Signal Peptide Research Articles

Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR-Cas12a.

CRISPR-Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR-Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR-Cas12a enzyme, a dual-mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high-risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high-risk pooled HPV subtypes. To enable low-cost point-of-care testing, the assay is incorporated into a paper-based microfluidic chip. Furthermore, the clinical performance of the paper-based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.

Multimodal Fusion-Based Lightweight Model for Enhanced Generalization in Drug-Target Interaction Prediction.

Predicting drug-target interactions (DTIs) with precision is a crucial challenge in the quest for efficient and cost-effective drug discovery. Existing DTI prediction models often require significant computational resources because of the intricate and exceptionally lengthy protein target sequences. This study introduces MMF-DTI, a lightweight model that uses multimodal fusion, to improve the generalizability of DTI predictions without sacrificing computational efficiency. The MMF-DTI model combines four distinct modalities: molecular sequence, molecular properties, target sequence, and target function description. This approach is noteworthy because it is the first to use natural language-based target function descriptions in predicting DTIs. The effectiveness of MMF-DTI has been confirmed through benchmark data sets, demonstrating its comparable performance in terms of generalizability, especially in scenarios with limited information about the drug or target. Remarkably, MMF-DTI accomplishes this using only half of the parameters and 17% of the VRAM compared with previous state-of-the-art models. This allows it to function even in constrained computational environments. The combination of performance and efficiency highlights the potential of multimodal data fusion in improving the overall applicability of models, providing promising opportunities for future drug discovery endeavors.

Targeting n-myristoyltransferases promotes a pan-Mammarenavirus inhibition through the degradation of the Z matrix protein.

Several Old World and New World Mammarenavirus are responsible for hemorrhagic fever in humans. These enveloped viruses have a bi-segmented ambisense RNA genome that encodes four proteins. All Mammarenavirus identified to date share a common dependency on myristoylation: the addition of the C14 myristic acid on the N-terminal G2 residue on two of their proteins. The myristoylation of the Z matrix protein is required for viral particle budding, while the myristoylation of the signal peptide to the envelope glycoproteins is important for the entry mechanism. Using Mopeia virus as a model, we characterized the interaction of the Z matrix protein with the N-Myristoyltransferases (NMT) 1 and 2, the two enzymes responsible for myristoylation in mammals. While both enzymes were capable to interact with Z, we showed that only NMT1 was important for the production of viral progeny, the endogenous expression of NMT2 being insufficient to make up for NMT1 in its absence. Using the high affinity inhibitors of NMTs, IMP1088 and DDD85646, we demonstrated a strong, dose dependent and specific inhibition at the nanomolar range for all Mammarenavirus tested, including the highly pathogenic Lassa, Machupo, Junin and Lujo viruses. Mechanistically, IMP1088 and DDD85646 blocked the interaction between Z and both NMTs, preventing myristoylation and further viral particle formation, egress and spread. Unexpectedly, we found that the matrix protein devoid of myristate, despite being fully translated, did not accumulate as the other viral proteins in infected cells but was instead degraded in a proteasome- and autophagy-independent manner. These molecules represent a new broad-spectrum class of inhibitors against Mammarenavirus.

Effects of anisometropic amblyopia on visual cognitive functions in children.

To investigate visual cognitive functions, including visual attention, executive function, and visual working memory, in children with anisometropic amblyopia versus those with normal vision. Thirty-five children with anisometropic amblyopia and 34 with normal vision participated. Visual acuity, stereoacuity, and contrast sensitivity were measured, followed by the Cambridge Neuropsychological Test Automated Battery's six subtests for cognitive evaluation. Visual attention was assessed using reaction time (RTI) and rapid visual information processing (RVP). Executive function was evaluated through the multitasking test (MTT). Visual working memory was assessed with spatial working memory (SWM), delayed matching to sample (DMS), and paired association learning (PAL), all under binocular conditions. The amblyopia group exhibited longer reaction and movement times in the RTI than the control group (p < 0.01). A trend towards lower RVP A' scores, reflecting reduced ability to detect target sequences, appeared in the amblyopia group (p = 0.056). Amblyopic children demonstrated a lower multitasking cost in the MTT compared with the control group (p = 0.04). As difficulty increased in the SWM (from four to six boxes), amblyopic children revisited more (p = 0.01). In the DMS task, while no differences were observed across all delay times (p = 0.55), amblyopic children performed significantly worse than the control group under the 12-second delay (p = 0.04). In the eight-pattern PAL condition, the amblyopia group made more errors (p = 0.01). Children with anisometropic amblyopia performed poorly on neuropsychological tests, particularly visual attention and working memory, but outperformed the control group in multitasking. These findings highlight the broader cognitive impacts of anisometropic amblyopia beyond vision.

