To examine how organizations assess their innovation readiness (IR) maturity using the Maastricht Innovation Readiness Approach (MIRA) Questionnaire. Cross-sectional study in 21 Dutch long-term care organizations. Health care professionals with insight into their organization's IR completed the questionnaire, including those in care, management, human resource management, communication, and client representation. Data were collected using the MIRA Questionnaire, based on the IR framework, containing 4 domains: strategic direction, organization of innovation, leadership for innovation, and learning climate. The response options include 5 IR positions: "not," "informal," "occasionally," "consistently," and "optimally," reflecting a progression in IR maturity and a "no insight" position. The MIRA Questionnaire was completed by 409 participants in 21 long-term care organizations. Across nearly all (n = 20) organizations, "consistently" was most frequently selected (mean [after which is M] = #21, 38%), particularly within the "strategic direction" domain (M = #21, 43%). In the "learning environment" domain, however, the "not" and "informal" positions were most frequently chosen (M = #21, 34%), suggesting that these factors were either not implemented or put in practice or only implemented without formalization. Questions about the active involvement of managers and intended end users (clients and family members) received more frequent responses in the "not and informal" positions (M = #21, 47%, 21%) and fewer responses in the "occasionally, consistently, and optimally" positions (M = #21, 25%, 59%). The main findings indicate that most health care organizations' attention goes to strategy and organizing for innovation but less to leadership and the learning climate. This might indicate a temporal order in organizing the IR factors. Improving IR requires balanced attention to all factors of the IR framework. MIRA can support organizations by identifying specific steps to improve IR. Additional research is necessary to longitudinally track whether and how organizations translate MIRA use into actions that improve their IR. Policymakers can utilize these findings to develop sector-wide programs that promote learning for sustainable IR.
Novel goose astrovirus (NGAstV) and goose circovirus (GoCV) are two major pathogens responsible for disease outbreaks in goslings, causing substantial economic losses to the goose farming industry. In this study, two duplex multienzyme isothermal rapid amplification (MIRA) assays were developed for the simultaneous detection of NGAstV and GoCV, and the specificity and sensitivity of these detection methods were evaluated. Both MIRA assays demonstrated high specificity for NGAstV and GoCV, with no cross-reactivity observed with six waterfowl pathogens, including duck enteritis virus, goose parvovirus, fowl adenovirus serotype 4, H9 subtype avian influenza virus, Muscovy duck reovirus and duck Tembusu virus. The basic duplex MIRA assay completed amplification within 25 min under a constant temperature of 25 °C, with minimum detection limits of 1 × 102 copies/µL for NGAstV and 1 × 103 copies/µL for GoCV. In contrast, the duplex MIRA-qPCR assay reduced the reaction time to 20 min at 39 °C, and increased the sensitivity to 1 × 101 copies/µL for NGAstV and 1 × 10² copies/µL for GoCV. Fluorescence imaging technology enables differentiation of infection types based on color variations: mixed infections appear yellow, single NGAstV infections show green fluorescence, and single GoCV infections exhibit red fluorescence. In the clinical sample testing, the detection rates of the two pathogens were relatively high, with a mixed infection rate of up to 18%. This method significantly improves pathogen detection efficiency and serves as an effective tool for the rapid identification of NGAstV and GoCV.
