The ethylene-responsive transcription factor ERF024 is a novel regulator of climacteric fruit ripening in melon.

Fruit ripening is an essential developmental stage in Angiosperms triggered by hormonal signals such as ethylene, a major player in climacteric ripening. Melon is a unique crop showing both climacteric and non-climacteric cultivars, offering an ideal model for dissecting the genetic mechanisms underpinning this process. The major quantitative trait locus ETHQV8.1 was previously identified as a key regulator of melon fruit ripening. Here, we narrowed down ETHQV8.1 to a precise genomic region containing a single gene, the transcription factor CmERF024. Functional validation using CRISPR/Cas9 knock-out plants unequivocally identified CmERF024 as the causal gene governing ETHQV8.1. The erf024 mutants exhibited suppression of ethylene production, leading to a significant delay and attenuation of fruit ripening. Integrative multi-omic analyses encompassing RNA-seq, DAP-seq, and DNase-seq revealed the association of CmERF024 with chromatin accessibility and gene expression dynamics throughout fruit ripening. Our data suggest CmERF024 as a novel regulator of climacteric fruit ripening in melon.

Lysine harvesting is an antioxidant strategy and triggers underground polyamine metabolism.

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p3-5 in maintaining oxidant resistance is unknown6. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.

Emergence of cefiderocol resistance during ceftazidime/avibactam treatment caused by a large genomic deletion, including ampD and piuCD genes, in Pseudomonas aeruginosa.

We report the emergence of cefiderocol resistance during the treatment of a ST312 Pseudomonas aeruginosa respiratory infection with ceftazidime/avibactam. whole genome sequencing (WGS) revealed that resistance was caused by a large genomic deletion, including PiuDC (iron transport system) and AmpD (ampC negative regulator), driven by the integration of phage DNA. Thus, our findings alert that this type of deletion could be an efficient (two mechanisms in one step) specific cefiderocol resistance mechanism that might occur nonspecifically upon treatment with ß-lactams that select for AmpC overexpression.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

T-Cell Immune Responses in Newborns and Long-Term Sequelae in Congenital Cytomegalovirus Infection (CYTRIC Study).

OBJECTIVE: The objective of this study was to assess the role of T-lymphocyte immune responses in newborns with congenital cytomegalovirus (CMV) infection (cCMV) and their potential association with the development of long-term sequelae. STUDY DESIGN: A multicenter, prospective study from 2017 to 2022 was conducted across 8 hospitals in Spain. Blood samples were collected within the first month of life from neonates diagnosed with cCMV. Intracellular cytokine staining was employed to evaluate the presence of CMV-specific interferon-gamma (IFN-γ)-producing CD8+ and CD4+ T lymphocytes (CMV-IFN-γ-CD8+/CD4+) using flow cytometry. The development of sequelae, including hearing loss and neurologic impairment, was assessed during follow-up. RESULTS: In total, 64 newborns were included; 42 infants (65.6%) had symptomatic cCMV. The median age at the last follow-up visit was 25.3 months (IQR 20.1-34.4). Eighteen infants had long-term sequelae (28.1%), predominantly hearing loss (20.3%) and neurologic disorders (15.6%). No relationship was observed between total count or percentage of CMV-specific IFN-γ-CD8+ or CD4+ lymphocytes and long-term sequelae. Multivariable analysis demonstrated an association between lower total lymphocyte count and long-term sequelae (aOR 0.549, 95% CI: 0.323-0.833), which requires further study. CONCLUSIONS: CMV-specific IFN-γ-CD4+ and CD8+ T-lymphocyte responses in neonates with cCMV were not predictive of long-term sequelae.

Letermovir use may impact on the Cytomegalovirus DNA fragmentation profile in plasma from allogeneic hematopoietic stem cell transplant recipients.