Genome-wide characterization of the sunflower kinome: classification, evolutionary analysis and expression patterns under different stresses

Protein kinases play a significant role in plant responses to biotic and abiotic stresses, as well as in growth and development. While the kinome has been extensively investigated in crops such as Arabidopsis thaliana, soybean, common bean, and cotton, studies on protein kinases in sunflower remain limited. Our objective is to explore protein kinases in sunflower to bridge the research gap and enhance the understanding of their functions. We identified a total of 2,583 protein kinases from sunflower, which were classified into 22 families and 121 subfamilies. By comparing the subfamily members between sunflower and other species, we found that three subfamilies in sunflower—RLK-Pelle_CrRLK1L-1, RLK-Pelle_SD-2b, and RLK-Pelle_WAK—had undergone significant expansion. We then investigated the chromosomal distribution, molecular weight, isoelectric point, transmembrane domain, signal peptide, and structural and evolutionary diversity of the protein kinases. Through these studies, we have obtained a basic understanding of protein kinases in sunflower. To investigate the role of protein kinases in sunflower’s response to biotic and abiotic stresses, we obtained 534 transcriptome datasets from various research groups, covering eight types of abiotic stress and two types of biotic stress. For the first time, we overcame the batch effects in the data and utilized a gene scoring system developed by our lab to perform a comprehensive analysis of multiple transcriptome datasets from different research groups. Ultimately, 73 key protein kinases were identified from numerous candidates, and functional annotation revealed that they are key members of signaling pathways such as ABA, MAPK, and SOS, actively participating in sunflower’s response to biotic and abiotic stresses. In summary, through the exploration of protein kinases in sunflower, we have filled the gap in protein kinase research and provided a substantial amount of foundational data. By using the new scoring method to eliminate batch effects between transcriptome datasets, we achieved the first comprehensive analysis of large-scale transcriptome data. This method allows for a more thorough and detailed identification of key protein kinases that are widely regulated under various stress conditions, providing numerous candidate genes for sunflower stress resistance research.

Development of a Versatile Plant-Derived Mitochondrial Targeting Sequence Based on a Reporter Protein Sorting Analysis and Biological Information.

Methods for the delivery of exogenous substances to specific organelles are important because each organelle functions according to its own role. Specifically, mitochondria play an important role in energy production. Recently, plant mitochondrial transformation via delivery methods to mitochondria has been actively researched. Mitochondrial targeting sequences (MTSs) are essential for transporting bioactive molecules, such as nucleic acids, to mitochondria. However, the selectivity and efficacy of MTSs as carrier molecules in plants are not yet sufficient. In this study, we developed an effective MTS in plants via a quantitative comparison of the targeting functions of several MTSs. The presequence of HSP60 from Nicotiana tabacum, which is highly similar to that of several other model plants, showed high mitochondrial-targeting ability among the MTSs tested. This result suggests the applicability of the HSP60 presequence for MTSs in various plants. We further investigated this HSP60 presequence through stepwise shortening on the basis of secondary structure prediction, aiming to simplify synthesis and increase the solubility of the peptides. As shown by assessment of the mitochondrial targeting ability, the 15 residues from the N-terminus of the HSP60 presequence for the MTS, which is particularly conserved among various model plants, retained a targeting efficacy comparable to that of the full-length HSP60 presequence. This developed sequence from the HSP60 sequence is a promising MTS for transfection into plant mitochondria.

Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach.

Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form. Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures. According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures. The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.

Characterization of a novel short-type peptidoglycan recognition protein from the sea cucumber Apostichopus japonicus.

