Vibrio vulnificus can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic V. vulnificus is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of V. vulnificus in aquatic products. Pathogenic V. vulnificus was detected using the virulence vvhA gene and the housekeeping gene gyrB gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for V. vulnificus, and no cross-reaction was observed with Vibrio spp. or other bacteria (n = 14). Furthermore, sensitive detection of V. vulnificus in oysters was achieved. The LODs of the gyrB and vvhA genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples (n = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic V. vulnificus in aquatic products and aquaculture water. Vibrio vulnificus is a Gram-negative bacterium that is widely distributed in ocean, river and aquatic products, and can accumulate in shellfish such as oysters  anA (~6 h)  for V. v


Escherichia coli as a sentinel. For this purpose, 244 samples of dogs and cats were collected from veterinary centres to assess antimicrobial susceptibility against a panel of 22 antibiotics with public health relevance. A total of 197 E. coli strains were isolated from asymptomatic dogs and cats. The results showed AMR against all the 22 antibiotics studied, including those critically important to human medicine. Moreover, almost 50% of the strains presented MDR. The present study revealed the importance of monitoring AMR and MDR trends in companion animals, as they could pose a risk due to the spread of AMR and its resistance genes to humans, other animals and the environment they cohabit. Failure in antibiotic therapies due to the increase in antimicrobial-resistant (AMR) bacteria is one of the main threats to public and animal health. In recent decades, the perception of companion animals has changed, from being considered as a work tool to a household member, creating a family bond and sharing spaces in their daily routine. Hence, the aim of this study is to assess the current epidemiological situation regarding the presence of AMR and multidrug resistance (MDR) in companion animals in the Valencia Region, using the indicator bacteria  The rise of AMR bacteria in both humans and animals leads to the failure of antibiotic therapies, posing a major threat to public and animal health. Thus, the World Health Organisation (WHO) includes antimicrobial resistance (AMR) among the top 10 threats to global public health [It is currently estimated that there are more than 340 million companion animals in European households, mainly 127 million cats and 104 million dogs, representing 70% of all companion animals in Europe , and peoHistorically, the acquisition of multidrug resistances (MDR), defined as being caused by microorganisms that are resistant to three or more antimicrobials commonly used in the treatment of infections caused by that microorganism, and that this resistance has clinical and epidemiological relevance , has beeMoreover, in human medicine, the WHO categorises antimicrobials as ‘critically important’, ‘highly important’ and ‘important’ to human health . In the Currently, to complement the present EARS-Net, the EU intends to launch a European Antimicrobial Resistance Surveillance Network in Veterinary Medicine (EARS-Vet), which aims to harmonise existing programmes and AMR surveillance trends in bacteria isolated from diseased animals and include animal species that have not been considered thus far, such as companion animals ,21. In SThe latest animals that are being included in AMR surveillance programmes are companion animals (dogs and cats) . The samEscherichia coli as a model, before setting the objectives in the EU scope.In this context, the importance of monitoring AMR in these animals lies not only in the amount of antibiotics they consume, but in the use of certain antibiotics considered critical for human health reserved to treat MDR hospital-acquired human infections, with the threat this entails to public health. Hence, the aim of this study was to assess the current epidemiological situation regarding the presence of AMR and MDR in companion animals (cats and dogs) in the Valencia Region, using the commensal sentinel bacteria In this study, a total of 244 animals were sampled . Regarding the distribution, 48.4%  of the samples were taken in Veterinary Hospitals, and 51.6%  of the samples were taken in Veterinary Clinics. E. coli were isolated. From the samples collected from cats, 84.1% (95/113) E. coli were isolated.From the collected canine samples, 77.9% (102/131) From all the animals sampled, a questionnaire with general and clinical information was drawn up in order to study the differences in the appearance of AMR depending on the epidemiological information. The canine population was distributed by sex, where 53.4% (70/131) were females and 46.6% (61/131) were males. The age of these animals ranged from 1.5 months to 16 years, where 24% (32/131) were puppies, 27% (35/131) were mature dogs, and 49% (64/131) were geriatric dogs. In addition, 45% (59/131) dogs cohabited in their households with other animals. However, all of them (131) went out daily, so they are in close contact with other animals outside the household.Regarding the clinical data collected from all the dogs sampled, 22.9% (30/131) of the animals presented a chronic disease and 77.1% (101/131) did not present any. The chronic diseases were classified depending on whether they were systemic  or musculoskeletal . In addition, regarding the daily medication taken by the animals, 22.1% (29/131) were on some kind of medication. Finally, of all dogs sampled, 83.2% (109/131) had received previous antibiotic therapy at some point in their lives, compared to 16.8% (22/131) that had never been treated with antibiotics. All data on when each animal was last treated with antibiotics and with which group of antibiotics are represented in The feline population was distributed by sex, where 55.8% (63/113) were females and 44.2% (50/113) were males. These animals ranged in age from 5 months to 17 years, where 42%  were kittens, 31% (35/113) were mature cats and 27% (31/113) were geriatric cats. In addition, 69.9% (79/113) of sampled cats cohabited in their households with other animals and only 12.4% (14/113) had contact with other animals outside the home.According to the clinical data collected from all the cats sampled, 26.5% (30/113) of the animals presented a chronic disease, all of them classified as systemic diseases, while 73.5% (83/113) did not present any disease. In addition, regarding the daily medication taken by the animals, 12.4% (14/113) were on some kind of medication. Finally, of all cats sampled, 51.3% (58/113) had received previous antibiotic therapy at some point in their lives, compared to 48.7% (55/113) that had never been treated with antibiotics. All data on when each animal was last treated with antibiotics and with which group of antibiotics are represented in p-value > 0.05). In contrast, in relation to the strains isolated from cats, lower AMR rates were found than in dogs, where 72.6% (69/95) were resistant to at least one of the antimicrobials tested and 34.7% (33/95) were MDR. In the feline population, no statistically significant differences were found between the different epidemiological groups studied and the occurrence of AMR and MDR .From all the strains isolated from dogs, 88.2% (90/102) were resistant to at least one of the 22 antimicrobials tested, and 47.1% (48/102) were considered MDR. However, no statistically significant differences were found between the different epidemiological groups described above . For example, the highest frequency of AMR in dogs was found to ampicillin  followed by ticarcillin , whereas in cats, the highest frequency was found against TIC  followed by AMP . As for the rest of the antibiotics studied, the differences were more striking, as in the case of amoxicillin/clavulanic acid, (AMC) where there was a huge difference between dogs  and cats , or in the case of gentamicin (GEN), where cat strains  showed a higher percentage of AMR than dog strains  . Regarding the CIAs  studied, high AMR levels were observed for this antibiotics group . Moreover, of all the antibiotics studied, following the WHO categorisation for important antibiotics, four of them belonged to category HPCIAs, as well as to category A according to the EMA categorisation (antibiotics reserved for human treatment only), with a high percentage of AMR being found for this type of antibiotics    showed high percentages of MDR in ectively . However% of MDR . Compari% of MDR , studies% of MDR , or othe% of MDR , further% of MDR , which mE. coli [E. coli and Klebsiella pneumoniae) that are resistant to β-lactam antibiotics , are becoming more frequent [Regarding the AMR observed to each antibiotic, three antibiotics from the penicillins group were those with the highest AMR in the present study: AMP, TIC and AMC. The first was AMP, the antibiotic with the highest resistances in dogs, and the second in cats. These results agree with those reported in other studies carried out in Europe, with the results from Italy being the most similar to those of the present study . Other aE. coli . Howeverfrequent . For thafrequent .E. coli [Following the AMR levels observed, TRS showed a higher resistance level in dogs than in cats, in line with previous studies ,62,63. TE. coli ,67,68. HE. coli . It is aE. coli ,70,71.In addition, four Category A antibiotics  were tesE. coli in dogs and cats showing high resistance rates, although a case of ETP-resistant Klebsiella pneumoniae has been reported in dogs in Belgium [E. coli isolated from calves [E. coli have been found to be 0% [Moreover, two of the Category A antibiotics studied were carbapenemases: ertapenem (ETP) and meropenem (MER). To the authors’ knowledge, this is the first study addressing the AMR observed against ETP in commensal  Belgium . Some otm calves , dairy cm calves  and humam calves , which mm calves ,80,81. Bto be 0% , posing to be 0% . Finallyto be 0% . This mato be 0% ,84. The present study revealed the importance of monitoring AMR and MDR trends in companion animals, as they could pose a direct threat to public health due to the spread of AMR genes. These results provide valuable information as a starting point and highlight the need for a One Health approach to implement new strategies in veterinary medicine for companion animals to control the alarming increase in AMR. However, more studies are needed to further study AMR in companion animals, not only phenotypically but also genotypically, as a more comprehensive analysis of this issue could help the scientific community better understand the dynamics of AMR genes between commensal and pathogenic bacteria from animals to humans and vice versa.The animal study was reviewed and approved by the Animal Ethics