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En las últimas décadas del siglo XIX, Eduardo Ladislao Holmberg emprende excursiones científicas y paseos de coleccionista por gran parte del territorio argentino. Entre los corolarios que se desprenden de esas experiencias se destaca la producción de textos que testimonian lo acontecido en sus exploraciones. Holmberg inicia su narrativa viajera con “Una excursión por el Río Luján”, diario de viaje publicado en El Naturalista Argentino, el primer periódico nacional consagrado al estudio de las ciencias naturales, escrito y dirigido por científicos autóctonos. Abordar este libro de viaje focalizándonos en la mirada del viajero naturalista que lo estructura constituye el objeto del presente trabajo.

Objectives: An increase in Streptococcus dysgalactiae subsp. equisimilis (SDSE) infections has been documented worldwide. This study aims to analyse invasive disease caused by SDSE (iSDSE) in adults over an 11-year period in Spain., Methods: We conducted a retrospective, laboratory-based study of iSDSE detected at Hospital Universitari de Bellvitge (HUB) from 2012 to 2022 (n=89) and isolates collected in three Spanish hospitals in 2018 (n=22). Clinical data from HUB was collected. Isolates were tested for antimicrobial susceptibility (EUCAST 2023), subjected to whole genome sequencing (WGS) and analysed for mobile genetic elements (MGEs). A mouse model was used to analyse virulence., Results: iSDSE episodes at HUB occurred predominantly in older patients with comorbidities (diabetes, chronic heart disease, malignancies, particularly). WGS revealed a high genetic diversity, with the most common lineages being CC15, CC17, and CC20. Various virulence factors, including the superantigen speG, were identified. Macrolides, lincosamides, and tetracyclines exhibited the highest resistance rates (above 27%) and changed over time, linked to multiple MGEs. The mouse model highlighted the virulence of the CC20-stG62647 lineage, but these results were discordant with clinical data., Conclusion: iSDSE incidence is increasing and is associated with older patients with comorbidities. Genetically, SDSE is diverse with a high capacity to integrate MGEs carrying resistance determinants. Mouse model studies showed the enhanced virulence of the CC20-stG62647 lineage. These findings underscore the need for ongoing surveillance of this emerging pathogen., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Ltd.)

Background: Streptococcus pneumoniae causes invasive pneumococcal disease (IPD) in adults. The introduction of pneumococcal conjugate vaccines (PCVs) has reduced vaccine serotypes but has also led to the rise of non-vaccine serotypes. The aim of this study was to analyse pneumococcal lineages and their association with recent changes in IPD among adults in Spain., Methods: Data from adult IPD cases (≥18 years) were collected from six Spanish hospitals in 2019-2021. Strains were serotyped, tested for antibiotic susceptibility and subjected to whole genome sequencing (WGS). Findings were compared with data from previous periods (2008-2016)., Results: A total of 655 IPD episodes were examined. Pneumonia was the main focus (515/655), and 366 episodes occurred in adults over 64 years. Although IPD incidence decreased during COVID-19 pandemic, the burden of disease caused by PCV13 serotypes was significant. Notably, serotype 3 persisted (GPSC12-ST180 and GPSC83-ST260), and a new serotype 4 lineage emerged (GPSC162-ST13022). Among non-PCV13 serotypes, serotype 8 expanded (GPSC3-ST53) and a new serotype 12F lineage emerged (GPSC55-ST8060). Most serotypes presented a dominant Global Pneumococcal Sequencing Cluster (GPSC) like GPSC16-ST67 of 9N or GPSC19-ST433 of 22F. Nevertheless, some GPSCs were associated with several serotypes, the most numerous were GPSC3 (serotypes 8, 11A, and 33F) and GPSC6 (serotypes 11A and 14). The overall penicillin non-susceptibility rate was 17.0 %, 14.6 % resistance for meningitis and 1.6 % for pneumonia (15.1 % susceptible at increased exposure [SIE]). Serotypes 11A and 14 (GPSC6-ST156/6521) and 19A (GPSC1-ST320) had penicillin MICs above 1 mg/L. Acquired resistance genes associated with macrolide and/or tetracycline resistance were present in 19.4 % of isolates, particularly among serotypes 6C (GPSC47-ST386/4310) and 19A (GPSC1-ST320)., Conclusions: The burden of PCV13 serotypes in adult IPD remains significant, and serotype 3 is the primary contributor. However, the rise of stable lineages associated with non-PCV13 serotypes, particularly 8, 9N, and 22F highlights a shifting epidemiology. The persistence of multidrug-resistant lineages, such as GPSC6-ST156 and GPSC1-ST320, emphasizes the need for continued surveillance. Vaccination of high-risk adults with current and broader coverage PCVs would help to control the burden of pneumonia and IPD among adults., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sara Calvo-Silveria declares that the Project was partially funded by PFIS predoctoral grant number FI22/00279. José Yuste declares grants or contracts paid to his institution from MSD, Pfizer and Meiji. Support for attending meetings and/or travel from MSD and Pfizer. Participation on a Data Safety Monitoring Board or Advisory Board from GSK, MSD and Pfizer. Carmen Ardanuy declares funding to the institution from Instituto de Salud Carlos III through PI18/0339; PI21/1000 and INT22/0096; CB06/06/0037. Grants or contracts paid to her institution from MSD, Pfizer and Menarini. Payment or honoraria for lectures, or educational events from MSD and Pfizer to her o her institution. Support for attending meetings and/or travel from MSD and Pfizer., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The immunosuppressive function of regulatory T (Treg) cells is essential for maintaining immune homeostasis. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27) methyltransferase, plays a key role in maintaining Treg cell function upon CD28 co-stimulation, and Ezh2 deletion in Treg cells causes autoimmunity. Here, we assess whether increasing H3K27me3 levels, by using an Ezh2 Y641F gain-of-function mutation, will improve Treg cell function. We find that Treg cells expressing Ezh2 Y641F display an effector Treg phenotype, are poised for improved homing to organ tissues, and can accelerate remission from autoimmunity. The H3K27me3 landscape and transcriptome of naive Ezh2 Y641F Treg cells exhibit a redistribution of H3K27me3 modifications that recapitulates the gene expression profile of activated Ezh2 WT Treg cells after CD28 co-stimulation. Altogether, increased H3K27me3 levels promote the differentiation of effector Treg cells that can better suppress autoimmunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: This study aimed to explore the prevalence of macrolide resistance and the underlying resistance mechanisms in Haemophilus influenzae (n = 2556) and Haemophilus parainfluenzae (n = 510) collected between 2018 and 2021 from Bellvitge University Hospital, Spain., Methods: Antimicrobial susceptibility was tested by microdilution. Whole-genome sequencing was performed using Illumina MiSeq and Oxford Nanopore technologies, and sequences were examined for macrolide resistance determinants and mobile genetic structures., Results: Macrolide resistance was detected in 67 H. influenzae (2.6%) and 52 (10.2%) H. parainfluenzae strains and associated with resistance to other antimicrobials (co-trimoxazole, chloramphenicol, tetracycline). Differences in macrolide resistance existed between the two species. Acquired resistance genes were more prevalent in H. parainfluenzae (35/52; 67.3%) than in H. influenzae (12/67; 17.9%). Gene mutations and amino acid substitutions were more common in H. influenzae (57/67; 85%) than in H. parainfluenzae (16/52; 30.8%). Substitutions in L22 and in 23S rRNA were only detected in H. influenzae (34.3% and 29.0%, respectively), while substitutions in L4 and AcrAB/AcrR were observed in both species. The MEGA element was identified in 35 (67.3%) H. parainfluenzae strains, five located in an integrative and conjugative element (ICE); by contrast, 11 (16.4%) H. influenzae strains contained the MEGA element (all in an ICE). A new ICEHpaHUB8 was described in H. parainfluenzae., Conclusions: Macrolide resistance was higher in H. parainfluenzae than in H. influenzae, with differences in the underlying mechanisms. H. parainfluenzae exhibits co-resistance to other antimicrobials, often leading to an extensively drug-resistant phenotype. This highlights the importance of conducting antimicrobial resistance surveillance., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Islatravir (ISL) is a deoxyadenosine analog that inhibits HIV-1 reverse transcription by multiple mechanisms. Lenacapavir (LEN) is a novel capsid inhibitor that inhibits HIV-1 at multiple stages throughout the viral life cycle. ISL and LEN are being investigated as once-weekly combination oral therapy for the treatment of HIV-1. Here, we characterized ISL and LEN in vitro to assess combinatorial antiviral activity, cytotoxicity, and the potential for interactions between the two compounds. Bliss analysis revealed ISL with LEN demonstrated additive inhibition of HIV-1 replication, with no evidence of antagonism across the range of concentrations tested. ISL exhibited potent antiviral activity against variants encoding known LEN resistance-associated mutations (RAMs) with or without the presence of M184V, an ISL RAM in reverse transcriptase (RT) . Static resistance selection experiments were conducted with ISL and LEN alone and in combination, initiating with either wild-type virus or virus containing the M184I RAM in RT to further assess their barrier to the emergence of resistance. The combination of ISL with LEN more effectively suppressed viral breakthrough at lower multiples of the compounds' IC 50 (half-maximal inhibitory concentration) values and fewer mutations emerged with the combination compared to either compound on its own. The known pathways for development of resistance with ISL and LEN were not altered, and no novel single mutations emerged that substantially reduced susceptibility to either compound. The lack of antagonism and cross-resistance between ISL and LEN support the ongoing evaluation of the combination for treatment of HIV-1., Competing Interests: T.L.D., S.L.G., W.N., S.R., M.X. (former), D.J.K., J.A.G. (former), and E.A.A. are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA (MSD), who may own stock and/or hold stock options in MSD.