Peptidoglycan recognition proteins (PGRPs) represent a key component of the family of pattern recognition receptors (PRRs). The functional mechanisms of PGRPs in innate immunity are poorly understood. In this study, we identified a novel short-type PGRP, AjPGRP-S2, from the sea cucumber Apostichopus japonicus. Our data showed that AjPGRP-S2 encoded an extracellular protein that possessed a signal peptide, a complete zinc (Zn2+) binding site, and a disulfide bond. A recombinant AjPGRP-S2 (rAjPGRP-S2) lacking the signal peptide was generated and demonstrated to exhibit amidase activity. Tissue expression analysis revealed that AjPGRP-S2 was highly expressed in coelomocytes and tube feet. Immune-responsive analysis indicated that AjPGRP-S2 was able to bind to various pathogen-associated molecular patterns (PAMPs) from bacteria and fungi, as well as to Gram-positive and -negative bacteria, and was majorly induced by DAP-PGN challenge. Basing on RNA-Seq and Pearson's correlation testing, RNA interference, and pull-down analysis, AjPGRP-S2 was found to be involved in transducing immune signals to the complement system and other PRRs, such as fibrinogen, by protein interactions to further recognize and kill pathogens. To respond comprehensively against pathogenic invasion, AjPGRP-S2 may also have the potential in transducing immune signals to key processes, such as cell adhesion, nerve conduction, apoptosis, and transcription by complex pathways that have yet to be elucidated. Our findings not only promote our understanding of the immune-related function and mechanisms of the PGRP family in A. japonicus, but also provide important data that will facilitate the identification of key evolutionary characteristics associated with invertebrate PGRPs.

Cas10 relieves host growth arrest to facilitate spacer retention during type III-A CRISPR-Cas immunity.

Cells from all kingdoms of life can enter growth arrest in unfavorable environmental conditions. Key to this process are mechanisms enabling recovery from this state. Staphylococcal type III-A CRISPR-Cas loci encode the Cas10 complex that uses a guide RNA to locate complementary viral transcripts and start an immune response. When the target sequence is expressed late in the viral lytic cycle, defense requires the activity of Csm6, a non-specific RNase that inhibits the growth of the infected cell. How Csm6 protects from infection and whether growth can be restored is not known. Here, we show that growth arrest provides immunity at the population level, preventing viral replication and allowing uninfected cells to propagate. In addition, the ssDNase activity of Cas10 is required for the regrowth of a subset of the arrested cells and the recovery of the infected host, presumably ending the immune response through degradation of the viral DNA.

Overexpression of the pullulan synthetase gene enhanced pullulan production and its molecular weight by a mutant of Aureobasidium melanogenum P16

The pullulan synthetase gene (PUL1), involved in pullulan biosynthesis in Aureobasidium species, remains poorly understood. The open reading frame (ORF) of the PUL1 gene from the high pullulan-producing yeast Aureobasidium melanogenum P16 strain was cloned and characterized. The ORF of the PUL1 gene was determined to be 592 bp in length, encoding 178 amino acid residues. It was observed that an intron of 55 bp disrupted the gene. The promoter of the PUL1 gene contained a CAAT box, a TATA box, and a 5’-HGATAR-3′ sequence. The deduced protein possessed a signal peptide comprising 18 amino acids and harbored five potential N-glycosylation sites. Following the disruption of the PUL1 gene in strain P16, the disruptant DP108 yielded 34.7 ± 0.3 g/L of pullulan from sucrose, significantly lower than the production by its wild-type strain P16. This discrepancy underscores the close association between the PUL1 gene and pullulan biosynthesis. The majority of the fused Gfp-Pul1 proteins were found to be localized in the cell membrane and on the surface of vacuoles within the yeast-like fungal cells, indicating that pullulan biosynthesis occurred at these subcellular sites. Following the overexpression of the PUL1 gene, strain G14 produced >72.0 g/L of pullulan from sucrose, surpassing the production of its wild-type counterpart strain P16, which yielded 65.5 g/L of pullulan under the identical conditions. This outcome demonstrated that the overexpression of the PUL1 gene significantly enhanced pullulan production. The apparent molecular mass of the purified pullulan increased to 4.4 × 105 Da. As an auxiliary protein, Pul1 was predicted to bind to AmAgs2, the key enzyme in pullulan biosynthesis, facilitating enhanced pullulan production.

