pH-Responsive Dynaplexes as Potent Apoptosis Inductors by Intracellular Delivery of Survivin siRNA.

Gene knockdown by siRNA offers an unrestricted choice of targets and specificity based on the principle of complementary Watson-Crick base pairing with mRNA. However, the negative charge, large molecular size, and susceptibility to enzymatic degradation of siRNA impede its successful transfection, hence limiting its potential for therapeutic use. The development of efficient and safe siRNA transfection agents is, therefore, critical for siRNA-based therapy. Herein, we developed a protein-based biodynamic polymer (biodynamer) that showed potential as a siRNA transfection vector, owing to its excellent biocompatibility, easy tunability, and dynamic polymerization under acidic environments. The positively charged biodynamers formed stable dynamic nanocomplexes (XL-DPs, hydrodynamic diameter of approximately 104 nm) with siRNA via electrostatic interactions and chemical cross-linking. As a proof of concept, the optimized XL-DPs were stable in physiological conditions with serum proteins and demonstrated significant pH-dependent size change and degradability, as well as siRNA release capability. The minimal cytotoxicity and excellent cellular uptake of XL-DPs effectively supported the intracellular delivery of siRNA. Our study demonstrated that the XL-DPs in survivin siRNA delivery enabled potent knockdown of survivin mRNA and induced notable apoptosis of carcinoma cells (2.2 times higher than a lipid-based transfection agent, Lipofectamine 2000). These findings suggested that our XL-DPs hold immense potential as a promising platform for siRNA delivery and can be considered strong candidates in the advancement of next-generation transfection agents.

Button Battery Ingestions cause the Majority of Severe Complications.

BACKGROUND: Foreign body ingestion in children is a clinically important reason for presentation to the emergency department. The individual outcome ranges from benign spontaneous courses to severe complications. Fatal outcomes occur rarely and complications are related to patient's age as well as type and location of the foreign body. The aim of our present study was to evaluate the outcome of children and adolescents with foreign body ingestion with a focus on complications, which mainly occurred after button battery ingestion. METHODS: We reviewed medical records of patients between 0 and 18 years of age who had presented to the paediatric emergency department of our hospital with suspected foreign body ingestion between January 2011 and March 2021 (123 months). Clinical, imaging, and endoscopic data as well as treatment modalities were analysed. RESULTS: In the ten10 year period under review, a total of 1,162 children and adolescents (6 months - 18 years) presented to our emergency room with suspected foreign body ingestion. Among those, 398 ingestions (34%) could be verified radiologically and/or endoscopically, while in the remaining 764 cases (66%) the suspicion could not be confirmed. The majority of patients with verified ingestion (n=324; 81%) presented with ingestion of a metallic foreign body. We observed 55 cases with verified ingestion of a button battery. Five of these cases had severe complications, with a near-fatal course in two patients who developed an oesophageal-tracheal fistula. CONCLUSION: In contrast to all other ingestions of foreign bodies in children, button battery ingestions lead to mucosal damage and severe complications in a significant number of children.

An Artificial In Vitro Metabolism to Angiopterlactone B Inspired by Traditional Retrosynthesis.

Nature's way to construct highly complex molecular entities as part of biosynthetic pathways is unmatched by any chemical synthesis. Yet, relying on a cascade of native enzymatic transformations to achieve a certain target structure, biosynthesis is also significantly limited in its scope. In this study, non-natural biocatalytic modules, a peroxidase-mediated Achmatowicz rearrangement and a dehydrogenase-catalyzed borrowing-hydrogen-type isomerization were successfully incorporated into an artificial metabolism, combining the benefits of traditional retrosynthesis with the elegance and efficacy of biosynthetic networks. In a highly streamlined process, the total synthesis of tricyclic angiopterlactone B was achieved in two steps operating entirely in an aqueous environment while relying mainly on enzymes as key reaction mediators.

Sandacrabins - Structurally Unique Antiviral RNA Polymerase Inhibitors from a Rare Myxobacterium.

Structure elucidation and total synthesis of five unprecedented terpenoid-alkaloids, the sandacrabins, are reported, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS-CoV-2 RNA-dependent RNA polymerase complex, highlighting them as structurally distinct non-nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition opens the possibility for their medicinal chemistry optimization towards selective inhibitors.

TLR7/8 regulates type I and type III interferon signalling in rhinovirus 1b-induced allergic asthma.