In this study, a novel nucleic acid detection method based on multienzyme isothermal rapid amplification (MIRA) combined with the Pyrococcus furiosus Argonaute (PfAgo) cleavage system (MIRA-PfAgo) was developed for the detection of Tembusu virus (TMUV), an important pathogen threatening the waterfowl industry. The method targets the highly conserved NS5 gene of TMUV. Target nucleic acids are rapidly enriched by MIRA under isothermal conditions at 39 °C, followed by PfAgo-mediated cleavage guided by sequence-specific guide DNA (gDNA), enabling visual detection through fluorescence signal output. Systematic optimization identified 0.5 μM gDNA, 1 μM PfAgo, and 5 mM Mn²⁺ as the optimal reaction conditions. The assay exhibited excellent specificity for TMUV, with no cross-reactivity observed with common waterfowl pathogens, including H9 subtype avian influenza virus (AIV-H9), duck enteritis virus (DEV), goose parvovirus (GPV), Muscovy duck reovirus (MDRV), Newcastle disease virus (NDV), and duck circovirus (DuCV). The sensitivity reached as low as 10⁰ copies/μL. In the analysis of 21 clinical samples, the results of the MIRA-PfAgo assay were fully consistent with those obtained by RT-PCR. This method does not require complex thermal cycling equipment and combines high sensitivity, strong specificity, and operational simplicity, providing a reliable and practical technical tool for rapid on-site monitoring of TMUV.
Global warming has resulted in frequent droughts worldwide, significantly affecting plant normal growth and development. Polyphenol oxidase (PPO), a copper-containing redox enzyme in plants, serves multiple functions. However, limited research has investigated the role of PPO in regulating plant drought adaptation. In this study, PmPPO was isolated from Prunus mira Koehne, the wild ancestor of cultivated peaches, which is a rare tree species known for its high stress tolerance under extreme conditions. Our results indicated that PmPPO was preferentially expressed in young roots, with its expression significantly induced by drought and abscisic acid (ABA). Overexpression of PmPPO in plants demonstrated enhanced tolerance to drought stress. Under drought stress, transgenic plants exhibited increased antioxidant enzyme activity, improved reactive oxygen species (ROS) scavenging capacity, and reduced cellular damage compared to wild-type (WT). Moreover, PmPPO overexpression facilitated anthocyanin accumulation by regulating the expression of genes involved in anthocyanin biosynthesis following drought treatment. Additionally, yeast two-hybrid (Y2H) and Luciferase complementation imaging (LCI) assays confirmed interactions between PmPPO and both PmRad23d and PmRGLG2. These findings introduce new molecular targets for genetic manipulation in peach germplasm improvement and present potential candidate genes for screening stress tolerance through molecular breeding.
Given the continuous threat posed by emerging and re-emerging infectious diseases worldwide, a rapid, sensitive, and practical molecular detection technology is urgently required for timely point-of-care (POC) diagnosis. Although the quantitative real-time PCR (qPCR) based technologies exhibit high sensitivity and specificity compared with traditional pathogen detection methods, they are not applicable for POC detection scenarios. Based on the photocontrolled principle, this study established a universal photoactivation strategy for LbCas12a by modifying four sites in the repeat region of LbCas12a crRNA with the photocleavable protecting group 6-nitropiperonyloxymethyl (NPOM). This strategy was integrated with multienzyme isothermal rapid amplification (MIRA) to develop a photocontrolled one-pot rapid detection method for monkeypox virus (MPXV), termed the temporally controlled MIRA-CRISPR/Cas12a (TC-MIRA-CRISPR/Cas12a) assay. The TC-MIRA-CRISPR/Cas12a assay exhibited a 100-fold improvement in detection sensitivity over the conventional one-step assay, achieving a limit of detection (LOD) of 3.9 copies per reaction, which was comparable to stepwise detection assay. The entire assay can be completed within 40 min, faster than the widely used qPCR. In clinical sample detection, this method showed good consistency with qPCR, with a Kappa coefficient of 0.980 (P < 0.001), a sensitivity of 98.4%, and a specificity of 100%. This method effectively avoids amplicon contamination while maintaining high sensitivity, and exhibits excellent universality and expandability. It enables detection of other pathogens simply by modifying the spacer sequence of crRNA, providing a novel technical approach and research perspective for POC detection of MPXV and other pathogens.
