis and psoriasis

Aims: The main aim of our study was identification of skin microbiome changes in patients with atopic dermatitis and psoriasis. The focus of our research were microorganisms of Staphylococcus spp. genus. Materials and methods: The study included 34 patients with atopic dermatitis and 41 patients with psoriasis. The enrollment period lasted for 3 months. The material for cultivation was taken from 4 skin areas: lesion and normal skin of dry body skin sites (medial surface of forearm for patients with atopic dermatitis and extensor surface of elbow joint for patients with psoriasis) and scalp. Further evaluation was carried out using cultural and molecular biological research methods. The obtained data were statistically processed. Results: The study included 34 patients with atopic dermatitis and 41 patients with psoriasis. Mean SCORAD for atopic dermatitis group was 51,5912,75 and mean PASI for psoriasis group was23,7214,14. Mean SCORAD for atopic dermatitis patients who carries toxigenic S.аureus strains was 57,439,75, in group without toxigenic S.аureus strains mean SCORAD was statistically significant lower - 37,908,63 (р=0,054). Mean SCORAD for atopic dermatitis patients who carries S.epidermidis commensal strain was 51,312,75, difference between this group and toxigenic S.аureus strains group of patients was insignificant (р=0,18). Mean PASI for psoriasis patients who carries toxigenic S.аureus strains was 26,412,76, mean PASI for psoriasis patients who carries S.epidermidis commensal strain was 20,99,07. Difference between S.epidermidis group and toxigenic S.аureus strains group of patients was statistically insignificant (р=0,53) with tendency to more severe disease in group of toxigenic S.аureus strains carriers. Conclusions: This study reveals some aspects of skin colonization by microorganisms belonging to Staphylococcus spp genus in patients with atopic dermatitis and psoriasis. We identify some correlations between severity of inflammatory disease and characteristics of microorganisms strain.

Isolation, Selection, and Identification of Keratinolytic Bacteria for Green Management of Keratin Waste.

The volume of difficult-to-process keratin waste is increasing as a result of rising global meat production. If not properly managed, this waste can contribute to environmental pollution and pose a threat to human and animal welfare. An interesting and more sustainable alternative is therefore the bioconversion of keratin using microorganisms and their enzymes. This work aimed to isolate bacteria from soil samples and zoonotic keratins and to evaluate their enzymatic capacity to degrade α- and β-keratin wastes. A total of 113 bacterial strains were isolated from environmental samples and subjected to taxonomic identification using the MALDI-TOF MS technique and to a two-step screening for proteolytic and keratinolytic activity. The ability to degrade a β-rich keratin substrate was observed in almost all of the strains isolated from soil and horsehairs. In contrast, when an α-rich keratin substrate was used, the highest levels of hydrolysis were observed only for Ker39, Ker66, Ker85, Ker100, and Ker101. Strains with the highest biodegradation potential were identified using molecular biology methods. Phylogenetic analysis of 16S rDNA gene sequences allowed the assignment of selected keratinolytic microorganisms to the genera Exiguobacterium, Priestia, Curtobacterium, Stenotrophomonas, Bacillus, Kocuria, or Pseudomonas. The results of this study are a promising precursor for the development of new, more sustainable methods of managing keratin waste to produce high-value hydrolysates.

Triclosan Dioxygenase: A Novel Two-component Rieske Nonheme Iron Ring-hydroxylating Dioxygenase Initiates Triclosan Degradation.

The emerging contaminant triclosan (TCS) is widely distributed both in surface water and in wastewater and poses a threat to aquatic organisms and human health due to its resistance to degradation. The dioxygenase enzyme TcsAB has been speculated to perform the initial degradation of TCS, but its precise catalytic mechanism remains unclear. In this study, the function of TcsAB was elucidated using multiple biochemical and molecular biology methods. Escherichia coli BL21(DE3) heterologously expressing tcsAB from Sphingomonas sp. RD1 converted TCS to 2,4-dichlorophenol. TcsAB belongs to the group IA family of two-component Rieske nonheme iron ring-hydroxylating dioxygenases. The highest amino acid identity of TcsA and the large subunits of other dioxygenases in the same family was only 35.50%, indicating that TcsAB is a novel dioxygenase. Mutagenesis of residues near the substrate binding pocket decreased the TCS-degrading activity and narrowed the substrate spectrum, except for the TcsAF343A mutant. A meta-analysis of 1492 samples from wastewater treatment systems worldwide revealed that tcsA genes are widely distributed. This study is the first to report that the TCS-specific dioxygenase TcsAB is responsible for the initial degradation of TCS. Studying the microbial degradation mechanism of TCS is crucial for removing this pollutant from the environment.