Cytomegalovirus (CMV) DNA in plasma is mainly unprotected and highly fragmented. The size of the amplicon largely explains the variation in CMV DNA loads quantified across PCR platforms. In this proof-of-concept study, we assessed whether the CMV DNA fragmentation profile may vary across allogeneic hematopoietic stem cell transplant recipients (allo-SCT), within the same patient over time, or is affected by letermovir (LMV) use. A total of 52 plasma specimens from 14 nonconsecutive allo-SCT recipients were included. The RealTime CMV PCR (Abbott Molecular), was used to monitor CMV DNA load in plasma, and fragmentation was assessed with a laboratory-designed PCR generating overlapping amplicons (around 90-110 bp) within the CMV UL34, UL80.5, and UL54 genes. Intrapatient, inter-patient, and LMV-associated qualitative and quantitative variations in seven amplicons were observed. These variations were seemingly unrelated to the CMV DNA loads measured by the Abbott PCR assay. CMV DNA loads quantified by UL34_4, UL54.5, and UL80.5_1 PCR assays discriminate between LMV and non-LMV patients. Our observations may have relevant implications in the management of active CMV infection in allo-SCT recipients, either treated or not with LMV, although the data need further validation.

SARS-CoV-2-Spike T-cell response after receiving one or two Wuhan-Hu-1-based mRNA COVID-19 vaccine booster doses in elderly nursing home residents.

The effect of COVID-19 booster vaccination on SARS-CoV-2 T-cell mediated immune responses in elderly nursing home residents has not been explored in depth. Thirty-nine elderly nursing home residents (median age, 91 years) were included, all fully vaccinated with mRNA vaccines. The frequency of and the integrated mean fluorescence (iMFI) for peripheral blood SARS-CoV-2-Spike reactive IFN-γ-producing CD4+ or CD8+ T cells before and after the first (Pre-3D and Post-3D) and second (Pre-4D and Post-4D) vaccine booster doses was determined using flow cytometry for an intracellular staining method. 3D increased significantly (p = 0.01) the percentage of participants displaying detectable SARS-CoV-2-T-cell responses compared with pre-3D (97% vs. 74%). The magnitude of the increase was statistically significant for CD8+ T cells (p = 0.007) but not for CD4+ T cells (p = 0.77). A trend towards higher frequencies of peripheral blood SARS-CoV-2-CD8+ T cells was observed post-3D compared with pre-3D (p = 0.06). The percentage of participants with detectable SARS-S-CoV-2 CD4+ T-cell responses decreased post-4D (p = 0.035). Following 4D, a nonsignificant decrease in the frequencies of both T cell subsets was noticed (p = 0.94 for CD8+ T cells and p = 0.06 for CD4+ T cells). iMFI data mirrored that of T-cell frequencies. The kinetics of SARS-CoV-2 CD8+ and CD4+ T cells following receipt of 3D and 4D were comparable across SARS-CoV-2-experienced and -naïve participants and between individuals receiving a homologous or heterologous vaccine booster. 3D increased the percentage of elderly nursing home residents displaying detectable SARS-CoV-2 T-cell responses but had a marginal effect on T-cell frequencies. The impact of 4D on SARS-CoV-2 T-cell responses was negligible; whether this was due to suboptimal priming or rapid waning could not be ascertained.

Biochemical Clusters as Substitutes of Bone Biopsies in Kidney Transplant Patients.

