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Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M-1s-1 to 20.3 ± 4.3 × 104 M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.

The Magnisi peninsula is a small portion of land located near the largest Italian petrol-chemical pole of Augusta–Priolo–Melilli (40 km2), which, since the 1950s, devastated the local environment and landscape and unloaded directly into the sea an impressive quantity of pollutants. Unlike the terrestrial part of the area, where a Natural Oriented Reserve (NOR) called “Saline di Priolo” was established in the 2000s, no concrete legislative action has been implemented or proposed for the marine environment. At the same time, the fauna of the marine environment has not been studied in the same way as that of the terrestrial environment. As concerns the molluscan fauna, most of the information dates back to the 1800s. These studies were exclusively focused on the shells of some mollusks in the area. Instead, no study related to this area has ever been carried out on the group of sea slugs. This study conducted between 2022 and 2023, through snorkeling activities, allowed to provide the first faunistic list of the sea slugs of this area, together with information on the biology and ecology of these gastropods, highlighting the potential biodiversity present in this small stretch of coastline affected by high industrial pollution.

Abstract Background Surfactant is a well-established therapy for preterm neonates affected by respiratory distress syndrome (RDS). The goals of different methods of surfactant administration are to reduce the duration of mechanical ventilation and the severity of bronchopulmonary dysplasia (BPD); however, the optimal administration method remains unknown. This study compares the effectiveness of the INtubate-RECruit-SURfactant-Extubate (IN-REC-SUR-E) technique with the less-invasive surfactant administration (LISA) technique, in increasing BPD-free survival of preterm infants. This is an international unblinded multicenter randomized controlled study in which preterm infants will be randomized into two groups to receive IN-REC-SUR-E or LISA surfactant administration. Methods In this study, 382 infants born at 24+0–27+6 weeks’ gestation, not intubated in the delivery room and failing nasal continuous positive airway pressure (nCPAP) or nasal intermittent positive pressure ventilation (NIPPV) during the first 24 h of life, will be randomized 1:1 to receive IN-REC-SUR-E or LISA surfactant administration. The primary outcome is a composite outcome of death or BPD at 36 weeks’ postmenstrual age. The secondary outcomes are BPD at 36 weeks’ postmenstrual age; death; pulse oximetry/fraction of inspired oxygen; severe intraventricular hemorrhage; pneumothorax; duration of respiratory support and oxygen therapy; pulmonary hemorrhage; patent ductus arteriosus undergoing treatment; percentage of infants receiving more doses of surfactant; periventricular leukomalacia, severe retinopathy of prematurity, necrotizing enterocolitis, sepsis; total in-hospital stay; systemic postnatal steroids; neurodevelopmental outcomes; and respiratory function testing at 24 months of age. Randomization will be centrally provided using both stratification and permuted blocks with random block sizes and block order. Stratification factors will include center and gestational age (24+0 to 25+6 weeks or 26+0 to 27+6 weeks). Analyses will be conducted in both intention-to-treat and per-protocol populations, utilizing a log-binomial regression model that corrects for stratification factors to estimate the adjusted relative risk (RR). Discussion This trial is designed to provide robust data on the best method of surfactant administration in spontaneously breathing preterm infants born at 24+0–27+6 weeks’ gestation affected by RDS and failing nCPAP or NIPPV during the first 24 h of life, comparing IN-REC-SUR-E to LISA technique, in increasing BPD-free survival at 36 weeks’ postmenstrual age of life. Trial registration ClinicalTrials.gov NCT05711966. Registered on February 3, 2023.

Neurotrophic tyrosine receptor kinase (NTRK) fusions are infrequent genetic events that can occur in various tumor types. Specifically, NTRK-rearranged sarcoma has been observed in pediatric mesenchymal tumors and, to a lesser extent, in adult mesenchymal tumors like fibrosarcoma. Recently, NTRK-rearranged uterine sarcoma (US) has been identified as a rare entity characterized by constitutive activation or overexpression of the TRK receptor, which plays a role in cell proliferation and differentiation. Since its initial description in 2018, only 46 cases of NTRK-rearranged US have been reported. In this context, herein we describe an exceptional case of an STRN3::NTRK3 fused US with histologically confirmed splenic metastasis. Notably, such localization has not been previously associated with pure uterine sarcomas in the literature. The fusion involved STRN3 (exon-3) and NTRK3 (exon-14) genes and was identified through next-generation sequencing analysis. Recognizing this specific molecular rearrangement is crucial, as it not only enables targeted therapy but also holds diagnostic significance in specific clinical scenarios.

Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in higher eukaryotes, including humans. NO-dependent signaling via sGC is associated with important physiological effects in the vascular, pulmonary, and neurological systems, and sGC itself is an established drug target for the treatment of pulmonary hypertension due to its central role in vasodilation. Despite isolation in the late 1970s, high-resolution structural information on full-length sGC remained elusive until recent cryo-electron microscopy structures were determined of the protein in both the basal unactivated state and the NO-activated state. These structures revealed large-scale conformational changes upon activation that appear to be centered on rearrangements within the coiled-coil (CC) domains in the enzyme. Here, a structure-guided approach was used to engineer constitutively unactivated and constitutively activated sGC variants through mutagenesis of the CC domains. These results demonstrate that the activation-induced conformational change in the CC domains is necessary and sufficient for determining the level of sGC activity.

Blast disease in cereal plants is caused by the fungus Magnaporthe oryzae and accounts for a significant loss in food crops. At the outset of infection, expression of a putative polysaccharide monooxygenase (MoPMO9A) is increased. MoPMO9A contains a catalytic domain predicted to act on cellulose and a carbohydrate-binding domain that binds chitin. A sequence similarity network of the MoPMO9A family AA9 showed that 220 of the 223 sequences in the MoPMO9A-containing cluster of sequences have a conserved unannotated region with no assigned function. Expression and purification of the full length and two MoPMO9A truncations, one containing the catalytic domain and the domain of unknown function (DUF) and one with only the catalytic domain, were carried out. In contrast to other AA9 polysaccharide monooxygenases (PMOs), MoPMO9A is not active on cellulose but showed activity on cereal-derived mixed (1→3, 1→4)-β-D-glucans (MBG). Moreover, the DUF is required for activity. MoPMO9A exhibits activity consistent with C4 oxidation of the polysaccharide and can utilize either oxygen or hydrogen peroxide as a cosubstrate. It contains a predicted 3-dimensional fold characteristic of other PMOs. The DUF is predicted to form a coiled-coil with six absolutely conserved cysteines acting as a zipper between the two α-helices. MoPMO9A substrate specificity and domain architecture are different from previously characterized AA9 PMOs. The results, including a gene ontology analysis, support a role for MoPMO9A in MBG degradation during plant infection. Consistent with this analysis, deletion of MoPMO9A results in reduced pathogenicity.

Abstract Heterozygosity-rich regions (HRR) are genomic regions of high heterozygosity, which may harbor loci related to key functional traits such as immune response, survival rate, fertility, and other fitness traits. This study considered 30 Italian and 19 worldwide goat breeds genotyped with the Illumina GoatSNP50k BeadChip. The aim of the work was to study inter-breed relationships and HRR patterns using Sliding Window (SW) and Consecutive Runs (CR) detection methods. Genetic relationships highlighted a clear separation between non-European and European breeds, as well as the north–south geographic cline within the latter. The Pearson correlation coefficients between the descriptive HRR parameters obtained with the SW and CR methods were higher than 0.9. A total of 166 HRR islands were detected. CHI1, CHI11, CHI12 and CHI18 were the chromosomes harboring the highest number of HRR islands. The genes annotated in the islands were linked to various factors such as productive, reproductive, immune, and environmental adaptation mechanisms. Notably, the Montecristo feral goat showed the highest number of HRR islands despite the high level of inbreeding, underlining potential balancing selection events characterizing its evolutionary history. Identifying a species-specific HRR pattern could provide a clearer view of the mechanisms regulating the genome modelling following anthropogenic selection combined with environmental interaction.

We analyzed the interaction of three materials, reduced graphene oxide (RGO), [6,6]-phenyl-C61-butyric acid methyl ester (PCBM), and poly(3-hexylthiphene) (P3HT), as well as the dependence of its photophysical properties within the temperature range of 90 to 300 K. The nanocomposite of the films was analyzed by optical absorption ultraviolet–visible (UV-Vis) and photoluminescence (PL) and emission ellipsometry (EE) as a function of sample temperature. The surface morphology was studied by atomic force microscopy (AFM). We noted that onset levels (Eonset) of the nanocomposite of P3HT and RGO are smaller than the others. The PL spectra showed the presence of anomalies in the emission intensities in the nanocomposite of P3HT and PCBM. It was also possible to determine the electron–phonon coupling by calculating the Huang–Rhys parameters and the temperature dependence of samples. Through EE, it was possible to analyze the degree of polarization and the anisotropy. We observed a high degree of polarized emission of the P3HT films, which varies subtly according to the temperature. For nanocomposites with RGO, the polarization degree in the emission decreases, and the roughness on the surface increases. As a result, the RGO improves the energy transfer between adjacent polymer chains at the cost of greater surface roughness. Then, the greater energy transfer may favor applications of this type of nanocomposite in organic photovoltaic cells (OPVCs) with enhancement in energy conversion efficiency.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

