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Abstract The formation of inclusion bodies (IBs) in microbial cell factories is a very common process occurring during recombinant protein production. Different protocols have been developed for the extraction of soluble proteins from IBs using several strategies, ranging from the use of harsh denaturing and high concentrations of chaotropic agents and reducing agents to the use of mild protocols based on the use of non-denaturing detergents. However, in recent years, the biological vision of IBs has changed and research studies have demonstrated that these protein aggregates contain biologically active and properly folded recombinant proteins. This drives us to redefine the methodologies currently used to obtain soluble protein using IB as a protein source. Hence, we propose the extraction of IB protein via the simple spontaneous solubilization of IB as a strategy broadly applicable to all kinds of recombinant proteins without the negative effects of detergents and chaotropic agents on final biological activity. We prove the wide applicability of spontaneous solubilization processes to different types of IBs and that protocols can be easily customized for each protein in terms of timing and incubation temperature by monitoring the protein activity of the solubilized fraction.

Abstract Background Objective assessment of skills after training is essential for safe implementation of lung point-of-care ultrasound (POCUS). In low-and middle-income countries (LMIC) there is a need for assessment tools without onsite experts to scale up POCUS access. Our objective is to develop a web-based assessment tool and evaluate trainees across different countries and at different time points after initial lung POCUS training. Methods We adapted the objective and validated lung ultrasound score (LUS-OSAUS) to a web-based tool with quiz and practical skills test. Trainees were evaluated after a short (4-day) standardized lung POCUS training and were classified in distinct groups according to (i) their geographical location (Benin vs. South-Africa) and (ii) time elapsed since training (Benin 0 months vs. Benin 6 months). The Benin 6 months group had minimal continuous education. Skills test images were read by two blinded experts. We report the overall success rates and then compare these rates based on location and timing since training, using the Fischer’s exact test. Results A total of 35 out of 43 participants completed the online LUS-OSAUS quiz and skills test. The overall success rate was 0.84 (95%CI 0.80–0.88), with lower success rates for “correct depth” 0.54 (0.37–0.71), “correct assessment of pleura” 0.63 (0.45–0.79) and “conclusion” 0.71 (0.54–0.85). There were no differences based on location, with respective rates of 0.86 (0.80–0.92) and 0.83 (0.75–0.91) (p-value = 0.125) for Benin and South Africa at 0 months, respectively. Similarly, there were no differences according to timing with success rates of 0.86 (0.80–0.92) and 0.82 (0.72–0.93) (p-value = 0.563) for Benin at 0 months and 6 months, respectively. Conclusion Web-based objective and structured assessment of lung POCUS skills in LMIC following a short-standardized training is feasible and has a good overall success rate with consistent results across regions and up to 6 months after training given minimal continuous education. Overall, technical and POCUS-based clinical conclusion skills are the most difficult to acquire.

Colostrum is rich in membranous vesicles of endocytic origin named exosomes, with proteins, lipids, RNA, and/or DNA cargos which can play different roles in physiological processes. Like other colostrum bioactive compounds, exosomes could be also influenced by individual characteristics. The objective of the study was to characterize miRNA cargo of colostrum exosomes from primiparous and multiparous cows in different farms. Twenty-seven colostrum samples of clinically healthy Holstein cows (11 primiparous and 16 multiparous) from 3 different farms were obtained and frozen. After thawing, exosomes were isolated following an ultracentrifugation protocol, and characterized morphologically. Particle size distribution and western immunoblotting were also analyzMaed. After RNA extraction, miRNAs were sequenced and analyzed to assess potential differences in profiles between primiparous and multiparous cows from different farms. Fourteen miRNA were upregulated and 11 miRNAs downregulated in primiparous compared with multiparous cows. Most of the miRNA differences between primiparous and multiparous cows regulate the gene expression of factors involved in mammary gland development and differentiation, and lipogenesis. In addition, miRNAs from one of the farms showed 8 miRNAs downregulated and 12 upregulated compared with the other 2 farms, independently of parity. Differences in miRNA between farms were mainly associated with immune and inflammatory-related genes. In conclusion, miRNA cargos of bovine colostrum exosomes differ in primiparous and multiparous cows, and some on-farm practices might also determine the content and activity of miRNA in colostrum exosomes.

This paper proposes a robust methodology for integrating process-specific data and domain expert knowledge into linked knowledge graphs. These graphs utilize an ontology that provides a standardized vocabulary for material science and facilitates the creation of semantic models for various processes along the digital process chain. A generic template for structuring processes is proposed, simplifying subsequent data retrieval. The templates of specific processes are designed collaboratively by domain and ontology experts, aided by a proposed interview template that bridges the knowledge gap. Following the digitalization of material data through semantic modeling, machine-readable data with contextual metadata is stored in a graph database, which can be efficiently queried using the SPARQL language, enabling seamless integration into data pipelines. To demonstrate this approach, a knowledge graph is developed to represent the process chain of AlSi10Mg objects manufactured via permanent mold casting, capturing their complete history from the initial manufacturing step to final non-destructive testing and mechanical characterization. This methodology enhances data interoperability and accessibility while providing context-rich data for training AI models, potentially accelerating new knowledge discovery in material science.