INTRODUCTION: Interferon (IFN) responses have been reported to be defective in rhinovirus (RV)-induced asthma. The heterodimeric receptor of type I IFN (IFN-α/ß) is composed of IFN-αR1 and IFN-αR2. Ligand binding to the IFN-α/ß receptor complex activates signal transducer and activator of transcription (STAT) proteins STAT1 and STAT2 intracellularly. Although type III IFN (IFN-λ) binds to a different receptor containing IFN-λR1 and interleukin-10R2, its triggering leads to activation of the same downstream transcription factors. Here, we analysed the effects of RV on IFN type I and III receptors, and asked about possible Toll-like receptor 7/8 (TLR7/8) agonist R848-mediated IFN-αR1 and IFN-λR1 regulation. METHODS: We measured IFN-α, IFN-ß and IFN-λ and their receptor levels in peripheral blood mononuclear cell (PBMC) supernatants and cell pellets stimulated with RV1b and R848 in two cohorts of children with and without asthma recruited at pre-school age (PreDicta) and at primary school age (AGENDAS) as well as in cell supernatants from total lung cells isolated from mice. RESULTS: We observed that R848 induced IFN-λR mRNA expression in PBMCs of healthy and asthmatic children, but suppressed IFN-αR mRNA levels. In murine lung cells, RV1b alone and together with R848 suppressed IFN-αR protein in T-cells compared with controls and in total lung IFN-λR mRNA compared with RV1b infection alone. CONCLUSIONS: In PBMCs from pre-school age children, IFN-αR mRNA was reduced and IFN-λR1 mRNA was induced upon treatment with the TLR7/8 agonist R848, thus suggesting new avenues for induction of antiviral immune responses in paediatric asthma.

Dual-source computed tomography of the lung with spectral shaping and advanced iterative reconstruction: potential for maximum radiation dose reduction.

BACKGROUND: Radiation dose at CT should be as low as possible without compromising diagnostic quality. OBJECTIVE: To assess the potential for maximum dose reduction of pediatric lung dual-source CT with spectral shaping and advanced iterative reconstruction (ADMIRE). MATERIALS AND METHODS: We retrospectively analyzed dual-source CT acquisitions in a full-dose group (FD: 100 kV, 64 reference mAs) and in three groups with spectral shaping and differing reference mAs values (Sn: 100 kV, 96/64/32 reference mAs), each group consisting of 16 patients (age mean 11.5 years, standard deviation 4.8 years, median 12.8 years, range 1.3-18 years). Advanced iterative reconstruction of images was performed with different strengths (FD: ADMIRE Level 2; Sn: ADMIRE Levels 2, 3 and 4). We analyzed dose parameters and measured noise. Diagnostic confidence and detectability of lung lesions as well as anatomical structures were assessed using a Likert scale (from 1 [unacceptable] to 4 [fully acceptable]). RESULTS: Compared to full dose, effective dose was reduced to 16.7% in the Sn 96 group, 11.1% in Sn64, and 5.5% in Sn32 (P<0.001). Noise values of Sn64ADM4 did not statistically differ from those in FDADM2 (45.7 vs. 38.9 Hounsfield units [HU]; P=0.132), whereas noise was significantly higher in Sn32ADM4 compared to Sn64ADM4 (61.5 HU; P<0.001). A Likert score >3 was reached in Sn64ADM4 regarding diagnostic confidence (3.2) and detectability of lung lesions (3.3). For detectability of most anatomical structures, no significant differences were found between FDAM2 and Sn64ADM4 (P≥0.05). CONCLUSION: In pediatric lung dual-source CT, spectral shaping together with ADMIRE 4 enable radiation dose reduction to about 10% of a full-dose protocol while maintaining an acceptable diagnostic quality.

Synthesis of New Cyclomarin Derivatives and Their Biological Evaluation towards Mycobacterium Tuberculosis and Plasmodium Falciparum.

Cyclomarins are highly potent antimycobacterial and antiplasmodial cyclopeptides isolated from a marine bacterium (Streptomyces sp.). Previous studies have identified the target proteins and elucidated a novel mode of action, however there are currently only a few studies examining the structure-activity relationship (SAR) for both pathogens. Herein, we report the synthesis and biological evaluation of 17 novel desoxycyclomarin-inspired analogues. Optimization via side chain modifications of the non-canonical amino acids led to potent lead structures for each pathogen.

Contribution of repeated infections in asthma persistence from preschool to school age: Design and characteristics of the PreDicta cohort.