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccines have been long overdue. Structure-based vaccine design created a new momentum in the last decade, and the first RSV vaccines have finally been approved in older adults and pregnant individuals. These vaccines are based on recombinant stabilized pre-fusion F glycoproteins administered as soluble proteins. Multimeric antigenic display could markedly improve immunogenicity and should be evaluated in the next generations of vaccines. Here we tested a new virus like particles-based vaccine platform which utilizes the direct fusion of an immunogen of interest to the structural human immunodeficient virus (HIV) protein Gag to increase its surface density and immunogenicity. We compared, in mice, the immunogenicity of RSV-F or hMPV-F based immunogens delivered either as soluble proteins or displayed on the surface of our VLPs. VLP associated F-proteins showed better immunogenicity and induced superior neutralizing responses. Moreover, when combining both VLP associated and soluble immunogens in a heterologous regimen, VLP-associated immunogens provided added benefits when administered as the prime immunization., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julia Blanco reports financial support and equipment, drugs, or supplies were provided by Merck Sharp & Dohme Corp. Anna Pons-Grifols reports financial support was provided by Government of Catalonia Agency for Administration of University and Research Grants and European Social Fund. Julia Blanco reports a relationship with AlbaJuna Therapeutics SL that includes: board membership, employment, and equity or stocks. Jorge Carillo reports a relationship with AlbaJuna Therapeutics SL that includes: board membership, employment, and equity or stocks. Bonaventura Clotet reports a relationship with AlbaJuna Therapeutics SL that includes: board membership, employment, and equity or stocks. Eberhard Durr reports a relationship with Merck Sharp & Dohme Corp that includes: employment. Gregory O’Donnell reports a relationship with Merck Sharp & Dohme Corp that includes: employment. Silveria Rodriguez reports a relationship with Merck Sharp & Dohme Corp that includes: employment. Joel Mane reports a relationship with Merck Sharp & Dohme Corp that includes: employment. Lan Zhang reports a relationship with Merck Sharp & Dohme Corp that includes: employment. Eberhard Durr reports a relationship with Merck & Co Inc that includes: equity or stocks. Gregory O’Donnell reports a relationship with Merck & Co Inc that includes: equity or stocks. Silveria Rodriguez reports a relationship with Merck & Co Inc that includes: equity or stocks. Joel Mane reports a relationship with Merck & Co Inc that includes: equity or stocks. Lan Zhang reports a relationship with Merck & Co Inc that includes: equity or stocks. Bonaventura Clotet has patent #EP1638234.4 “Virus-like particles with high density coating for the production of neutralizing antibodies” licensed to Irsicaixa. Jorge Carrillo has patent #EP1638234.4 “Virus-like particles with high density coating for the production of neutralizing antibodies” licensed to Irsicaixa. Julia Blanco has patent #EP1638234.4 “Virus-like particles with high density coating for the production of neutralizing antibodies” licensed to Irsicaixa. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Invasive pneumococcal disease due to serotype 3 (S3-IPD) is associated with high mortality rates and long-term adverse effects. The introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the Spanish paediatric immunisation programme has not led to a decrease in the adult S3-IPD. We aimed to analyse the incidence, clinical characteristics and genomics of S3-IPD in adults in Spain., Methods: Adult IPD episodes hospitalized in a Southern Barcelona hospital were prospectively collected (1994-2020). For genomic comparison, S3-IPD isolates from six Spanish hospitals (2008-2020) and historical isolates (1989-1993) were analysed by WGS (Illumina and/or MinION)., Findings: From 1994 to 2020, 270 S3-IPD episodes were detected. When comparing pre-PCV (1994-2001) and late-PCV13 (2016-2020) periods, only modest changes in S3-IPD were observed (from 1.58 to 1.28 episodes per 100,000 inhabitants year). In this period, the incidence of the two main lineages shifted from 0.38 to 0.67 (CC180-GPSC12) and from 1.18 to 0.55 (CC260-GPSC83). The overall 30-day mortality remained high (24.1%), though a decrease was observed between the pre-PCV (32.4%; 95.0% CI, 22.0-45.0) and the late-PCV13 period (16.7%; 95.0% CI, 7.5-32.0) (p = 0.06). At the same time, comorbidities increased from 77.3% (95.0% CI, 65.0-86.0) to 85.7% (95.0% CI, 71.0-94.0) (p = 0.69). There were no differences in clinical characteristics or 30-day mortality between the two S3 lineages. Although both lineages were genetically homogeneous, the CC180-GPSC12 lineage presented a higher SNP density, a more open pan-genome, and a major presence of prophages and mobile genetic elements carrying resistance genes., Interpretation: Adult S3-IPD remained stable in our area over the study period despite PCV13 introduction in children. However, a clonal shift was observed. The decrease in mortality rates and the increase in comorbidities suggest a change in clinical management and overall population characteristics. The low genetic variability and absence of clinical differences between lineages highlight the role of the S3 capsule in the disease severity., Funding: This study has been funded by Instituto de Salud Carlos III (ISCIII) "PI18/00339", "PI21/01000", "INT22/00096", "FI22/00279", CIBER "CIBERES-CB06/06/0037", "CIBERINFEC-CB21/13/00009" and MSD grant "IISP 60168"., Competing Interests: C.A. has been a scientific adviser for, and/or has received research funding from, Merck Sharp & Dohme Corp and Pfizer. J.Y. has been a scientific adviser for, and/or has received research funding from, Merck Sharp & Dohme Corp, Pfizer and GSK. All other authors declare that they have no conflicts of interest regarding this research., (© 2024 The Author(s).)