Bone and mineral metabolism abnormalities are frequent in kidney transplant recipients and have been associated with cardiovascular morbidity. The primary aim of this study was to analyse the association between routine clinically available biochemical evaluation, non-routine histomorphometric bone evaluation, and vascular disease in kidney transplanted patients. A cross-sectional analysis was performed on 69 patients, 1-year after kidney transplantation. Laboratory analysis, radiography of hands and pelvis, bone biopsy, bone densitometry, and coronary CT were performed. One-year post-transplantation, nearly one-third of the patients presented with hypercalcemia, 16% had hypophosphatemia, 39.3% had iPTH levels > 150 pg/mL, 20.3% had BALP levels > 40 U/L, and 26.1% had hypovitaminosis D. Evaluation of extraosseous calcifications revealed low Adragão and Agatston scores. We divided patients into three clusters, according to laboratory results routinely used in clinical practice: hypercalcemia and hyperparathyroidism (Cluster1); hypercalcemia and high BALP levels (Cluster2); hypophosphatemia and vitamin D deficiency (Cluster 3). Patients in clusters 1 and 2 had higher cortical porosity (p = 0.001) and osteoid measurements, although there was no difference in the presence of abnormal mineralization, or low volume. Patients in cluster 2 had a higher BFR/BS (half of the patients in cluster 2 had high bone turnover), and most patients in cluster 1 had low or normal bone turnover. Cluster 3 has no differences in volume, or turnover, but 60% of the patients presented with pre-osteomalacia. All three clusters were associated with high vascular calcifications scores. Vascular calcifications scores were not related to higher bone mineral density. Instead, an association was found between a higher Adragão score and the presence of osteoporosis at the femoral neck (p = 0.008). In conclusion, inferring bone TMV by daily clinical biochemical analysis can be misleading, and bone biopsy is important for assessing both bone turnover and mineralization after kidney transplantation, although hypophosphatemia combined with vitamin D deficiency is associated with abnormal mineralization. The presence of hypercalcemia with high levels of PTH or high levels of BALP, or hypophosphatemia and vitamin D deficiency should remind us to screen vascular calcification status of patients.Clinical Research: ClinicalTrials.gov ID NCT02751099.

Effect of chronic maternal L-Glu intake during gestation and/or lactation on oxidative stress markers, AMPA Glu1 receptor and adenosine A1 signalling pathway from foetal and neonatal cerebellum.

L-Glutamate (L-Glu) is an amino acid present in the diet that plays a fundamental role in the central nervous system, as the main excitatory neurotransmitter participating in learning and memory processes. In addition, the nucleoside adenosine has a crucial role in L-Glu metabolism, by regulating the liberation of this neurotransmitter through four different receptors: A1, A2A, A2B and A3, which activate (A2A and A2B) or inhibit (A1 and A3) adenylate cyclase pathway. L-Glu at high concentrations can act as a neurotoxin and induce oxidative stress. The study of the oxidative stress correlated with an excess of L-Glu consumption during maternity is key to understand its effects on foetuses and neonates. Previous studies have shown that there is a change in the receptor levels in the brain of pregnant rats and their foetuses when mothers are administered L-Glu during gestation; however, its effect on the cerebellum is unknown. Cerebellum is known to be responsible for motor, cognitive and emotional functions, so its possible involvement after L-Glu consumption is an important issue to study. Therefore, the aim of the present work was to study the effect of L-Glu exposure during gestation and lactation on oxidative stress biomarkers and neurotransmitter receptors from the cerebellum of foetuses and neonates. After maternal L-Glu intake during gestation, oxidative stress was increased, as the ionotropic L-Glu receptors, and GluR1 AMPA subunit levels were altered in foetuses. A1 adenosine receptor suffered changes after L-Glu treatment during gestation, lactation or both, in lactating neonate cerebellum, while adenylate cyclase activity remain unaltered. Further studies will be necessary to elucidate the importance of L-Glu intake and its possible excitotoxicity in the cerebellum of Wistar rats during the pregnancy period and their involvement in long-term neurodegeneration.

Abbott realtime MTB assay for detecting Mycobacterium tuberculosis complex in respiratory specimens: a cost-benefit analysis.

PURPOSE: Molecular screening for Mycobacterium tuberculosis (MTB) can lead to rapid empirical treatment inception and reduce hospitalization time and complementary diagnostic tests. However, in low-prevalence settings, the cost-benefit balance remains controversial due to the high cost. METHODS: We used a Markov model to perform an economic analysis to evaluate the profit after implementing molecular MTB screening (Period B) compared with conventional culture testing (Period A) in respiratory samples from 7,452 consecutive subjects with presumed tuberculosis (TB). RESULTS: The proportion of positivity was comparable between both periods (P > 0.05), with a total of 2.16 and 1.78 samples/patient requested in periods A and B, respectively (P < 0.001). The mean length of hospital stay was 8.66 days (95%CI: 7.63-9.70) in Period B and 11.51 days (95%CI: 10.15-12.87) in Period A (P = 0.001). The healthcare costs associated with diagnosing patients with presumed TB were reduced by €717.95 per patient with PCR screening. The probability of remaining hospitalized and the need for a greater number of outpatient specialty care visits were the variables with the most weight in the model. CONCLUSION: Employing PCR as an MTB screening method in a low-prevalence setting may increase the profits to the system.