BACKGROUND: The PreDicta cohort was designed to prospectively evaluate wheeze/asthma persistence in preschoolers in association with viral/microbial exposures and immunological responses. We present the cohort design and demographic/disease characteristics and evaluate unsupervised and predefined phenotypic subgroups at inclusion. METHODS: PreDicta is a 2-year prospective study conducted in five European regions, including children 4-6 years with a diagnosis of asthma as cases and healthy age-matched controls. At baseline, detailed information on demographics, asthma and allergy-related disease activity, exposures, and lifestyle were recorded. Lung function, airway inflammation, and immune responses were also assessed. Power analysis confirmed that the cohort is adequate to answer the initial hypothesis. RESULTS: A total of 167 asthmatic children (102 males) and 66 healthy controls (30 males) were included. Groups were homogeneous in respect to most baseline characteristics, with the exception of male gender in cases (61%) and exposure to tobacco smoke. Comorbidities and number and duration of infections were significantly higher in asthmatics than controls. 55.7% of asthmatic children had at least one positive skin prick test to aeroallergens (controls: 33.3%, P = .002). Spirometric and exhaled nitric oxide values were within normal limits; only baseline FEV0.5 and FEV1 reversibility values were significantly different between groups. Viral infections were the most common triggers (89.2%) independent of severity, control, or atopy; however, overlapping phenotypes were also common. Severity and control clustered together in an unsupervised analysis, separating moderate from mild disease. CONCLUSIONS: The PreDicta cohort presented no differences in non-asthma related measures; however, it is well balanced regarding key phenotypic characteristics representative of "preschool asthma".

Synthesis of modified ß-methoxyphenylalanines via diazonium chemistry and their incorporation in desoxycyclomarin analogues.

The marine natural products cyclomarins have remarkable anti-mycobacterial and antiplasmodial activities. The heptapeptic structure of this compound class comprisis four highly interesting non-canonical amino acids, including a rather unusual syn ß-methoxyphenylalanine. To get a deeper insight into the structure-activity realtionship of cyclomarines, a straightforward protocol for the stereoselective synthesis of this building block was developed, based on diazonium chemistry.

Successful eradication of newly acquired MRSA in six of seven patients with cystic fibrosis applying a short-term local and systemic antibiotic scheme.

BACKGROUND: In individuals with cystic fibrosis (CF), colonization with methicillin-resistant Staphylococcus aureus (MRSA) was reported to be associated with a deterioration of pulmonary disease as reflected by an accelerated decline in lung function. Thus, an early eradication of MRSA could be beneficial in these patients. Here, we report on an intensified MRSA eradication protocol. METHODS: Since 2012 a protocol for the eradication of newly acquired MRSA has been used in our CF Clinic, combining oral rifampicin and fusidic acid, inhaled vancomycin, nasal mupirocin, local antiseptic treatment and hygienic directives all of which are applied for only 7 days during an inpatient hospital stay. RESULTS: Since 2012 seven patients (3 male, 4 female; age range 4 to 30 years) newly acquired MRSA. In 6 of the 7 patients (86%) successful eradication of MRSA was achieved upon first treatment using the protocol described above. In one patient a second course of treatment was performed which, however, also failed to eliminate the colonizing MRSA. CONCLUSIONS: Our protocol led to an eradication rate of 86%. The impact of each individual component of the protocol remains to be determined.

IFN-α/IFN-λ responses to respiratory viruses in paediatric asthma.

We analysed the influence of rhinovirus (RV) in nasopharyngeal fluid (NPF) on type I and III interferon (IFN) responses (e.g. IFN-α and IFN -: λ) and their signal transduction, at baseline and during disease exacerbation, in cohorts of pre-school children with and without asthma.At the time of recruitment into the Europe-wide study PreDicta, and during symptoms, NPF was collected and the local RV colonisation was analysed. Peripheral blood mononuclear cells (PBMCs) were challenged in vitro with RV or not. RNA was analysed by quantitative real-time PCR and gene arrays. Serum was analysed with ELISA for IFNs and C-reactive protein.We found that PBMCs from asthmatic children infected in vitro with the RV1b serotype upregulated MYD88, IRF1, STAT1 and STAT2 mRNA, whereas MYD88, IRF1, STAT1 and IRF9 were predominantly induced in control children. Moreover, during symptomatic visits because of disease exacerbation associated with RV detection in NPF, IFN-α production was found increased, while IFN-λ secretion was already induced by RV in asthmatic children at baseline.During asthma exacerbations associated with RV, asthmatic children can induce IFN-α secretion, indicating a hyperactive immune response to repeated respiratory virus infection.