The aim of this study was to evaluate the effect of two plaque disclosing agents on the color stability of four different restorative materials. Twenty disc-shaped specimens (8 mm diameter, 2 mm thickness) were prepared for each material: Z250 (3M ESPE), Charisma Diamond (Heraeus Kulzer), Estelite Universal Flow Medium (Tokuyama Dental Corporation Inc.), and Beautifil II (Shofu Inc.). Baseline color measurements were conducted using the VITA Easyshade Compact after numbering the specimens and immersing them in distilled water at 37°C for 24 hours. Two plaque disclosing agents were applied to the specimens: Mira-2-Ton (Hager & Werken) to 10 specimens and ProPind plaque indicator gel (Promida Co.), containing erythrosin dye, to the remaining 10 specimens. Ten seconds after the application, each specimen was rinsed under running water for 30 seconds and air-dried. Color changes in the specimens were calculated using the CIEDE2000 formula. Statistical analysis was performed using One-way ANOVA, Kruskal-Wallis, and Mann-Whitney U tests (p<0.05). The color changes were perceptible (ΔE00 > 0.8) in all experimental groups. Clinically unacceptable color changes (ΔE00 >1.8) were observed in all groups following Mira-2-Ton application, with Charisma Diamond exhibiting the highest color change (7.18 ± 2.65) and Z250 the lowest (2.98 ± 1.13). In contrast, all groups stained with ProPind gel showed clinically acceptable color changes (ΔE00 <1.8), with no statistically significant differences among materials (p>0.05). Plaque disclosing agents can significantly impact the color stability of restorative materials. Understanding the chemical composition of plaque disclosing agents is essential for clinicians to ensure the long term aesthetic stability of restorative materials. Further in vitro and in vivo studies are needed to assess the underlying mechanisms of discoloration caused by disclosing agents.
Large language models (LLMs) show great potential for clinical decision-making, yet most applications remain narrow, task-specific chat tools rather than systems integrated into clinical workflows1,2. However, building physician copilots will require models that operate within the electronic health record (EHR), with governed access to patient data and the ability to initiate permitted EHR actions within defined safety constraints. Yet it remains unproven whether such a system can manage patient cases with physician-level performance. Here we show that MIRA (Medical Intelligence for Reasoning and Action), an autonomous artificial intelligence agent operating in a sandboxed EHR environment, can navigate a large clinical action space to obtain patient histories; order and interpret laboratory, imaging and microbiology tests; generate differential diagnoses; and formulate treatment plans such as prescribing medications, scheduling surgical procedures and planning admissions. In simulations on real patient cases spanning multiple diagnoses, MIRA outperformed physicians in diagnostic accuracy and made guideline-concordant, medication-safe and appropriate admission decisions. Compared with previous LLM applications that addressed isolated subtasks or provided free-text advice, these results suggest that an EHR-integrated artificial intelligence agent can turn clinical intent into structured, actionable EHR operations, possibly making it a more effective decision-support partner for physicians. Further work is needed to establish generalization, safety and governance through prospective, real-world studies.
In recent years, the detection rates of bovine astrovirus (BoAstV) and bovine norovirus (BNoV) in Chinese farms have continued to rise, resulting in significant economic losses to the livestock industry. To meet the need for rapid on-site screening, this study established a dual nucleic acid detection system capable of simultaneously detecting BoAstV and BNoV using the multi-enzyme isothermal rapid amplification technology. Its specificity, sensitivity, and repeatability were further validated using clinical samples. The experimental results showed that the established MIRA method specifically amplified the target nucleic acids of BoAstV and BNoV, while exhibiting no cross-reaction with other common diarrhea-associated pathogens, such as bovine rotavirus (BRV), bovine viral diarrhea virus, and bovine coronavirus, indicating good specificity. The method demonstrated high sensitivity, with detection limits of 920 copies/μL for BoAstV and 6,100 copies/μL for BNoV. In the repeatability tests, all the coefficients of variation were below 10%, confirming the high stability of the method. In clinical sample testing, the method showed strong agreement with conventional dye-based quantitative PCR results (Kappa value = 0.887), while offering shorter detection time and higher detection rates. In summary, the detection method established in this study demonstrated high sensitivity, strong specificity, and a short turnaround time, making it suitable for the rapid detection of bovine astrovirus and bovine norovirus in clinical samples of cattle.
