Neutrophil immune profile guides spinal cord regeneration in zebrafish.

Spinal cord injury triggers a strong innate inflammatory response in both non-regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet, their role in the repair process, particularly in a regenerative context, remains largely unknown. Here, we show that, following rapid recruitment to the injured spinal cord, neutrophils mostly reverse migrate throughout the zebrafish body. In addition, promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil-specific RNA-seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf-α expression in macrophage/microglia populations. Conversely, blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether, these findings provide new insights into the role of neutrophils in spinal cord regeneration, emphasizing the significant impact of their immune profile on the outcome of the repair process.

Potential Environmental Reservoirs of Candida auris: A Systematic Review.

Candida auris, a multidrug-resistant yeast, poses significant challenges in healthcare settings worldwide. Understanding its environmental reservoirs is crucial for effective control strategies. This systematic review aimed to review the literature regarding the natural and environmental reservoirs of C. auris. Following the PRISMA guidelines, published studies until October 2023 were searched in three databases: PubMed, Web of Science, and Scopus. Information regarding the origin, sampling procedure, methods for laboratory identification, and antifungal susceptibility was collected and analyzed. Thirty-three studies published between 2016 and 2023 in 15 countries were included and analyzed. C. auris was detected in various environments, including wastewater treatment plants, hospital patient care surfaces, and natural environments such as salt marshes, sand, seawater, estuaries, apples, and dogs. Detection methods varied, with molecular techniques often used alongside culture. Susceptibility profiles revealed resistance patterns. Phylogenetic studies highlight the potential of environmental strains to influence clinical infections. Despite methodological heterogeneity, this review provides valuable information for future research and highlights the need for standardized sampling and detection protocols to mitigate C. auris transmission.

Brave New World of Artificial Intelligence: Its Use in Antimicrobial Stewardship-A Systematic Review.

Antimicrobial resistance (AMR) is a growing public health problem in the One Health dimension. Artificial intelligence (AI) is emerging in healthcare, since it is helpful to deal with large amounts of data and as a prediction tool. This systematic review explores the use of AI in antimicrobial stewardship programs (ASPs) and summarizes the predictive performance of machine learning (ML) algorithms, compared with clinical decisions, in inpatients and outpatients who need antimicrobial prescriptions. This review includes eighteen observational studies from PubMed, Scopus, and Web of Science. The exclusion criteria comprised studies conducted only in vitro, not addressing infectious diseases, or not referencing the use of AI models as predictors. Data such as study type, year of publication, number of patients, study objective, ML algorithms used, features, and predictors were extracted from the included publications. All studies concluded that ML algorithms were useful to assist antimicrobial stewardship teams in multiple tasks such as identifying inappropriate prescribing practices, choosing the appropriate antibiotic therapy, or predicting AMR. The most extracted performance metric was AUC, which ranged from 0.64 to 0.992. Despite the risks and ethical concerns that AI raises, it can play a positive and promising role in ASP.

A zebrafish xenotransplant model of anaplastic thyroid cancer to study tumor microenvironment and innate immune cell interactions in vivo.

Anaplastic thyroid cancer (ATC) is of the most aggressive thyroid cancer. While ATC is rare, it accounts for a disproportionately high number of thyroid cancer-related deaths. Here, we developed an ATC xenotransplant model in zebrafish larvae, where we can study tumorigenesis and therapeutic response in vivo. Using both mouse (T4888M) and human (C643)-derived fluorescently labeled ATC cell lines, we show these cell lines display different engraftment rates, mass volume, proliferation, cell death, angiogenic potential, and neutrophil and macrophage recruitment and infiltration. Next, using a PIP-FUCCI reporter to track proliferation in vivo, we observed cells in each phase of the cell cycle. Additionally, we performed long-term non-invasive intravital microscopy over 48 h to understand cellular dynamics in the tumor microenvironment at the single-cell level. Lastly, we tested two drug treatments, AZD2014 and a combination therapy of dabrafenib and trametinib, to show our model could be used as an effective screening platform for new therapeutic compounds for ATC. Altogether, we show that zebrafish xenotransplants make a great model to study thyroid carcinogenesis and the tumor microenvironment, while also being a suitable model to test new therapeutics in vivo.

Exploring the dynamic behavior of leukocytes with zebrafish.