The method for assessing the specificity of developing CAR therapies

The effectiveness of antitumor chimeric antigen receptor (CAR) therapy mainly dealt with an elevated sensitivity of CAR cells to target cells. However, CAR therapies are associated with nonspecific side effects: on-target off-tumor toxicity. Sensitivity and specificity of CAR cells are the most important properties of the recognition process of target cells among other cells. Current developments are mainly concentrated on exploring molecular biology methods for designing CAR cells with the highest sensitivity, while the problem of the CAR cell specificity is rarely considered. For the assessment of CAR cell specificity, we suggest that, in addition to an elevated level of CAR-antigen affinity, the ability of CARs for clustering should be taken into account. We assume that the CAR cell cytotoxicity is determined by CAR clustering. The latter is treated within the framework of nucleation theory. The master equation for the probability of CAR cell cytotoxicity is derived. The size of a critical CAR cluster is found to be one of two most essential parameters. The conditions for necessary sensitivity and sufficient specificity are explored. Relevant parametric diagrams are derived. Possible applications of the method for assessing the specificity of developing CAR therapies are discussed.

Metabolome and Its Mechanism Profiling in the Synergistic Toxic Effects Induced by Co-Exposure of Tenuazonic Acid and Patulin in Caco-2 Cells.

Tenuazonic acid (TeA), usually found in cereals, fruits, vegetables, oil crops, and their products, was classified as one of the highest public health problems by EFSA as early as 2011, but it has still not been regulated by legislation due to the limited toxicological profile. Moreover, it has been reported that the coexistence of TeA and patulin (PAT) has been found in certain agricultural products; however, there are no available data about the combined toxicity. Considering that the gastrointestinal tract is the physiological barrier of the body, it would be the first target site at which exogenous substances interact with the body. Thus, we assessed the combined toxicity (cell viability, ROS, CAT, and ATP) in Caco-2 cells using mathematical modeling (Chou-Talalay) and explored mechanisms using non-targeted metabolomics and molecular biology methods. It revealed that the co-exposure of TeA + PAT (12.5 μg/mL + 0.5 μg/mL) can induce enhanced toxic effects and more severe oxidative stress. Mechanistically, the lipid and amino acid metabolisms and PI3K/AKT/FOXO signaling pathways were mainly involved in the TeA + PAT-induced synergistic toxic effects. Our study not only enriches the scientific basis for the development of regulatory policies but also provides potential targets and treatment options for alleviating toxicities.

Schizophrenia-Like Deficits and Impaired Glutamate/Gamma-aminobutyric acid Homeostasis in Zfp804a Conditional Knockout Mice.

Zinc finger protein 804A (ZNF804A) was the first genome-wide associated susceptibility gene for schizophrenia (SCZ) and played an essential role in the pathophysiology of SCZ by influencing neurodevelopment regulation, neurite outgrowth, synaptic plasticity, and RNA translational control; however, the exact molecular mechanism remains unclear. A nervous-system-specific Zfp804a (ZNF804A murine gene) conditional knockout (cKO) mouse model was generated using clustered regularly interspaced short palindromic repeat/Cas9 technology and the Cre/loxP method. Multiple and complex SCZ-like behaviors, such as anxiety, depression, and impaired cognition, were observed in Zfp804a cKO mice. Molecular biological methods and targeted metabolomics assay validated that Zfp804a cKO mice displayed altered SATB2 (a cortical superficial neuron marker) expression in the cortex; aberrant NeuN, cleaved caspase 3, and DLG4 (markers of mature neurons, apoptosis, and postsynapse, respectively) expressions in the hippocampus and a loss of glutamate (Glu)/γ-aminobutyric acid (GABA) homeostasis with abnormal GAD67 (Gad1) expression in the hippocampus. Clozapine partly ameliorated some SCZ-like behaviors, reversed the disequilibrium of the Glu/GABA ratio, and recovered the expression of GAD67 in cKO mice. Zfp804a cKO mice reproducing SCZ-like pathological and behavioral phenotypes were successfully developed. A novel mechanism was determined in which Zfp804a caused Glu/GABA imbalance and reduced GAD67 expression, which was partly recovered by clozapine treatment. These findings underscore the role of altered gene expression in understanding the pathogenesis of SCZ and provide a reliable SCZ model for future therapeutic interventions and biomarker discovery.

