Spore Proteins Research Articles

Graphite‐Based Bio‐Mimetic Nanopores for Protein Sequencing and Beyond

AbstractProtein sequencing using nanopores represents the next frontier in bio‐analytics. However, linearizing unfolded proteins and controlling their translocation speed through solid‐state nanopores pose significant challenges in protein sequencing. In order to address these issues, this work proposes a biomimetic graphite‐based nanopore construction. These nanopores feature a nanometer‐sized pore with a constriction zone, mimicking the structure of the α‐hemolysin protein pore. Our all‐atom Molecular Dynamics simulations demonstrate the high practical potential of these nanopores by revealing how their charge state renders them complete ion‐selective and generates an electro‐osmotic flow. This study shows that this nanopore construction can detect peptides at the single amino acid level by analyzing the ionic current traces generated as peptides traverse the nanopore. The novelty of the proposed nanopore lies in its ability to modulate the hydrodynamic drag induced by electro‐osmotic flow, relative to the electro‐phoretic force. This investigation reveals that tuning these forces helps to linearize translocating peptides and extend the residence time of individual amino acids at the constriction zone of the pore. This significantly enhances the detection and sequencing efficiency of the pore. Furthermore, the high relevance of the proposed nanopores is underscored for seawater desalination through electrodialysis and extends to ion separation under salinity gradients.

Neutrophil-activating protein in Bacillus spores inhibits casein allergy via TLR2 signaling

BackgroundMilk allergy commonly occurs in children, mainly caused by bovine-derived casein (CAS) protein. Neutrophil-activating protein (NAP) of Helicobacter pylori plays an immunomodulatory role with potential to suppress Th2-type immune responses. Bacillus subtilis (B. subtilis) spores are commonly used as oral vectors for drug delivery.ObjectiveTo investigate whether recombinantly expressed NAP on B. subtilis spores could be an effective treatment for CAS allergy in mouse.MethodsAfter CAS sensitization, mice were orally administered B. subtilis spores expressing recombinant NAP for 6 weeks. Allergic symptoms and parameters were evaluated after CAS challenge oral gavage, including allergic inflammation, splenic cytokines, and serum-specific antibodies. Protein levels of Toll-like receptor 2 (TLR2) and c-JUN in the jejunum tissue were measured by western blot. Bone marrow-derived macrophages (BMDMs) were stimulated with inactivated NAP spores to measure the influence on cytokine profiles in vitro.ResultsNAP recombinant spore treatment significantly reduced allergic symptoms and intestinal inflammation. Interleukin-12 and interferon-gamma levels increased, whereas serum CAS-specific IgG1 and IgE levels decreased. TLR2 and c-JUN expression levels were elevated in the jejunal tissue. Inactivated NAP spores polarized BMDMs to the M1 phenotype and enhanced cytokine expression, which were inhibited by a TLR2 neutralizing antibody.ConclusionNAP offers a new strategy in the treatment of CAS allergy by inhibiting the Th2 response, while eliciting macrophages to promote Th1 immune responses.

Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells, which was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomic analysis of the whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile.

Stochastic Sensing of Chloride Anions Using an α-Hemolysin Pore with a semiaza-Bambusuril Adapter.

Pores containing molecular adapters provide internal selective binding sites, thereby allowing the stochastic sensing of analytes. Herein, we demonstrate that semiaza-bambusuril (BU) acts as a non-covalent molecular adapter when lodged within the lumen of the wild-type α-hemolysin (WT-αHL) protein pore. Because the bambusurils are recognized as anion receptors, the anion binding site within the adapter-nanopore complex allows the detection of chloride anions, thus converting a non-selective pore into an anion sensor.