Local livestock breeds play a crucial role in global biodiversity, connecting natural and human-influenced environments and contributing significantly to ecosystem services. While commercial breeds dominate industrial systems, local livestock breeds in developing countries, like Barbarine sheep in Tunisia, are vital for food security and community maintenance. The Tunisian Barbarine sheep, known for its adaptability and distinctive fat-tailed morphology, faces challenges due to historical crossbreeding. In this study, the Illumina Ovine SNP50K BeadChip array was used to perform a genome-wide characterization of Tunisian Barbarine sheep to investigate its genetic diversity, the genome structure, and the relationship within the context of Mediterranean breeds. The results show moderate genetic diversity and low inbreeding. Runs of Homozygosity analysis find genomic regions linked to important traits, including fat tail characteristics. Genomic relationship analysis shows proximity to Algerian thin-tailed breeds, suggesting crossbreeding impacts. Admixture analysis reveals unique genetic patterns, emphasizing the Tunisian Barbarine’s identity within the Mediterranean context and its closeness to African breeds. Current results represent a starting point for the creation of monitoring and conservation plans. In summary, despite genetic dilution due to crossbreeding, the identification of genomic regions offers crucial insights for conservation. The study confirms the importance of preserving unique genetic characteristics of local breeds, particularly in the face of ongoing crossbreeding practices and environmental challenges. These findings contribute valuable insights for the sustainable management of this unique genetic reservoir, supporting local economies and preserving sheep species biodiversity.

The Medical Device Regulation 2017/745 (MDR) enables the development of a wide range of innovative products. With respect to Directive 93/42, the MDR explicitly identifies the so-called “medical devices made of substances” (MDMS) through specific requirements. In addition, the MDR expands the definition of medical device (MD) by including the “modification of a physiological or pathological state” as a medical purpose specific to devices. This clarifies that materials interacting with the human body in such a way as to modify its “state” are medical devices. Natural materials, such as vegetal matrices, are characterized by the presence of both functional and structural interactions between their components; they can thus be described as “network/s" and interact with the human body in a coordinated, complex way. Since the “state” of the human body is a network of biological functions, the “network/s over a network” interaction between the natural material and the human body is likely to modify the “state” of the human body. Thus, therapeutic products consisting of natural materials, such as vegetal matrices, seem to fit perfectly into the definition of a medical device. Here we analyze the main characteristics of medicinal products, of medical devices made of substances and of medical devices consisting of natural materials. We see that medicinal products and medical devices made of substances have the common characteristic of being based on substances, either synthetic or derivatives of natural materials, but differ in their mechanism of action. On the other hand, medical devices constituted of natural materials relate to the general category of medical devices and cannot be characterized by any single component, identified as an active component. We also discuss how these characteristics relate to the mechanism of action of each type of product. This analysis should allow to identify the most appropriate path for each product, a necessary step to promote research and development of innovative therapies for a large number of unmet medical needs.

Replacing the native porphyrin cofactor in haem proteins has led to the development of novel designer proteins for a variety of applications. In most cases, haem analogues bind in a way that is comparable to the iron porphyrin, but this is not necessarily the case for complexes bearing non-exchangeable ligands. This study probes how a P[double bond, length as m-dash]O corrole binds to functionally disparate hemoproteins: a haem-dependent oxygen sensor (H-NOX) and a haem-scavenging protein (HasA). The results demonstrate that the protein-cofactor interactions are distinct from the native, haem-bound holoprotein. In H-NOX, the P[double bond, length as m-dash]O unit primarily hydrogen bonds with the haem-ligating histidine (H102), rather than the hydrogen-bonding network that stabilises the Fe(ii)-O2 complex in the native protein. In the absence of H102, the protein is still able to bind the corrole, albeit at reduced levels. Molecular dynamics simulations were utilised to determine the flexibility of apo H-NOX and revealed the coupled motion of key residues necessary for corrole binding. In the case of HasA, the P[double bond, length as m-dash]O unit does not primarily interact with either the haem-ligating histidine (H32) or tyrosine (Y75). Instead, histidine 83, the hydrogen-bonding partner for Y75, is critical for P[double bond, length as m-dash]O corrole binding. The conformation of HasA is interrogated by site-specifically labelling the protein and exploiting Förster resonance energy transfer (FRET) to determine the dye-cofactor distance. HasA reconstituted with the P[double bond, length as m-dash]O corrole exhibits an extended, apo-like conformation. Together, these results demonstrate that non-natural cofactors can bind to proteins in unexpected ways and highlight the need to uncover these interactions for the further development of designer haem proteins.

Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr (cell wall remodeling), encodes two linked genes, cwr-1 and cwr-2. Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the catalytic (PMO) domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein-protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR-1 PMO domain.