Factors Determining Adherence to Hand Antisepsis by Healthcare Workers during Pandemic Infection Spread (as exemplified by COVID­-19)

Relevance. The healthcare personnel’s adherence to hand antisepsis is а pressing problem during any epidemic outbreak. In the context of high biological hazards, the possibility of performing proper antiseptic procedures is reduced while nosocomial pathogen transmission and healthcare-associated infection (HAI) rate growth are promoted. Aims. Studying the factors determining the healthcare workers’ adherence to hand antisepsis during a pandemic infection spread (as exemplified by COVID-19). Materials and methods. The study was conducted during the entire COVID-19 pandemic. To estimate the rate of adherence rate by the healthcare personnel to hand hygiene, we conducted an anonymous questionnaire survey of the staff in infectious disease hospitals (2021–2022) as well as assessed the viral and bacterial contamination rates of the personnel’s hands and the hospital environment items (2021–2023). Epidemiological (descriptive-evaluation), bacteriological (conventional culture, AMR determination using a VITEK2 analyzer), molecular biological (RT-PCR, Sanger sequencing) and statistical (a questionnaire survey, building machine learning models) methods were used in the study. Results. Based on the results of the survey, the main factors influencing the formation of attitudes towards antiseptics among the staff of medical organizations with medical and non-medical education were identified. It was found that the pandemic spread of infections negatively affects the level of contamination of the hands of infectious diseases hospital staff, which amounted to 35.3% of doctors, 28.8% of nurses, and 25% of hospital cleaners. The spectrum of isolated pathogens included viruses (SARS-CoV-2) and opportunistic microorganisms (Enterococcus faecalis, Enterococcus faecium, Pantoea agglomerans, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli), including those with a wide profile of antimicrobial resistance. Conclusion. During the COVID-19 epidemic, the rate of adherence to hand antisepsis in the infectious disease hospital was 82.3% in the staff with a medical background and 71.2% in the staff without one. The results confirm the significant role of staff hands in the in-hospital transmission of dangerous pathogens and indicate the need for systematic monitoring of the level of knowledge on antiseptics among all employees of a medical organization, regardless of specialization and level of education.

Difficulty in diagnosing intracranial infection caused by Mycobacterium avium in an AIDS patient: case report and review of the literature

BackgroundMycobacterium avium complex (MAC) is an uncommon clinical pathogen, especially in the central nervous system (CNS), and carries a poor prognosis. MAC infections commonly present as immune reconstitution disease (IRD) in HIV patients. Herein, we report a case of intracranial infection caused by MAC in an AIDS patient without disseminated MAC (DMAC) and immune reconstitution inflammatory syndrome (IRIS).Case presentationA 31-year-old HIV-positive male presented us with progressively worsening CNS symptoms, and neuroimaging revealed ring-enhancing lesions. The intracranial lesions worsened after the empirical therapy for toxoplasma encephalitis and fungal infection. Due to the rapid progression of the disease, the patient died. Mycobacterium avium was the only pathogen in brain tissue after cultures and molecular biology tests.ConclusionMAC infection in CNS is challenging to diagnose in HIV patients. Our findings emphasize that obtaining tissue samples and applying molecular biology methods is essential to help diagnose the patient as soon as possible to receive adequate treatment.

Inhibition of carbon steel corrosion caused by Pseudomonas aeruginosa biofilms using the eco-friendly ε-Polylysine antimicrobial peptide

This study evaluated the effect of the ε-Polylysine (E-PL) antimicrobial peptide on the corrosion behavior of P. aeruginosa attached to carbon steel surfaces. The results showed that E-PL exhibited good antibacterial and antibiofilm activity, further effectively inhibited the corrosion behavior of P. aeruginosa by biofilm thickness, surface morphology, corrosion product, EIS and molecular biology method. In addition, the transcriptome and qPCR data showed that E-PL significantly downregulated the expression of key genes in flagella, quorum sensing, biofilm formation, and phenazines for resisting microbiologically influenced corrosion. This study has theoretical significance for evaluating the corrosion of marine equipment.