Committee of UCH-CEU University (research permit nº. CEEA 22/04). Veterinary Hospitals (VH) and Veterinary Clinics (VC) distributed throughout the Valencia Region were asked to voluntarily participate in this study. Of these, three VH and five VC were willing to cooperate. Thus, eight veterinary centres agreed to collaborate voluntarily. Three are characterised by being three large reference VH, where cases from the entire Valencia Region are referred, and five VC distributed throughout the region of Valencia also participated. To gather epidemiological data on the animals sampled, an epidemiological questionnaire was filled in for each animal. The questionnaire was divided into 3 parts. The first section referred to information on the source of the animals (origin details), and included the informed consent signed by the owners. The second included general information on the animal: sex, age and whether other animals cohabited at home. To categorise the age range into groups, dogs were classified as puppies and young dogs (≤2 years), mature dogs (3–7 years), and senior and geriatric dogs (≥8 years) . In contE. coli. To assess the antimicrobial profile, a rectal swab was taken from asymptomatic animals by introducing a swab into the rectum approximately 3 cm [Between October 2022 and June 2023, samples from companion animals (dogs and cats) were taken in order to isolate commensal ely 3 cm ,88, usinE. coli identification, Tryptone Bile X-glucuronide agar , and incubated at 37 ± 1 °C for 24 h. After incubation, colonies with compatible morphology with E. coli were selected and inoculated into a nutrient agar plate  and incubated at 37 ± 1 °C for 24 h. Finally, a biochemical test was performed to confirm E. coli . Rectal swabs were pre-enriched in buffered peptone water , in 1:10 vol/vol proportion, and incubated at 37 ± 1 °C for 24 ± 2 h. All the pre-enriched samples were inoculated onto selective culture agar for https://www.eucast.org/ast_of_bacteria/calibration_and_validation. Accessed on 12 September 2023). To detect AMR microorganisms, the phenotypic resistance should be studied in vitro. When the microorganism under study shows acquired resistance to at least one agent from three or more antimicrobial classes, it is defined as an MDR microorganism [To establish the epidemiological situation, an antimicrobial susceptibility test was carried out with antibiotics of importance in public health. AMR was evaluated using Minimum Inhibition Concentration (MIC) assay in EUGNF Gram Negative Sensititre Plate . Finally, Sensititre plate results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) established breakpoints 2022, available on the European Society of Clinical Microbiology and Infectious Diseases website . The suspensions were mixed and adjusted, adding colonies until a 0.5 McFarland score was reached using a Nephelometer . Then, 30 μL of this suspension were added to a vial containing 11 mL of Mueller–Hinton broth  and mixed. From that suspension, 50 μL of the vial contents were transferred into each well of the Sensititre plate. After inoculation, the plates were sealed with plate film and incubated at 37 ± 1 °C for 24 h. The plates were read manually using a Sensititre Vizion .E. coli AMR profile, a commercial panel of 22 antibiotics with relevance in public health were used. The antibiotics are summarised in To assess Under the One Health concept, this commercial Sensititre plate was selected because 12 of these antibiotics belong to those that EARS-Vet wishes to monitor , and the other 10 belong to antibiotics used in human medicine .p-value ≤ 0.05 was considered to indicate a statistically significant difference. Data are presented as least squares means ± standard error of the least squares means. Statistical analyses were carried out using the R software packages EMMs [A Generalised Linear Model (GLM) using the probit link function, which assumed a binomial distribution for the influence of external factors in AMR and MDR patterns, was fitted to the data to determine whether there was an association with the categorical variables . Also, for microbiological results, a GLM using the probit link function, which assumed a binomial distribution for AMR patterns in commensal bacteria of dogs, and a GLM using the probit link function, which assumed a binomial distribution for AMR patterns in commensal bacteria of cats, were performed. A ges EMMs , car [90ges EMMs .The results obtained in this study highlight the need to control the administration of antibiotics, not only in food-producing animals, but also in companion animals, which cohabit and interact with humans, domestic and wild animals and their environment, posing a risk in the dissemination of AMR and resistance genes. Furthermore, the similar AMR patterns observed between dogs and cats support the hypothesis of its transmission. However, no statistical differences were found between epidemiological groups, which may be more alarming, as AMR is widespread even if the animals have not been previously treated with antibiotics. These results are of special concern regarding CIAs, as high AMR levels are observed.


The escalating impacts of the climate crisis, zoonotic spill-over, and antibiotic resistance have positioned molecular medicine at the forefront of pioneering translational research. With the rapid advances in modern medicine, the constantly evolving technologies of molecular pathology, diagnostics, and therapeutics are driving the transformation towards precision medicine. By exploring the molecular intricacies of diseases, these inter-related disciplines offer novel insights into disease origins and progressions and promise the identification of more effective therapeutic targets. Consequently, the growing integration of these fields has the potential to revolutionise healthcare by accelerating the advent of personalised, targeted, and more effective treatments. This combination also plays a pivotal role in improving the ability to make informed choices for managing infectious and metabolic diseases. Molecular pathology drives the development and validation of biological diagnostic assays, facilitating the identification of disease-specific biomarkers or pathogens. Molecular diagnostics, in turn, helps guide treatment decisions, influences the most appropriate molecular therapeutics for an individual patient’s unique profile, and is an essential guardian of public health. As molecular therapeutics and treatment responses are recorded, valuable insights are fed into molecular pathobiology and diagnostics research, refining biomarker discovery and therapeutic strategies.Molecular pathology focuses on the study and interpretation of molecular and genetic changes in tissues and cells to aid in the diagnosis, pathogenesis, prognosis, and treatment of diseases. By analysing genetic mutations, gene expression patterns, epigenetic modifications, and protein alterations, molecular pathologists aim to understand the underlying mechanisms driving disease development and progression. Hence, molecular pathology differs from molecular diagnostics in its broader scope and application. While molecular diagnostic mainly focus on detecting specific biomarkers, molecular pathologists work closely with clinicians, oncologists, and other healthcare professionals to correlate specific molecular alterations with histopathological findings, biomarkers, clinical data, and patient outcomes. This comprehensive interaction aims to provide a solid foundation for the accurate diagnosis, prognostication, and prediction of treatment responses, thereby significantly improving patient outcomes.Molecular pathology plays a critical role in bridging the gap between basic research and clinical applications. The identification of disease-specific biomarkers and genetic alterations provides insight into pathogenic mechanisms and enables the development of targeted therapies and personalised treatment regimens. The ability to classify diseases and stratify patients precisely, based on their molecular profiles, facilitates the implementation of tailored therapeutic approaches, thereby maximising treatment efficacy and minimising adverse effects. Additionally, molecular pathology supports advancements in research and drug development by elucidating molecular targets and pathways that can be exploited for novel therapeutic interventions.Molecular pathology is witnessing rapid advances in technology and automation, enabling higher-throughput analysis and improved accuracy. Integrating data from various ‘-omics’ technologies will yield a more comprehensive understanding of disease pathogenesis. Furthermore, artificial intelligence and machine learning algorithms are anticipated to revolutionise disease classification and prognosis, empowering clinicians with more precise and personalised treatment recommendations.The first involves applying molecular techniques to detect and analyse genetic and molecular biomarkers in patient samples, thus defining the era of personalised medicine. By identifying disease-specific biomarkers, molecular diagnostics plays a pivotal role in early detection, accurate diagnosis, prognosis, and treatment monitoring and has started to transform the landscape of disease detection and management.The second involves diagnosing and screening infectious diseases, where rapid and sensitive molecular tests have proven particularly vital in the COVID-19 pandemic. The ability to identify SARS-CoV-2 as its causative agent quickly and accurately was instrumental in developing tests, targeted therapeutics, and vaccines. More generally, traditional methods for infectious disease diagnosis are time-consuming and may lack sensitivity, leading to delays in treatment and potential disease transmission. Molecular diagnostics offer the potential for the rapid and accurate detection of infectious agents, allowing for timely intervention, infection control, and improved patient outcomes.The third involves applying molecular biomarkers in the diagnosis and screening for diseases in the hepatic and gastrointestinal systems, including neoplasms, often hiding behind non-specific symptoms, where the application of biomarkers enables a rapid, non-invasive, and accurate diagnosis, as well as continuous monitoring of therapeutic interventions and patient outcomes.Molecular diagnostics is crucial in guiding targeted therapies, predicting treatment responses, and improving overall patient care through its precise and rapid testing capabilities. It has two main applications:Molecular biomarkers encompass a wide array of molecules, including nucleic acids, proteins, lipids, and metabolites, all of which are potential indicators of disease status or therapeutic responses. Techniques like the polymerase chain reaction (PCR) allow for the rapid and sensitive detection of target genetic