Performance of the Roche Elecsys® IGRA SARS-CoV-2 test for the detection and quantification of virus-reactive T cells in COVID-19-vaccinated immunosuppressed patients and healthy subjects.

PURPOSE: Comparing the performance of commercially available SARS-CoV-2 T-cell immunoassay responses may provide useful information for future observational or intervention studies as well as to their potential customers. METHOD: Whole blood was collected from a total of 183 subjects fully vaccinated against COVID-19: 55 healthy controls (Group 1), 50 hematological patients (Group 2), 50 chronic kidney disease patients (Group 3), and 28 elderly nursing home residents (Group 4). Samples were tested with the Roche Elecsys® IGRA (Interferon-gamma release assay) SARS-CoV-2 test (Roche Diagnostics, Rotkreuz, Switzerland), the Euroimmun SARS-CoV-2 test (Euroimmun, Lubeck, Germany), the SARS-CoV-2 T Cell Analysis Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and a flow-cytometry for intracellular cytokine (IFN-γ) staining-based immunoassay (FC-ICS). RESULTS: Overall, the Roche Elecsys® assay returned the highest number of positive results (151/179; 84.3%), followed by the Euroimmun test (127/183; 69%), and the FC-ICS (135/179; 75%). The Kappa coefficient of agreement was best between IGRAs (0.64). Most discordant results across assays involved patients from Group 2. Overall, IFN-γ concentrations measured by both IGRAs correlated strongly (rho = 0.78; 95% CI 0.71-0.84; P < 0.001) irrespective of the study group. The frequencies of SARS-CoV-2-reactive IFN-γ T cells and IFN-γ concentrations measured by the IGRAs correlated moderately for CD4+ T cells, however, weakly for CD8+ T cells. SARS-CoV-2-experienced participants displayed stronger responses than SARS-CoV-2-naïve when IGRAs, rather than FC-ICS, were used. CONCLUSION: The SARS-CoV-2 immunoassays evaluated in the present study did not return interchangeable qualitative or quantitative results either in seemingly healthy individuals or in immunosuppressed patients.

Outcome of Human Parainfluenza Virus infection in allogeneic stem cell transplantation recipients: possible impact of ribavirin therapy.

BACKGROUND: This retrospective study focused on analyzing community-acquired respiratory virus (CARV) infections, in particular human parainfluenza virus (hPIV) after allogeneic stem cell transplant (allo-SCT) in adults recipients. It aimed to assess the impact of ribavirin treatment, clinical characteristics, and risk factors associated with lower respiratory tract disease (LRTD) progression and all-cause mortality. PATIENTS AND METHODS: The study included 230 allo-SCT recipients diagnosed with hPIV between December 2013 and June 2023. Risk factors for the development of LRTD, disease severity, and mortality were analyzed. Ribavirin treatment was administered at physician discretion in 61 out of 230 cases (27%). RESULTS: Risk factors for LRTD progression in multivariate analysis were corticosteroids > 30 mg/day (Odds ratio (OR) 3.5, 95% Confidence Interval (C.I.) 1.3-9.4, p = 0.013), fever at the time of hPIV detection (OR 3.89, 95% C.I. 1.84-8.2, p < 0.001), and absolute lymphocyte count (ALC) < 0.2 × 109/L (OR 4.1, 95% C.I. 1.42-11.9, p = 0.009). In addition, the study found that ribavirin therapy significantly reduced progression to LRTD [OR 0.19, 95% C.I. 0.05-0.75, p = 0.018]. Co-infections (OR 5.7, 95% C.I. 1.4-23.5, p = 0.015) and ALC < 0.2 × 109/L (OR 17.7, 95% C.I. 3.6-87.1, p < 0.001) were independently associated with higher day + 100 after hPIV detection all-cause mortality. There were no significant differences in all-cause mortality and infectious mortality at day + 100 between the treated and untreated groups. CONCLUSION: ALC, corticosteroids, and fever increased the risk for progression to LRTD while ribavirin decreased the risk. However, mortality was associated with ALC and co-infections. This study supports further research of ribavirin therapy for hPIV in the allo-HSCT setting.

