Advancing stem cell technologies for conservation of wildlife biodiversity.

Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species.

Ten simple rules to make computable knowledge shareable and reusable.

Computable biomedical knowledge (CBK) is: "the result of an analytic and/or deliberative process about human health, or affecting human health, that is explicit, and therefore can be represented and reasned upon using logic, formal standards, and mathematical approaches." Representing biomedical knowledge in a machine-interpretable, computable form increases its ability to be discovered, accessed, understood, and deployed. Computable knowledge artifacts can greatly advance the potential for implementation, reproducibility, or extension of the knowledge by users, who may include practitioners, researchers, and learners. Enriching computable knowledge artifacts may help facilitate reuse and translation into practice. Following the examples of 10 Simple Rules papers for scientific code, software, and applications, we present 10 Simple Rules intended to make shared computable knowledge artifacts more useful and reusable. These rules are mainly for researchers and their teams who have decided that sharing their computable knowledge is important, who wish to go beyond simply describing results, algorithms, or models via traditional publication pathways, and who want to both make their research findings more accessible, and to help others use their computable knowledge. These rules are roughly organized into 3 categories: planning, engineering, and documentation. Finally, while many of the following examples are of computable knowledge in biomedical domains, these rules are generalizable to computable knowledge in any research domain.

Assessing potential human health and ecological implications of PFAS from leave-in dental products.

Contaminated water and food are the main sources of documented per- and polyfluoroalkyl substances (PFAS) exposure in humans. However, other sources may contribute to the overall PFAS intake. While several studies documented the presence of PFAS in consumer products, PFAS evaluation in dental products has been limited to floss and tape to date. This study estimated PFAS exposures from a convenience sample of leave-in dental products (night guards and whitening trays), which remain in contact with the mouth for longer durations than previously evaluated dental products. This analysis evaluated whether consumer usage of these dental products meaningfully contributes to oral exposure of PFAS. Leaching of PFAS upon disposal of products was also considered. Out of 24 PFAS measured, perfluorobutanoic acid (PFBA; 3.24-4.17 ng/product or 0.67-0.83 ng/g) and perfluorooctanesulfonic acid (PFOS; 7.25-16.45 ng/product or 1.2-2.3 ng/g) were detected in night guards, and no PFAS were detected in whitening trays. Non-targeted analysis showed additional possible PFAS, which could not be characterized. The findings showed that PFOS and/or PFBA present in night guards were unlikely to pose a health concern. From an ecological perspective, the dental products examined were shown to constitute a negligible contribution to environmental PFAS. In conclusion, the examined dental products do not represent a significant source of exposure to PFAS for humans or the environment. The study demonstrates how risk assessment can be integrated by the industry into product stewardship programs to evaluate the potential health and environmental impacts of chemicals in consumer products.

Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease.

Chagas disease is caused by the parasite Trypanosoma cruzi. In humans, it evolves into a chronic disease, eventually resulting in cardiac, digestive, and/or neurological disorders. In the present study, we characterized a novel T. cruzi antigen named Tc323 (TcCLB.504087.20), recognized by a single-chain monoclonal antibody (scFv 6B6) isolated from the B cells of patients with cardiomyopathy related to chronic Chagas disease. Tc323, a ~323 kDa protein, is an uncharacterized protein showing putative quinoprotein alcohol dehydrogenase-like domains. A computational molecular docking study revealed that the scFv 6B6 binds to an internal domain of Tc323. Immunofluorescence microscopy and Western Blot showed that Tc323 is expressed in the main developmental forms of T. cruzi, localized intracellularly and exhibiting a membrane-associated pattern. According to phylogenetic analysis, Tc323 is highly conserved throughout evolution in all the lineages of T. cruzi so far identified, but it is absent in Leishmania spp. and Trypanosoma brucei. Most interestingly, only plasma samples from patients infected with T. cruzi and those with mixed infection with Leishmania spp. reacted against Tc323. Collectively, our findings demonstrate that Tc323 is a promising candidate for the differential serodiagnosis of chronic Chagas disease in areas where T. cruzi and Leishmania spp. infections coexist.