sequences and quantification of gene expression signatures, as well as enabling the identification of pathogens with high sensitivity and specificity. Next-generation sequencing technologies have further advanced the ability to comprehensively analyse the genetic makeup of pathogens, providing crucial insights into their virulence, resistance mechanisms, and epidemiological patterns. Enzyme-linked immunosorbent assays (ELISAs) and mass spectrometry enable the quantification and profiling of additional biomarkers in patient samples, providing valuable information about disease progression and treatment efficacy.The significance of molecular diagnostics lies in its ability to detect diseases at their earliest stages, enabling timely intervention and improved patient outcomes. Early detection can prevent disease progression, reduce treatment complexities, and lead to more successful therapeutic interventions. Moreover, molecular diagnostics offers a powerful tool for monitoring treatment response and detecting potential relapse, guiding clinicians to adjust treatment regimens accordingly. It also empowers genetic counsellors to provide personalised risk assessments, thus facilitating informed decision-making for individuals with inherited genetic conditions. Molecular diagnostic testing also enables the detection of drug-resistant strains of infectious agents, aiding in selecting appropriate antimicrobial therapies. At the same time, identifying genetic markers associated with drug resistance allows clinicians to tailor treatments to optimise patient outcomes and minimise the development of further resistance.The future of molecular diagnostics is poised for remarkable advancements driven by emerging technologies and innovative methodologies. Liquid biopsies, which involve analysing circulating tumour DNA, RNA, and other biomolecules, hold immense promise for non-invasive early cancer detection and monitoring. Furthermore, point-of-care molecular diagnostic devices are likely to become more accessible and widely adopted, particularly in resource-limited settings, thereby democratising precision medicine. As the field continues to evolve, integrating digital health platforms and data-sharing initiatives will streamline the exchange of molecular diagnostic information, fostering collaboration and enabling evidence-based decision making.In the context of genetic analysis, integrating genomic data with electronic health records and machine learning algorithms holds great promise for precision medicine. This integration will enable the identification of personalised treatment options based on individual genetic profiles, leading to improved therapeutic outcomes and reduced adverse effects.In infectious diseases, the continuous development of novel molecular techniques will contribute to faster and more widespread pathogen detection, facilitating the early intervention and containment of outbreaks. Moreover, ongoing research into host–pathogen interactions and immune responses will advance our understanding of infectious disease pathogenesis, leading to identifying new therapeutic targets and developing effective antiviral and antimicrobial agents.Molecular therapeutics describes a wide range of therapeutic approaches that utilise our understanding of molecular pathways, genetics, and biomarkers to develop targeted treatments for various diseases, including personalised medicine and targeting some infectious diseases. This can involve targeted therapies, such as monoclonal antibodies or small molecule inhibitors, that specifically act on key molecules or pathways involved in disease processes. In the context of infectious diseases, it can be aimed at disrupting the entry and replication of pathogens or allowing the development of vaccines targeting specific antigens, as for SARS-CoV-2. Unlike traditional therapies that often result in non-specific systemic effects, molecular therapeutics focuses on targeting the root cause of the disease, preserving healthy tissues and minimising adverse reactions. The significance of molecular therapeutics lies in its potential to usher in a new era of customised medicine. By tailoring treatments to individual patients based on their unique molecular profile, molecular therapeutics maximise treatment efficacy while minimising the risk of treatment resistance and side effects. This approach has shown tremendous promise in managing various diseases, particularly cancer. By specifically targeting molecular aberrations in cancer cells, targeted therapies have shown remarkable success in prolonging survival, improving quality of life, and achieving remission in previously untreatable cases. Beyond cancer, molecular therapeutics holds potential for managing autoimmune disorders, rare genetic diseases, and infectious diseases, heralding a new era of precision medicine with tailored treatments for individual patients.The future of molecular therapeutics promises exciting developments in precision drug design and delivery. Advances in pharmacogenomics will enable the identification of patient-specific drug responses, avoiding treatments that may prove ineffective or harmful. Combinatorial therapies involving a combination of targeted agents, immunotherapies, and conventional treatments are expected to emerge as powerful treatment regimens with synergistic effects. Moreover, gene editing technologies have the potential to correct genetic defects and treat genetic disorders at the molecular level, opening up new therapeutic avenues. Finally, the success of RNA vaccines against diseases like COVID-19 has demonstrated the power of molecular therapeutics in rapidly responding to infectious disease outbreaks.This Special Issue can only touch on a small section of the vast and diverse landscape that this field encompasses. Nonetheless, the issue explores a broad spectrum, from mechanosensing through fibroids, cancer, and inflammatory conditions to myocardial infarction. This Special Issue also takes us beyond traditional medical territories, delving into the realm of mindfulness-based interventions. The exciting explorations of how mindfulness can influence molecular biomarkers open new doors for integrative approaches to managing mental health and fostering overall well-being. The 16 papers presented in this Special Issue represent a remarkable collection of contributions highlighting the richness and complexity of molecular pathology, diagnostics, and therapeutics.The first review deals with infectious diseases, particularly mycobacterium tuberculosis, the cause of tuberculosis (TB), a highly contagious airborne disease affecting billions worldwide . One of The second review deals with solid organ transplantation (SOT) , a life-Tourkochristou and colleagues  highlighThe next review discusses mechanosensing, which describes the ability of a cell to sense and respond to mechanical cues of its microenvironment . It playYang and Al-Hendy’s review summarises the recent progress in ascertaining the biological functions and regulatory mechanisms in Uterine fibroids (UFs) . These aThe following three reviews discuss how new therapeutic strategies for cancer treatment, particularly targeted therapies, have improved patient survival. One of the main challenges remains the lack of understanding of the mechanisms of resistance to conventional therapies, with primary and secondary drug resistance remaining a substantial problem for many patients. Acquired resistance is often determined using the overexpression of efflux pumps, and the review by Labbozzetta  discusseThe second of the three articles reviews the development of new treatment options for HCC , the mosThe third review provides an update on photodynamic therapy (PDT) for colorectal cancer (CRC) and explores potential future directions . PDT is The next four reviews deal with diverse inflammatory conditions. Axial spondyloarthritis  is a multifactorial disease affecting the sacroiliac joints and spine, leading to inflammation, bone reabsorption, and ankylosis, and its pathogenesis is not yet fully understood . The disAsthma is a chronic inflammatory lung disease that displays diverse clinical features and phenotypes with varying phenotypes and affects up to 18% of the population of all age groups . A subseThe “Gut-Liver Axis” refers to the interaction between the gut and its microbiota and the liver. This plays a vital role in maintaining health through immune tolerance . It invoThe final review discussing inflammation looks at the potential role of EVs in periodontitis, a chronic infectious disease that damages periodontal tissues, leading to inflammation and bone loss . CurrentA nicely speculative review emphasises the importance of phospholipids in cell membranes with critical functions and their potential as therapeutic targets for cardiovascular diseases, particularly acute myocardial infarction . Given tThe final three reviews deal with more esoteric and rather important topics. The first looks at the role of metals in disorders of consciousness (DoC) . These rThe last review is a meta-analysis that deals with mindfulness-based interventions (MBIs) . These hThe final review analyses the role of age and sex in the fatal outcome of COVID-19  by invesThe combination of molecular pathology, diagnostics, and therapeutics marks a paradigm shift in medical practice. As precision medicine continues to gain momentum, it has the potential to revolutionise patient care, offering tailored treatments and improved outcomes for individuals grappling with diverse diseases. The integration of these three fields holds the promise of not only enhancing the accuracy and efficacy of disease management but also paving the way for novel discoveries and therapeutic approaches in the pursuit of healthier lives. As technology continues to advance and our understanding of molecular mechanisms deepens, the future outlook for precision medicine is undoubtedly one of the immense possibilities.Finally, artificial intelligence (AI) is becoming a critical partner. AI-powered algorithms will analyse genomic, proteomic, transcriptomic and metabolomic data and identify disease biomarkers and molecular signatures. They will predict disease progression, therapeutic responses, and potential adverse reactions, leading to more accurate diagnoses and treatment plans. They are likely to be able to detect and quantify subtle molecular alterations for early disease detection and better patient outcomes. Crucially, AI will also accelerate therapeutic development by identifying drug targets, predicting responses, and even designing novel agents, thus facilitating targeted therapies based on individual patient profiles. As AI technology evolves, its integration into molecular medicine will drive significant improvements, shaping the future of healthcare for precision and personalised care.