Consciousness: a quantum optical effect in fluorescent protein pathways

This paper presents a physical mechanism of embodied consciousness based on the dynamic organicity theory. We use the entropic pilot wave theory to describe the quantum dipole oscillations from dipole-bound delocalized (quasi-free) electrons in nonpolar nanocavities of aromatic amino acid residues (benzene moiety /cyclic hydrocarbons that contain resonance structures) and their fluorescent pathways. These pathways contain cavity quasipolariton condensates composed of quantized polarization waves of entangled photons. The behavior of oscillating molecular dipoles is influenced by the quantum nature of dynamic organicity, which causes energy fluctuations necessary to move delocalized electrons and create bare polaritons (quantized polarization waves). These waves correspond to photon quasiparticles interacting with water molecules to form quasipolaritons, softened by interacting with hydroxide ions (OH-) within crystal lattices of interfacial water H30. When [H3O+]=[OH−], the solution is neutral in hydrophobic nanocavities. In such nonpolar cavities, evanescent photons (non-radiative transitions in the absence of any source) are emitted while protons (H+) diffuse due to recombination with hydroxide (OH-) ions, acting as protonic analogs of ‘holes’ or excitons. In protein pores, proton motion strongly coupled with π-electron delocalization is a conduit for exciton-photon (polariton) and its polarization wave component. Internal energy fluctuations comprise both quantum potential energy and negative quantum kinetic energy. We explore how quantum potential energy influences the negentropic effect of a 'repulsive force' guided by entropic pilot waves through pilot wave force (negentropic force), functioning as an information-based action of the polarization wave as a conduit of the cavity quasipolariton. When the rate of change of quantum entropy is zero, and momentum of the polarization wave is zero the internal energy transduction between quantum potential energy to quantum kinetic energy manifests as the negentropic force and facilitates the movement of information-based action for information encoding when negative quantum kinetic energy happens at certain times. Experiments have shown that biphoton entanglement interferes with anaesthetics. We postulate that the disruption of cavity polaritonic condensate through its polarization wave component disrupts the decoding of information, suggesting consciousness is attributed to a quantum optical effect. Therefore, we further theorize that consciousness is attributed to the negative quantum kinetic energy of the polarization wave, which provides an informational structure of multiscale redundancy when negentropic action reduces information redundancy. This has profound implications for understanding consciousness as nonmechanical action channeled through negentropic force when the rate of change of quantum entropy is zero.

Research Progress on Saccharide Molecule Detection Based on Nanopores.

Saccharides, being one of the fundamental molecules of life, play essential roles in the physiological and pathological functions of cells. However, their intricate structures pose challenges for detection. Nanopore technology, with its high sensitivity and capability for single-molecule-level analysis, has revolutionized the identification and structural analysis of saccharide molecules. This review focuses on recent advancements in nanopore technology for carbohydrate detection, presenting an array of methods that leverage the molecular complexity of saccharides. Biological nanopore techniques utilize specific protein binding or pore modifications to trigger typical resistive pulses, enabling the high-sensitivity detection of monosaccharides and oligosaccharides. In solid-state nanopore sensing, boronic acid modification and pH gating mechanisms are employed for the specific recognition and quantitative analysis of polysaccharides. The integration of artificial intelligence algorithms can further enhance the accuracy and reliability of analyses. Serving as a crucial tool in carbohydrate detection, we foresee significant potential in the application of nanopore technology for the detection of carbohydrate molecules in disease diagnosis, drug screening, and biosensing, fostering innovative progress in related research domains.

Ionic liquid-assisted sample preparation mediates sensitive proteomic analysis of Bacillus subtilis spores

Endospore-forming bacteria are ubiquitous. Bacterial endospores are multilayered proteinaceous structures that protects the bacterial genome during stress conditions. They are also responsible for a wide range of critical clinical infections in humans. Precise analysis of spore-forming pathogens remains a major challenge in the field of proteomics because spore structures are highly resistant to conventional solubilizers and denaturing agents, such as sodium dodecyl sulfate and urea. We present an ionic liquid-assisted (i-soln) technique of sample preparation, called pTRUST, which enables shotgun analysis of Bacillus subtilis spores even when the starting materials are in the sub-microgram range. In proteomic analysis, this technique shows 50–2000-fold higher sensitivity than other conventional gel-based or gel-free methods (including one-pot sample processing). Using this technique, we identified 445 proteins with high confidence from trace amounts of highly pure spore preparations, including 52 of the 79 proteins (approximately 70%) previously demonstrated to be localized in spores in the SubtiWiki database and detected through direct protein analysis. Consequently, 393 additional proteins were identified as candidates for spore constitutive proteins. Twenty of these newly identified candidates were produced as green fluorescent protein fusion proteins, and each was evaluated for authenticity as a spore constituent using fluorescence microscopy analysis. The pTRUST method's sensitivity and reliability using the i-soln system, together with hitherto unreported proteins in spores, will enable an array of spore research for biological and clinical applications.