DNA damage alters nuclear mechanics through chromatin reorganization.

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

A Digital-First Health Care Approach to Managing Pandemics: Scoping Review of Pandemic Self-triage Tools.

BACKGROUND: During the COVID-19 pandemic, many patient-facing digital self-triage tools were designed and deployed to alleviate the demand for pandemic virus triage in hospitals and physicians' offices by providing a way for people to self-assess their health status and get advice on whether to seek care. These tools, provided via websites, apps, or patient portals, allow people to answer questions, for example, about symptoms and contact history, and receive guidance on appropriate care, which might be self-care. OBJECTIVE: This scoping review aimed to explore the state of literature on digital self-triage tools that direct or advise care for adults during a pandemic and to explore what has been learned about the intended purpose, use, and quality of guidance; tool usability; impact on providers; and ability to forecast health outcomes or care demand. METHODS: A literature search was conducted in July 2021 using MEDLINE, Embase, Scopus, PsycINFO, CINAHL, and Cochrane databases. A total of 1311 titles and abstracts were screened by 2 researchers using Covidence, and of these, 83 (6.76%) articles were reviewed via full-text screening. In total, 22 articles met the inclusion criteria; they allowed adults to self-assess for pandemic virus, and the adults were directed to care. Using Microsoft Excel, we extracted and charted the following data: authors, publication year and country, country the tool was used in, whether the tool was integrated into a health care system, number of users, research question and purpose, direction of care provided, and key findings. RESULTS: All but 2 studies reported on tools developed since early 2020 during the COVID-19 pandemic. Studies reported on tools that were developed in 17 countries. The direction of care advice included directing to an emergency room, seeking urgent care, contacting or seeing a physician, being tested, or staying at home and self-isolating. Only 2 studies evaluated tool usability. No study demonstrated that the tools reduce demand on the health care system, although at least one study suggested that data can predict demand for care and that data allow monitoring public health. CONCLUSIONS: Although self-triage tools developed and used around the world have similarities in directing to care (emergency room, physician, and self-care), they differ in important ways. Some collect data to predict health care demand. Some are intended for use when concerned about health status; others are intended to be used repeatedly by users to monitor public health. The quality of triage may vary. The high use of such tools during the COVID-19 pandemic suggests that research is needed to assess and ensure the quality of advice given by self-triage tools and to assess intended or unintended consequences on public health and health care systems.

Standardized Calcium Replacement in Massive Transfusion: A Quality Improvement Project.

BACKGROUND: Hypocalcemia is common in trauma patients receiving massive transfusion protocol and often leads to worsening coagulopathies. Despite the identified problem and recommendations for replacement, few institutions have implemented a standardized calcium replacement protocol. OBJECTIVE: This study aims to assess whether a revised massive transfusion protocol, including standardized calcium replacement, increases the incidence of calcium administration in trauma patients receiving massive transfusion protocol. METHODS: This quality improvement project used a retrospective pre-/postdesign to study the revision of the current facility's massive transfusion protocol to include calcium replacement and ionized calcium monitoring at an urban Level I academic trauma center. Pre- and postintervention data were collected from January 2022 through October 2022 to determine the number of times massive transfusion protocol was ordered, ionized calcium monitoring, and calcium administration rates. Feedback regarding the protocol was collected throughout the monitoring period and was utilized in the final analysis. RESULTS: A total of 40 patients received massive transfusion protocol, preintervention, 18 of 23 (78%) received calcium supplementation, postintervention, 15 of 16 (98%) were treated. The majority of protocol activations occurred in the trauma bay (79%) and postintervention; ionized calcium monitoring dropped by 14%. CONCLUSION: This study found that the addition of standardized calcium replacement improved administration rates of calcium in this patient population. Ongoing research will ensure the recommended changes improve the identified shortcomings and that patients maintain adequate ionized calcium levels with the current dosing parameters.