Heme proteins have proven to be a convenient platform for the development of designer proteins with novel functionalities. This is achieved by substituting the native iron porphyrin cofactor with a heme analogue that possesses the desired properties. Replacing the iron center of the porphyrin with another metal provides one inroad to novel protein function. A less explored approach is substitution of the porphyrin cofactor with an alternative tetrapyrrole macrocycle or a related ligand. In general, these ligands exhibit chemical properties and reactivity that are distinct from those of porphyrins. While these techniques have most prominently been utilized to develop artificial metalloenzymes, there are many other applications of this methodology to problems in biochemistry, health, and medicine. Incorporation of synthetic cofactors into protein environments represents a facile way to impart water solubility and biocompatibility. It circumvents the laborious synthesis of water-soluble cofactors, which often introduces substantial charge that leads to undesired bioaccumulation. To this end, the incorporation of unnatural cofactors in heme proteins has enabled the development of designer proteins as optical oxygen sensors, MRI contrast agents, spectroscopic probes, tools to interrogate protein function, antibiotics, and fluorescent proteins.Incorporation of an artificial cofactor is frequently accomplished by denaturing the holoprotein with removal of the heme; the refolded apoprotein is then reconstituted with the artificial cofactor. This process often results in substantial protein loss and does not necessarily guarantee that the refolded protein adopts the native structure. To circumvent these issues, our laboratory has pioneered the use of the RP523 strain of E. coli to incorporate artificial cofactors into heme proteins using expression-based methods. This strain lacks the ability to biosynthesize heme, and the bacterial cell wall is permeable to heme and related molecules. In this way, heme analogues supplemented in the growth medium are incorporated into heme proteins. This approach can also be leveraged for the direct expression of the apoprotein for subsequent reconstitution.These methodologies have been exploited to incorporate non-native cofactors into heme proteins that are resistant to harsh environmental conditions: the heme nitric oxide/oxygen binding protein (H-NOX) from Caldanaerobacter subterraneus (Cs) and the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). The exceptional stability of these proteins makes them ideal scaffolds for biomedical applications. Optical oxygen sensing has been accomplished using a phosphorescent ruthenium porphyrin as the artificial heme cofactor. Paramagnetic manganese and gadolinium porphyrins yield high-relaxivity, protein-based MRI contrast agents. A fluorescent phosphorus corrole serves as a heme analogue to produce fluorescent proteins. Iron complexes of nonporphyrin cofactors bound to HasA inhibit the growth of pathogenic bacteria. Moreover, HasA can deliver a gallium phthalocyanine into the bacterial cytosol to serve as a sensitizer for photochemical sterilization. Together, these examples illustrate the potential for designer heme proteins to address burgeoning problems in the areas of health and medicine. The concepts and methodologies presented in this Account can be extended to the development of next-generation biomedical sensing and imaging agents to identify and quantify clinically relevant metabolites and other key disease biomarkers.

The Mediterranean Sea is a hotspot for the introduction of non-indigenous species. Among them, invasive alien species can seriously affect the areas they colonize, not only by altering the marine food webs and community structure, but also by harming certain economic activities, such as fishing, tourism, aquaculture and marine infrastructure. Rugulopteryx okamurae is a brown macroalga considered the fourth most harmful species among the 10 most invasive species reported in the Mediterranean; in fact, it has recently been included in the list of invasive alien species of Union concern due to its serious impact on biodiversity and socioeconomic implications. This species was reported for the first time in the Mediterranean in 2002 in Thau Lagoon (France), introduced by the aquaculture of the Japanese oyster Magallana gigas. Since then, this species has spread in both the Mediterranean and Atlantic Ocean with a strong invasive behavior, particularly along the Strait of Gibraltar. Rugulopteryx okamurae was also recently reported for the first time in Italian waters, in the Gulf of Palermo (Sicily). In this study, we report a record of this species further to the east, in the Ionian Sea, where it has not previously been recorded, and provide possible explanations of the spreading and arrival routes in this area. The rapid expansion of R. okamurae is raising concerns; thus, in order to safeguard native communities, it is crucial to keep monitoring this invasive species so that it is possible to regularly update its distribution and follow up on its spread dynamics in the Mediterranean basin.

Nitric oxide (NO) has long been known to be an intermediate in bacterial pathways of denitrification. Only in the middle to late 1980s was it found to play a central role in a much broader biological context. For example, it is now well established that NO acts as a signaling agent in metazoans, including humans, yet NO is toxic and very reactive under biological conditions. How is the biology in which NO plays a role controlled? How is NO used and the inherent toxicity avoided? Looking back at the initial discovery time, to the present, and on to the future provides many answers to questions such as those listed above.