SpoIIQ-dependent localization of SpoIIE contributes to septal stability and compartmentalization during the engulfment stage of Bacillus subtilis sporulation.

During spore development in bacteria, a polar septum separates two transcriptionally distinct cellular compartments, the mother cell and the forespore. The conserved serine phosphatase SpoIIE is known for its critical role in the formation of this septum and activation of compartment-specific transcription in the forespore. Signaling between the mother cell and forespore then leads to activation of mother cell transcription and a phagocytic-like process called engulfment, which involves dramatic remodeling of the septum and requires a balance between peptidoglycan synthesis and hydrolysis to ensure septal stability and compartmentalization. Using Bacillus subtilis, we identify an additional role for SpoIIE in maintaining septal stability and compartmentalization at the onset of engulfment. This role for SpoIIE is mediated by SpoIIQ, which anchors SpoIIE in the engulfing membrane. A SpoIIQ mutant (SpoIIQ Y28A) that fails to anchor SpoIIE, results in septal instability and miscompartmentalization during septal peptidoglycan hydrolysis, when other septal stabilization factors are absent. Our data support a model whereby SpoIIE and its interactions with the peptidoglycan synthetic machinery contribute to the stabilization of the asymmetric septum early in engulfment, thereby ensuring compartmentalization during spore development.IMPORTANCEBacterial sporulation is a complex process involving a vast array of proteins. Some of these proteins are absolutely critical and regulate key points in the developmental process. Once such protein is SpoIIE, known for its role in the formation of the polar septum, a hallmark of the early stages of sporulation, and activation of the first sporulation-specific sigma factor, σF, in the developing spore. Interestingly, SpoIIE has been shown to interact with SpoIIQ, an important σF-regulated protein that functions during the engulfment stage. However, the significance of this interaction has remained unclear. Here, we unveil the importance of the SpoIIQ-SpoIIE interaction and identify a role for SpoIIE in the stabilization of the polar septum and maintenance of compartmentalization at the onset of engulfment. In this way, we demonstrate that key sporulation proteins, like SpoIIQ and SpoIIE, function in multiple processes during spore development.

Stochastic Sensing of Chloride Anions Using an α‐Hemolysin Pore with a semiaza‐Bambusuril Adapter

AbstractPores containing molecular adapters provide internal selective binding sites, thereby allowing the stochastic sensing of analytes. Herein, we demonstrate that semiaza‐bambusuril (BU) acts as a non‐covalent molecular adapter when lodged within the lumen of the wild‐type α‐hemolysin (WT‐αHL) protein pore. Because the bambusurils are recognized as anion receptors, the anion binding site within the adapter‐nanopore complex allows the detection of chloride anions, thus converting a non‐selective pore into an anion sensor.

Adsorptive removal of aflatoxin B1 via spore protein from Aspergillus luchuensis YZ-1

Aflatoxin B1 (AFB1) is the most toxic mycotoxin commonly found in the environment. Finding efficient and environmentally friendly ways to remove AFB1 is critical. In this study, Aspergillus luchuensis YZ-1 demonstrated a potent ability to adsorb AFB1 for the first time, and the binding of AFB1 to YZ-1 is highly stable. Spores exhibited higher adsorption efficiency than mycelia, adsorbing approximately 95 % of AFB1 within 15 min. The spores were comprehensively characterized using scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and atomic force microscopy. Various adsorption kinetic models (pseudo-first and pseudo-second order), adsorption isotherm models (Freundlich and Langmuir), Fourier transform infrared, and X-ray photoelectron spectroscopy were used to investigate the adsorption properties and mechanisms. The adsorption capacity of spores decreased with heating, urea, and SDS treatments, indicating that spore proteins may be the primary substance for AFB1 adsorption. Subsequent experiments showed that proteins with molecular weights greater than 50 kDa played a key role in the adsorption. Additionally, the spores possess excellent storage properties and are valuable for adsorbing AFB1 from vegetable oils. Therefore, the YZ-1 spores hold promise for development into a novel biosorbent for AFB1 removal.