The concentrations of neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and plasma have become key biomarkers of many neurodegenerative diseases, including Huntington's Disease (HD). However, the relationship between the dynamics of NfL concentrations in CSF and the time-course of neurodegeneration (whole brain atrophy) has not yet been described in a quantitative and mechanistic manner. Here, we present a novel semi-mechanistic model, which postulates that the amount of NfL entering the CSF corresponds to the amount of NfL released from damaged neurons, whose degeneration results in a decrease in brain volume. In mathematical terms, the model expresses the NfL concentration in CSF in terms of the NfL concentration in brain tissue, the rate of change of whole brain volume and the CSF flow rate. To test our model, we used a non-linear mixed effects approach to analyze NfL and brain volume data from the HD-CSF study, a 24-month prospective study of individuals with premanifest HD, manifest HD and healthy controls. The time-course of whole brain volume, obtained from MRI, was represented empirically by a 2nd order polynomial, from which its rate of change was computed. CSF flow rates in healthy and HD populations were taken from recent literature data. By estimating the NfL concentration in brain tissue, the model successfully described the time-course of the NfL concentration in CSF in both HD subjects and healthy controls. Furthermore, the model-derived estimate of NfL concentration in brain agreed well with recent direct experimental measurements. The consistency of our model with the NfL and brain volume data suggests that the NfL concentration in CSF reflects the rate, rather than the extent, of neurodegeneration and that the increase in NfL concentration over time is a measure of the accelerating rate of neurodegeneration associated with aging and HD. For HD subjects, the degree of acceleration was found to increase markedly with the number of CAG repeats on their HTT gene. The application of our semi-mechanistic NfL model to other neurodegenerative diseases is discussed.

Palmoplantar keratoderma (PPK) is a multi-faceted skin disorder characterized by the thickening of the epidermis and abrasions on the palms and soles of the feet. Among the genetic causes, biallelic pathogenic variants in the FAM83G gene have been associated with PPK in dogs and humans. Here, a novel homozygous variant (c.794G>C, p.Arg265Pro) in the FAM83G gene, identified by whole exome sequencing in a 60-year-old female patient with PPK, is reported. The patient exhibited alterations in the skin of both hands and feet, dystrophic nails, thin, curly and sparse hair, long upper eyelid eyelashes, and poor dental enamel. FAM83G activates WNT signalling through association with ser/thr protein kinase CK1α. When expressed in FAM83G−/− DLD1 colorectal cancer cells, the FAM83GR265P variant displayed poor stability, a loss of interaction with CK1α and attenuated WNT signalling response. These defects persisted in skin fibroblast cells derived from the patient. Our findings imply that the loss of FAM83G-CK1α interaction and subsequent attenuation of WNT signalling underlie the pathogenesis of PPK caused by the FAM83GR265P variant.

Background and Objectives: Direct transport to a cardiac arrest centre following out-of-hospital cardiac arrest may be associated with higher survival. However, there is limited evidence available to support this within the New Zealand context. This study used a propensity score-matched cohort to investigate whether direct transport to a cardiac arrest centre improved survival in New Zealand. Methods: A retrospective cohort study was conducted using the Aotearoa New Zealand Paramedic Care Collection (ANZPaCC) database for adults treated for out-of-hospital cardiac arrest of presumed cardiac aetiology between 1 July 2018 to 30 June 2023. Propensity score-matched analysis was used to investigate survival at 30-days post-event according to the receiving hospital being a cardiac arrest centre versus a non-cardiac arrest centre. Results: There were 2,297 OHCA patients included. Propensity matching resulted in 554 matched pairs (n = 1108). Thirty-day survival in propensity score-matched patients transported directly to a cardiac arrest centre (56%) versus a non-cardiac arrest centre (45%) was not significantly different (adjusted Odds Ratio 0.78 95%CI 0.54, 1.13, p = 0.19). Shockable presenting rhythm, bystander CPR, and presence of STEMI were associated with a higher odds of 30 day survival (p

Abstract Antimicrobial resistance (AMR) is an escalating global health crisis, driven by the overuse and misuse of antibiotics. Multidrug‐resistant Gram‐negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are particularly concerning due to their high morbidity and mortality rates. In this context, endolysins, derived from bacteriophages, offer a promising alternative to traditional antibiotics. This study introduces LysJEP8, a novel endolysin derived from Escherichia phage JEP8, which exhibits remarkable antimicrobial activity against key Gram‐negative members of the ESKAPE group. Comparative assessments highlight LysJEP8's superior performance in reducing bacterial survival rates compared to previously described endolysins, with the most significant impact observed against P. aeruginosa, and notable effects on A. baumannii and K. pneumoniae. The study found that LysJEP8, as predicted by in silico analysis, worked best at lower pH values but lost its effectiveness at salt concentrations close to physiological levels. Importantly, LysJEP8 exhibited remarkable efficacy in the disruption of P. aeruginosa biofilms. This research underscores the potential of LysJEP8 as a valuable candidate for the development of innovative antibacterial agents, particularly against Gram‐negative pathogens, and highlights opportunities for further engineering and optimization to address AMR effectively.

Abstract Small noncoding RNAs play an important role in various disease states, including cancer. PIWI proteins, a subfamily of Argonaute proteins, and PIWI-interacting RNAs (piRNAs) were originally described as germline-specific molecules that inhibit the deleterious activity of transposable elements. However, several studies have suggested a role for the piRNA-PIWI axis in somatic cells, including somatic stem cells. Dysregulated expression of piRNAs and PIWI proteins in human tumors implies that, analogously to their roles in undifferentiated cells under physiological conditions, these molecules may be important for cancer stem cells and thus contribute to cancer progression. We provide an overview of piRNA biogenesis and critically review the evidence for the role of piRNA-PIWI axis in cancer stem cells. In addition, we examine the potential of piRNAs and PIWI proteins to become biomarkers in cancer.