Clostridioides difficile infection rates collected faecal samples from anonymous patients which were discarded after C. difficile testing over a period of 9 to 15 months. Metagenomic DNA was extracted from these samples and sequenced using an Illumina NovaSeq 6000 platform. The resulting sequencing reads were analysed for taxonomic composition and for the presence of AMR genes. Among 683 faecal metagenomes we found huge variation between individuals in terms of taxonomic diversity (Shannon Index range 0.10–3.99) and carriage of AMR genes . We found no statistically significant differences in diversity (median Shannon index 2.16 (IQR 1.71–2.56), 2.15 (IQR 1.62–2.50) and 2.26 (IQR 1.55–2.51)) or carriage of AMR genes (median 1.37 genes/cell/sample (IQR 0.70–3.24), 1.70 (IQR 0.70–4.52) and 1.43 (IQR 0.55–3.71)) at the three trusts respectively. This was also the case across the sample collection period within the trusts. While we have not demonstrated differences over place or time using metagenomic sequencing of faecal discards, other sampling frameworks may be more suitable to determine whether organisational level differences in antibiotic use are associated with individual-level differences in burden of AMR carriage.Antimicrobial resistance (AMR) is a threat to global public health. However, unsatisfactory approaches to directly measuring the AMR burden carried by individuals has hampered efforts to assess interventions aimed at reducing selection for AMR. Metagenomics can provide accurate detection and quantification of AMR genes within an individual person’s faecal flora (their gut “resistome”). Using this approach, we aimed to test the hypothesis that differences in antimicrobial use across different hospitals in the United Kingdom will result in observable differences in the resistome of individual patients. Three National Health Service acute Hospital Trusts with markedly different antibiotic use and  Antimicrobial resistance (AMR) is a global threat to public health  but obtaMetagenomic analyses, using short-read next-generation sequencing data, have the ability to quantify thousands of resistance genes in a single sample. Metagenomics can provide accurate detection and quantification of AMR genes within an individual person’s faecal flora (their gut “resistome”) . This apThe term “antimicrobial stewardship” is used to describe organisational level strategies to optimise antimicrobial use and minimise selection for AMR, and studies which evaluate different stewardship strategies typically compare organisations rather than individual patients . Even wiClostridioides difficile testing from unselected adult medical admissions to three large acute hospitals in England.In this study, we aimed to test the hypothesis that substantial differences in hospital-level use of antibiotics will result in detectable differences in the faecal resistome of patients treated at those hospitals. If so, this would indicate that assessment of the faecal resistome could be an appropriate direct measure of harms associated with antibiotics in studies evaluating antimicrobial stewardship interventions. We therefore undertook metagenomic analyses of microbial diversity, AMR gene burden and AMR gene diversity in faecal samples submitted for C. difficile test results for the acute medical inpatients, as described previously [C. difficile took part in the sub-study. Each was an acute NHS hospital trust based in different cities of southern England with distinct patterns of antimicrobial use, and sampling and data analysis was undertaken for distinct time periods for each Trust as defined in The study was performed as a sub-study of the Antibiotic Review Kit (ARK)–Hospital trial , registreviously . Three AC. difficile (within -1/+90 days of admission) was calculated per 100 acute medical hospital admissions per month.Overall use of systemic antibiotics was defined by DDDs per acute medical admission and antibiotics were categorised using the World Health Organisation AWaRe categorisation adapted for use in England . The perC. difficile infection. The only meta-data available for samples was therefore the date of sampling and hospital site. This approach was taken to avoid the burden of, and biases inherent in, taking informed consent. For each site samples were provided determined by the time of their involvement in the ARK-hospital cluster randomised controlled and availability of clinical laboratory staff to process samples and this varied between Jul 2017 and Oct 2019 , either 103 ng or 20 ng, was added to Stool Transport And Recovery (S.T.A.R.) Buffer . 1 ml of the T. thermophilus-S.T.A.R buffer was pipetted into Lysing Matrix E tubes  and weighed to the nearest mg to obtain the tube weight. Faecal sample was added to the weighed Lysing Matrix E tubes with the T. thermophilus-S.T.A.R. buffer and then weighed again to the nearest mg to obtain the sample weight. The samples were then bead beaten using Tissue Lyser LT (Qiagen) at 50 Hz for 10 mins. 1 ml of the bead beaten sample was then transferred to the InhibitEX buffer from the QIAamp Fast DNA Stool kit and heated at 95°C for 5 mins, and then centrifuged at 13,000 rpm for 3 mins. 600 μl of the supernatant from the above InhibitEX step was added to 45 μl proteinase K, then 600 μl Buffer AL was added to the mix. The kit protocol was then followed; briefly, the above mix was incubated at 70°C for 10 mins, before 600 μl ethanol was added. The resulting lysate was loaded onto a spin column, then the columns were washed with 500 μl each Buffer AW1 and Buffer AW2. The bound DNA was eluted using 100 μl molecular grade water before the DNA concentration was determined using Qubit dsDNA HS Assay . A number of samples could not be taken to the next step due to insufficient DNA concentration  according to the manufacturer’s protocol with additional steps and modifications as follows: A known concentration of ntration .Samples were sequenced to a minimum depth of 10 million reads (including human and non-human). Sequencing was conducted by the Oxford Genomics Centre  using their High Multiplex Whole Genome/MMM library preparation for sequencing on a NovaSeq 6000 platform (Illumina), generating 150 bp paired-end reads.Taxonomic classification was performed using Kraken2 , and samDifferences in species diversity and AMR gene carriage were tested for statistical significance by pair-wise Chi-squared tests and reported when P<0.05.A total of 683 metagenomes were included in the analysis from 2,654 samples in total, a median of 18 per site per calendar month (interquartile range (IQR)15-22, range 0–42) . For sitThe overall species alpha-diversity of samples varied widely (Shannon Index range 0.10–3.99) but the median and IQR were very similar at the three hospitals: median 2.16 (IQR 1.71–2.56), 2.15 (IQR 1.62–2.50) and 2.26 (IQR 1.55–2.51) for sites A, B and C respectively . There wThe overall burden of AMR genes per bacterial cell in faecal samples also varied widely from 0.03–48.55 with a median of 1.50 genes per cell per sample overall. The median (IQR) AMR genes carried were very similar at the three hospitals: median 1.37 (IQR 0.70–3.24), 1.70 (IQR 0.70–4.52) and 1.43 (IQR 0.55–3.71) for sites A, B and C respectively . There wThe alpha-diversity present within the faecal resistome was broadly comparable to the overall species alpha-diversity, ranging from 0.62–4.46. Again, there was no evidence of variation between sites, with median 3.17 (IQR 2.63–3.54), 3.17 (IQR 2.74–3.56) and 3.05 (IQR 2.64–3.58) for sites A, B and C respectively . There wIn terms of resistance to major antibiotic classes, there was no evidence that the percentage of samples with genes conferring resistance to each antibiotic class varied across the three sites . This reC. difficile testing in adult medical patients receiving treatment at three large acute hospitals in England. This has allowed us to characterise the diversity and burden of AMR gene carriage among these patients but also make comparisons across time and place in a way that has not been attempted in humans previously. The most striking findings from our analyses are firstly the huge variation that exists in AMR gene diversity between patient samples and secondly the overall consistency of inpatient resistomes over time and place in terms of the burden and nature of AMR gene carriage.Here, we have conducted metagenomic analysis of 683 faecal metagenomes sequenced to median depth of ~16 million reads obtained from specimens submitted for C. difficile infection. Nevertheless, we found no suggestion in our data of any detectable hospital-level difference in diversity or AMR gene carriage in the faecal samples obtained from patients.Our estimates of overall and AMR resistance gene alpha diversity are consistent with previous studies of healthy individuals ,16. HoweC. difficile testing and therefore likely to be biased towards patients with recent antibiotic exposure, and towards a sub-group of patients with diarrhoea which might have also impacted on taxonomic and resistance gene distributions. While this has the potential to dilute our ability to measure the individual patient-level impact of antibiotic exposure, it would not be expected to detract from the validity of our comparisons which are of organisation-level antibiotic use and C. difficile colonisation rates. Site-related differences in resistance carriage might plausibly be smaller in such patients than in the wider population. Finally, the sensitivity of metagenomic data for the profiling of AMR genes in polymicrobial samples has been shown to be closely linked to sequencing depth [Our study has important limitations. We generated metagenomic data on a very limited number of admitted patients (<1%/month), and it is possible that pooling larger numbers of samples to evaluate the wider inpatient-level resistome might better reflect hospital-level prescribing, that the impact of prescribing on faecal metagenomes can be more reliably ascertained in certain population sub-groups or that there is a threshold effect through which above a certain level of antibiotic use differences are small. Our samples were uncoupled from any associated metadata including age, gender, ethnicity, medical history, previous antibiotic exposure and time from admission, all of which may influence AMR gene burden. If samples were taken from patients recently admitted to hospital then the faecal resistome may potentially be more likely to reflect community antibiotic use, for which data are not available, rather than hospital prescribing. Similarly, some samples may have been taken from patients who were not on antibiotics. Notably however, the samples analysed were discards after ng depth , and we C. difficile testing could be used to assess the impact of antibiotic practice on carried resistance at an hospital-level. Further studies investigating whether more detailed sampling, or pooled sampling, would be of benefit in detecting prescribing signatures might be considered.In summary, we found no evidence that evaluating the faecal resistome of <1% of hospital inpatients using discarded samples submitted for S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