Imipenem/Relebactam Resistance in Clinical Isolates of Extensively Drug Resistant Pseudomonas aeruginosa: Inhibitor-Resistant ß-Lactamases and Their Increasing Importance.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are a major clinical challenge. Many isolates are carbapenem resistant, which severely limits treatment options; thus, novel therapeutic combinations, such as imipenem-relebactam (IMI/REL), ceftazidime-avibactam (CAZ/AVI), ceftolozane-tazobactam (TOL/TAZO), and meropenem-vaborbactam (MEM/VAB) were developed. Here, we studied two extensively drug-resistant (XDR) P. aeruginosa isolates, collected in the United States and Mexico, that demonstrated resistance to IMI/REL. Whole-genome sequencing (WGS) showed that both isolates contained acquired GES ß-lactamases, intrinsic PDC and OXA ß-lactamases, and disruptions in the genes encoding the OprD porin, thereby inhibiting uptake of carbapenems. In one isolate (ST17), the entire C terminus of OprD deviated from the expected amino acid sequence after amino acid G388. In the other (ST309), the entire oprD gene was interrupted by an ISPa1328 insertion element after amino acid D43, rendering this porin nonfunctional. The poor inhibition by REL of the GES ß-lactamases (GES-2, -19, and -20; apparent Ki of 19 ± 2 µM, 23 ± 2 µM, and 21 ± 2 µM, respectively) within the isolates also contributed to the observed IMI/REL-resistant phenotype. Modeling of REL binding to the active site of GES-20 suggested that the acylated REL is positioned in an unstable conformation as a result of a constrained Ω-loop.

Diffusion-weighted MR spectroscopy (DW-MRS) is sensitive to LPS-induced changes in human glial morphometry: A preliminary study.

BACKGROUND: Low-dose lipopolysaccharide (LPS) is a well-established experimental method for inducing systemic inflammation and shown by microscopy to activate microglia in rodents. Currently, techniques for in-vivo imaging of glia in humans are limited to TSPO (Translocator protein) PET, which is expensive, methodologically challenging, and has poor cellular specificity. Diffusion-weighted magnetic resonance spectroscopy (DW-MRS) sensitizes MR spectra to diffusion of intracellular metabolites, potentially providing cell-specific information about cellular morphology. In this preliminary study, we applied DW-MRS to measure changes in the apparent diffusion coefficients (ADC) of glial and neuronal metabolites to healthy participants who underwent an LPS administration protocol. We hypothesized that the ADC of glial metabolites will be selectively modulated by LPS-induced glial activation. METHODS: Seven healthy male volunteers, (mean 25.3 ± 5.9 years) were each tested in two separate sessions once after LPS (1 ng/Kg intravenously) and once after placebo (saline). Physiological responses were monitored during each session and serial blood samples and Profile of Mood States (POMS) completed to quantify white blood cell (WBC), cytokine and mood responses. DW-MRS data were acquired 5-5½ hours after injection from two brain regions: grey matter in the left thalamus, and frontal white matter. RESULTS: Body temperature, heart rate, WBC and inflammatory cytokines were significantly higher in the LPS compared to the placebo condition (p < 0.001). The ADC of the glial metabolite choline (tCho) was also significantly increased after LPS administration compared to placebo (p = 0.008) in the thalamus which scaled with LPS-induced changes in POMS total and negative mood (Adj R2 = 0.83; p = 0.004). CONCLUSIONS: DW-MRS may be a powerful new tool sensitive to glial cytomorphological changes in grey matter induced by systemic inflammation.

Direct Ink 3D Printing of Porous Carbon Monoliths for Gas Separations.

Additive manufacturing or 3D printing is the advanced method of manufacturing monolithic adsorbent materials. Unlike beads or pellets, 3D monolithic adsorbents possess the advantages of widespread structural varieties, low heat and mass transfer resistance, and low channeling of fluids. Despite a large volume of research on 3D printing of adsorbents having been reported, such studies on porous carbons are highly limited. In this work, we have reported direct ink 3D printing of porous carbon; the ink consisted of commercial activated carbon, a gel of poly(4-vinylphenol) and Pluronic F127 as plasticizer, and bentonite as the binder. The 3D printing was performed in a commercial 3D printer that has been extensively modified in the lab. Upon 3D printing and carbonization, the resultant 3D printed porous carbon demonstrated a stable structure with a BET area of 400 m2/g and a total pore volume of 0.27 cm3/g. The isotherms of six pure-component gases, CO2, CH4, C2H6, N2, CO, and H2, were measured on this carbon monolith at 298 K and pressure up to 1 bar. The selectivity of four gas pairs, C2H6/CH4, CH4/N2, CO/H2, and CO2/N2, was calculated by Ideally Adsorbed Solution Theory (IAST) and reported. Ten continuous cycles of adsorption and desorption of CO2 on this carbon confirmed no loss of working capacity of the adsorbent.