The Natural Products Discovery Center: Release of the First 8490 Sequenced Strains for Exploring Actinobacteria Biosynthetic Diversity.

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ~7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal's strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.

The many facets of sulfur incorporation in natural product biosynthesis.

Sulfur-containing natural products (S-containing NPs) exhibit diverse chemical structures and biosynthetic machineries. Unraveling the intricate chemistry of S-incorporation requires innovative and multidisciplinary approaches. In this review, we surveyed the landscape of S-containing NP biosynthetic machineries, classified the S-incorporation chemistry into four distinct classes, and highlighted each of the four classes with representative examples from recent studies. All highlighted chemistry has been correlated to the genes encoding the biosynthetic machineries of the S-containing NPs, which open new opportunities to discover S-containing NPs through genome mining. These examples should inspire the community to explore uncharted territories in NP research, promoting further advancements in both novel S-containing NP discovery and S-incorporation chemistry.

Comparative analysis of RSV-related hospitalisations in children and adults over a 7 year-period before, during and after the COVID-19 pandemic.

BACKGROUND: RSV is an important cause for respiratory illness in children and the elderly. We analysed RSV seasons since 2016 in both age groups for differences, similarities and timely associations before, during and after the COVID-19 pandemic. METHODS: We studied epidemiological and clinical features of seven consecutive RSV seasons since 2016 retrospectively in children and adults who were systematically monitored for RSV infections by PCR when hospitalized in Regensburg, Germany. RESULTS: Data from 1903 RSV positive, hospitalised patients were analysed (1446 children, 457 adults). We observed a complete absence of RSV associated hospitalizations in season 2020/2021. While in the season of 2021/2022, RSV associated hospitalizations in children returned to considerable numbers earlier than expected, hospitalizations in the elderly were still mitigated during that season in temporal association with the continuation of NPI measures for COVID-19 in the elderly until summer of 2022. Overall, children were hospitalized more often for RSV, while the elderly showed more severe outcomes. RSV hospitalisations continuously increase in both age groups, following a bi-annual pattern of severe and less severe seasons, which was not altered by the COVID-19 pandemic. CONCLUSION: We demonstrate the relation between RSV waves in children and the elderly. NPI measures may protect the elderly from RSV infections and epidemiological data could be used to predict RSV waves early enough to prepare countermeasures.

Application of a Biocatalytic Strategy for the Preparation of Tiancimycin-Based Antibody-Drug Conjugates Revealing Key Insights into Structure-Activity Relationships.

Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.

Vitamin D3 resolved human and experimental asthma via B lymphocyte-induced maturation protein 1 in T cells and innate lymphoid cells.

Background: Vitamin D3 (VitD3) is known to have immunomodulatory functions, and VitD3 deficiency is associated with more severe asthma. Objective: We aimed to assess the immunoregulatory effects of VitD3 food supplementation on asthma manifestation, with particular focus on T cells and type 2 innate lymphoid cells. Methods: Preschool children and adult asthmatic cohorts were analyzed in the context of VitD3 supplementation and serum levels. In a murine model of ovalbumin-induced asthma, effects of diet VitD3 sufficiency and deficiency on T cells and type 2 innate lymphoid cells immune mechanisms were investigated. Results: We found less severe and better-controlled asthma phenotypes along with reduced need for steroid medication in preschool children and asthmatic adults with VitD3 supplementation. VitD3 serum levels correlated with B lymphocyte-induced maturation protein 1 (Blimp-1) expression in blood peripheral mononuclear cells. VitD3-supplement-fed mice showed decreased asthmatic traits, with a decrease in IgE serum levels, reduced airway mucus, and increased IL-10 production by lung cells. Furthermore, we discovered an upregulation of effector T cells and Blimp-1+ lung tissue-resident memory T cells as well as induction of anti-inflammatory Blimp-1+ lung innate lymphoid cells producing IL-10. Conclusion: Supplementing VitD3 resulted in amelioration of clinical asthma manifestations in human studies as well as in experimental allergic asthma, indicating that VitD3 shifts proinflammatory immune responses to anti-inflammatory immune responses via upregulating Blimp-1 in lung innate lymphoid cells and tissue-resident memory cells.