Mechanistic insight into roles of α/β-type small acid-soluble proteins, RecA, and inner membrane proteins during bacterial spore inactivation by ohmic heating.

Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores. We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/β-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50V/cm. In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.

Functional Analysis of Stress Resistance of Bacillus cereus SCL10 Strain Based on Whole-Genome Sequencing.

A Gram-positive, rod-shaped, aerobic, motile, and spore-forming bacterium, designated SCL10, was isolated from Acaudina molpadioides exposure to Co-60 radiation. In this study, whole-genome sequencing was performed to identify the strain as Bacillus cereus and functional characterization, with a focus on stress resistance. The genome of the B. cereus SCL10 strain was sequenced and assembled, revealing a size of 4,979,182 bp and 5167 coding genes. The genes involved in biological functions were annotated by using the GO, COG, KEGG, NR, and Swiss-Prot databases. The results showed that genes related to alkyl hydroperoxide reductase (ahpC, ahpF), DNA-binding proteins from starved cells (dps), spore and biofilm formation (spoVG, spo0A, gerP), cold shock-like protein (cspC, cspE), ATP-dependent chaperone (clpB), and photolyase, small, acid-soluble spore protein (SASP) and DNA repair protein (recA, radD) could explain the stress resistance. These findings suggest that antioxidant activity, sporulation, biofilm formation, and DNA protection may be considered as the main resistance mechanisms under exposure to radiation in the B. cereus SCL10 strain.

Electro-osmotic Flow Generation via a Sticky Ion Action.

Selective transport of ions through nanometer-sized pores is fundamental to cell biology and central to many technological processes such as water desalination and electrical energy storage. Conventional methods for generating ion selectivity include placement of fixed electrical charges at the inner surface of a nanopore through either point mutations in a protein pore or chemical treatment of a solid-state nanopore surface, with each nanopore type requiring a custom approach. Here, we describe a general method for transforming a nanoscale pore into a highly selective, anion-conducting channel capable of generating a giant electro-osmotic effect. Our molecular dynamics simulations and reverse potential measurements show that exposure of a biological nanopore to high concentrations of guanidinium chloride renders the nanopore surface positively charged due to transient binding of guanidinium cations to the protein surface. A comparison of four biological nanopores reveals the relationship between ion selectivity, nanopore shape, composition of the nanopore surface, and electro-osmotic flow. Guanidinium ions are also found to produce anion selectivity and a giant electro-osmotic flow in solid-state nanopores via the same mechanism. Our sticky-ion approach to generate electro-osmotic flow can have numerous applications in controlling molecular transport at the nanoscale and for detection, identification, and sequencing of individual proteins.

Chronic Unpredictable Stress Induces Mitochondrial Dysfunction in Hypothalamic Pituitary Adrenal Axis Regions

Background: Stress elicits physiological and behavioral responses via adaptive responses like the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS) activation. Under chronic stress, however, these responses become dysfunctional leading to pathological changes in behavior and health. Mitochondria are dynamic organelles that provide cellular energy, activate immune responses, and synthesize glucocorticoids in the adrenal glands in response to stress. Thus, the mitochondrion is a key candidate linking the effects of chronic stress and health, however, the effects of chronic stress on mitochondrial function in the HPA axis, particularly with respect to sex differences, are largely unexplored. Hypothesis: Chronic unpredictable stress (CUS) decreases mitochondrial function and dynamics in key regions within the HPA axis. Methods: Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of CUS or handling (non-CUS) with weekly weighing. Mice were euthanized 24h after the last stress exposure and the brain and adrenal glands dissected and weighed. Mitochondrial function was assessed using high resolution respirometry in hypothalamus and adrenal tissues. Protein expression was assessed by immunoblot. Results: CUS decreased mitochondrial respiration within key stress centers, with stressed males exhibiting decreased respiration in the amygdala (20%, p=0.05), and stressed females exhibiting decreased respiration in the hypothalamus (20%, p=0.004) and adrenal glands (17%, p=0.06). CUS increased mitochondrial complex protein in male adrenal gland (47%, p=0.01) and mitochondrial mass in hypothalamus (p=0.007). Finally, cleaved Gasdermin D expression was increased nearly 2-fold in male hypothalamus (p=0.03). Conclusions: Our data show sex- and region-dependent decreases in HPA axis mitochondrial function concurrent with upregulated protein expression and pore formation. These observations point to sex-specific effects of CUS to induce mitochondrial dysfunction. This work was supported by P20GM109091 (F.H.), R01HL130972-01A1, R01HL5949, BX000168-10A1, BX005320 (F.G.S.), R01DK132948 (C.W. & F.P.) R01 MH129798 (SKW); VA VISN7 RDA (F.H.), 1U54HL169191-01 & BX002604 (M.J.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the abstract. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Distinct proteomes and allergen profiles appear across the life-cycle stages of Alternaria alternata