The immunosuppressive function of regulatory T (Treg) cells is essential for maintaining immune homeostasis. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27) methyltransferase, plays a key role in maintaining Treg cell function upon CD28 co-stimulation, and Ezh2 deletion in Treg cells causes autoimmunity. Here we assessed whether increased EZH2 activity in Treg cells would improve Treg cell function. Using an Ezh2 gain-of-function mutation, Ezh2 Y641F , we found that Treg cells expressing Ezh2 Y641F displayed an increased effector Treg phenotype and were poised for improved homing to organ tissues. Expression of Ezh2 Y641F in Treg cells led to more rapid remission from autoimmunity. H3K27me3 profiling and transcriptomic analysis revealed a redistribution of H3K27me3, which prompted a gene expression profile in naïve Ezh2 Y641F Treg cells that recapitulated aspects of CD28-activated Ezh2 WT Treg cells. Altogether, increased EZH2 activity promotes the differentiation of effector Treg cells that can better suppress autoimmunity., Highlights: EZH2 function promotes effector differentiation of Treg cells.EZH2 function promotes Treg cell migration to organ tissues.EZH2 function in Treg cells improves remission from autoimmunity.EZH2 function poises naïve Treg cells to adopt a CD28-activated phenotype.

Regulatory T cells (Treg) can suppress antitumor immune responses, and their presence in tumors is associated with worse prognoses in most cancers. Strategies to neutralize Treg-mediated suppression in tumors without immune-related adverse events, however, are challenging due to the essential role of Tregs in maintaining immune homeostasis. In this issue, Pinioti and colleagues identify fucosylation as a critical regulator of Treg function in tumors that can be targeted therapeutically without impacting immune homeostasis. See related article by Pinioti et al., p. 1611 (3) ., (©2023 American Association for Cancer Research.)

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 (" i nducer of ce ll d eath-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Islatravir (ISL) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) that inhibits human immunodeficiency virus (HIV) reverse transcription by blocking reverse transcriptase (RT) translocation on the primer:template. ISL is being developed for the treatment of HIV-1 infection. To expand our knowledge of viral variants that may confer reduced susceptibility to ISL, resistance selection studies were conducted with wild-type (WT) subtype A, B, and C viruses. RT mutations encoding M184I and M184V were the most frequently observed changes. Selection studies were also initiated with virus containing a single known resistance-associated mutation in RT (K65R, L74I, V90I, M184I, or M184V), and no additional mutations were observed. Antiviral activity assays were performed on variants that emerged in selection studies to determine their impact. M184I and M184V were the only single-codon substitutions that reduced susceptibility >2-fold compared to WT. A114S was an emergent substitution that when combined with other substitutions further reduced susceptibility >2-fold. Viruses containing A114S in combination with M184V did not replicate in primary blood mononuclear cells (PBMCs), consistent with the rare occurrence of the combination in clinical samples. While A114S conferred reduced susceptibility to ISL, it increased susceptibility to approved nucleoside reverse transcriptase inhibitors (NRTIs). This differential impact of A114S on ISL, an NRTTI, compared to NRTIs likely results from the different mechanisms of action. Altogether, the results demonstrate that ISL has a high barrier to resistance and a differentiated mechanism compared to approved NRTIs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19., Competing Interests: Competing interest statement: All authors are employees of Merck and Company, Inc. A provisional patent application on the discoveries of this work has been filed., (Copyright © 2021 the Author(s). Published by PNAS.)

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rβ, and common γ [γ c ]). IL-2Rα, which is highly expressed on regulatory T (T Reg ) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward T Reg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating T Reg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8 + and CD4 + effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.