Genotypic study of Chlamydia trachomatis for lymphogranuloma venereum diagnosis in rectal specimens from men who have sex with men: a cost-effectiveness analysis.

PURPOSE: The significant proportion of asymptomatic patients and the scarcity of genotypic analysis of lymphogranuloma venereum (LGV), mainly among men who have sex with men (MSM), triggers a high incidence of underdiagnosed patients, highlighting the importance of determining the most appropriate strategy for LGV diagnosis, at both clinical and economical levels. MATERIALS AND METHODS: We conducted L1-L3 serovar detection by molecular biology in stored Chlamydia trachomatis-positive samples from MSM patients with HIV, another STI or belonging to a Pre-exposure prophylaxis program, to make a cost effectiveness study of four diagnostic strategies with a clinical, molecular, or mixed approach. RESULTS: A total of 85 exudates were analyzed: 35urethral (31 symptomatic/4 positive) and 50 rectal (22 symptomatic/25 positive), 70/85 belonging to MSM with associated risk factors. The average cost per patient was €77.09 and €159.55 for clinical (Strategy I) and molecular (Strategy IV) strategies respectively. For molecular diagnosis by genotyping of all rectal exudate samples previously positive for CT (Strategy II), the cost was €123.84. For molecular diagnosis by genotyping of rectal and/or urethral exudate samples from all symptomatic patients (proctitis or urethritis) with a previous positive result for CT (Strategy III), the cost was €129.39. The effectiveness ratios were 0.80, 0.95, 0.91, and 1.00 for each strategy respectively. The smallest ICER was €311.67 for Strategy II compared to Strategy I. CONCLUSIONS: With 30% asymptomatic patients, the most cost-effective strategy was based on genotyping all rectal exudates. With less restrictive selection criteria, thus increasing the number of patients with negative results, the most sensitive strategies tend to be the most cost-effective, but with a high incremental cost-effectiveness ratio.

Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Implementing a challenge-based learning experience in a bioinstrumentation blended course.

BACKGROUND: Bioinstrumentation is essential to biomedical engineering (BME) undergraduate education and professional practice. Several strategies have been suggested to provide BME students with hands-on experiences throughout the curriculum, promoting their preparedness to pursue careers in industry and academia while increasing their learning and engagement. This paper describes the implementation of challenge-based learning (CBL) in an undergraduate bioinstrumentation blended course over the COVID-19 pandemic. METHODS: The CBL experience was implemented in a third-year bioinstrumentation course from the BME program at Tecnologico de Monterrey. Thirty-nine students enrolled in two sections formed fourteen teams that tackled blended learning activities, including online communication, lab experiments, and in-person CBL activities. Regarding the latter, students were challenged to design, prototype, and test a respiratory or cardiac gating device for radiotherapy. An institutional student opinion survey was used to assess the success of our CBL implementation. RESULTS: Student responses to the end-of-term survey showed that they strongly agreed that this course challenged them to learn new concepts and develop new skills. Furthermore, they rated the student-lecturer interaction very positively despite the blended format. Overall, students assessed their learning experience positively. However, implementing this CBL experience required a substantial time increase in planning, student tutoring, and constant communication between lecturers and the industry partner. CONCLUSION: This work provides an effective instance of CBL for BME education to improve students' learning experience despite decreased resource efficiency. Our claim is supported by the student's performance and the positive feedback from our industrial partner.

Post-transplant dynamics and clinical significance of CMV-specific neutralizing antibodies in kidney transplant recipients treated with T-cell-depleting agents.