In this study, morphology and in vitro response of electroconductive composite nanofibers were explored for biomedical use. The composite nanofibers were prepared by blending the piezoelectric polymer poly(vinylidene fluoride–trifluorethylene) (PVDF-TrFE) and electroconductive materials with different physical and chemical properties such as copper oxide (CuO), poly(3-hexylthiophene) (P3HT), copper phthalocyanine (CuPc), and methylene blue (MB) resulting in unique combinations of electrical conductivity, biocompatibility, and other desirable properties. Morphological investigation via SEM analysis has remarked some differences in fiber size as a function of the electroconductive phase used, with a reduction of fiber diameters for the composite fibers of 12.43% for CuO, 32.87% for CuPc, 36.46% for P3HT, and 63% for MB. This effect is related to the peculiar electroconductive behavior of fibers: measurements of electrical properties showed the highest ability to transport charges of methylene blue, in accordance with the lowest fibers diameters, while P3HT poorly conducts in air but improves charge transfer during the fiber formation. In vitro assays showed a tunable response of fibers in terms of viability, underlining a preferential interaction of fibroblast cells to P3HT-loaded fibers that can be considered the most suitable for use in biomedical applications. These results provide valuable information for future studies to be addressed at optimizing the properties of composite nanofibers for potential applications in bioengineering and bioelectronics. Electroconductive polymers have garnered significant attention in recent years due to their unique combination of electrical conductivity, mechanical, and chemical properties. This makes them suitable for a wide range of applications in fields such as electronics, energy storage, and sensing ,2,3. In Despite these efforts, there are still many questions that need to be addressed, particularly with regard to the optimization of polymer properties for specific applications such as bioelectronics. Furthermore, the development of new electroconductive polymers and materials with improved performance is of ongoing importance. This is particularly true for applications in demanding environments, where high levels of performance and stability are required ,9,10.In the last years, electroactive materials such as copper oxide (CuO) ,12, polyThe unique properties of these electroactive materials have enabled their use in a wide range of bioelectronics and biological applications. These applications include biosensors for monitoring various biomolecules and metabolites, tissue engineering for the regeneration of damaged tissues, and drug delivery for targeted and controlled release of therapeutic agents. In this context, the use of these materials embedded into nanofibers has the potential to revolutionize the biotechnology field. It can provide a foundation for future research efforts aimed at optimizing the properties of electroconductive polymers with piezoelectric platforms and expanding their use in various fields. Herein, a comparative study on composite piezoelectric PVDF-TrFE fibers embedded with electroactive phases, i.e., namely, CuO, P3HT, CuPc, and MB, was proposed in order to investigate the correlations among electroconductive properties, morphology, and in vitro response.Nanofibers were fabricated using PVDF-TrFE  dissolved in tetrahydrofuran (THF) at a 13 wt% solution, as published elsewhere.  The actiThe morphology of the nanofibers was investigated by using scanning electron microscopy, SEM (JEOL JSM-IT-100). Briefly, a properly sized nanofiber sample was mounted on a sample holder by carbon double adhesive tape. Selected images were used for quantitative analyses. In detail, fiber diameters were measured from 100 nanofibers randomly using the SEM built-in measurement system. Meanwhile, energy dispersive spectroscopy (JEOL JSM-IT-100: EDS) analysis was used for a qualitative estimation of the elemental composition of the nanofibers.Electrical properties of the composites were evaluated by drop casting the composite solutions over interdigitated gold electrodes to reveal the bulk electrical conductivity in air, at laboratory conditions. The electrical characterization was performed by three sweeps: from 0 V to 5 V, 5 V to −5 V, and −5 V to 0. This sweep shows possible charge traps and redox properties in the drop-casted films promoted by the characteristic properties of the conductive phase and the randomness of the electroactive molecules in the piezoelectric polymer.2 cell culture flask in Dulbecco’s Modified Eagle Medium  supplemented with 10% of fetal bovine serum , antibiotic solution , and 2 mM of L-glutamine . The cells were incubated at 37 °C in a humidified atmosphere with 5% of CO2 and 95% air. Before the assays, samples were cut and placed into 96-well cell culture and sterilized with UV light for 30 min.For in vitro assays, L929 cell line  were used. The L929 cells were cultured in a 75 cm5 cells per well onto PVDF, P3HT, MB, CuO, and CuPc fibers. The cell culture plate (TCP) was used as a control. At each scheduled time, the culture medium was replaced with 100 μL of fresh medium and 50 mL XTT solution was added, followed by 4 h of incubation. After the incubation time, the supernatant was measured using a plate reader  at 450 nm of absorbance. The XTT assays were based on the reduction of tetrazolium salt XTT by mitochondrial enzymes into soluble formazan dye by living cells. In order to evaluate morphology and cell spreading, L929 were investigated by confocal laser scanning microscopy . Cell Tracker™ Green CMFDA in phenol red-free medium at standard conditions. Then, samples were washed with PBS  solution and incubated for 1 h in complete medium. After the incubation, samples were washed three times and cells were fixed with 4% of PFA. Before imaging, samples were stained with 1 μg/mL DAPI  for 5 min, and then washed. For proliferation tests, 6 × 103 cells per well were seeded onto P3HT, MB, and CuPc electrospun fibers to then perform cell proliferation at 1, 3, and 7 days by using XTT assay. The resulting amount of formazan dye was directly proportional to the number of viable cells for each electroactive culture.The cytotoxicity of fibers was evaluated by using the XTT assay protocol  at 24 and 72 h. The L929 cells were seeded at a density of 1 × 10The combination of electroactive components with piezoelectric matrices represents an interesting strategy to fabricate innovative devices. These devices have customizable electroconductive properties as a function of the peculiar properties of the dispersing phases. In this context, electrospinning offers the opportunity to emphasize this effect by creating electrostatic interactions among polar groups at the surface interface.Current–voltage (I–V) curves shown in Methylene blue shows the highest ability to transport charges, following CuPc, in accordance with fibers diameters being the thinnest for MB, and followed similarly by P3HT and CuPc. While P3HT poorly conducts in air, it improves charge transfer during the fiber formation, enhancing the formation and the decrease in fiber diameter ,28.The electrical conductivity of electroactive materials and their composites is determined by the movement of charge carriers through the material. The conductivity is affected by factors such as the material’s crystal and molecular structure, doping level, percolation, and presence of impurities or defects. Copper oxide exhibits semiconducting properties, and its conductivity mechanism involves the movement of electrons through the material. Poly(3-hexylthiophene) is a conjugated polymer with semiconductive properties, and its conductivity mechanism involves the movement of holes through the polymer chains. Copper phthalocyanine is a small molecule organic semiconductor, and its conductivity mechanism involves the movement of ambipolar charge carriers through the material. Methylene blue acts as a redox-active dye, and its conductivity mechanism involves the transfer of electrons between the dye molecules and the electrodes via a hopping mechanism. The conductivity mechanisms of these materials can be enhanced by increasing their degree of crystallinity, improving the alignment and packing of the molecules, and facilitating charge carrier hopping. Additionally, the conductivity of the studied composites is influenced by the percolation of the active material into the composite and the properties of the PVDF-TrFE.When PVDF-TrFE is added to these materials, it can have various effects on their conductivity mechanisms, depending on the nature of the interaction between them, as a function of the specific properties of active components used. In particular, the addition of PVDF-TrFE acts as a binder that mechanically facilitates the formation of the nanofibers and a well-connected network of particles or molecules in the composite, which can improve charge transport through the material. Additionally, PVDF-TrFE can also act as a dopant, introducing additional charge carriers into the material and increasing its conductivity. The presence of PVDF-TrFE can also modify the surface chemistry of the electrodes, leading to changes in the energy-level alignment at the interface and affecting the electron injection and charge transfer processes.In the case of composite PVDF-TrFE fibers, the electrical conductivity can be influenced by the morphology and microstructure of the composite material. The presence of PVDF-TrFE can promote the formation of a more ordered crystalline structure, which can improve charge transport and enhance the overall conductivity of the composite material . AdditioTaking into consideration the different response of composite PVDF-TrFE nanofibers in terms of morphology and electrical properties, in vitro studies were performed in order to validate their biocompatibility and suggest the best formulation for potential biomedical uses.In vitro tests were performed on PVDF-TrFE and their composites embedded with CuO, P3HT, CuPc, and MB fibers firstly to investigate cell adhesion at 24 and 72 h . The resIn vitro response was also confirmed by the morphological investigation performed via confocal microscopy a–c. The The trend was also confirmed by in vitro tests performed to evaluate cell proliferation . In thisIn other terms, cell viability and proliferation assays showed that L929 cultured on P3HT and MB composites had higher viability and proliferation rates compared with those cultured on CuO and CuPc composites. These results are consistent with previous studies that have shown that P3HT and MB can promote cell adhesion and proliferation due to their surface properties and charge transfer mechanisms ,32. In cThese promising results demonstrated a good biocompatibility of composite meshes, able to successfully support adhesion and proliferation of fibroblasts. Accordingly, further investigations could be performed in order to evaluate also the antibacterial response for a potential application of PVDF-TrFE composite nanofibers for physical filtration and/or static entrapment of bacteria.PVDF-TrFE electrospun nanofibers are recently emerging as interesting substrates with piezo/electroconductive properties suitable for a wide range of applications in the biomedical area. Indeed, the use of PVDF-TrFE provides the strength, flexibility, and mechanical stability needed to optimize the morphology of nanofibers for scaffolds and/or bio-functional platforms. The piezoelectric and triboelectric properties of PVDF allow for the generation of electrostatic charges, which can be used to trap particles ,35, moleIn conclusion, the present study explored the morphology and in vitro response of electroconductive composite nanofibers for biomedical applications. Blending PVDF-TrFE with different electroconductive materials such as CuO, P3HT, CuPc, and MB resulted in unique combinations of electrical conductivity, biocompatibility, and other desirable properties. Morphological analysis revealed that the fiber size was affected by the type of electroconductive phase used. The in vitro assays demonstrated a tunable response of the fibers in terms of cell viability and proliferation, with P3HT-loaded fibers showing the most suitable interaction with fibroblast cells. These findings provide valuable information for researchers and engineers in the field of biomedical engineering and electronics, which can help optimize the properties of composite nanofibers and expand their use in bioelectronics. Additionally, further investigations could be performed to evaluate the antibacterial response of PVDF-TrFE composite nanofibers for physical filtration and/or the static entrapment of bacteria. Overall, the study highlights the potential of electroconductive composite nanofibers as a promising material for a broad application in the biomedical area, from tissue engineering to biosensors, to biofiltration.