Proteomic and lipidomic analysis of the mechanism underlying astragaloside IV in mitigating ferroptosis through hypoxia-inducible factor 1α/heme oxygenase 1 pathway in renal tubular epithelial cells in diabetic kidney disease

Ethnopharmacological relevanceThe limitations of modern medicine in mitigating the pathological process of diabetic kidney disease (DKD) necessitate novel, precise, and effective prevention and treatment methods. Huangqi, the root of Astragalus membranaceus Fisch. ex Bunge has been used in traditional Chinese medicine for various kidney ailments. Astragaloside IV (AS-IV), the primary pharmacologically active compound in A. membranaceus, is involved in lipid metabolism regulation; however, its potential in ameliorating renal damage in DKD remains unexplored. Aim of the studyTo elucidate the specific mechanism by which AS-IV moderates DKD progression. Materials and methodsA murine model of DKD and high glucose-induced HK-2 cells were treated with AS-IV. Furthermore, multiomics analysis, molecular docking, and molecular dynamics simulations were performed to elucidate the mechanism of action of AS-IV in DKD, which was validated using molecular biological methods. ResultsAS-IV regulated glucose and lipid metabolism in DKD, thereby mitigating lipid deposition in the kidneys. Proteomic analysis identified 12 proteins associated with lipid metabolism regulated by AS-IV in the DKD renal tissue. Additionally, lipid metabolomic analysis revealed that AS-IV upregulated and downregulated 4 beneficial and 79 harmful lipid metabolites, respectively. Multiomics analysis further indicated a positive correlation between the top-ranked differential protein heme oxygenase (HMOX)1 and the levels of various harmful lipid metabolites and a negative correlation with the levels of beneficial lipid metabolites. Furthermore, enrichment of both ferroptosis and hypoxia-inducible factor (HIF)-1 signaling pathways during the AS-IV treatment of DKD was observed using proteomic analysis. Validation results showed that AS-IV effectively reduced ferroptosis in DKD-affected renal tubular epithelial cells by inhibiting HIF-1α/HMOX1 pathway activity, upregulating glutathione peroxidase-4 and ferritin heavy chain-1 expression, and downregulating acyl-CoA synthetase long-chain family member-4 and transferrin receptor-1 expression. Our findings demonstrate the potential of AS-IV in mitigating DKD pathology by downregulating the HIF-1α/HMOX1 signaling pathway, thereby averting ferroptosis in renal tubular epithelial cells. ConclusionsAS-IV is a promising treatment strategy for DKD via the inhibition of ferroptosis in renal tubular epithelial cells. The findings of this study may help facilitate the development of novel therapeutic strategies.

Current Status of Genetic/Molecular Abnormality Analysis and Prognosis Prediction of Meningioma

Biological molecular studies of meningiomas have also developed with the development of molecular biological methods. In 2013, Clark et al. reported that driver genetic mutations other than NF2, including TRAF7, KLF4, AKT1, and SMO, were associated with meningioma development. In 2017, Sahm et al. proposed a classification of meningiomas based on global methylation status, which was more accurate in predicting prognosis than conventional WHO grading. In 2022, based on this classification, various groups reported an integrated classification that comprehensively included some biological molecular abnormalities, such as DNA mutations, copy number alterations, and RNA sequences. This field is expected to elucidate the mechanism of meningioma development and further research is expected to lead to the development of effective molecularly targeted therapeutics and biomarkers of radiosensitivity in the future. In this article, we summarize the current status and prospects of these biological molecular studies.

Subsequent maternal sleep deprivation aggravates cognitive impairment by modulating hippocampal neuroinflammatory responses and synaptic function in maternal isoflurane-exposed offspring mice.

Pregnant women may need to undergo non-obstetric surgery under general anesthesia owing to medical needs, and pregnant women frequently experience sleep disturbances during late gestation. Preclinical studies demonstrated that maternal isoflurane exposure (MISO) or maternal sleep deprivation (MSD) contributed to cognitive impairments in offspring. Research studies in mice have revealed that SD can aggravate isoflurane-induced cognitive deficits. However, it remains unclear whether MSD aggravates MISO-induced cognitive deficits in offspring. The purpose of this research was to explore the combined effects of MSD and MISO on offspring cognitive function and the role of neuroinflammation and synaptic function in the process of MSD+MISO. Pregnant mice were exposed to 1.4% isoflurane by inhalation for 4h on gestational day (GD) 14. Dams were then subjected to SD for 6h (12:00-18:00h) during GD15-21. At 3 months of age, the offspring mice were subjected to the Morris water maze test to assess cognitive function. Then the levels of inflammatory and anti-inflammatory markers and synaptic function-related proteins were assessed using molecular biology methods. The results of this study demonstrated that MISO led to cognitive dysfunction, an effect that was aggravated by MSD. In addition, MSD exacerbated the maternal isoflurane inhalation, leading to an enhancement in the expression levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha and a reduction in the hippocampal levels of IL-10, synaptophysin, post-synaptic density-95, growth-associated protein-43, and brain-derived neurotrophic factor. Our findings revealed that MSD aggravated the cognitive deficits induced by MISO in male offspring mice, and these results were associated with neuroinflammation and alternations in synaptic function.

