Single-cell brain organoid screening identifies developmental defects in autism.

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1-4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous extracellular matrix.

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.

Kaposi's sarcoma herpesvirus latency-associated nuclear antigen broadly regulates viral gene expression and is essential for lytic infection.

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.

Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

Pulmonary valve replacement in tetralogy of Fallot - who and how?

BACKGROUND AND AIM: Pulmonary regurgitation is the most common complication in repaired tetralogy of Fallot patients. Severe chronic pulmonary regurgitation can be tolerated for decades, but if not treated, it can progress to symptomatic, irreversible right ventricular dilatation and dysfunction. We investigated clinical associations with pulmonary valve replacement among patients with significative pulmonary regurgitation and how interventional developments can change their management. METHODS: All adult patients with repaired tetralogy of Fallot who were followed at an adult CHD Clinic at a single centre from 1980 to 2022 were included on their first outpatient visit. Follow-up was estimated from the time of correction surgery until one of the following events occurred first: pulmonary valve replacement, death, loss to follow-up or conclusion of the study. RESULTS: We included 221 patients (116 males) with a median age of 19 (18-25). At a median age of 33 (10) years old, 114 (51%) patients presented significant pulmonary regurgitation. Among patients with significant pulmonary regurgitation, pulmonary valve replacement was associated with male gender, older age at surgical repair, and longer QRS duration in adulthood. Pulmonary valve replacement was performed in 50 patients, including four transcatheter pulmonary valve implantations, at a median age of 34 (14) years. CONCLUSION: Pulmonary regurgitation affects a large percentage of tetralogy of Fallot adult patients, requiring a long-term clinical and imaging follow-up. Sex, age at surgical repair and longer QRS are associated with the need of PVR among patients with significative pulmonary regurgitation. Clinical practice and current literature support TPVI as the future gold standard intervention.

Cardiovascular magnetic resonance in muscular dystrophies: looking ahead.

Cardiac magnetic resonance (CMR) is an established tool for risk stratification in several cardiomyopathies, and its role in muscular dystrophies (MuD) looks promising. We sought to assess how CMR performs in predicting cardiac events in a real cohort of MuD patients. A prospective single-center study with the enrollment of consecutive adult MuD patients referred to cardiac screening from 2012 to 2018 with the collection of clinical and CMR data. During follow-up (FUP), major adverse cardiac events were considered a composite of device implantation, ventricular tachycardia (VT), hospitalization due to heart failure, and death. Sixty-five patients were included (mean age of 32±16, 51% female); the majority had myotonic dystrophy (34; 52.3%); most were asymptomatic (60; 92.3%) and at sinus rhythm (64; 98.5%). CMR was abnormal in 23 (43.3%) patients: left ventricle ejection fraction (LVEF) <55% was found in 7 patients, and late gadolinium enhancement (LGE) was present in 23 patients, mainly intra-myocardial or subepicardial (10 and 8 patients, respectively). During a median FUP of 77 months (interquartile range: 33), there were 7 deaths, 8 implanted devices, and one sustained VT. LVEF<55% and the presence of LGE were associated with the occurrence of all events (log rank test, p=0.002 and p=0.045, respectively). LVEF<55% was associated with a 6-fold higher risk of events (crude hazard ratio of 6.15; 95% confidence interval of 1.65-22.93), that remained significant after adjusting for LGE presence (adjusted hazard ratio of 4.81, 95% confidence interval of 1.07-15.9). In our cohort, CMR LVEF<55% and the presence of LGE were significantly associated with adverse events during follow-up, reinforcing the role of this technique on risk stratification of MuD populations.

First-degree atrioventricular block in hypertrophic cardiomyopathy patients: an easy and worthy prognostic marker?

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Recently, a connection has been observed between the presence of first-degree atrioventricular block (FDAVB) and cardiovascular outcomes, although the pathophysiology of this association remains poorly understood. Considering the period 2000-2023, we retrospectively included HCM patients at sinus rhythm at the first appointment and sought possible interactions of FDAVB (defined as PR interval >200 ms) with different clinical and imaging variables and with the occurrence of cardiovascular events, including atrial fibrillation (AF). A total of 97 patients were included, of whom 57 (58.8%) were men, with a mean age of 51±19 years, and 14 (14.4%) had FDAVB. During a median of 4.29 (P25 1.92, P75 7.67) years of follow-up, 35 cardiovascular events occurred, including 13 de novo diagnoses of AF, 8 hospitalizations due to heart failure, 8 new-onset strokes, 4 myocardial infarctions, and 2 implantations of cardio defibrillators in secondary prevention; no HCM-related death occurred. We did not find any association between outcomes and the presence of FDAVB. The role of FDAVB as a prognostic marker in HCM patients requires further investigation. We found that FDAVB patients were older, more frequently reported dyspnea, had a larger QRS duration, a higher E/e' ratio, and lower maximal left ventricle wall thickness by magnetic resonance (p<0.05). After multivariable analysis, FDAVB was independently associated with a higher echocardiographic E/e' ratio (p=0.039) (odds ratio=1.588). This is the first paper to document an independent association between FGAVB and a higher E/e' ratio in HCM patients.