Cell migration is a complex and intricate network of physical, chemical, and molecular events that ultimately leads to cell motility. This phenomenon is involved in both physiological and pathological processes such as proper immune and inflammatory responses. Dysregulation of cell migration machinery in immune cells can have a tremendous impact on the trajectory of inflammation, infection, and resolution. The small vertebrate, the zebrafish, has a remarkable capacity for genetic and pharmacological manipulation aligned to transparency that enables modulation and visualization of cell migration in vivo noninvasively. Such characteristics revolutionized the field of leukocyte biology, particularly neutrophils. In this review, we will focus on leukocyte migration and highlight findings made in the zebrafish that demonstrate how this small vertebrate system is a unique model to perform in vivo imaging and study mechanisms that regulate the dynamic behavior of immune cells in their native environment under homeostasis or upon challenge.

A zebrafish xenotransplant model of anaplastic thyroid cancer to study the tumor microenvironment and innate immune cell interactions in vivo.

Anaplastic thyroid cancer (ATC) is a rare malignant subtype of thyroid cancer. While ATC is rare it accounts for a disproportionately high number of thyroid cancer-related deaths. Here we developed an ATC xenotransplant model in zebrafish larvae, where we can study tumorigenesis and therapeutic response in vivo. Using both mouse (T4888M) and human (C643) derived fluorescently labeled ATC cell lines we show these cell lines display different engraftment rates, mass volume, proliferation, and angiogenic potential. Next, using a PIP-FUCCI reporter to track proliferation in-vivo we observed cells in each phase of the cell cycle. Additionally, we performed long-term non-invasive intravital microscopy over 48 hours to understand cellular dynamics in the tumor microenvironment at the single cell level. Lastly, we tested a well-known mTOR inhibitor to show our model could be used as an effective screening platform for new therapeutic compounds. Altogether, we show that zebrafish xenotransplants make a great model to study thyroid carcinogenesis and the tumor microenvironment, while also being a suitable model to test new therapeutics in vivo.

Effect of Vapor-Phase Oregano Essential Oil on Resistant Candida Species Biofilms: Mechanisms of Action.

Vulvovaginal candidiasis (VVC) is one of the most prevalent vaginal infectious diseases. The increasing incidence of drug-resistant Candida strains and the limited therapeutic options make the discovery of effective alternative therapies fundamental. Essential oils (EOs) have been suggested as a promising alternative, and interestingly, vapor-phase essential oils (VP-EOs) present more advantages than their direct application. Thus, this study aims to evaluate the effect of oregano VP-EO (VP-OEO) on biofilms of antifungal-resistant vaginal isolates of Candida species (Candida albicans and Candida glabrata) and determine its mode of action. CFU, membrane integrity, and metabolic activity were evaluated. Furthermore, a reconstituted vaginal epithelium was used to mimic vaginal conditions and evaluate the effect of VP-OEO on Candida species infection, analyzed by DNA quantification, microscopy, and lactate dehydrogenase activity. The results revealed high VP-OEO antifungal activity. There was a significant reduction (>4 log CFU) in Candida species biofilms. Furthermore, the results show that the mechanisms of action of VP-OEO are related to membrane integrity and metabolic activity. The epithelium model confirms the effectiveness of VP-OEO. This study suggests that VP-EO can be considered a first approach for the development of an alternative form of VVC treatment. IMPORTANCE This work presents a new approach to the application of essential oils, exposure to the vapor phase, which can be considered a first approach for the development of a complementary or alternative form of vulvovaginal candidiasis (VVC) treatment. VVC is a significant infection caused by Candida species and remains a common disease that affects millions of women every year. The great difficulty in treating VVC and the extremely limited effective therapeutic options make the development of alternative treatments crucial. In this scope, this study aims to contribute to the development of effective, inexpensive, and nontoxic strategies for the prevention and treatment of this infectious disease, based on natural products. Moreover, this new approach has several advantages for women, such as lower costs, easy access, an easier mode of application, avoidance of skin contact, and, therefore, fewer negative impacts on women's health.

Development of a nutritional risk screening tool for cancer patients undergoing outpatient treatment.