Analysis of the Molecular Structure of Lipase-Dependent Chaperone from Ralstonia pickettii Strain BK6

Several biotechnology industries are exploring the characteristics of lipase-dependent chaperones due to their distinctive biochemical traits. This study aimed to employ bioinformatics to analyze the molecular structure of Ralstonia picketii BK6's lipase-dependent chaperon (LipRM). The sequence mapping and amino acid distribution were examined using BioEdit (version 7.0.9.1). SignalP 5.0 and Interpro are employed for signal peptide detection, whereas Swiss-Model and VMD 1.9.2 are used for molecular dynamics modelling. The results showed that the Shine-Dalgarno sequence was discovered in the LipRM promoter, seven nucleotides upstream of the initiation codon (AUG) with the 5'-AGGAGA-3', and has a terminator region that facilitates the formation of a secondary structure. The protein's 3D structure prediction results indicate differences in the alpha helix chains (residues 166-174 and 254-271) between LipRM and the reference lipase. LipRM's molecular structure comprises a detachable signal peptide, and with variations in helix alpha chain conformation and ligand geometry.

Development of a loop-mediated isothermal amplification method for the rapid detection of Clostridium botulinum serotypes E and F.

Botulinum neurotoxin serotypes E and F (BoNT/E and BoNT/F) produced by the bacteria Clostridium botulinum (C. botulinum) found in a wide variety of foods cause poisoning in humans with high mortality rates. Mouse bioassays (MBAs), the gold standard method for BoNT detection, have a low detection limit; however, require experienced personnel and take a long time to obtain results. Therefore, it has been gradually replaced by nucleic acid amplification tests (NAATs) with primers targeting species-specific genes. In this study, for each serotype E and F, six LAMP primers were designed based on multiple sequence alignments of the conserved regions of the bont/E and bont/F genes collected from 180 serotype E strains and 23 serotype F strains published in NCBI. In silico PCR amplification with the outer primer pairs showed successful amplification of the target fragments. To validate the LAMP method, we constructed two synthetic plasmids containing the target sequences extended approximately 10-50bp to both ends. The specificity of the primers was further evaluated using six different Clostridium species and eight strains belonging to other common food poisoning-related bacterial species. Employing the synthetic plasmids, the optimal temperatures and limits of detection (LODs) were determined for bont/E (63°C, LOD ≤ 101 copies/reaction) and bont/F (65°C, LOD ≤ 102 copies/reaction) within 30min. In addition, the LAMP primer set for BoNT/F was redesigned with degenerate nucleotides that improved the coverage from 15 to 45%. For future directions, applications of the established method, especially with the degenerate primers, could be used as an alternative assay for the rapid and sensitive detection of C. botulinum.

A Sensitive, Specific and Fast Electrochemical‐based Nanobiosensor Diagnostic for Xanthomonas albilineans, the Cause of Sugarcane Leaf Scald Disease

AbstractLeaf scald (LS) caused by Xanthomonas albilineans (Xalb), is a major bacterial disease of sugarcane. The unreliable symptom expressions make traditional visual detection challenging. The molecular methods of detection require expensive equipment, labor‐intensive, and time‐consuming. This study proposes a novel electrochemical (EC)‐approach, that is relatively easy to use and less expensive to detect Xalb DNA in LS‐infected sugarcane leaves, meristematic tissue, and xylem sap samples. This method involves three key steps: i) DNA isolation from sugarcane samples via boiling lysis; ii) magnetic purification of target sequences from the lysate using magnetic bead‐bound capture probes; and iii) EC detection of the target DNA. The method shows excellent detection sensitivity (10 cells µL−1), reproducibility (Standard deviation, SD &lt;5%, for n = 3), and a wide linear dynamic range (1 nM–1 fM or 106–10° copies µL−1, r = 0.99). The EC assay has a strong negative correlation with quantitative polymerase chain reaction (qPCR) results (r = −0.95–0.97, n = 24, p &lt; 0.001), and weak or no correlation with the varietal resistance ratings. This EC‐based assay can be a commercially viable alternative, providing a DNA isolation/purification‐free solution, and can potentially be adapted into a handheld device for on‐farm detection and quantification of the LS‐causing pathogen.