Abstract Background Lower respiratory tract infections (LRTIs) are among the most frequent infections and a significant contributor to inappropriate antibiotic prescription. Currently, no single diagnostic tool can reliably identify bacterial pneumonia. We thus evaluate a multimodal approach based on a clinical score, lung ultrasound (LUS), and the inflammatory biomarker, procalcitonin (PCT) to guide prescription of antibiotics. LUS outperforms chest X-ray in the identification of pneumonia, while PCT is known to be elevated in bacterial and/or severe infections. We propose a trial to test their synergistic potential in reducing antibiotic prescription while preserving patient safety in emergency departments (ED). Methods The PLUS-IS-LESS study is a pragmatic, stepped-wedge cluster-randomized, clinical trial conducted in 10 Swiss EDs. It assesses the PLUS algorithm, which combines a clinical prediction score, LUS, PCT, and a clinical severity score to guide antibiotics among adults with LRTIs, compared with usual care. The co-primary endpoints are the proportion of patients prescribed antibiotics and the proportion of patients with clinical failure by day 28. Secondary endpoints include measurement of change in quality of life, length of hospital stay, antibiotic-related side effects, barriers and facilitators to the implementation of the algorithm, cost-effectiveness of the intervention, and identification of patterns of pneumonia in LUS using machine learning. Discussion The PLUS algorithm aims to optimize prescription of antibiotics through improved diagnostic performance and maximization of physician adherence, while ensuring safety. It is based on previously validated tests and does therefore not expose participants to unforeseeable risks. Cluster randomization prevents cross-contamination between study groups, as physicians are not exposed to the intervention during or before the control period. The stepped-wedge implementation of the intervention allows effect calculation from both between- and within-cluster comparisons, which enhances statistical power and allows smaller sample size than a parallel cluster design. Moreover, it enables the training of all centers for the intervention, simplifying implementation if the results prove successful. The PLUS algorithm has the potential to improve the identification of LRTIs that would benefit from antibiotics. When scaled, the expected reduction in the proportion of antibiotics prescribed has the potential to not only decrease side effects and costs but also mitigate antibiotic resistance. Trial registration This study was registered on July 19, 2022, on the ClinicalTrials.gov registry using reference number: NCT05463406. Trial status Recruitment started on December 5, 2022, and will be completed on November 3, 2024. Current protocol version is version 3.0, dated April 3, 2023.

Abstract Background Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications. Results To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250–300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates. Conclusions These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.

Clay minerals have been applied in biomedicine for thousands of years. Laponite is a nanostructured synthetic clay with the capacity to retain and progressively release drugs. In recent years there has been a resurgence of interest in Laponite application in various biomedical areas. This is the first paper to review the potential biomedical applications of Laponite in ophthalmology. The introduction briefly covers the physical, chemical, rheological, and biocompatibility features of different routes of administration. After that, emphasis is placed on 1) drug delivery for antibiotics, anti-inflammatories, growth factors, other proteins, and cancer treatment; 2) bleeding prevention or treatment; and 3) tissue engineering through regenerative medicine using scaffolds in intraocular and extraocular tissue. Although most scientific research is not performed on the eye, both the findings and the new treatments resulting from that research are potentially applicable in ophthalmology since many of the drugs used are the same, the tissue evaluated in vitro or in vivo is also present in the eye, and the pathologies treated also occur in the eye. Finally, future prospects for this emerging field are discussed.

This study was designed as a randomized, placebo-controlled, double-blinded, cross-over trial performed to investigate the effects of a dietary supplement containing undenatured type II collagen (UCII®) and Boswellia Serrata on mobility, pain and joint metabolism in mild moderate osteoarthritis (OA) in dogs. A total of 60 dogs with mobility problems were evaluated and enrolled in the study. Seventeen of these dogs with mild/moderate OA were randomized to receive the product A (UCII® + Boswellia Serrata supplement-UCII®-BW) or product B (Placebo -PL), 1 chew per day for 8 weeks by oral route, and repeated in a crossover design after 4 weeks of washout period. All the subjects had veterinary evaluations during the trial and owners were requested to fill out a questionnaire on mobility impairment using the Liverpool Osteoarthritis in dogs scale (L.O.A.D.) at each time of the study. Objective tools were used to assess mobility, activity, and pain. Metabolomic analysis was performed on synovial fluid of most affected joint at the beginning and the end of the study. The results proved that UCII®+Boswellia serrata supplemented group over a period of eight weeks results in an improvement of mobility impairment, already at 4 weeks of administration, according to the owner´s evaluation. In contrast, its absence increased the risk of OA crisis and decreased the pain threshold on the most affected joint. Furthermore, the synovial fluid metabolic profile showed moderate differences between the beginning and the end of the supplementation period, with a particular influence associated to the time of UCII®-BW administration.

To create a chronic glaucoma animal model by a single intracameral injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (Ms) co-loaded with dexamethasone and fibronectin (MsDexaFibro). MsDexaFibro were prepared by a water-in-oil-in-water emulsion method including dexamethasone in the organic phase and fibronectin in the inner aqueous phase. To create the chronic glaucoma model, an interventionist and longitudinal animal study was performed using forty-five Long Evans rats (4-week-old). Rats received a single intracameral injection of MsDexafibro suspension (10%w/v) in the right eye. Ophthalmological parameters such as clinical signs, intraocular pressure (IOP), neuro-retinal functionality by electroretinography (ERG), retinal structural analysis by optical coherence tomography (OCT), and histology were evaluated up to six months. According to the results obtained, the model proposed was able to induce IOP increasing in both eyes over the study, higher in the injected eyes up to 6 weeks (p

Abstract The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.