Assessment of formaldehyde exposures under contemporary embalming conditions in U.S. funeral homes.

The funeral service profession has used formaldehyde-containing embalming solutions for the preparation of decedents since the early 1900s. The available literature regarding funeral director exposure to formaldehyde largely consists of data collected prior to 2000, with most studies reporting task-length exposure concentrations rather than full-shift time-weighted average concentrations. As formaldehyde undergoes review in the U.S. Environmental Protection Agency Toxic Substances Control Act (TSCA) risk evaluation process, accurately characterizing long-term exposure potential in this profession is critical. This study presents passive badge sampling and air change rate measurement results conducted at 13 funeral home locations across the United States. Full-shift (approximately 8-hr) samples were collected on one embalmer per day in each funeral home and on one occupational non-user (ONU), e.g., a receptionist. Additionally, task-length samples were collected during each embalming that occurred during the shift, were one to occur. Full-shift concentrations ranged from 0.007 to 1.1 ppm and 0.007 to 0.042 ppm for embalmers and ONUs, respectively. Task-length formaldehyde concentrations ranged from 0.058 to 1.4 ppm, with the average embalming taking 72.8 min to complete. Air change rates in the preparation rooms ranged from 2.8 to 28.3 air changes per hour; however, no correlation between task-length formaldehyde concentrations and air change rate was observed. Following empirical data collection, a Monte Carlo analysis of estimated annual 8-hr time-weighted average (TWA) exposure was conducted to determine the potential exposure distribution for embalmers employed at private funeral homes. Inputs to the simulation were derived from responses to a National Funeral Directors Association survey and from empirical measurements collected during the study. With respect to the reconstructed 8-hr TWAs, the median 8-hr TWA was 0.037 ppm, with 93.6% of the predicted concentrations below 0.1 ppm. This study provides a robust characterization of contemporary formaldehyde exposures in the funeral service profession. Further, it provides a strategy for interpreting the results along with surveyed responses regarding embalming frequency to better inform risks associated with formaldehyde exposure in this profession.

Competition between two high- and low-affinity protein-binding sites in myosin VI controls its cellular function.

Myosin VI is involved in many cellular processes ranging from endocytosis to transcription. This multifunctional potential is achieved through alternative isoform splicing and through interactions of myosin VI with a diverse network of binding partners. However, the interplay between these two modes of regulation remains unexplored. To this end, we compared two different binding partners and their interactions with myosin VI by exploring the kinetic properties of recombinant proteins and their distribution in mammalian cells using fluorescence imaging. We found that selectivity for these binding partners is achieved through a high-affinity motif and a low-affinity motif within myosin VI. These two motifs allow competition among partners for myosin VI. Exploring how this competition affects the activity of nuclear myosin VI, we demonstrate the impact of a concentration-driven interaction with the low-affinity binding partner DAB2, finding that this interaction blocks the ability of nuclear myosin VI to bind DNA and its transcriptional activity in vitro We conclude that loss of DAB2, a tumor suppressor, may enhance myosin VI-mediated transcription. We propose that the frequent loss of specific myosin VI partner proteins during the onset of cancer leads to a higher level of nuclear myosin VI activity.

COVID-19 in transplant recipients: The Spanish experience.