Firefighters are routinely exposed to combustion-derived toxicants. This exploratory study examined associations between occupational exposure, DNA methylation, and reproductive hormones. Genome-wide methylome profiling using MIRA-seq was performed on pooled blood samples from six male firefighters and four matched controls. Candidate genes were validated by bisulfite sequencing. Jurkat and U937 cells were exposed to benzo[a]pyrene and a phthalate mixture to assess methylation and gene expression. Plasma reproductive hormones were measured in 17 firefighters and 17 matched controls. Firefighters showed 960 differentially methylated genes enriched in reproductive pathways. KLHL17 promoter hypomethylation and increased expression were reproduced in vitro. Hormone analyses demonstrated elevated anti-Müllerian hormone, service year-dependent increases in follicle-stimulating hormone, and reduced testosterone and estradiol. Occupational exposure in firefighters is associated with epigenetic and endocrine alterations potentially affecting reproductive health in firefighters.
Previous efforts to link Palaeolithic cultural records to specific populations through DNA analysis have focused on materials from archaeological floor deposits such as bones, sediments, and artefacts. In this study, we explore whether rock art, a spatially distinct expression of human activity, can also preserve DNA traces from its creators. We analyse DNA preservation in pigment samples collected in and around 24 rock art panels from 11 caves across Spain and Portugal, including simple marks (from nine sites), hand stencils (Maltravieso Cave, Extremadura, Spain), and figurative paintings (Cave of Altamira, Cantabria, Spain). We recover traces of ancient human mitochondrial and nuclear DNA, unaccompanied by faunal DNA, from a pigmented calcite crust at Escoural Cave (Portugal), as well as from an unpigmented cave wall sample from the same site. The absence of faunal DNA in both samples suggests direct DNA deposition through human contact. In contrast, three additional unpigmented samples, from Escoural and Covarón Cave (Asturias, Spain), yielded mixtures of human and faunal DNA, suggesting indirect deposition. Although our results do not conclusively link ancient human DNA preservation to the generation of cave art, we show that traces of human DNA can persist on cave walls for thousands of years.
COVID-19 pandemic has significantly impacted global health, particularly evident in the detection of lung lesions via chest computed tomography scans. This study investigates the role of biomarkers in lung injury among COVID-19 patients and patients with idiopathic pulmonary fibrosis (IPF). This single-center prospective study included 154 hospitalized and 10 outpatient COVID-19 patients, 62 IPF patients, and 40 healthy volunteers, and evaluated biomarkers of alveolar epithelial cell dysfunction, extracellular matrix (ECM) and fibroblast dysfunction, and macrophage/monocyte activation (Galectin-3, CXCL13). Findings were further validated in an independant confirmation cohort (DisCoVeRy trial). COVID-19 patients had higher levels of biomarkers compared to healthy controls, except for SP-D. Compared to IPF patients, COVID-19 patients had significantly higher plasma levels of OPN, Galectin-3 and CXCL13 at admission. Elevated CXCL13 and Galectin-3 levels in COVID-19 were associated with greater lung injury. Multivariate logistic regression and Kaplan Meier analysis confirmed the role of CXCL13 in predicting severe lung injury (odd ratio 3.17, 95% CI 1.03-9.76) and in-hospital mortality (p = 0.04), with consistent results observed in the confirmation cohort. COVID-19 is characterized by increased ECM and macrophage activation biomarkers compared with IPF. CXCL13 may serve as a relevant biomarker for assessing lung injury and improving risk stratification at hospital admission.