Background and Objectives: Airway management is crucial for emergency care in critically ill patients outside the hospital setting. An Airway Registry is useful in providing essential information for quality improvement purposes. Therefore, this study aimed to develop an out-of-hospital airway registry and describe airway management practices in Aotearoa New Zealand (AoNZ). Methods: Data from the Aotearoa New Zealand Paramedic Care Collection (ANZPaCC) database were used in a retrospective cohort study covering July 2020 to June 2021. All patients receiving airway interventions were included. An airway intervention was defined as one or more of the following: non-drug assisted endotracheal intubation (NDA-ETI), drug-assisted endotracheal intubation (DA-ETI; where a combination of paralytic agent and sedative were used to aid in intubation), laryngeal mask airway (LMA), oropharyngeal airway (OPA), nasopharyngeal airway (NPA), surgical airway (cricothyroidotomy), suction, jaw thrust. Descriptive statistics were analysed using Chi-Square and logistic regression modelling investigated the relationship between advanced airway success and patient characteristics. Results: The study included 4,529 patients who underwent 7,779 airway interventions. Basic airway interventions were used most frequently: OPA (45.1%), NPA (29.3%), LMA (28.9%), suction (19.9%) and jaw thrust (17.6%). Advanced airway interventions were used less frequently: NDA-ETI (19.8%), DA-ETI (8.7%), and surgical airways (0.2%). The success rate for ETI (including both NDA-ETI and DA-ETI) was 89.4%, with NDA-ETI success at 85.8% and DA-ETI success at 97.7%. ETI first-pass success rates were significantly lower for males (aOR 0.65, 95%CI 0.48–0.87, p

Recent studies have demonstrated that immune-related recombinant proteins can enhance immune function, increasing host survival against infectious diseases in salmonids. This research evaluated inclusion bodies (IBs) of antimicrobial peptides (CAMPIB and HAMPIB) and a cytokine (IL1βIB and TNFαIB) as potential immunostimulants in farmed salmonids. For this purpose, we produced five IBs (including iRFPIB as a control), and we evaluated their ability to modulate immune marker gene expression of three IBs in the RTS11 cell line by RT–qPCR. Additionally, we characterized the scale-up of IBs production by comparing two different scale systems. The results showed that CAMPIB can increase the upregulation of tnfα, il1β, il8, and il10, HAMPIB significantly increases the upregulation of tnfα, inos, and il10, and IL1βIB significantly upregulated the expression of tnfα, il1β, and cox2. A comparison of IL1βIB production showed that the yield was greater in shake flasks than in bioreactors (39 ± 1.15 mg/L and 14.5 ± 4.08 mg/L), and larger nanoparticles were produced in shake flasks (540 ± 129 nm and 427 ± 134 nm, p < 0.0001, respectively). However, compared with its shake flask counterpart, the IL1βIB produced in a bioreactor has an increased immunomodulatory ability. Further studies are needed to understand the immune response pathways activated by IBs and the optimal production conditions in bioreactors, such as a defined medium, fed-batch production, and mechanical bacterial lysis, to increase yield.

Glaucoma is a multifactorial pathology involving the immune system. The subclinical immune response plays a homeostatic role in healthy situations, but in pathological situations, it produces imbalances. Optical coherence tomography detects immune cells in the vitreous as hyperreflective opacities and these are subsequently characterised by computational analysis. This study monitors the changes in immunity in the vitreous in two steroid-induced glaucoma (SIG) animal models created with drug delivery systems (microspheres loaded with dexamethasone and dexamethasone/fibronectin), comparing both sexes and healthy controls over six months. SIG eyes tended to present greater intensity and a higher number of vitreous opacities (p < 0.05), with dynamic fluctuations in the percentage of isolated cells (10 µm2), non-activated cells (10–50 µm2), activated cells (50–250 µm2) and cell complexes (>250 µm2). Both SIG models presented an anti-inflammatory profile, with non-activated cells being the largest population in this study. However, smaller opacities (isolated cells) seemed to be the first responder to noxa since they were the most rounded (recruitment), coinciding with peak intraocular pressure increase, and showed the highest mean Intensity (intracellular machinery), even in the contralateral eye, and a major change in orientation (motility). Studying the features of hyperreflective opacities in the vitreous using OCT could be a useful biomarker of glaucoma.

Abstract This study compares four different animal models of chronic glaucoma against normal aging over 6 months. Chronic glaucoma was induced in 138 Long–Evans rats and compared against 43 aged-matched healthy rats. Twenty-five rats received episcleral vein sclerosis injections (EPIm cohort) while the rest were injected in the eye anterior chamber with a suspension of biodegradable microspheres: 25 rats received non-loaded microspheres (N-L Ms cohort), 45 rats received microspheres loaded with dexamethasone (MsDexa cohort), and 43 rats received microspheres co-loaded with dexamethasone and fibronectin (MsDexaFibro cohort). Intraocular pressure, neuroretinal function, structure and vitreous interface were evaluated. Each model caused different trends in intraocular pressure, produced specific retinal damage and vitreous signals. The steepest and strongest increase in intraocular pressure was seen in the EPIm cohort and microspheres models were more progressive. The EPIm cohort presented the highest vitreous intensity and percentage loss in the ganglion cell layer, the MsDexa cohort presented the greatest loss in the retinal nerve fiber layer, and the MsDexaFibro cohort presented the greatest loss in total retinal thickness. Function decreased differently among cohorts. Using biodegradable microspheres models it is possible to generate tuned neurodegeneration. These results support the multifactorial nature of glaucoma based on several noxa.