We report the nationwide experience with solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients diagnosed with coronavirus disease 2019 (COVID-19) in Spain until 13 July 2020. We compiled information for 778 (423 kidney, 113 HSCT, 110 liver, 69 heart, 54 lung, 8 pancreas, 1 multivisceral) recipients. Median age at diagnosis was 61 years (interquartile range [IQR]: 52-70), and 66% were male. The incidence of COVID-19 in SOT recipients was two-fold higher compared to the Spanish general population. The median interval from transplantation was 59 months (IQR: 18-131). Infection was hospital-acquired in 13% of cases. No donor-derived COVID-19 was suspected. Most patients (89%) were admitted to the hospital. Therapies included hydroxychloroquine (84%), azithromycin (53%), protease inhibitors (37%), and interferon-ß (5%), whereas immunomodulation was based on corticosteroids (41%) and tocilizumab (21%). Adjustment of immunosuppression was performed in 85% of patients. At the time of analysis, complete follow-up was available from 652 patients. Acute respiratory distress syndrome occurred in 35% of patients. Ultimately, 174 (27%) patients died. In univariate analysis, risk factors for death were lung transplantation (odds ratio [OR]: 2.5; 95% CI: 1.4-4.6), age >60 years (OR: 3.7; 95% CI: 2.5-5.5), and hospital-acquired COVID-19 (OR: 3.0; 95% CI: 1.9-4.9).

Benznidazole in Cerebrospinal Fluid: a Case Series of Chagas Disease Meningoencephalitis in HIV-Positive Patients.

Chagas disease reactivation in HIV-positive people is an opportunistic infection with 79 to 100% mortality. It commonly involves the central nervous system (CNS). Early treatment with trypanocidal drugs such as benznidazole (BNZ) is crucial for this severe manifestation of Trypanosoma cruzi infection. However, limited BNZ clinical pharmacology data are available, especially its concentration in the CNS. We report a series of HIV-positive patients undergoing treatment for T. cruzi meningoencephalitis, their clinical response, and cerebrospinal fluid (CSF) and plasma BNZ concentrations. Measurements were carried out using leftover samples originally obtained for routine medical care. A high-performance liquid chromatography/tandem mass spectrometry bioanalytical method designed for BNZ plasma measurements was adapted and validated for CSF samples. Six patients were enrolled in this study from 2015 to 2019. A total of 6 CSF and 19 plasma samples were obtained. Only three of the CSF samples had detectable BNZ levels, all under 1 µg/ml. Fifteen plasma samples had detectable BNZ, and 13 were above 2 µg/ml, which is the putative trypanocidal level. We observed BNZ concentrations in human CSF and plasma. CSF BNZ concentrations were low or not measurable in all patients, suggesting that the usual BNZ doses may be suboptimal in HIV-positive patients with T. cruzi meningoencephalitis. While drug-drug and drug-disease interactions may be in part responsible, the factors leading to low CSF BNZ levels remain to be studied in detail. These findings highlight the potential of therapeutic drug monitoring in BNZ treatment and suggest that the use of higher doses may be useful for Chagas disease CNS reactivations.

The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity.

The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Among the controls determining polarity are the PAR proteins, PAR6, aPKCι and PAR3, regulating both known and unknown effectors. Here, we identify FARP2 as a 'RIPR' motif-dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain-kinase domain interaction and detachment promoted by aPKCι-dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42, which is consistent with it being involved in upstream regulation of the polarising PAR6-aPKCι complex. However, we show that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι-FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors.This article has an associated First Person interview with the first author of the paper.

Mycoplasma hyopneumoniae Surveillance in Pig Populations: Establishing Sampling Guidelines for Detection in Growing Pigs.

Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae Thereafter, one tracheal sample, four serum samples, and one oral fluid sample were collected from every pen at 2-week intervals. Tracheal and oral fluid samples were tested for M. hyopneumoniae DNA and serum samples for M. hyopneumoniae antibody. Test results were modeled using a hierarchical Bayesian model, based on a latent spatial piecewise exponential survival model, to estimate the probability of detection by within-pen prevalence, number of positive pens in the barn, sample allocation, sample size, and sample type over time. Analysis showed that tracheal samples provided the earliest detection, especially at large sample sizes. While serum samples are more commonly collected and are less expensive to test, high probability of detection estimates were only obtained 30 days postexposure at large sample sizes. In all scenarios, probability of detection estimates for oral fluids within 30 days were significantly lower than those for tracheal and serum samples. Ultimately, the choice of specimen type, sample number, and assay will depend on testing objectives and economics, but the estimates provided here will assist in the design of M. hyopneumoniae surveillance and monitoring programs for different situations.