Polydactyly-derived chondrocyte (PD) sheets are being developed as an allogeneic cell sheet therapy for cartilage defects associated with knee osteoarthritis. Although intact PD sheets can promote hyaline cartilage repair, transplantation requires invasive open procedures. Injectable fragments of PD sheets (PD sheet-minis) may reduce invasiveness. However, their in vivo repair capacity after intra-articular administration remains unclear. PD cells were isolated from discarded cartilage tissue obtained during polydactyly surgery. PD sheets and PD sheet-minis were fabricated for 14 days using temperature-responsive culture inserts and RepCell plates, respectively. PD cells, PD sheets, and PD sheet-minis were compared by cell counting, viability assays, flow cytometry, soluble factor assays, and quantitative polymerase chain reaction. A full-thickness cartilage defect was created in the femoral trochlea of male athymic nude rats. The animals were randomized by body weight into vehicle, PD cell suspension, PD sheet-mini suspension, or intact PD sheet transplantation groups (n = 6 each). Hindlimb weight-bearing was assessed after surgery, and cartilage repair was evaluated histologically 4 weeks after transplantation. The viable cell numbers administered per knee were comparable between PD cells, PD sheet-minis, and intact PD sheets. The PD sheet-minis and PD sheets showed similar surface marker profiles, whereas PD cells showed significantly lower CD26 and higher CD146, CD49a, and CD166 expression. Compared to PD cells, PD sheet-minis secreted more melanoma inhibitory activity and dickkopf-related protein 1 and less transforming growth factor-β, monocyte chemoattractant protein-1, and matrix metalloproteinase-3. Gene expression analysis revealed lower COL1A1 and RUNX2 expression and higher MMP3 expression in PD sheet-minis than in PD cells; COL2A1 and COL10A1 were not detected. Weight-bearing showed no sustained intergroup difference. Histologically, intact PD sheets produced consistent Safranin O-positive, type II collagen-positive, and human vimentin-positive repair tissue, whereas PD cells and PD sheet-minis mainly produced fibrous repair tissue. The mean Osteoarthritis Research Society International (OARSI) scores were 11.4 ± 5.4, 10.7 ± 6.2, 9.3 ± 7.1, and 4.4 ± 3.2 in the vehicle, PD cell, PD sheet-mini and PD sheet groups, respectively. PD sheet-minis retained several sheet-like in vitro characteristics; however, single intra-articular administration did not reproduce the cartilage repair achieved with intact PD sheets. These findings suggest that lesion-specific retention and persistence, rather than injectability alone, may be important determinants of structural cartilage repair by PD cell-sheet products.
To evaluate the association between the newly proposed preclinical and clinical obesity framework and the long-term risk of major adverse liver outcomes (MALO). This retrospective cohort study included 7,462,871 individuals who participated in the 2009-2010 National Health Screening Program. Individuals were divided into three groups: without excess adiposity, preclinical obesity, and clinical obesity. Excess adiposity was determined by body mass index, waist circumference, and waist-to-height ratio. Participants were followed up through December 31, 2023. MALO was defined as hepatocellular carcinoma, liver cirrhosis-related events, and liver-related death, including liver transplantation. Cox proportional hazards regression analysis was conducted to estimate hazard ratios (HRs) and 95% confidence intervals (CI). During a median follow-up of 14.0 years, 105,812 incident cases of MALO were identified. Preclinical obesity and clinical obesity were associated with significantly higher risks of MALO compared to individuals without excess adiposity. The adjusted HRs (95% CI) were 1.08 (1.06-1.10) for preclinical obesity and 1.09 (1.07-1.10) for clinical obesity. When clinical obesity was further stratified, the adjusted HR (95% CI) was 17.64 (16.61-18.73) for clinical obesity with underlying hepatic dysfunction and 1.04 (1.03-1.06) for those without. The clinical obesity framework offers a clinical approach in predicting long-term liver outcomes. Integrating this framework into clinical and public health practice could reduce the burden of obesity-related liver disease.