Point-of-care ultrasound (POCUS) is an increasingly accessible skill, allowing for the decentralization of its use to nonspecialist healthcare workers to guide routine clinical decision-making. The advent of ultrasound-on-a-chip has transformed the technology into a portable mobile health device. Because of its high sensitivity to detect small consolidations, pleural effusions, and subpleural nodules, POCUS has recently been proposed as a sputum-free likely triage tool for tuberculosis (TB). To make an objective assessment of the potential and limitations of POCUS in routine TB management, we present a Strengths, Weaknesses, Opportunities and Threats (SWOT) analysis based on a review of the relevant literature and focusing on Sub-Saharan Africa (SSA). We identified numerous strengths and opportunities of POCUS for TB management, e.g., accessible, affordable, easy to use and maintain, expedited diagnosis, extrapulmonary TB detection, safer pleural/pericardial puncture, use in children/pregnant women/people living with HIV, targeted screening of TB contacts, monitoring TB sequelae, and creating artificial intelligence decision support. Weaknesses and external threats such as operator dependency, lack of visualization of central lung pathology, poor specificity, lack of impact assessments and data from SSA must be taken into consideration to ensure that the potential of the technology can be fully realized in research as in practice.

Accurate analysis of mitochondrial DNA is important for mitochondrial disease clinical research and diagnostics. Here, authors present a method using Cas9 cleavage, nanopore sequencing and a custom pipeline to identify pathogenic variants, deletions and accurately quantify heteroplasmy to below 1%.

Objectives Owing to its ease-of-use and excellent diagnostic performance for the assessment of respiratory symptoms, point-of-care lung ultrasound (POC-LUS) has emerged as an attractive skill in resource-low settings, where limited access to specialist care and inconsistent radiology services erode health equity.To narrow down the research to practice gap, this study aims to gain in-depth insights in the perceptions on POC-LUS and computer-assisted POC-LUS for the diagnosis of lower respiratory tract infections (LRTIs) in a low-income and middle-income country (LMIC) of sub-Saharan Africa.Design and setting Qualitative study using face-to-face semi-structured interviews with three pneumologists and five general physicians in a tertiary centre for pneumology and tuberculosis in Benin, West Africa. The center hosts a prospective cohort study on the diagnostic performance of POC-LUS for LRTI. In this context, all participants started a POC-LUS training programme 6 months before the current study. Transcripts were coded by the interviewer, checked for intercoder reliability by an independent psychologist, compared and thematically summarised according to grounded theory methods.Results Various barriers− and facilitators+ to POC-LUS implementation were identified related to four principal categories: (1) hospital setting (eg, lack of resources for device renewal or maintenance−, need for POC tests+), (2) physician’s perceptions (eg, lack of opportunity to practice−, willingness to appropriate the technique+), (3) tool characteristics (eg, unclear lifespan−, expedited diagnosis+) and (4) patient’s experience (no analogous image to keep−, reduction in costs+). Furthermore, all interviewees had positive attitudes towards computer-assisted POC-LUS.Conclusions There is a clear need for POC affordable lung imaging techniques in LMIC and physicians are willing to implement POC-LUS to optimise the diagnostic approach of LRTI with an affordable tool. Successful integration of POC-LUS into clinical routine will require adequate responses to local challenges related to the lack of available maintenance resources and limited opportunity to supervised practice for physicians.

In recent years, the prevalence of acquired brain injury (ABI) has been on the rise, leading to impaired gait functionality in affected individuals. Traditional gait exoskeletons are typically rigid and bilateral and lack adaptability. To address this, the STELO, a pioneering modular gait-assistive device, was developed. This device can be externally configured with joint modules to cater to the diverse impairments of each patient, aiming to enhance adaptability and efficiency. This study aims to assess the safety and usability of the initial functional modular prototype, STELO, in a sample of 14 ABI-diagnosed participants. Adverse events, device adjustment assistance and time, and gait performance were evaluated during three sessions of device use. The results revealed that STELO was safe, with no serious adverse events reported. The need for assistance and time required for device adjustment decreased progressively over the sessions. Although there was no significant improvement in walking speed observed after three sessions of using STELO, participants and therapists reported satisfactory levels of comfort and usability in questionnaires. Overall, this study demonstrates that the STELO modular device offers a safe and adaptable solution for individuals with ABI, with positive user and therapist feedback.

Abstract The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.

Abstract Background CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). Results In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger. Conclusions The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

Background: Undenatured type-II collagen (UC-II®) has demonstrated its benefits in degenerative joint disease (DJD) management. Aim: Exploratory clinical study to investigate the beneficial effects of, FLEXADIN® Advanced in dogs. Methods: Multicentre clinical prospective study, 110 client-owned dogs with a clinical diagnosis of DJD. Dogs received FLEXADIN® Advanced during 6 months. Pain, general condition, appetite, mobility and lameness was assessed monthly by veterinarians and owners for 6 months. Compliance was assessed at 3 and 6 months. Statistical analysis was performed with non-parametric tests (significance, P< 0.05). Results: Parameters assessed were significantly lower comparing inclusion with all subsequent months (p < 0.0001). Compliance at 3 months showed a good score but was significantly lower (p < 0.0001) at six months. Conclusion: FLEXADIN® Advanced, containing UC-II®, can be considered a good complementary feed to provide joint support in dogs with mild to moderate DJD.

Abstract Combining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the different domains affects their final protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences affects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram-positive and Gram-negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA2 by shorter active fragments of both peptides improves the final immunomodulatory (IL-8 secretion) and antimicrobial function of the multidomain protein against antimicrobial-resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic effects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specific immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence.