INTRODUCTION: Malnutrition is a common reality in many hospitals, especially in cancer patients. In order to avoid its late diagnosis, there are screening instruments that help professionals detect nutritional risk early, thus avoiding further damage to the nutritional status. OBJECTIVE: To develop a nutritional screening tool for cancer patients undergoing outpatient treatment. MATERIAL AND METHODS: This cross-sectional, observational study was carried out in cancer patients assisted at an outpatient clinic for nutritional care. Data were collected from nutritional care records, including bioelectrical and body composition data (by electrical bioimpedance spectroscopy), anthropometry (adductor pollicis muscle thickness, circumferences, and body mass index), routine clinical biochemical tests, and dynamometry. Malnourished patients were identified through screening using the NRS-2002 method, and all participants were classified according to the GLIM criterion as the control group (no risk and no malnutrition) and the malnourished group. The logistic regression method was used to select the variables that were more sensitive to nutritional risk, thus composing the final screening instrument. RESULTS: The study sample consisted of 72 patients, 58% male, with a mean age of 63 years (±13.3). The malnourished group had a higher frequency of sarcopenia, worse Karnofsky Performance Status (KPS), and lower values for weight, adductor pollicis muscle thickness (APMT), body mass index (BMI), calf circumference (CC), dynamometry (HAND), and fat mass index (FMI) than the control group. After multivariate analyses and analysis of covariance, the final model was devised in the form of an equation containing two variables, which were more associated with malnutrition, the BMI and the KPS. CONCLUSION: The screening instrument developed in this study resulted in an equation for screening nutritional risk, which included the variables KPS and BMI with 84% assertiveness.

Senescence-Independent Anti-Inflammatory Activity of the Senolytic Drugs Dasatinib, Navitoclax, and Venetoclax in Zebrafish Models of Chronic Inflammation.

Telomere shortening is the main molecular mechanism of aging, but not the only one. The adaptive immune system also ages, and older organisms tend to develop a chronic pro-inflammatory status with low-grade inflammation characterized by chronic activation of the innate immune system, called inflammaging. One of the main stimuli that fuels inflammaging is a high nutrient intake, triggering a metabolic inflammation process called metainflammation. In this study, we report the anti-inflammatory activity of several senolytic drugs in the context of chronic inflammation, by using two different zebrafish models: (i) a chronic skin inflammation model with a hypomorphic mutation in spint1a, the gene encoding the serine protease inhibitor, kunitz-type, 1a (also known as hai1a) and (ii) a non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) model with inflammation induced by a high-fat diet. Our results show that, although these models do not manifest premature aging, the senolytic drugs dasatinib, navitoclax, and venetoclax have an anti-inflammatory effect that results in the amelioration of chronic inflammation.

Biodegradable Bone Implants as a New Hope to Reduce Device-Associated Infections-A Systematic Review.

Bone fractures often require fixation devices that frequently need to be surgically removed. These temporary implants and procedures leave the patient more prone to developing medical device-associated infections, and osteomyelitis associated with trauma is a challenging complication for orthopedists. In recent years, biodegradable materials have gained great importance as temporary medical implant devices, avoiding removal surgery. The purpose of this systematic review was to revise the literature regarding the use of biodegradable bone implants in fracture healing and its impact on the reduction of implant-associated infections. The systematic review followed the PRISMA guidelines and was conducted by searching published studies regarding the in vivo use of biodegradable bone fixation implants and its antibacterial activity. From a total of 667 references, 23 studies were included based on inclusion and exclusion criteria. Biodegradable orthopedic implants of Mg-Cu, Mg-Zn, and Zn-Ag have shown antibacterial activity, especially in reducing infection burden by MRSA strains in vivo osteomyelitis models. Their ability to prevent and tackle implant-associated infections and to gradually degrade inside the body reduces the need for a second surgery for implant removal, with expectable gains regarding patients' comfort. Further in vivo studies are mandatory to evaluate the efficiency of these antibacterial biodegradable materials.

Efficacy of Acupuncture on Quality of Life, Functional Performance, Dyspnea, and Pulmonary Function in Patients with Chronic Obstructive Pulmonary Disease: Protocol for a Randomized Clinical Trial.

Chronic obstructive pulmonary disease (COPD) is a respiratory disease characterized by the presence of chronic airflow obstruction. Previous studies have evaluated the effect of acupuncture treatment (AT) in patients with COPD. Nevertheless, these studies show a great deal of heterogeneity in treatment protocols, having sample sizes that are too small to estimate and clarify effect size and heterogeneity in patients' baseline. The aim of this study is to evaluate the effectiveness of acupuncture on quality of life, functional performance, dyspnea, and pulmonary function in patients with COPD. As such, patients will go through the following three phases: Phase I-pretreatment: period of subject selection and inclusion in the protocol, with an interview and performance of exams and tests as follows: Mini-Cog, dual-energy X-ray absorptiometry, spirometry, the Patient-Generated Index, Saint George's Respiratory Questionnaire, the six-minute walk test, the London Chest Activity of Daily Living, and the COPD Assessment Test. Phase II-8 weeks of treatment, with AT 3 times a week, with two parallel groups: Group I-with 50 subjects-AT according to the recommended technical standards; Group II-with 50 subjects-Control, without acupuncture. Phase III-Continuation of AT for 8 weeks, maintaining the subjects in the previously allocated groups and following the same methodology.