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary ß3-subunit.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated ß-subunits. The ß3-subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart-specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full-length ß3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric ß3-subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α-subunit and the effects upon these of the ß3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the ß3-subunit, has not been previously investigated. Using super-resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α-subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the ß3-subunit is not required for this clustering but ß3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the ß3-subunit increases the number of PLA-positive signals generated by anti-(Nav1.5 α-subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the ß3-subunit introduces a significant change in the relative alignment of individual Nav1.5 α-subunits, but the clustering itself depends on other factors. We also show that these structural and higher-order changes induced by the ß3-subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α-subunits. Our data provide new insights into the role of the ß3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

Single-Point Mutations in Qß Virus-like Particles Change Binding to Cells.

Virus-like particles (VLPs) constitute large, polyvalent platforms onto which a wide variety of functional units can be grafted. Their use in biological settings often depends on their specific binding to cells or receptors of interest; this can be compromised by excessive nonspecific association with other cells. We found that lysine residues mediate such nonspecific interactions, presumably by virtue of protonation and interaction with anionic membrane lipid headgroups and/or complementary residues of cell surface proteins and polysaccharides. Chemical acylation of surface-exposed amines of the Qß VLP led to a significant reduction in the association of particles with mammalian cells. Single-point mutations of particular lysine residues to either glutamine, glutamic acid, tryptophan, or phenylalanine were mostly well-tolerated and formed intact capsids, but the introduction of double and triple mutants was far less forgiving. Introduction of glutamic acid at position 13 (K13E) led to a dramatic increase in cellular binding, whereas removal of the lysine at position 46 (K46Q) led to an equally striking reduction. Several plasma membrane components were found to specifically interact with the Qß capsid irrespective of surface charge. These results suggest that specific cellular interactions are engaged or obviated by such mutations and provide us with more "benign" particles to which can be added binding functionality for targeted delivery applications.

The clinical relevance of the microbiome when managing paediatric infectious diseases-Narrative review.

In recent years, the field of infectious diseases has been hit by the overwhelming amount of information generated while the human microbiome is being disentangled. Based on the interaction between the microbiota and the immune system, the implications regarding infectious diseases are probably major and remain a challenge. AIMS: This review was conceived as a comprehensive tool to provide an overview of the available evidence regarding the influence of the microbiome on infectious diseases in children. METHODS: We present the main findings aroused from microbiome research in prevention, diagnosis and treatment of infectious disease under a paediatric perspective, to inform clinicians of the potential relevance of microbiome-related knowledge for translation to clinical practice. RESULTS AND CONCLUSION: The evidence shown in this review highlights the numerous research gaps ahead and supports the need to move forward to integrating the so-called microbiome thinking into our routine clinical practice.

Solid-phase synthesis of branched oligonucleotides containing a biologically relevant dCyd341 interstrand crosslink DNA lesion.

Branched oligonucleotides containing a biologically relevant DNA lesion, dCyd341, which involves an interstrand crosslink between a cytosine base on one strand and a ribose moiety on the opposite strand, were prepared in a single automated solid-phase synthesis. For this, we first prepared the phosphoramidite analogue of dCyd341 bearing an orthogonal levulinyl protecting group. Then, following the synthesis of the first DNA strand containing dCyd341, the levulinic group was removed and the synthesis was then continued from the free base hydroxyl group at the branching point, using traditional phosphoramidites. The synthesized oligonucleotides were fully characterized by MALDI-TOF/MS and were enzymatically digested, and the presence of the lesion was confirmed by HPLC-MS/MS and the sequence was finally controlled upon exonuclease digestion followed by MALDI-TOF/MS analysis. The developed strategy was successfully employed for the preparation of several short linear and branched oligonucleotides containing the aforementioned lesion.