Alternaria alternata is associated with allergic respiratory diseases, which can be managed with allergen extract-based diagnostics and immunotherapy. It is not known how spores and hyphae contribute to allergen content. Commercial allergen extracts are manufactured by extracting proteins without separating the different forms of the fungus. We sought to determine differences between spore and hyphae proteomes and how allergens are distributed in Aalternata. Data-independent acquisition mass spectrometry was used to quantitatively compare the proteomes of asexual spores (nongerminating and germinating) with vegetative hyphae. We identified 4515 proteins in nongerminating spores, germinating spores, and hyphae; most known allergens are more abundant in nongerminating spores. On comparing significant protein fold-change differences between nongerminating spores and hyphae, we found that 174 proteins were upregulated in nongerminating spores and 80 proteins in hyphae. Among the spore proteins are ones functionally involved in cell wall synthesis, responding to cellular stress, and maintaining redox balance and homeostasis. On comparing nongerminating and germinating spores, 25 proteins were found to be upregulated in nongerminating spores and 54 in germinating spores. Among the proteins specific to germinating spores were proteases known to be virulence factors. One of the most abundant proteins in the spore proteome is sialidase, which has not been identified as an allergen but may be important in the pathogenicity of this fungus. Major allergen Alt a 1 is present at low levels in spores and hyphae and appears to be largely secreted into growth media. Spores and hyphae express overlapping but distinct proteomes. Most known allergens are found more abundantly in nongerminating spores.

Tobacco aquaporin NtAQP1 and human aquaporin hAQP1 contribute to single cell photosynthesis in Synechococcus.

Aquaporins are H2O-permeable membrane protein pores. However, some aquaporins are also permeable to other substances such as CO2. In higher plants, overexpression of such aquaporins has already led to an enhanced photosynthetic performance due to improved CO2 mesophyll conductance. In this work, we investigated the effects of such aquaporins on unicellular photosynthetically active organisms, specifically cyanobacteria. Overexpression of aquaporins NtAQP1 or hAQP1 that might have a function to improve CO2 membrane permeability lead to increased photosynthesis rates in the cyanobacterium Synechococcus sp. PCC7002 as concluded by the rate of evolved O2. A shift in the Plastoquinone pool state of the cells supports our findings. Water permeable aquaporins without CO2 permeability, such as NtPIP2;1, do not have this effect. We conclude that also in single cell organisms like cyanobacteria, membrane CO2 conductivity could be rate limiting and CO2-porins reduce the respective membrane resistance. We could show that besides the tobacco aquaporin NtAQP1 also the human hAQP1 most likely functions as CO2 diffusion facilitator in the photosynthesis assay.

Effect of Starch Types on the Textural and Rehydration Properties of Extruded Peanut Protein Pore Gel Particles.

In this study, the effect of different starches from corn, potato and pea containing varying amylose/amylopectin ratios on the textural and rehydration properties of extruded peanut protein gel particles were investigated. Results showed that textural and rehydration properties of peanut protein extruded with corn starch, potato starch and amylopectin are slightly inferior to those of peanut protein with pea starch extrudates. The addition of pea starch led to an increase in the pore structure of the peanut protein extrudates and improved their water absorption index, simultaneously reducing the hardness and density. Pea starch, as a natural water-absorbing expansion material, helped peanut protein to form cross-linked gel polymers that bind more water molecules, in addition to further polymerization with peanut protein, which made the protein secondary structure became disordered. These changes directly affected the textural properties of the extrudates. In addition, the blended system of starches and peanut protein tended to form more elastic solids, which affected the expansion of the extrudates. These findings indicate that starch can effectively improve the poor expansion of proteins, making it suitable for use in the production of plant protein-based foods.