Same-day discharge after elective percutaneous closure of patent foramen ovale.

Percutaneous closure of the patent foramen ovale (PFO) is increasingly performed in specific patients with cryptogenic stroke or clinical evidence of a paradoxical embolism. This study was performed to determine the safety of same-day discharge (SDD) following such procedures. This is a prospective, observational study of patients undergoing elective percutaneous PFO closure in a single tertiary center in Portugal between January 2020 and July 2023. AmplatzerTM devices (St. Jude Medical, St. Paul, MN, USA) and NobblestichTM EL (HeartStitch, Inc., Fountain Valley, CA, USA) were used. After 6 months, the following events were looked at: post-procedural paroxysmal atrial fibrillation, stroke, unplanned cardiac re-hospitalization, urgent cardiac surgery, major vascular complications, pericardial effusions, device embolization, and death. We studied 122 consecutive patients (52% female, 68; 48±12 years old) who had elective percutaneous closure with success and no complications. Forty-nine (40%) had SDD. AmplatzerTM devices were used more frequently in the SDD group, while NobblestichTM EL was more common in the overnight group. During the overnight group's follow-up period, there was one non-cardiovascular death; there were no further events. SDD after elective percutaneous closure of PFO was shown to be a safe and successful patient management method, including NobblestichTM, which we describe for the first time. Our results prove the safety of this same-day discharge strategy. We hypothesize that in the near future, in selected cases, PFO closure might become an ambulatory procedure.

20-year follow-up of rheumatic mitral stenosis patients after percutaneous mitral commissurotomy: invasive transmitral gradient differential as a predictor of events.

Percutaneous mitral valve commissurotomy (PMC) is a viable alternative to mitral valve (MV) surgery in the treatment of patients with rheumatic mitral stenosis (RMS). In this single-center retrospective study of consecutive patients with RMS submitted to PMC from 1991 to 2008, we analyzed clinical, echocardiographic, and hemodynamic data and events during follow-up (FUP) until December 2021. Major adverse cardiovascular events (MACE) were a combined endpoint of all-cause death, cardiovascular hospitalization, and MV re-intervention. A total of 124 patients were enrolled: 108 (87.1%) were female, with a mean age at PMC of 46 [standard deviation (SD) 11] years. PMC was successful in 91.1%, with a mean reduction in invasive transmitral pressure gradient (TMPG) of 8 (SD 7) mmHg at PMC time. During the mean FUP of 20 (SD 6) years, 51 (41.1%) patients had MV re-intervention (86.3% surgery and 13.7% redo-PMC), 37 (29.8%) were hospitalized, and 30 (24.2%) died. Approximately 75% of patients remained MACE-free after 10 years, and this percentage decreased to around 40% after 20 years; at this time mark, about 8 in 10 patients were alive. A reduction of <5 mmHg in TMPG at PMC time was associated with a 2.7-fold greater rate of MACE compared to a reduction of ≥5 mmHg, independent of MV regurgitation after PMC and moderate disease of other valves (adjusted hazard ratio 2,7; 95% confidence interval 1.395-5.298, p=0.003). In this cohort with favorable long-term results after PMC, a reduction of <5 mmHg in TMPG at PMC time was associated with MACE during FUP. More studies are needed to validate this independent predictor.

Celiac disease presenting as hemorrhagic shock: a rare complication.

Celiac disease (CD) is a systemic autoimmune disorder triggered by ingested gluten in patients with genetic susceptibility, with rising incidence in adults. In this population, symptoms can be atypical. We report the case of a 71-year-old woman that presented with coagulopathy due to severe vitamin K deficiency and hemorrhagic shock, as well as cholestatic hepatitis and chronic diarrhea. Serological studies were negative for CD, but a biopsy demonstrated duodenal atrophy and HLA typing was positive for DQ2.5, pointing to this diagnosis. The patient responded favorably to a gluten free diet. This case highlights the challenge in diagnosing CD in elderly patients due to its unusual manifestations, which can range from mild and unspecific to potentially life-threatening. The exclusion of other causes of duodenal atrophy is key in establishing the diagnosis, especially in seronegative cases, permitting the establishment of an adequate therapy and follow-up strategy.