Therapeutic targeting of the inflammasome in myeloid malignancies.

Even though genetic perturbations and mutations are important for the development of myeloid malignancies, the effects of an inflammatory microenvironment are a critical modulator of carcinogenesis. Activation of the innate immune system through various ligands and signaling pathways is an important driver of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The DAMPs, or alarmins, which activate the inflammasome pathway via the TLR4/NLR signaling cascade causes the lytic cell death of hematopoietic stem and progenitor cells (HSPCs), ineffective hematopoiesis, and ß-catenin-induced proliferation of cancer cells, leading to the development of MDS/AML phenotype. It is also associated with other myeloid malignancies and involved in the pathogenesis of associated cytopenias. Ongoing research suggests the interplay of inflammasome mediators with immune modulators and transcription factors to have a significant role in the development of myeloid diseases, and possibly therapy resistance. This review discusses the role and importance of inflammasomes and immune pathways in myeloid malignancies, particularly MDS/AML, to better understand the disease pathophysiology and decipher the scope of therapeutic interventions.

The Impact of Angiotensin-Converting Enzyme 2 (ACE2) Expression Levels in Patients with Comorbidities on COVID-19 Severity: A Comprehensive Review.

Angiotensin-Converting Enzyme 2 (ACE2) has been proved to be the main host cell receptor for the binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic. The SARS-CoV-2 spike (S) protein binds to ACE2 to initiate the process of replication. This enzyme is widely present in human organ tissues, such as the heart and lung. The pathophysiology of ACE2 in SARS-CoV-2 infection is complex and may be associated with several factors and conditions that are more severe in COVID-19 patients, such as age, male gender, and comorbidities, namely, cardiovascular diseases, chronic respiratory diseases, obesity, and diabetes. Here we present a comprehensive review that aims to correlate the levels of expression of the ACE2 in patients with comorbidities and with a poor outcome in COVID-19 disease. Significantly higher levels of expression of ACE2 were observed in myocardial and lung tissues in heart failure and COPD patients, respectively. An age-dependent increase in SARS2-CoV-2 receptors in the respiratory epithelium may be also responsible for the increased severity of COVID-19 lung disease in elderly people. Although the role of ACE2 is highlighted regarding the damage that can arise upon the SARS-CoV-2 invasion, there was no association observed between renin-angiotensin-aldosterone system (RAAS) inhibitors and the severity of COVID-19.

Analysis of Liver Microenvironment during Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish.

Liver cancer is currently the third leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Excessive R-loops trigger an inflammatory cascade leading to increased HSPC production.

Hematopoietic stem and progenitor cells (HSPCs) arise during embryonic development and are essential for sustaining the blood and immune systems throughout life. Tight regulation of HSPC numbers is critical for hematopoietic homeostasis. Here, we identified DEAD-box helicase 41 (Ddx41) as a gatekeeper of HSPC production. Using zebrafish ddx41 mutants, we unveiled a critical role for this helicase in regulating HSPC production at the endothelial-to-hematopoietic transition. We determined that Ddx41 suppresses the accumulation of R-loops, nucleic acid structures consisting of RNA:DNA hybrids and ssDNAs whose equilibrium is essential for cellular fitness. Excess R-loop levels in ddx41 mutants triggered the cGAS-STING inflammatory pathway leading to increased numbers of hemogenic endothelium and HSPCs. Elevated R-loop accumulation and inflammatory signaling were observed in human cells with decreased DDX41, suggesting possible conservation of mechanism. These findings delineate that precise regulation of R-loop levels during development is critical for limiting cGAS-STING activity and HSPC numbers.

Species Distribution and Antifungal Susceptibility Profiles of Isolates from Women with Nonrecurrent and Recurrent Vulvovaginal Candidiasis.