We measured cytomegalovirus (CMV)-specific antibodies that neutralize epithelial cell infection (CMV-AbNEIs) in 101 CMV-seropositive kidney transplant recipients (KTRs) at baseline and post-transplant months 3 and 6. All the patients received antithymocyte globulin and 3-month valganciclovir prophylaxis. There were no significant differences in pre-transplant AbNEIs titers between KTRs that developed or did not develop any-level CMV infection or the composite of high-level infection and/or disease. One-year CMV infection-free survival was comparable between KTRs with or without pre-transplant CMV-AbNEIs. No differences were observed by months 3 and 6 either. We observed no protective role for CMV-AbNEIs among CMV-seropositive KTRs undergoing T-cell-depleting induction.

Broad protection against invasive fungal disease from a nanobody targeting the active site of fungal b-1,3-glucanosyltransferases.

Invasive fungal disease accounts for ~3.8 million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotype D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insights about the role of b-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.

Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement.

Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.

Evaluation of a Quantitative Dual-Target EBV DNA Test on a Fully Automated Molecular Testing System.

The measurement of Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) is key to diagnosing and managing EBV-associated complications in transplant recipients. The performance of the new Conformité Européenne (CE) and Food and Drug Administration (FDA)-cleared quantitative Roche cobas EBV real-time PCR assay was determined by using EDTA-plasma dilution panels and clinical samples that were spiked with either the World Health Organization's EBV international standard or high-titer EBV lambda stock. Correlation with the Abbott Realtime EBV assay was assessed in clinical specimens and conducted at two independent laboratories. An in silico analysis revealed that the dual-target test (EBNA1 and BMRF2) was 100% inclusive for the known diversity of EBV. The overall limit of detection (LoD) was 16.6 IU/mL for genotype 1 (GT1). GT2 LoD was verified at 18.8 IU/mL. The linear ranges were from 1.40 × 101 to 2.30 × 108 IU/mL and from 2.97 × 101 to 9.90 × 107 IU/mL for GT1 and GT2, respectively. Accuracy was confirmed across the linear range (mean difference not exceeding ±0.18 log10). Precision was not influenced by the factors analyzed (standard deviation of 0.02 to 0.17 log10), including the presence of potentially interfering endogenous or exogenous substances. Plasma samples were stable under several conditions (variable time points, storage, and freeze/thaw cycles). In clinical EBV DNA-positive samples, correlation between the cobas EBV test and the comparator was high (n = 126 valid results; R2 = 0.96) with a 0.1 mean log10 titer difference. The cobas EBV test is an accurate, sensitive, specific, and reproducible assay for the detection of EBV DNAemia in plasma. In general, high levels of automation and calibration to the international standard will lead to improvements in the harmonization of quantitative EBV DNA test results across laboratories.

Multi-site performance evaluation of the Alinity m Molecular assay for quantifying Epstein-Barr virus DNA in plasma samples.

Detection and monitoring of acute infection or reactivation of Epstein-Barr virus (EBV) are critical for treatment decision-making and to reduce the risk of EBV-related malignancies and other associated diseases in immunocompromised individuals. The analytical and clinical performance of the Alinity m EBV assay was evaluated at two independent study sites; analytical performance was assessed by evaluating precision with a commercially available 5-member EBV verification panel, while the clinical performance of the Alinity m EBV assay was compared to the RealTime EBV assay and a laboratory-developed test (LDT) as the routine test of record (TOR). Analytical analysis demonstrated standard deviation (SD) between 0.08 and 0.13 Log IU/mL. A total of 300 remnant plasma specimens were retested with the Alinity m EBV assay, and results were compared to those of the TOR at the respective study sites (n = 148 with the RealTime EBV assay and n = 152 with the LDT EBV assay). Agreement between Alinity m EBV and RealTime EBV or LDT EBV assays had kappa values of 0.88 and 0.84, respectively, with correlation coefficients r of 0.956 and 0.912, while the corresponding observed mean bias was -0.02 and -0.19 Log IU/mL. The Alinity m EBV assay had a short median onboard turnaround time of 2:40 h. Thus, the Alinity m system can shorten the time to results and, therefore, to therapy.