Lung abscess in a non-compliant patient with Crohn's disease.

A 66-year-old man was admitted to the emergency department due to malaise, fatigue and anorexia for the last 2 weeks. He presented no fever, no respiratory or gastrointestinal symptoms. The patient had been previously diagnosed with Crohn's Disease (CD) (A2L1L4B1 of Montreal Classification) 10 years before, when he presented complaints of watery diarrhea and unexplained weight loss. Despite refusing to start treatment, in the last staging exams performed 5 years before the admission (colonoscopy and magnetic resonance imaging) the patient was in deep remission. Nevertheless, he frequently missed his medical appointments and his disease had not been monitored since then. He denied previous use of corticosteroids, past abdominal surgery or previous CD related hospital admissions. He also denied smoking habits or chronic lung disease.

First description of Portuguese patients with cardiac amyloidosis and p.Val142Ile: more evidence of an "African variant" in Caucasians.

Objectives. Hereditary transthyretin amyloidosis caused by the (ATTRv) p. Val142Ile variant is a common cause of cardiac amyloidosis among Western African countries and Afro-Americans populations. However, in recent years, Caucasian patients have been identified in greater numbers, raising the question of whether this variant has been undeappreciated in this population. We now have new cases of cardiac amyloidosis caused by the p.Val142Ile from a center in northern Portugal. In addition, we reviewed and discussed the published data concerning p.Val142Ile in Caucasians. Design. Patients diagnosed with cardiac amyloidosis underwent genetic testing using TTR gene sequencing and their relatives were recommended for genetic counsellingand testing if a pathogenic TTR variant was found. In our center, we reviewed the clinical data of patients who had the p.Val142Ile variant. A review of published cases of p.Val142Ile in Caucasians was also performed, to which our data was compared. Results. We found three ATTRv patients with the p.Val142Ile variant (one homozygotic), all Caucasian males with a median age at diagnosis of 69 years old. All of them had heart failure and arrhythmias. During the follow-up period, two patients died. There were 47 unrelated unrelated Caucasian cases of ATTRv p.Val142Ile variant reported worldwide until May 2022. Conclusions. Our findings add to the mounting evidence that the global prevalence of p.Val142Ile is likely understated. This highlights the importance of the systematic screening of the TTR gene in amyloidosis and phenocopies, as well as larger epidemiologic studies to determine the true ATTRv p.Val142Ile prevalence in non-African communities.

Interactions between Ionic Cellulose Derivatives Recycled from Textile Wastes and Surfactants: Interfacial, Aggregation and Wettability Studies.

Interactions between polymers (P) and surfactants (S) in aqueous solution lead to interfacial and aggregation phenomena that are not only of great interest in physical chemistry but also important for many industrial applications, such as the development of detergents and fabric softeners. Here, we synthesized two ionic derivatives-sodium carboxymethylcellulose (NaCMC) and quaternized cellulose (QC)-from cellulose recycled from textile wastes and then explored the interactions of these polymers with assorted surfactants-cationic (CTAB, gemini), anionic (SDS, SDBS) and nonionic (TX-100)-commonly used in the textile industry. We obtained surface tension curves of the P/S mixtures by fixing the polymer concentration and then increasing the surfactant concentration. In mixtures where polymer and surfactant are oppositely charged (P-/S+ and P+/S-), a strong association is observed, and from the surface tension curves, we determined the critical aggregation concentration (cac) and critical micelle concentration in the presence of polymer (cmcp). For mixtures of similar charge (P+/S+ and P-/S-), virtually no interactions are observed, with the notable exception of the QC/CTAB system, which is much more surface active than the neat CTAB. We further investigated the effect of oppositely charged P/S mixtures on hydrophilicity by measuring the contact angles of aqueous droplets on a hydrophobic textile substrate. Significantly, both P-/S+ and P+/S- systems greatly enhance the hydrophilicity of the substrate at much lower surfactant concentrations than the surfactant alone (in particular in the QC/SDBS and QC/SDS systems).

Acute myocardial infarction during late COVID-19 era: patient characteristics, presentation and outcomes.