Purpose: To evaluate alterations of the choroid in patients with a neurodegenerative disease versus healthy controls, a custom algorithm based on superpixel segmentation was used. Design: A cross-sectional study was conducted on data obtained in a previous cohort study. Subjects: Swept-source optical coherence tomography (OCT) B-scan images obtained using a Triton (Topcon, Japan) device were compiled according to current OSCAR IB and APOSTEL OCT image quality criteria. Images were included from three cohorts: multiple sclerosis (MS) patients, Parkinson disease (PD) patients, and healthy subjects. Only patients with early-stage MS and PD were included. Methods: In total, 104 OCT B-scan images were processed using a custom superpixel segmentation (SpS) algorithm to detect boundary limits in the choroidal layer and the optical properties of the image. The algorithm groups pixels with similar structural properties to generate clusters with similar meaningful properties. Main outcomes: SpS selects and groups the superpixels in a segmented choroidal area, computing the choroidal optical image density (COID), measured as the standard mean gray level, and the total choroidal area (CA), measured as px2. Results: The CA and choroidal density (CD) were significantly reduced in the two neurodegenerative disease groups (higher in PD than in MS) versus the healthy subjects (p < 0.001); choroidal area was also significantly reduced in the MS group versus the healthy subjects. The COID increased significantly in the PD patients versus the MS patients and in the MS patients versus the healthy controls (p < 0.001). Conclusions: The SpS algorithm detected choroidal tissue boundary limits and differences optical density in MS and PD patients versus healthy controls. The application of the SpS algorithm to OCT images potentially acts as a non-invasive biomarker for the early diagnosis of MS and PD.

Multiple sclerosis (MS) and Alzheimer’s disease (AD) cause retinal thinning that is detectable in vivo using optical coherence tomography (OCT). To date, no papers have compared the two diseases in terms of the structural differences they produce in the retina. The purpose of this study is to analyse and compare the neuroretinal structure in MS patients, AD patients and healthy subjects using OCT. Spectral domain OCT was performed on 21 AD patients, 33 MS patients and 19 control subjects using the Posterior Pole protocol. The area under the receiver operating characteristic (AUROC) curve was used to analyse the differences between the cohorts in nine regions of the retinal nerve fibre layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL) and outer nuclear layer (ONL). The main differences between MS and AD are found in the ONL, in practically all the regions analysed (AUROCFOVEAL = 0.80, AUROCPARAFOVEAL = 0.85, AUROCPERIFOVEAL = 0.80, AUROC_PMB = 0.77, AUROCPARAMACULAR = 0.85, AUROCINFERO_NASAL = 0.75, AUROCINFERO_TEMPORAL = 0.83), and in the paramacular zone (AUROCPARAMACULAR = 0.75) and infero-temporal quadrant (AUROCINFERO_TEMPORAL = 0.80) of the GCL. In conclusion, our findings suggest that OCT data analysis could facilitate the differential diagnosis of MS and AD.

The length of hospital stay and its implications have a significant economic and human impact. As a consequence, the prediction of that key parameter has been subject to previous research in recent years. Most previous work has analysed length of stay in particular hospital departments within specific study groups, which has resulted in successful prediction rates, but only occasionally reporting predictive patterns. In this work we report a predictive model for length of stay (LOS) together with a study of trends and patterns that support a better understanding on how LOS varies across different hospital departments and specialties. We also analyse in which hospital departments the prediction of LOS from patient data is more insightful. After estimating predictions rates, several patterns were found; those patterns allowed, for instance, to determine how to increase prediction accuracy in women admitted to the emergency room for enteritis problems. Overall, concerning these recognised patterns, the results are up to 21.61% better than the results with baseline machine learning algorithms in terms of error rate calculation, and up to 23.83% in terms of success rate in the number of predicted which is useful to guide the decision on where to focus attention in predicting LOS.

Abstract Background Inclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties. Results Three different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content. Conclusions This study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.

Abstract Background Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity. Results A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM. Conclusions Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.

Abstract In this paper, we show a comparison of different definitions of the topological charge on the lattice. We concentrate on one small-volume ensemble with 2 flavours of dynamical, maximally twisted mass fermions and use three more ensembles to analyze the approach to the continuum limit. We investigate several fermionic and gluonic definitions. The former include the index of the overlap Dirac operator, the spectral flow of the Wilson–Dirac operator and the spectral projectors. For the latter, we take into account different discretizations of the topological charge operator and various smoothing schemes to filter out ultraviolet fluctuations: the gradient flow, stout smearing, APE smearing, HYP smearing and cooling. We show that it is possible to perturbatively match different smoothing schemes and provide a well-defined smoothing scale. We relate the smoothing parameters for cooling, stout and APE smearing to the gradient flow time $$\tau $$ τ . In the case of hypercubic smearing the matching is performed numerically. We investigate which conditions have to be met to obtain a valid definition of the topological charge and susceptibility and we argue that all valid definitions are highly correlated and allow good control over topology on the lattice.

Despite the advances achieved in reconstructive surgery, amputation is still the only option after some severe traumas. Preservation of the knee joint is considered a significant functional advantage. We present the case of a 39-year-old man with a comminuted Gustilo type IIIC open tibia fracture with massive bone loss. To achieve a well-fashioned amputation stump and preserve the knee joint, a free osteocutaneous fillet flap was performed, including the distal tibia and fibula, talus, and calcaneus bones. As a result, a sensate and long amputation stump covered with thick skin from the sole of the foot provided a stable coverage with an excellent functional result and adjustment to prosthesis.