Detection of Species DNA in Chicken Meat Processed Food Products Using GH (Growth Hormone) Genes as Molecular Markers

The species DNA detection test in processed chicken meat food products using genetic growth hormone (GH) gene markers is a novel approach for authenticity test regarding the presence of a particular species in a product. This research aimed to test the ability of the GH genetic marker to detect the presence of chicken species in processed chicken meat food products. The method used in this research is a molecular biology method using real-time PCR with the SYBR Green kit. The genetic marker primers used were designed independently from the NCBI website. Based on DNA isolation data, the DNA concentration value is 43.60 ng/µL – 45.20 ng/µL with an average of 44.11. The purity value of isolated DNA is 1.855 – 1.925 with an average of 1.906. Chicken species DNA was detected in all samples using real-time PCR analysis, with a Ct value of 24.50 and a Tm value of 78.10. However, the Ct and Tm values were not detected in the negative control (NTC) and specificity (internal) control samples. In the positive control LOD, the Ct value was detected at 27.20 with a Tm value of 79.40. In the positive control, a Ct value of 21.10 and a Tm value of 78.10 were detected. The results indicate that all samples contained the GH gene, and hence, chicken species DNA was detected in all samples. This suggests that the GH genetic marker can be potentially used for species DNA identification testing.

Intercellular adhesion molecule-1 suppresses TMZ chemosensitivity in acquired TMZ-resistant gliomas by increasing assembly of ABCB1 on the membrane

AimsDespite aggressive treatment, the recurrence of glioma is an inevitable occurrence, leading to unsatisfactory clinical outcomes. A plausible explanation for this phenomenon is the phenotypic alterations that glioma cells undergo aggressive therapies, such as TMZ-therapy. However, the underlying mechanisms behind these changes are not well understood. MethodsThe TMZ chemotherapy resistance model was employed to assess the expression of intercellular adhesion molecule-1 (ICAM1) in both in vitro and in vivo settings. The potential role of ICAM1 in regulating TMZ chemotherapy resistance was investigated through knockout and overexpression techniques. Furthermore, the mechanism underlying ICAM1-mediated TMZ chemotherapy resistance was examined using diverse molecular biological methods, and the lipid raft protein was subsequently isolated to investigate the cellular subcomponents where ICAM1 operates. ResultsAcquired TMZ resistant (TMZ-R) glioma models heightened production of intercellular adhesion molecule-1 (ICAM1) in TMZ-R glioma cells. Additionally, we observed a significant suppression of TMZ-R glioma proliferation upon inhibition of ICAM1, which was attributed to the enhanced intracellular accumulation of TMZ. Our findings provide evidence supporting the role of ICAM1, a proinflammatory marker, in promoting the expression of ABCB1 on the cell membrane of TMZ-resistant cells. We have elucidated the mechanistic pathway by which ICAM1 modulates phosphorylated moesin, leading to an increase in ABCB1 expression on the membrane. Furthermore, our research has revealed that the regulation of moesin by ICAM1 was instrumental in facilitating the assembly of ABCB1 exclusively on the lipid raft of the membrane. ConclusionsOur findings suggest that ICAM1 is an important mediator in TMZ-resistant gliomas and targeting ICAM1 may provide a new strategy for enhancing the efficacy of TMZ therapy against glioma.

Exposure to PFAS contaminated urban wetland water causes similar metabolic alterations to laboratory-based exposures in the freshwater amphipod Austrochiltonia subtenuis

Assessing the harm caused by pollutants in urban ecosystems remains a significant challenge. Traditional ecotoxicological endpoints are often not sensitive enough to detect the effects of toxicants at environmentally relevant concentrations (≤ng/L). A potential solution is using molecular biology methods to look at small biochemical changes caused by exposure to ng/L concentrations of contaminants. This has been tested in the lab but not conclusively demonstrated in the field. We exposed the freshwater amphipod (Austrochiltonia subtenuis) to water from an urban wetland containing known concentrations of per-and polyfluoroalkyl substances (as well as very low concentrations of pesticides) for 14 days and analyzed their metabolite profiles. Mannose, Myo-inositol, and Isopropyl propionate were found to change in PFAS exposed amphipods, a similar response to that previously observed in laboratory exposures to the same PFAS, but not pesticides. The results give a better understanding of PFAS toxicity at environmentally relevant concentrations and conditions.

A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.