Recurrent vulvovaginal candidiasis (RVVC) is caused by Candida spp., a vaginal colonizer. Despite the clinical importance of RVVC, little is known regarding the characteristics of the disease in Portugal. Thirty-six clinical cases were analyzed, comprising 93 yeast vulvovaginal isolates obtained from women attending a gynecologic consultation at a private clinic. Of these, 18 women were diagnosed with RVVC, while other 18 women had a sporadic episode of infection (nonrecurrent vulvovaginal candidiasis [NR-VVC]). Species identification was performed with CHROMagar chromogenic medium and by analysis of biochemical profiles. In addition, antifungal susceptibility testing for two azole compounds was performed by broth microdilution. We found that Candida albicans was isolated from both NR-VVC and RVVC cases, being highly predominant; C. glabrata and C. tropicalis were also isolated. Resistance to at least one antifungal was detected in up to 65% of the isolates, and resistance to both antifungals reached a frequency of 25%. Moreover, azole-resistant isolates were distributed among all species identified. We conclude that in the studied group of patients, C. albicans is in fact the major player both in NR-VVC and in RVVC, C. glabrata being more frequently associated with recurrence (p < 0.05). In addition, we found a high proportion of azole-resistant strains.

FKS1 mutation associated with decreased echinocandin susceptibility of Aspergillus fumigatus following anidulafungin exposure.

Invasive aspergillosis (IA) is a potentially lethal infection that affects mostly immunocompromised patients caused by Aspergillus fumigatus. Echinocandins are a second-line therapy against IA, used as a salvage therapy as well as for empirical or prophylactic therapy. Although they cause lysis of growing hyphal tips, they are considered fungistatic against molds. In vivo echinocandins resistance is uncommon; however, its wide clinical use could shortly lead to the emergence of A. fumigatus resistance. The aims of the present work was to assess the development of reduced echinocandins susceptibility phenotype by a A. fumigatus strain and to unveil the molecular mechanism underlying such phenotype. We induced in vitro cross-resistance to echinocandins following exposure of A. fumigatus to anidulafungin. Stability of the resistant phenotype was confirmed after removal of anidulafungin pressure. The FKS1 gene was partially sequenced and a E671Q mutation was found. A computational approach suggests that it can play an important role in echinocandin resistance. Given the emerging importance of this mechanism for clinical resistance in pathogenic fungi, it would be prudent to be alert to the potential evolution of this resistant mechanism in Aspergillus spp infecting patients under echinocandins therapeutics.

DnaJ-PKAc fusion induces liver inflammation in a zebrafish model of fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that affects adolescents and young adults. Genomic analysis of FLC has revealed a 400 kb deletion in chromosome 19 that leads to the chimeric transcript DNAJB1-PRKACA (DnaJ-PKAc), comprised of the first exon of heat shock protein 40 (DNAJB1) and exons 2-10 of the catalytic subunit of protein kinase A (PRKACA). Here, we report a new zebrafish model of FLC induced by ectopic expression of zebrafish Dnaja-Pkaca (zfDnaJa-Pkaca) in hepatocytes that is amenable to live imaging of early innate immune inflammation. Expression of zfDnaJa-Pkaca in hepatocytes induces hepatomegaly and increased hepatocyte size. In addition, FLC larvae exhibit early innate immune inflammation characterized by early infiltration of neutrophils and macrophages into the liver microenvironment. Increased Caspase-a (the zebrafish homolog for human caspase-1) activity was also found in the liver of FLC larvae, and pharmacological inhibition of Tnfα and caspase-a decreased liver size and inflammation. Overall, these findings show that innate immune inflammation is an early feature in a zebrafish model of FLC and that pharmacological inhibition of TNFα or caspase-1 activity might be targets to treat inflammation and progression in FLC patients.This article has an associated First Person interview with the first author of the paper.

Candida albicans Antifungal Resistance and Tolerance in Bloodstream Infections: The Triad Yeast-Host-Antifungal.

Candida albicans represents the most frequent isolated yeast from bloodstream infections. Despite the remarkable progress in diagnostic and therapeutic approaches, these infections continue to be a critical challenge in intensive care units worldwide. The economic cost of bloodstream fungal infections and its associated mortality, especially in debilitated patients, remains unacceptably high. Candida albicans is a highly adaptable microorganism, being able to develop resistance following prolonged exposure to antifungals. Formation of biofilms, which diminish the accessibility of the antifungal, selection of spontaneous mutations that increase expression or decreased susceptibility of the target, altered chromosome abnormalities, overexpression of multidrug efflux pumps and the ability to escape host immune defenses are some of the factors that can contribute to antifungal tolerance and resistance. The knowledge of the antifungal resistance mechanisms can allow the design of alternative therapeutically options in order to modulate or revert the resistance. We have focused this review on the main factors that are involved in antifungal resistance and tolerance in patients with C. albicans bloodstream infections.