Bacillus amyloliquefaciens: a versatile antimicrobial Firmicute for the suppression of wilt caused by Ralstonia pseudosolanacearum in tomato

ABSTRACT Rhizosphere bacteria based biological control strategy is an eco-friendly approach to protect crops from various plant diseases. Among these, spore-forming Bacillus, abundant in the rhizosphere, provide unique advantages for plant health. This study focused on characterising two tomato rhizosphere-associated Bacillus amyloliquefaciens strains (Trb7 and Trb11), highly effective against the bacterial wilt pathogen Ralstonia pseudosolanacearum in vitro. Species identification was based on partial 16S rRNA gene sequencing, supported by phylogenetic analysis. Five antimicrobial peptide (AMP) genes (surfactin, fengycin, bacilysin, subtilin, and spore protein) were detected in both the isolates of B. amyloliquefqciens using specific molecular markers. Greenhouse trials showed that prophylactic application of these isolates led to an 83.4% reduction in tomato bacterial wilt, with only 16.6% wilt incidence compared to the 100% in the R. pseudosolanacearum-inoculated control. Moreover, these B. amyloliquefaciens isolates exhibited plant-growth-promoting traits in vitro. Interestingly, they also showed significant inhibition of mycelial growth against Sclerotium rolfsii and Alternaria alternata, major fungal pathogens in tomatoes. Overall, our findings highlight the potent bactericidal role of B. amyloliquefaciens isolates in suppressing R. pseudosolanacearum in tomatoes. Key highlights Two among the 100 Bacillus displayed antibacterial activity against Ralstonia pseudosolanacearum. Antibacterial firmicutes was identified as Bacillus amyloliquefaciens through the 16S rRNA gene sequence analysis. Five AMP genes (srfAA, fenD, bacA, spas, and spoVG) were identified in the isolates. Delivery of B. amyloliquefaciens drenching on rhizosphere significantly reduced wilt. Additionally, B. amyloliquefaciens showed the antifungal activity on Sclerotium rolfsii and Alternaria alternata.

A novel RyR2 mutation associated with co-morbid catecholaminergic polymorphic ventricular tachycardia (CPVT) and benign epilepsy with centrotemporal spikes (BECTS)

In this case report, we describe a 14-year-old patient with a novel RyR2 gene mutation (c.6577G > T/p.Val2193Leu), identified through a comprehensive review of medical history, examination findings, and follow-up data. The pathogenic potential of this mutation, which results in the loss of some interatomic forces and compromises the closure of the RyR2 protein pore leading to calcium leakage, was analyzed using the I-TASSER Suite to predict the structural changes in the protein. This mutation manifested clinically as co-morbid catecholaminergic polymorphic ventricular tachycardia (CPVT) and benign epilepsy with centrotemporal spikes (BECTS), a combination not previously documented in the same patient. While seizures were successfully managed with levetiracetam, the patient's exercise-induced syncope episodes could not be controlled with metoprolol, highlighting the complexity and challenge in managing CPVT associated with this novel RyR2 variation.

Sensing PEGylated Peptide Conformations Using a Protein Nanopore.

Membrane pores are exploited for the stochastic sensing of various analytes, and here, we use electrical recordings to explore the interaction of PEGylated peptides of different sizes with a protein pore, CymA. This wide-diameter natural pore comprises densely filled charged residues, facilitating electrophoretic binding of polyethylene glycol (PEG) tagged with a nonaarginine peptide. The small PEG 200 peptide conjugates produced monodisperse blockages and exhibited voltage-dependent translocation across the pores. Notably, the larger PEG 1000 and 2000 peptide conjugates yielded heterogeneous blockages, indicating a multitude of PEG conformations hindering their translocation through the pore. Furthermore, a much larger PEG 5000 peptide occludes the pore entrance, resulting in complete closure. The competitive binding of different PEGylated peptides with the same pore produced specific blockage signals reflecting their identity, size, and conformation. Our proposed model of sensing distinct polypeptide conformations corresponds to disordered protein unfolding, suggesting that this pore can find applications in proteomics.