Polyneuropathies are common and often require specialist expertise for accurate diagnosis. This study evaluated the diagnostic performance of ChatGPT-4o on real-world polyneuropathy cases, comparing it to peripheral neuropathy specialists and non-specialist neurologists. One hundred cases were selected from two tertiary centers in Milan, Italy. Standardized summaries included clinical, laboratory, and electrophysiological data. ChatGPT-4o was prompted to provide a leading diagnosis, two differentials, and a confirmatory test. Neurologists reviewed the same cases and generated comparable outputs, then could revise their responses after viewing ChatGPT-4o's suggestions. ChatGPT-4o achieved 65.5% leading diagnosis accuracy, comparable to non-specialists (63.0%) but lower than specialists (74.0%, p = 0.002). For differential diagnoses, it outperformed non-specialists (82.0% vs. 77.5%, p = 0.043) and recommended more appropriate tests (68.0% vs. 53.0%, p < 0.001). After reviewing ChatGPT-4o outputs, non-specialists revised their assessments in 21.8% of cases, improving accuracy. ChatGPT-4o shows potential as a diagnostic aid, particularly in non-specialist or resource-limited settings.
Improving people's well-being is one of the most important goals of public policy, as well as one of the tasks of psychology as an applied branch of knowledge. To effectively implement these tasks, it is necessary to clearly understand what the well-being of people of different ages consists of. The study of the well-being of the elderly and senile people is especially relevant due to the entry of society into the phase of super-aging, also due to the fact that psychology goes beyond the ideas of aging as regression and decline. Analysis of the emotional component of the image of well-being in elderly and senile people allows us to identify the most subjectively significant components of well-being. Objective - to identify the features of the emotional component of the image of well-being in the perceptions of elderly and senile people. The study involved 264 elderly and senile people. The data were collected using the method of limited associations using the following instructions: provide 9 associations for the word «well-being» (3 words in the form of a verb, noun, adjective). The resulting associations were examined through the prism of the thesaurus of emotive vocabulary by L.G.Babenko. As a result, the most frequent associations were identified, from which only associates related to emotive vocabulary were subsequently selected. Comparison of older and younger people shows that the most frequent associations of emotive vocabulary are similar. Differences are found in less frequent associations. Semantic groups of associations are unique to elderly and senile people: successful, kindness, cheerful, live, vigorous, communicate. Emotive vocabulary in the thesaurus of elderly and senile people is less represented than in young people, which indicates a lower intensity of needs. The features of the emotional component of the image of well-being, manifested in the associations of elderly and senile people, are described. The emotional component of the image of elderly people is represented mainly by positive emotions. The emotions of the core zone of the image are happiness, joy, calm; the emotions of the middle zone are friendship, love, attraction; the emotions of the peripheral zone of the image are kindness and anxiety. Повышение благополучия людей является одной из важнейших целей государственной политики, а также одной из задач психологии как прикладной отрасли знания. Для эффективной реализации этих задач необходимо ясно понимать, в чем состоит благополучие людей разного возраста. Исследование благополучия у людей пожилого и старческого возраста особенно актуально в силу вхождения общества в фазу сверхстарения, а также в силу того, что психология выходит за пределы представлений о старении как о регрессии и упадке. Анализ эмоциональной составляющей образа благополучия у людей пожилого и старческого возраста позволяет выделить наиболее субъективно значимые компоненты благополучия. Цель работы — выявить особенности эмоциональной составляющей образа благополучия в представлениях людей пожилого и старческого возраста. В исследовании участвовали 244 человека пожилого и старческого возраста. Был применен метод ограниченных ассоциаций по инструкции — привести девять ассоциаций на слово «благополучие» (по три слова в форме глагола, существительного, прилагательного). Полученные ассоциации рассматривали через призму тезауруса эмотивной лексики Л.Г.Бабенко. В результате были выделены наиболее частотные ассоциации, среди которых впоследствии были отобраны только ассоциаты, относящиеся к эмотивной лексике. Сравнение людей пожилого и старческого возраста и молодых людей показывает, что наиболее частотные ассоциации эмотивной лексики у них сходны. Различия обнаруживаются в менее частотных ассоциациях. Уникальными для людей пожилого и старческого возраста являются семантические группы ассоциаций: успешный, доброта, веселый, жить, бодрый, общаться. Эмотивная лексика в тезаурусе людей пожилого и старческого возраста представлена меньше, чем у молодежи, что, возможно, указывает на меньшую напряженность потребностей. Эмоциональная составляющая образа благополучия у людей пожилого и старческого возраста представлена преимущественно положительными эмоциями. В качестве эмоций ядерной зоны образа благополучия выступают счастье, радость, спокойствие; средней зоны — дружба, любовь, влечение; периферийной зоны — доброта и беспокойство.