COVID-19 pandemic has unquestionably influenced care of acute myocardial infarction (AMI). Still, its impact on patients (pts) characteristics, presentation, treatment, and outcomes remains not well established in late pandemic times. To address this issue, we performed a prospective study of type-1 AMI patients admitted in a tertiary care hospital. Pts were enrolled during 6-months in 2019 [n=122; pre-COVID-19 (PC) group] and in 2021 [n=196; late-COVID-19 (C) group]. Data was based on pts interview and review of medical records. Age and gender distribution, as well as ST/non-ST-elevation myocardial infarction (STEMI/NSTEMI) proportion and access to coronariography and revascularization were similar between groups. Group C patients presented more pre-existing established cardiovascular disease (CVD) (43% vs 30%; p=0.03); more frequent description of typical chest pain (94% vs 84%; p=0,002); higher levels of pain intensity, in a 0-10 scale (8±2 vs 7±2; p=0.02); higher frequencies of AMI complications (27% vs 15%; p=0.01) and worse Killip (K) class evolution (K≥2 in 22% C vs13% PC patients; p=0.05). In conclusion, late pandemic AMI patients presented worse in-hospital outcomes in our study, though pre-hospital and hospital care were comparable to pre-pandemic times. COVID patients had a higher burden of pre-existing established CVD and a more typical and intense symptom presentation. Therefore, it can be hypothesized that "sicker" patients continued to look for help when presenting AMI symptoms, while "less sick" patients and the ones with less typical and intense symptoms possibly avoided contact with health care services during late pandemic period.

Gastric ulcer with liver penetration.

A 54-year-old male with previous history of chronic hepatitis C complained of postprandial epigastric discomfort and weight loss. Esophagogastroduodenoscopy revealed a large gastric ulcer on the lesser antral curvature, with biopsies showing granulation tissue and inflammatory activity without other significant changes. Despite therapy with double-dose proton pump inhibitor, the ulcer had not healed or diminished in size at reevaluation endoscopy eight weeks later. Biopsies were repeated and again only revealed granulation tissue and inflammation. The patient undergone partial gastrectomy where fibrotic adhesions to liver and peritoneum were noted. Remarkably, macroscopical analysis of the surgical specimen revealed an hepatic fragment adherent to the ulcer and histopathological examination was consistent with liver penetration. Liver penetration is a rare but important complication that must be considered in the differential diagnosis of medically refractory gastric ulcers, even in the absence of hepatic tissue in endoscopic biopsies.

Seroprevalence of anti-SARS-CoV-2 antibodies in COVID-19 patients and healthy volunteers up to 6 months post disease onset.

SARS-CoV-2 has emerged as a human pathogen, causing clinical signs, from fever to pneumonia-COVID-19-but may remain mild or asymptomatic. To understand the continuing spread of the virus, to detect those who are and were infected, and to follow the immune response longitudinally, reliable and robust assays for SARS-CoV-2 detection and immunological monitoring are needed. We quantified IgM, IgG, and IgA antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) or the Spike (S) protein over a period of 6 months following COVID-19 onset. We report the detailed setup to monitor the humoral immune response from over 300 COVID-19 hospital patients and healthcare workers, 2500 University staff, and 198 post-COVID-19 volunteers. Anti-SARS-CoV-2 antibody responses follow a classic pattern with a rapid increase within the first three weeks after symptoms. Although titres reduce subsequently, the ability to detect anti-SARS-CoV-2 IgG antibodies remained robust with confirmed neutralization activity for up to 6 months in a large proportion of previously virus-positive screened subjects. Our work provides detailed information for the assays used, facilitating further and longitudinal analysis of protective immunity to SARS-CoV-2. Importantly, it highlights a continued level of circulating neutralising antibodies in most people with confirmed SARS-CoV-2.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata.

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

YEASTRACT: an upgraded database for the analysis of transcription regulatory networks in Saccharomyces cerevisiae.

The YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT-www.yeastract.com) information system has been, for 11 years, a key tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in Saccharomyces cerevisiae. Since its last update in June 2017, YEASTRACT includes approximately 163000 regulatory associations between transcription factors (TF) and target genes in S. cerevisiae, based on more than 1600 bibliographic references; it also includes 247 specific DNA binding consensus recognized by 113 TFs. This release of the YEASTRACT database provides new visualization tools to visualize each regulatory network in an interactive fashion, enabling the user to select and observe subsets of the network such as: (i) considering only DNA binding evidence or both DNA binding and expression evidence; (ii) considering only either positive or negative regulatory associations; or (iii) considering only one set of related environmental conditions. A further tool to observe TF regulons is also offered, enabling a clear-cut understanding of the exact meaning of the available data. We believe that with this new version, YEASTRACT will improve its role as an open web resource instrumental for Yeast Biologists and Systems Biology researchers.

A Transcriptomics Approach To Unveiling the Mechanisms of In Vitro Evolution towards Fluconazole Resistance of a Candida glabrata Clinical Isolate.

Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the C. glabrataPDR1 gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a C. glabrata population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.