Antibiotic resistance has exponentially increased during the last years. It is necessary to develop new antimicrobial drugs to prevent and treat infectious diseases caused by multidrug- or extensively-drug resistant (MDR/XDR)-bacteria. Host Defense Peptides (HDPs) have a versatile role, acting as antimicrobial peptides and regulators of several innate immunity functions. The results shown by previous studies using synthetic HDPs are only the tip of the iceberg, since the synergistic potential of HDPs and their production as recombinant proteins are fields practically unexplored. The present study aims to move a step forward through the development of a new generation of tailored antimicrobials, using a rational design of recombinant multidomain proteins based on HDPs. This strategy is based on a two-phase process, starting with the construction of the first generation molecules using single HDPs and further selecting those HDPs with higher bactericidal efficiencies to be combined in the second generation of broad-spectrum antimicrobials. As a proof of concept, we have designed three new antimicrobials, named D5L37βD3, D5L37D5L37 and D5LAL37βD3. After an in-depth exploration, we found D5L37D5L37 to be the most promising one, since it was equally effective against four relevant pathogens in healthcare-associated infections, such as methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis (MRSE) and MDR Pseudomonas aeruginosa, being MRSA, MRSE and P. aeruginosa MDR strains. The low MIC values and versatile activity against planktonic and biofilm forms reinforce the use of this platform to isolate and produce unlimited HDP combinations as new antimicrobial drugs by effective means.

Despite the promising outcome of modular distance learning, its implementation is challenging. Thus, this study aimed to examine the usefulness and challenges of the implemented clustered self-directed learning modules in entrepreneurship for senior high school distance learning amidst the emerging pandemic using the descriptive-survey research method. This study was conducted at Espiritu Santo Parochial School of Manila, Inc., during the school year 2021-2022, among Grade 12 students using a stratified proportional random sampling technique. The study used both quantitative and qualitative methods of analyzing the data, including descriptive statistics and thematic analysis. As a result, all the modules were inferred to be very effective and very useful in terms of their usefulness for distance learning. On the other hand, there were six emergent themes from twenty-five challenges, as follows: (a) difficulties and limitations in theoretical and practical processes; (b) concerns about time constraints and academic workloads; (c) technical issues in learning modules; (d) problems with communication, lessons, and instructions; (e) issues with online learning, connectivity, and learning management systems; and (f) external and personal challenges as a student. These challenges were the basis in framing the recommendations of the students in addressing the aforementioned emergent themes.

Intravenous foreign bodies following trauma to the hand are relatively uncommon with sparse reports of this condition being published in the literature. They have been reported to migrate as far as the thoracic cavity and the heart. In the following case report, we describe a case of an intravenous foreign body following hand trauma, and the treatment and potential complications are also discussed.

Background. To evaluate the neuroretina and retinal vasculature of fibromyalgia (FM) patients and calculate a linear discriminant function (LDF) to improve retinal parameters’ contribution to FM diagnosis. Methods. Fifty FM patients and 232 healthy controls underwent retinal evaluation using swept-source optical coherence tomography (SS-OCT) angiography (Triton plus; Topcon) and spectral domain OCT (SD-OCT) (Spectralis; Heidelberg). The macular (m) and peripapillary (p) retinal nerve fibre layer (RNFL) and ganglion cell layer (GCL) were assessed, as was the macular vascular density. A logistic regression analysis was performed, and an LDF was calculated to evaluate OCT’s contribution to FM diagnosis. Results. With Triton OCT, the patients presented pRNFL thinning in the temporal sector (p=0.006). Spectralis OCT measurements showed decreased pRNFL in patients in the following sectors: superonasal, p=0.001; nasal, p=0.001; inferonasal, p=0.006; temporal, p=0.001; and inferotemporal, p=0.001. No significant differences were observed in the macular vascular plexus between patients and controls. However, vascular density in the superior sector showed a strong inverse correlation with disease duration (r = −0.978, p=0.022). The LDF calculated for Spectralis OCT yielded an area under the ROC curve of 0.968. Conclusions. FM patients present RNFL thinning observable using SS- and SD-OCT. However, these patients show similar vascular density in the macular area to healthy controls. The LDF that combines several RNFL parameters obtained using Spectralis OCT gives this device a powerful ability to differentiate between healthy individuals and individuals with FM.

Background: For older Latinos, some benefits of leisure-time physical activity (LTPA) include enhanced cognitive functioning, decreased loneliness, and reduced premature mortality. Despite LTPA benefits, adults [greater than or equal to]50 years are one of the most inactive age groups in the United States. Methods: This qualitative study aimed to add to the limited evidence of LTPA in older Latino adults by exploring the barriers and facilitators for fitness class uptake and park use. Guided by a Social-Ecological Model of Health and Social Determinants of Health theoretical and a phenomenological research design, qualitative research data were collected via 27 personal interviews with Latino adults using a semistructured interview guide. Participants were recruited using purposive sampling strategies in collaboration with community partners. Bicultural data collectors conducted the semistructured interviews over Zoom. Thematic analysis was performed using Dedoose, following an inductive and deductive approach. Results: The main barriers to fitness class attendance and park use were family and/or work commitments, perceived safety, and perceived discrimination. Conversely, the critical facilitators for participation were socialization into a group, social connectedness with group members, fitness instructor characteristics, and exercise history. Conclusions: Findings from this study hold important implications for the design of fitness programming and park-based recreation, including the need to increase the cultural diversity of recreation staff and culturally relevant programming. Future research is needed on time poverty related to family/work commitments and whether discriminatory practices at fitness centers, like ageism and race/ethnicity, influence physical activity behavior.

We report the case of a thenar eminence arteriovenous malformation presenting with continuous growth and pain that was treated with surgical excision after embolization. Extracapsular resection compromised thenar muscles which function was reconstructed with extensor indicis proprius transfer for opposition and abduction, and neurotized free gracilis muscle flap for opposition and adduction, as well as thenar eminence reconstruction.