Moss bag biomonitoring has been applied to investigate airborne organic pollutants. Two moss species, Hypnum cupressiforme and Sphagnum girgensohnii, were exposed for two months within different-sized urban areas - metropolis, city, and town, and a rural site during winter and summer seasons. The bags were exposed at nine regulatory Air Quality monitoring stations, which represent different land use classes within the urban areas. After exposure, 16 polycyclic aromatic hydrocarbons (PAHs) were determined in the moss samples by gas chromatography‒mass spectrometry (GC-MS). At the urban sites, the mosses primarily accumulated three to six-ring PAHs: Phen, Fl, and Pyr, followed by B[k]F, B[a]P, I[cd]P, B[ghi]P, etc., while Nap was recorded in the summer samples. Both moss species identified the sites with the highest and lowest PAH levels in the air - typical urban sites with heavy traffic and those with thermal power plants (TPP) and seasonal residential heating as additional pollution sources, while the rural site was recognized as a proper control one. Mosses showed a strong seasonal fluctuation in PAHs with markedly higher prevalence in the cold winter season. Combustion-derived PAHs (COMPAHs) accounted for about 70% in the winter season, while in the summer, they fell to 40%. These results suggest activation of residential heating systems/units. Correlation analysis, principal component analysis, and diagnostic ratios confirmed that PAH emissions vary between seasons. Moss bag biomonitoring showed the same temporal distribution patterns as the instrumental measurements, but with similar spatial distribution patterns, for available comparative B[a]P and ∑PAH measurements.
Probiotics offer a promising alternative to antibiotics in animal husbandry. This study evaluated the inhibitory potential of live cells and cell-free supernatants (CFS) from 31 putative probiotic strains against two multidrug-resistant (MDR) enterotoxigenic Escherichia coli (ETEC) strains. Methods included antibiogram profiling, zone of inhibition assays, hemolytic activity, and auto-aggregation tests. Results highlighted strain-specific antimicrobial heterogeneity. Lactobacillus acidophilus ATCC 4356 and Pediococcus acidilactici CNCTC 6986 exhibited the strongest contact-dependent inhibition against SP11 and SP31, respectively, while Lactiplantibacillus plantarum FW0BV0887 displayed superior CFS-mediated activity. Notably, L. acidophilus ATCC 4356 and L. helveticus ATCC 15009 showed consistent efficacy in both assays. Safety assessments confirmed non-hemolytic phenotypes for most strains, excluding Enterococcus faecium. Furthermore, at the 24-h plateau, L. crispatus FB141-CAN-1 achieved the highest auto-aggregation capacity (88.03 ± 8.60%), followed closely by L. acidophilus ATCC 4356 (75.88 ± 2.62%), indicating putative adhesive potential. Overall, these findings identify specific probiotics with distinct antimicrobial mechanisms and preliminary safety profiles based on hemolytic activity, supporting their potential as biocontrol agents and warranting further in vivo validation.