Kaposi sarcoma (KS) is an acquired immunodeficiency syndrome-defining condition that primarily manifests as mucocutaneous lesions; however, other organs have been implicated in disseminated disease. Fortunately, since the development of antiretroviral therapy, the incidence of KS among patients with human immunodeficiency virus has significantly declined. We report an atypical case of a rapidly progressing pulmonary KS to highlight the importance of prompt recognition of this condition, which can be challenging to distinguish from other pulmonary infectious diseases in immunocompromised individuals, as well as discuss the current treatment for this disease. Kaposi sarcoma (KS) is a malignancy caused by human herpesvirus 8 (HHV8), which was first identified in its association with human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) in the 1980s . KS is r3, viral load 22,100 copies/mL) who was non-compliant with bictegravir/emtricitabine/tenofovir (Biktarvy). He initially presented to the hospital due to shortness of breath, productive cough, and intermittent fever for several weeks. He also noted the development of raised, purple lesions on the left forearm and abdomen four months prior. A computerized tomography (CT) of the chest showed extensive bilateral lung perilymphatic nodularity, nodular interlobular septal thickening in a flame-shaped morphology (Figure We present the case of a 33-year-old male with a recent diagnosis of HIV/AIDS (CD4 383 mmy Figure , and bily Figure , suspiciHe was subsequently discharged from the emergency department due to hemodynamic stability with plans for outpatient follow-up with pulmonology. He was instructed to be compliant with his ART and prophylactic trimethoprim-sulfamethoxazole and valacyclovir.One month later, he underwent outpatient flexible bronchoscopy with biopsy, which revealed numerous macular, erythematous endobronchial lesions within the mainstem Figure  and bila3, hemoglobin of 8.5 g/dL, platelet count of 74 × 103/mL, elevated lactate dehydrogenase of 336 U/L, lactate of 2.2 mmol/L, and procalcitonin of 3.02 ng/mL. His cryptococcal antigen and respiratory panel were negative. Initial chest X-ray revealed multifocal bilateral airspace opacities (Figure Three weeks later, he presented to the hospital due to worsening respiratory status and high-grade fevers. Upon arrival, respiratory rate was 30-40 breaths/minute, and heart rate was 100 beats/minute. Labs were significant for CD4 of 73 mmCT of the chest revealed severe diffuse ground-glass opacities Figure  and bilaHome Biktarvy along with prophylactic trimethoprim-sulfamethoxazole and valacyclovir were continued. Hematology/Oncology was consulted to treat KS, who advised ruling out an infectious component before starting chemotherapy.Pneumocystis jirovecii, Legionella pneumophila, and fungal cultures were negative. He was extubated to a high-flow nasal cannula but continued with significant respiratory distress. Repeat CD4 was 70 mm3. As an infectious process was ruled out, Hematology/Oncology recommended initiating chemotherapy. However, after discussing his poor prognosis, he ultimately decided not to proceed with chemotherapy and was discharged to home hospice.His hospital course was quickly complicated by increasing oxygen requirements, for which he was intubated and underwent a repeat bronchoscopy to obtain a bronchoalveolar lavage for ruling out an underlying infection. Respiratory culture, cytology, acid-fast bacillus, 3 [3. The patient presented twice with vague pulmonary complaints initially attributed to a more likely cause, such as an infection. This prompted a complete infectious workup which led to a delay in diagnosing pulmonary Kaposi and, eventually, a delay in therapy.KS is an opportunistic malignancy, occurring in patients with a CD4 count less than 200 mm3 ,6,8. In 3 or ≥two-fold rise in baseline CD4+ cell count [3.The diagnosis of pulmonary KS can usually be confirmed through a combination of clinical, laboratory, radiography, bronchoscopy, and transbronchial biopsy ,10. Simill count . Our patThere are two main treatment pillars for managing KS, namely, ongoing ART and concurrent chemotherapy agents such as liposomal doxorubicin and paclitaxel . Some stWhile ART has played a role in preventing and treating KS, there is still much to be learned regarding further disease management. The pathogenesis of KS is still not well understood. One pathway which is being studied is vascular endothelial growth factor inhibition and the use of checkpoint inhibitors ,3. PrognHere, we present a unique case of rapidly progressing pulmonary KS. Pulmonary Kaposi has a median survival of 1.6 years; however, our case quickly progressed over the course of six months. Due to its increased morbidity, it is imperative to raise awareness for the potential pulmonary involvement of KS in patients with AIDS. As in our case, it is also important to rule out any underlying infectious process before starting chemotherapy. The early recognition of pulmonary KS is crucial to reach an early diagnosis and ensuring timely and successful treatment to prevent fatal outcomes.


Heterocapsa circularisquama, which selectively kills bivalve mollusks. This species has been detected exclusively in southwestern Japan. The completely unexpected outbreak of H. circularisquama in the northern region is believed to have been caused by the contamination of purchased seedlings with this species. The water quality and nutrient data collected by our group from July through October over the past 10 years revealed that the environment of Lake Kamo has not changed significantly. However, in the open water around Sado Island, where Lake Kamo is located, the water temperature has increased by 1.80 °C in the last 100 years, which is equivalent to 2–3-fold the world average. This has resulted in a rise in the sea level, which is expected to further deteriorate the water exchange between Lake Kamo and the open sea and low dissolved oxygen in the bottom layer of the Lake and the associated dissolution of nutrients from the bottom sediment. Therefore, seawater exchange has become insufficient and the lake has become nutrient rich, making it prone to the establishment of microorganisms, such as H. circularisquama, once they have been introduced. We developed a method to mitigate the damage caused by the bloom by spraying sediments containing the H. circularisquama RNA virus (HcRNAV), which infects H. circularisquama. After ∼10 years of performing various verification tests, including field trials, this method was used at the Lake in 2019. During the 2019 H. circularisquama growth season, a small amount of sediment containing HcRNAV was sprayed on the lake three times, which resulted in a decrease in H. circularisquama and an increase in HcRNAV, indicating that this method is effective in diminishing the bloom.Lake Kamo is an enclosed, low-inflow estuary connected to the open sea that is famous for oyster farming in Japan. In the fall of 2009, this lake experienced its first bloom of the dinoflagellate  Heterocapsa circularisquama Horiguchi, which is one of the most harmful microalgae, forms harmful algal blooms (HABs) that specifically kill bivalves  can infect H. circularisquama  in Lake Kamo (38°03′32″N latitude to 138°26′39″E longitude) . Lake KaH. circularisquama cells in one mL of unfixed water sample was counted under 100-fold magnification using a light microscope  immediately after sampling. A glass slide with a boundary line was used for the count . The remaining samples were kept at −20 °C in the laboratory until HcRNAV enumeration. The amount of HcRNAV was quantified by the MPN assay up to 2015 (see ‘MPN Assay’) and by mRT-qPCR after 2016, as reported by Lake water was collected from depths of 0 m (the surface) and 1 m above the bottom of the lake (B-1 m) using a bucket and a Kitahara water-sampling bottle . Water temperature and dissolved oxygen were measured using a Portable Dissolved Oxygen Meter . Salinity was measured with a Portable Electrical Conductivity Meter DKKCM-31P  and the calibration was performed manually before each survey in accordance with the manufacturer’s instructions. The number of Sediment samples were collected from the surface layer (the upper ∼30 mm) using an Ekman–Birge grab and maintained at −20 °C until the experiment was performed. The subsequent series of steps, such as RNA extraction and cDNA synthesis, were processed within 6 months after the samples were frozen.For analysis, 10 mL of the sample was filtered with a Membrane Filter Unit Dismic® Series, with a pore size of 0.22 µm  to determine the nutrient levels in the water, and kept at −20 °C until the measurements were performed. The concentrations of dissolved inorganic nitrogen , dissolved inorganic phosphorus (DIP), and dissolved silicon (DSi) in the water samples were measured using an AutoAnalyzer .H. circularisquama blooms occur frequently and HcRNAV accumulation is expected. The sediment was stored at −20 °C until the spraying was carried out, which killed harmful and toxic algal cysts.The sediment used in this study was collected from the thin surface layer (upper ∼30 mm) using an Ekman–Birge grab at St. 7 in Lake Kamo  in NovemH. circularisquama populations to demonstrate the effectiveness of the spraying of sediment containing HcRNAV in 2011, 2016, 2018. In 2019, a pilot spreading of sediment containing HcRNAV was conducted at Lake Kamo, to check each of the work procedures in the field. Sediment was collected and frozen the previous year. First, approximately 5 kg (wet weight) of the sediment was naturally thawed at room temperature the day before the test. On the day of application, approximately 5 L of lake water containing H. circularisquama was collected in a bucket, and the thawed bottom sediment was added and stirred well. After incubation for ∼3 h, we traveled to a fixed point using a research boat and sprayed three locations in the order of St. 6, 2, and 7. For spraying, a 10-L bucket was used, and seawater containing sediment was poured into the bucket while moving slowly in the research boat. Sediment spraying was conducted on July 30, August 16, and September 25, 2019. Regarding the evaluation of sediment dispersal, usual monitoring was performed, i.e., by comparing the density change of H. circularisquama and HcRNAV with past data. H. circularisquama was quantified by direct counting under a light microscope  immediately after sampling, and HcRNAV was assessed using mRT-qPCR , according to the manufacturer’s instructions.For RNA extraction from water samples, aqueous samples (10 mL) were centrifuged at 100,000 × For RNA extraction from sediments samples, 2 g of wet weight from each sediment sample was used for RNA extraction using the RNA PowerSoil Total RNA Isolation Kit , in accordance with the manufacturer’s instructions.