Abstract Background Bruch membrane opening–minimum rim width (BMO–MRW) assessment offers a new diagnostic use in glaucoma patients of the Glaucoma Module Premium Edition (GMPE) available for the Spectralis optical coherence tomography (OCT) system. The objective of our research was to evaluate the diagnostic benefits of examining BMO–MRW and peripapillary retinal nerve fibre layer (pRNFL) readings acquired with Spectralis OCT to distinguish between healthy and mild glaucoma patients, comparing those readings with the standard pRNFL application. Moreover, we investigated whether using a particular combination of BMO–MRW and pRNFL parameters with a linear discriminant function (LDF) could further enhance glaucoma diagnosis. Methods One hundred thirty-six eyes from 136 individuals were incorporated into this observational, prospective cross-sectional study: 68 mild primary open-angle glaucoma (POAG) patients according to the Hodapp-Parrish-Anderson criteria (mean deviation between 0 and − 6 dB) and 68 healthy control subjects selected by Propensity Score Matching. MRW and pRNFL thickness around the disc (diameters: 3.5 mm, 4.1 mm, and 4.7 mm) were obtained using the BMO–MRW protocol, and pRNFL thickness at 3.5 mm was obtained with the standard glaucoma application. The group data were contrasted. One sample was chosen at random to develop the LDF (teaching set: 34 healthy subjects and 34 POAG patients) using a combination of MRW and pRNFL parameters (acquired with the BMO–MRW protocol); the other sample provided a test of how the LDF performed on an independent group (validating set: 34 healthy subjects and 34 POAG patients). The receiver operating curves (ROCs) were plotted for every measurement and contrasted with the proposed LDF. The OCT parameters with the best area under the receiver operating characteristic curve (AUC) were determined. Results Global MRW and pRNFL thicknesses were significantly thinner in the POAG group (p

BackgroundThe CAPRICE trial was designed to specifically evaluate neoadjuvant pegylated liposomal doxorubicin (PLD) in elderly patients or in those with other cardiovascular risk factors in whom conventional doxorubicin was contraindicated. The primary analysis of the study showed a pathological complete response (pCR) of 32% and no significant decreases in LVEF during chemotherapy. Here, we report important secondary study objectives: 5-year cardiac safety, disease-free survival (DFS), overall survival (OS) and breast cancer specific survival (BCSS).MethodsIn this multicentre, single-arm, phase II trial, elderly patients or those prone to cardiotoxicity and high risk stage II-IIIB breast cancer received PLD (35 mg/m2) plus cyclophosphamide (600 mg/m2) every 4 weeks for 4 cycles, followed by paclitaxel for 12 weeks as neoadjuvant chemotherapy (NAC). Left ventricular ejection fraction (LVEF) monitorization, electrocardiograms and cardiac questionnaires were performed at baseline, during treatment and at 9, 16, 28 and 40 weeks thereafter. The primary endpoint was pCR and 5-year cardiac safety, DFS, BCSS and OS were also analyzed.ResultsBetween Oct 2007, and Jun 2010, 50 eligible patients were included. Median age was 73 (35-84) years, 84% were older than 65; 64% of patients suffered from hypertension, and 10% had prior cardiac disease. Most of tumors (88%) were triple negative. No significant decreases in LVEF were observed. The mean baseline LVEF was 66.6% (52-86) and after a median follow-up of 5 years, mean LVEF was 66 (54.5-73). For intention to treat population, 5-year DFS was 50% (95% CI 40.2-68.1) and 5-year OS was 56% (95%CI 41.2-68.4). There were 8 non-cancer related deaths, achieving a 5 years BCSS of 67.74% (CI 95%:54.31%- 81.18%).ConclusionAt 5-year follow-up, this PLD-based NAC regimen continued to be cardiac-safe and effective in a population of very high-risk breast cancer patients. This scheme should be considered as an option in elderly patients or in those with other risks of developing cardiotoxicity.Trial Registration NumberClinicalTrials.gov reference NCT00563953.

Abstract In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha‐galactosidase A (GLA) or N‐sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV‐GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR‐labelled EVs were localized in brain parenchyma 1 h after intra‐arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV‐GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.

tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) deficiency causes an early onset potentially reversible acute liver failure, so far reported in less than 30 patients. We describe two new unrelated patients with an acute liver failure and a neuroimaging compatible with Leigh syndrome (LS) due to TRMU deficiency, a combination not previously reported. Our report enlarges the phenotypical spectrum of TRMU disease.

The radial dimension expands during central nervous system development after the proliferative neuroepithelium is molecularly patterned. The process is associated with neurogenesis, radial glia scaffolding, and migration of immature neurons into the developing mantle stratum. Radial histogenetic units, defined as a delimited neural polyclone whose cells share the same molecular profile, are molded during these processes, and usually become roughly stratified into periventricular, intermediate, and superficial (subpial) strata wherein neuronal cell types may differ and be distributed in various patterns. Cell-cell adhesion or repulsion phenomena together with interaction with local intercellular matrix cues regulate the acquisition of nuclear, reticular, or layer histogenetic forms in such strata. Finally, the progressive addition of inputs and outputs soon follows the purely neurogenetic and radial migratory phase. Frequently there is heterochrony in the radial development of adjacent histogenetic units, apart of peculiarities in differentiation due to non-shared aspects of the respective molecular profiles. Tangential migrations may add complexity to radial unit cytoarchitecture and function. The study of the contributions of such genetically controlled radial histogenetic units to the emerging complex neural structure is a key instrument to understand central nervous system morphology and function. One recent example in this scenario is the recently proposed radial model of the mouse pallial amygdala. This is theoretically valid generally in mammals (Garcia-Calero et al., 2020), and subdivides the nuclear complex of the pallial amygdala into five main radial units. The approach applies a novel ad hoc amygdalar section plane, given the observed obliquity of the amygdalar radial glial framework. The general relevance of radial unit studies for clarifying structural analysis of all complex brain regions such as the pallial amygdala is discussed, with additional examples.