High-throughput screening of protein domains enables the systematic discovery of protein sequences that encode specific cellular functions. Fluorescence-activated cell sorting-based assays have long been the standard readout for such screens but remain time- and resource-intensive, imposing practical limits on library size and coverage. Here we describe a scalable magnetic separation-based workflow that provides an alternative to fluorescence-activated cell sorting for screening large protein libraries in mammalian cells. We engineered a modular synthetic surface marker, consisting of a fusion between the fragment crystallizable (Fc) region of human immunoglobulin G and the transmembrane domain of platelet-derived growth factor receptor-β, that allows cells to be magnetically separated on the basis of surface reporter expression using Protein G-coated magnetic beads. The procedure covers pooled library cloning, lentiviral delivery, magnetic separation and sequencing-based quantification, enabling reproducible screening of more than 100,000 protein domain variants. The approach is suitable for the identification of functional protein domains capable of transcriptional and post-transcriptional RNA regulation and may lead to the selection of improved transmembrane domains for efficient protein surface display. The entire workflow, from library design to data analysis, can be completed in 4-6 weeks and requires skills in cell culture, molecular cloning and computational techniques. This scalable and accessible Protocol enables researchers to systematically measure protein domain functions across biological contexts, thus accelerating both biological discovery and protein engineering.
From preclinical promise to clinical translation in intervertebral disc degeneration (IDD). Robust preclinical evidence supports a range of biologic therapies, with consistent improvements in imaging, extracellular matrix composition, inflammation, and behavior. However, a substantial translational gap persists, driven by key challenges including the discordance between IDD and pain, patient heterogeneity, inadequate phenotyping, placebo effects, limitations of preclinical models, hostile disc microenvironment, and practical/clinical trial barriers. A precision medicine framework is crucial to overcome these limitations, based on improved patient stratification, definition of molecular and clinical endotypes linked to targeted therapies, integration of advanced biomarkers, development of more sensitive outcome measures, and optimization of clinical trial design, ultimately aiming to enable successful clinical translation. Biologic therapies for intervertebral disc degeneration (IDD) have shown strong preclinical promise, yet clinical translation for discogenic low back pain (LBP) remains limited. This gap reflects the inconsistent relationship between degeneration and pain, compounded by heterogeneous patient populations, insufficient phenotyping, and inadequate selection of truly discogenic LBP patients in current trials. Emerging evidence indicates that IDD and LBP comprise distinct biological endotypes, requiring matched therapeutic strategies, or “theratypes”, rather than uniform approaches. In this narrative review, we critically examined the current evidence on biologic therapies for IDD, focusing on the translational gap between preclinical success and clinical outcomes. We analyzed key methodological and biological limitations, including patient heterogeneity, pain misclassification, inadequate phenotyping, and suboptimal trial design. Additionally, we reviewed emerging concepts such as disease phenotyping, mechanism‐based patient selection, and stage‐adapted therapeutic approaches. Multiple barriers hinder clinical translation of biologic therapies for IDD, including incomplete understanding of disc biology, limited intrinsic regenerative capacity, and a hostile tissue microenvironment. Preclinical models frequently fail to replicate the complexity of human pathology and pain, while clinical trials rely heavily on subjective outcomes and are often confounded by strong placebo effects. Furthermore, regulatory, logistical, and economic challenges limit widespread clinical adoption. Progress in the field will require a shift toward precision medicine approaches to align therapies with specific endotypes, pain phenotypes, and stages of IDD. Greater standardization, transparency, and community‐wide reporting practices are also essential to improve reproducibility. Advancing biologic therapies for IDD will depend on the development of more precise and biologically informed clinical strategies. Improved patient stratification, better alignment between therapeutic mechanisms and disease biology, and the adoption of standardized and clinically meaningful outcome measures are critical. Despite recent progress, key pathophysiological mechanisms remain poorly understood, underscoring the need for rigorous and multidisciplinary research efforts.