® qPCR RT Master Mix with gDNA remover , as per the manufacturer’s instructions. The concentration of the extracted RNA to be used for cDNA synthesis was confirmed using a Bio Spec-nano spectrophotometer , as the kits require an RNA range between 0.5 pg and 0.5 µg for efficient cDNA synthesis.The purified RNA was reverse transcribed using the ReverTra AceThe density of HcRNAV was quantified, as described by 0–10−8 in SWM3 medium. Subsequently, aliquots were added to eight wells of a 96-well microtiter plate containing exponentially growing specific host algae, as described below. The plates were incubated for 7 d and checked daily for cell lysis , HCLG-1 , and 05HC06 . The algal cells were incubated in a modified SWM3 medium enriched with 2 nM Na2SeO3 (−2 s−1) was provided by cool white fluorescent illumination.Seawater samples were mixed well and serially diluted from 10ll lysis . The dilll lysis . In this Na2SeO3  at 20 °CH. circularisquama in Lake Kamo was examined using data obtained from 2009 to 2021. ANOVA and GLM analyses were performed for the aforementioned measurements in years when H. circularisquama occurred, as well as in other years, in addition to simple correlation analysis. The analyses were performed using the SPSS computer software .The relationship between water temperature, dissolved oxygen, salinity, DIN, DIP, and DSi concentrations and the occurrence of H. circularisquama was first observed in Lake Kamo in October 2009, and has been occurring annually in the lake since H. circularisquama emerges around June or July, proliferates in the lower layers (below 6 m), reaches one or two growth peaks, and is usually undetectable by early November; moreover, it has not been detected from December to May. Two patterns of H. circularisquama proliferation were observed .Lake Kamo is in-flowed by four rivers and is connected to the open sea by a channel . HoweverH. circularisquama reportedly cannot grow at temperatures <10 °C and may overwinter by forming temporary cysts ; in fact, the bottom layer was highly dense at the beginning of the proliferation period, in July .During the 10-year study period, 2015 was an exceptional year. In this year, the density of H. circularisquama. Furthermore, when water conditions, including low oxygen, are unfavorable for competing species, such as diatoms, it is likely that H. circularisquama would become dominant, i.e., the relationship between the poor oxygenation derived from the closed conditions of the Lake and competing species has a significant impact on the dominance of H. circularisquama, which was artificially introduced into the Lake through the transfer of oyster seedlings, and on the constancy of the blooms of this species.Closed saltwater lakes, such as Lake Kamo, where seawater exchange is slow and water temperature and salinity are stable, are considered suitable for the growth of microorganisms such as H. circularisquama bloom occurred, the seawater was collected in a small container, such as a bucket, and the sediment with HcRNAV was thawed and added to the seawater, followed by incubation for over 3 h. Subsequently, the HcRNAVs in the sediment began to infect H. circularisquama, i.e., this seawater with the sediment included a mixture of HcRNAV-infected H. circularisquama and HcRNAV-containing sediments, which can be spread to increase the virus. As a result, HcRNAVs in the seawater serially infected H. circularisquama, eventually leading to the decline of the H. circularisquama bloom. This method was effective and safe and had low cost in that it used autochthonous sediment containing enormous amounts of HcRNAV and artificially accelerated natural events. In general, several issues need to be addressed before biological methods can be applied practically. Previous studies have revealed the following issues: the HcRNAV-containing sediment is sufficiently infectious, the frozen bottom sediment remains infectious for at least 3 years (−1), the timing of spraying should be more effective when H. circularisquama density is low, and the spreading of autochthonous sediment is harmless to other aquatic organisms   . Based oH. circularisquama development, to suppress its proliferation. In the case of Lake Kamo, St. 6, located at the inner end of the lake, is the initial outbreak point of H. circularisquama; St. 2 is the deepest point and easily anoxic; and St. 7 is an area where plankton gathers easily because of the wind; thus, spraying at these three points is efficient. In addition, it is important for the fishermen in the field to be able to handle it easily. In other words, efficiency must be improved. The two-step inoculation method used in this mitigation study (spraying with increasing amounts of HcRNAV) can be effective because it minimizes the amount of sediment with HcRNAV that needs to be collected and spread , it is likely that, when the lower layers of Lake Kamo become anoxic, competing species cannot survive, and H. circularisquama can proliferate and become dominant.Regarding water temperature, it is unlikely that low temperature promoted the termination of the blooms in Lake Kamo, because of Japan , and bloH. circularisquama can grow at much lower DIN and DIP based on past emergence trends in western Japan. H. circularisquama cultures and found that it had the lowest values among dinoflagellates. This indicates that H. circularisquama has a greater ability to adapt to environments with lower nutrient concentrations compared with other phytoplankton species. These observations suggest that the contribution of HcRNAV to H. circularisquama decline is relatively large.Regarding nutrients, H. circularisquama bloom before spraying, to allow for the propagation of HcRNAV, The use of a polyethylene tank allowed the omission of the steps of transferring between containers and mixing with a stick to prepare for spraying. Furthermore, preparation in polyethylene tanks facilitated the transport and spread of seawater with sediment containing HcRNAV.For this viral control method to be sustainable in the field, the process needs to be simple and efficient for fishermen. The spraying performed in 2020 was improved by the fishermen. As a preliminary preparation, sediment containing HcRNAV was mixed with seawater containing −2 (−2 of clay was used to control Cochlodinium polykrikoides blooms in Korea (Even in field tests, there were concerns about the environmental impact of sediment dispersal during the practical application of this method. During the dispersal performed in 2019, approximately 1.5 kg per station was spread., At St.2, St.6, and St.7 the dispersal was performed simultaneously. For comparison, the clay dosage used at aquaculture sites in Japan was 110–400 t km−2 , whereasin Korea . CompareH. circularisquama more easily and successfully by spreading frozen sediment with accumulated HcRNAV produced in Lake Kamo several times from the early stage of H. circularisquama development. Although it is difficult to rigorously evaluate the effectiveness of field methods because it is not possible to set up controls, the effectiveness and efficiency of the method could be improved by considering the relationship between HcRNAV in nature and environmental factors, such as DO and competitors, and the characteristics of HcRNAV when spraying sediments.This viral control method was finally put into practical use in 2019. Together with previous results, we showed that it is possible to control H. circularisquama and environmental factors, the results of this study suggest that low oxygen conditions are suitable for the growth of H. circularisquama and that H. circularisquama is more likely to dominate because of a decrease in competing species, which may have contributed to the establishment of H. circularisquama. Under these circumstances, we have established and implemented a method to reduce the damage caused by the H. circularisquama bloom by spraying sediments containing HcRNAV, which infects and kills this dinoflagellate. During the 2019 H. circularisquama growth season, a small amount of sediment containing HcRNAV was dispersed in Lake Kamo, which resulted in a decrease in H. circularisquama density. The synchronous increase in HcRNAV indicated that this method was effective in diminishing the blooms in the field.Long-term increases in water temperature and water level in ambient waters connected to Lake Kamo have modified the lake, which is enclosed and has a poor seawater circulation rate. Hence, the lake has become stagnant, its DO has decreased, and nutrients have become concentrated. Although there were no significant correlations between the occurrence of 10.7717/peerj.14813/supp-1Supplemental Information 1H. circularisquama and HcRNAV from July to October in 2010–2020.The density of Click here for additional data file.10.7717/peerj.14813/supp-2Supplemental Information 2The temporal changes recorded from July to October in 2010–2020 for water temperature, salinity, and dissolved oxygenClick here for additional data file.10.7717/peerj.14813/supp-3Supplemental Information 3The temporal changes recorded from July to October in 2013–2020 for DIN, SIP and DSiClick here for additional data file.


Although the influence of consumer reviews is increasing in weight, far-flung consumer comments on social networks are a retrogressive problem, disturbing users' attention in reviews studies by interpreting them as misleading messages. The need for investigating the unknown meaning layers of fake reviews in the stepwise topic movement of conversations and examining the effects of the fake reviews on consumers’ shopping attitudes encouraged us to adopt an integrated approach to marketing and discourse analyses.•identification of topic opening; then, topic closing procedure; and finally, the topic switch toward topic drift.Qualitative analysis: To qualitatively investigate the stepwise topic movement of fake reviews in each sampled conversation of the research, three phases were taken into consideration: firstly, •Quantitative investigation: we develop a questionnaire using multidisciplinary research variables. Then, the reliability and validity of the questionnaire were assessed using Cronbach's alpha and convergent and discriminant values, respectively. After that, the questionnaire was evaluated among a research sample. The data was analysed based on structural equation modeling (SEM) and machine learning (ML).•Conclusion: It was found that fake reviews using topic coherence and grammatical-lexical cohesion mechanisms had positive effects on shopping attitudes. Moreover, fake reviews using topic drift mechanisms influenced consumers’ shopping attitudes.  Specifications TableConsumer-based information in the form of online reviews is valuable for potential customers and, therefore, represents an essential element in the shopping attitude . In receCoherency as a dynamic process arises from mutual efforts of the communicator and addressee on a topic . This phAddressing fake reviews is crucial for maintaining the credibility of online review systems and ensuring consumers can make informed choices based on reliable information. In this context, discourse-based methodologies, guided by a discursive lens, offer novel avenues for grasping marketing as both an academic discipline and how marketing scholars perceive and investigate topics within this domain of study. Accordingly, our research is primarily focused on examining how fake reviews within online conversations impact consumers' shopping attitudes. We place a particular emphasis on delving into the previously unexplored layers of meaning embedded within these fake reviews and their contribution to the progression of topics in online discussions. Therefore, we are aimed to investigate which factors of topic coherence and lexical-grammatical cohesion are used by users with fake accounts that affect users' shopping attitudes in online written conversations and also which topic drift elements are applied by users with fake accounts in online reader-reader interactions that affect users’ purchasing attitude. Our study addresses a notable knowledge gap related to the influence of fake reviews, leveraging mechanisms like topic coherence, grammatical-lexical cohesion, and topic drift, on consumers' shopping attitudes. In summary, our investigation aims to reveal concealed aspects of fake reviews and their consequential impact on shopping attitudes, thereby enriching our understanding of their role in online discourse and consumer decision-making processes. In this regard, our research employs an integrated approach, incorporating both qualitative and quantitative methods, along with structural equation modeling (SEM) and machine learning (ML). This integration is relevant because it allows us to comprehensively explore the multifaceted aspects of our research topic, enhancing the depth and robustness of our analysis.This study was conducted from October 2021 to April 2022, when Instagram marketing in Iran grew following the outbreak of the coronavirus. Instagram was less filtered during than other social networks the post-corona period; hence, it was more widely used among groups and organizations in the Islamic Republic of Iran.The methodological approach taken in this study is a mixed methodology. For quantitative analysis, a language-based model derived from the multidisciplinary integrated approach to marketing and discourse analysis was obtained by investigating the purposed sample in order to extract different sub-classes. Accordingly, data sampling has been purposely performed based on the publicly published reviews of Digikala as the most famous Holding company in Iran–up to 3000 reviews- on Instagram.In this regard, we employed several knowledge-based techniques allowing us to identify fake accounts using criteria ranging from following/like ratios, followers' quality, Instagram account owner reliability, and others. Then, for quantitative investigation, a questionnaire was developed according to multidisciplinary research variables. The reliability and validity of the questionnaire were assessed using Cronbach's alpha and convergent and discriminant values, respectively.After measuring the validity and reliability of the questionnaire, it was distributed among 369 individuals. Finally, the data were analysed both SEM and ML.identification of topic opening; then, topic closing procedure (coherence and cohesion in topic movement); and finally, the topic switch toward topic drift [Topic opening occurred when the brand shared designated content, prompting users to contribute their perspectives. As the conversation advances, topic movement occur when new discussions emerge among participants, yet it maintains continuity with the recently introduced subject. Furthermore, there are instances of boundary topical movement, where the conclusion of one topic gives way to the initiation of another, even including responses related to unrelated reviews. Topic drift takes place when the central topic is temporarily neglected or even permanently abandoned. The discourse variables of coherence, including cohesion and topic drift, were investigated following the further clarification of the multidisciplinary research variable of consumer attitude.To qualitatively investigate the stepwise topic movement of fake reviews in each sampled conversation of the research, three phases were taken into consideration: firstly, ic drift . Topic oWhile Halliday  emphasizA stepwise movement is the development of a topic, whereas boundary movement concerns the limits between topics. Sacks  distinguCohesion has been proposed to be the structural relations on the text surface, while coherence is the structural relations underlying the surface . There aTopic drift occurs when the global topic is temporarily neglected or even permanently abandoned. Discourse then develops according to what is associatively closest or “easiest to say next”  rather than to what the speaker's projected goals of the global topic demand. In topic drift, each segment coheres with the preceding and the following segment, but where the conversation ends up is quite far from where it started. Topic drift occurs by means of successive small modifications in the topic .According to Hobbs , the subIn social networks, when consumers are more involved with the product, they should be more worried about the consumer attitude toward shopping  and haveThe current research employed a combination of structural equation modeling (SEM) and machine learning (ML) approaches. The initial phase of our results focused on utilizing SEM as a powerful tool for predicting the research model. Subsequently, we applied the ML approach to cluster various consumer segments. It's worth noting that both of these clustering algorithms fall under unsupervised machine learning methods. The integration of SEM and ML presents practical opportunities, enabling businesses to make well-informed decisions and enhance their strategies through data-driven consumer insights.Based on the theoretical background just presented, the figure below shows the model proposed and submitted for the empirical test. The independent variables include Topic Coherence, Lexical-grammatical Cohesion, and Topic drift, and the dependent variable is the shopping attitude.Presumably, the first main hypothesis was proposed as “topic coherence device used by users with fake accounts affects users' shopping attitudes.” Furthermore, our findings show our main second hypothesis stating that “grammatical-lexical cohesion mechanisms used by users with fake accounts affect users' shopping attitudes.” Regarding the subcomponents of grammatical-lexical cohesion, it can be stated that this hypothesis involves several secondary hypotheses suggesting that each of these devices, i.e., reference, substitution, conjunction, simple repetition, complex repetition, synonymy, hyponymy, meronymy, co-hyponymy and order set collocation, utilized by the users with fake accounts have a positive effect on users' shopping attitudes. However, other devices, including ellipsis, contrast, activity-related collocation, and elaborative collocation, used by users with fake accounts have adverse effects on users' shopping attitudes.Finally, we believe that our research has probably usefully employed a third main hypothesis which states, “topic drift mechanisms used by users with fake accounts affect users' shopping attitudes.” As for the sub-categories of this hypothesis, it has to be noted that semantic parallelism, chained explanation, metatalk, and also break have had a negative effect on users' shopping attitudes.The research variables employed in this study have been subject to expert evaluation. The questionnaire comprises 22 questions, each corresponding to specific research variables, namely topic coherence, lexical-grammatical cohesion, topic drift, and shopping attitude. These variables are instrumental in investigating the nuances of consumer behavior within the digital realm, thereby yielding invaluable insights for the optimization of marketing strategies and the enhancement of the holistic consumer experience. In formulating the research inquiries, we utilized a 5-option Likert scale, a widely recognized measurement tool. This scale presents respondents with a series of statements and response options, enabling them to express their level of agreement or disagreement with specific subjects or concepts, whether positively or negatively. In essence, this scale functions as a comprehensive assessment tool, streamlining the evaluation of respondents' attitudes and beliefs.The questionnaire was analyzed in terms of their reliability, by means of the internal consistency  and also validity, considering convergent and discriminant validity. Reliability, initially assessed by Cronbach's alpha, was computed in two stages: considering the original items of each scale and after that, the exclusion of those with lower correlations with the scale. To uphold the content validity of our research instrument, we initiated the questionnaire development process by extensively reviewing pertinent literature. Subsequently, leveraging expert judgment, we meticulously refined the questionnaire by incorporating essential adjustments. During this critical phase, the questionnaire was meticulously reviewed and scrutinized by a panel of six experts, who provided invaluable insights into the suitability of each item for effectively measuring the intended constructs.The statistical population of the present study consists of all people who are members of the social network Instagram. Respondents were instructed to consider reality while answering the questions with transparency and loyalty.The current research employed a combination of structural equation modeling (SEM), and machine learning (ML) approaches.SEM is analysed and interpreted in two phases, namely, the assessment of the measurement model where indicator items are examined and the assessment of the structural model, which involves examining the significance of the path coefficients, model of predictive relevance, and the like ,33.The tables below show the measurement model of coherence and cohesion and topic drift in shopping attitudes, which explains the factor loadings and reliability of the constructs based on the comparative fit index (CFI). CFI is an index of “good fit,” ranging from 0 to 1, which quantifies the proportional improvement in structural equation model fit over a “null” model .As can be seen, the study examined the mediating effect of topic coherence and lexical-grammatical cohesion, and topic drift on the influence of attitude on shopping and employed the internal consistency approach to determine the reliabilities of all the items of the constructs used in the study by using CFI. The covariance and correlations of the data indicate that all constructs differ from one another consequently; thus, discriminant validity is supported See , Table 3The table above shows that the relationship between the latent variables is significant.Furthermore, the table below displays the standardized correlation coefficient of the relationships between the variables. In general, the standardized correlation coefficient of the relationships between the variables, which is often 0.5, indicates the high intensity of this relationship.A structural model represented, estimated, and tested a theoretical network or (mostly) linear relation between variables . Also, wThe structural model above illustrates the internal relationship between research variables. The indices of the latent variable “Topic Coherence and Lexical Grammatical Cohesion” were evaluated with 14 independent variables. Among these indicators, Topic Drift with one independent variable, Lexical Cohesion with seven independent variables, and Grammatical Cohesion with six variables were measured. Also, the indicators of Topic Drift as a latent variable were measured by four independent variables. Finally, the latent variable of Shopping attitude was examined by four independent variables.In general, it can be observed that the relationship between research variables is positive and direct. Furthermore, there is a positive and direct correlation between the latent variable of “Topic Coherence and Lexical-grammatical Cohesion” with “Shopping Attitude,” “Topic Drift” with “Topic Coherence and Lexical-grammatical Cohesion,” and also “Topic Drift” with “Shopping Attitude.” It has to be noted that the correlation between the two latent variables of “Topic Coherence and Lexical-grammatical Cohesion” and “Shopping Attitude” is more intense.age, level of education, the extent of users' activity on Instagram, and gender. No preprocessing steps were required as there was no missing value in the data set.This study also aimed to examine the relationship between 369 Instagram users and their feedback on this social network using 26 questions, four of which addressed the users' personal information, including Accordingly, unsupervised learning uses artificial intelligence (AI) algorithms to identify patterns in datasets containing data points that are neither classified nor labeled. In this regard, clustering as a data mining technique groups unlabeled data by their similarities or differences. Meanwhile, exclusive clustering is a form of grouping stipulating a data point existing only in one cluster . After eTo measure the distinctness of clusters, the K-means clustering model was used to divide the participants into two groups with labels 0 and 1, containing 316 and 53 members, respectively.Moreover, the correlation among the concerned variables is calculated and demonstrated in the following Heatmap .Fig. 6Hethe extent of users' activity on Instagram and the following variables coherence, cohesion , topic drift , user's attitude, and gender. However, the intensity of the relationships is limited by the displayed values. Furthermore, there is an inverse relationship between gender and user's attitude and between age and user's attitude. Although the relationships between user's attitude and coherence and between attitude and gender are significant, their relationships are not intensive. Finally, the relationship between the rest of the variables is obtained directly and significantly.This figure shows the inverse relationships between Fake reviews are deliberately fabricated expressions that have the potential to lead to financial repercussions and a decline in customer loyalty. These reviews are not always straightforwardly binary, as some may contain elements of truth alongside falsehoods. Previous research has primarily focused on binary classification and has examined the impact of fake reviews using content analysis models and marketing approaches. However, these investigations have yet to fully leverage an integrated model that encompasses various discourse-based approaches, such as analyzing topic coherence, cohesion, and topic drift, to comprehensively understand their influence on consumer shopping attitudes.Even though Fiedler and Kissling  points osemantic parallelism, chained explanations, metatalk, and break were found to have a negative effect on users' shopping attitudes. Thus, to create a gap in the conversation, fake reviews mention what has already been mentioned in the reviews, repeat a sentence during the conversation, mention a new issue, or completely change the subject of the conversation.Finally, the most striking result from evaluating the third hypothesis is that fake reviews using topic drift mechanisms can affect users' shopping attitudes. Simply speaking, all of the topic drift mechanisms, including In essence, these strategies are employed to manipulate online discussions, sway public opinion, or create the illusion of a more diverse and genuine conversation when, in reality, it may be orchestrated by a single entity with a specific agenda. It's important for readers to be aware of these tactics and critically assess online content, especially when encountering reviews or comments that appear suspicious or overly orchestrated.This article was supported by the Iran National Science Foundation and Alzahra University, Tehran, Iran with a number of 99,016,793.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.