The current state and prospects of peptides use in cosmeceuticals

Aim. To analyze the range of peptides in cosmeceuticals. Materials and methods. The study materials were publications in scientific periodicals. The study used methods of content analysis, comparative, logical, analytical methods and generalization of information. Results. The analysis of scientific literature data has shown that bioactive peptides of various origins are widely used as active ingredients in cosmeceuticals. In 2022, the global market for the synthesis of cosmetic peptides was estimated in 195.3 million USD. The market is expanding due to the growing consumer demand for skin care and anti-ageing products. Consumers are shifting towards a holistic approach to beauty, appreciating products that not only provide instant results, but also support the skin’s natural processes. Peptides, with their various benefits, perfectly match this approach by promoting skin elasticity from the inside, providing a long-lasting positive result. Peptides can be used to address specific skin problems, such as hyperpigmentation, acne and inflammation. According to the mechanism of action, biologically active peptides are classified as carrier peptides, signal peptides, neurotransmitter inhibitor peptides and enzyme inhibitor peptides. Carrier peptides facilitate the transport of cofactors, such as copper and manganese, across the skin barrier. Both cofactors are essential for enzymatic reactions involved in preventing aging and wound healing. Copper tripeptide-1 and manganese tripeptide-1 are examples of carrier peptides that have been successfully used to reduce fine lines, wrinkles and hyperpigmentation associated with photoaging. Signal peptides or matrikines are derived from extracellular matrix proteins, such as collagen, elastin and fibronectin. Matrikines interact with specific receptors that stimulate the synthesis, repair and remodeling of the extracellular matrix. These peptides also regulate the activity of certain key enzymes involved in the aging process, such as collagenase, elastase, tyrosinase and hyaluronidase. Neurotransmitter inhibitor peptides are the latest cosmeceutical peptides. Similar to botulinum toxins, these peptides prevent the release of acetylcholine, a neurotransmitter responsible for muscle contraction. Inhibiting this process relaxes facial muscles, preventing the formation of fine lines and wrinkles. Neurotransmitter inhibitor peptides are a safer alternative to traditional botulinum toxin treatment with fewer potential side effects. Enzyme inhibitor peptides have become popular active ingredients in anti-ageing cosmetics. This class of peptides inhibits the activity of certain enzymes involved in various biochemical processes of the skin. Their application in cosmeceuticals is to control or inhibit those processes that cause skin damage, aging or loss of elasticity. Conclusions. The study of peptides for use in cosmeceuticals has opened new prospects in skin care and aesthetic medicine. Bioactive peptides of various origins – from plants, animals, marine organisms, and edible insects – exhibit a wide range of properties, including antiaging, antioxidant, anti-inflammatory, and antimicrobial effects. Peptides open new horizons in the creation of innovative cosmeceuticals, especially when combined with other active components, such as antioxidants or retinoids. Their safety, ability to biodegrade and spot impact make peptides an attractive ingredient for solving various dermatological problems without the side effects of more aggressive skin correction methods.

The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations

Repair of double-stranded breaks generated by CRISPR/Cas9 is highly dependent on the flanking DNA sequence. To learn about interactions between DNA repair and target sequence, we measure frequencies of over 236,000 distinct Cas9-generated mutational outcomes at over 2800 synthetic target sequences in 18 DNA repair deficient mouse embryonic stem cells lines. We classify the outcomes in an unbiased way, finding a specialised role for Prkdc (DNA-PKcs protein) and Polm in creating 1 bp insertions matching the nucleotide on the protospacer-adjacent motif side of the break, a variable involvement of Nbn and Polq in the creation of different deletion outcomes, and uni-directional deletions dependent on both end-protection and end-resection. Using our dataset, we build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of Cas9-generated mutations.

Unraveling dual fusion mechanisms in BmNPV GP64: critical roles of CARC motifs and signal peptide retention.

Viral membrane fusion is a critical process enabling viruses to invade host cells, driven by viral membrane fusion proteins (MFPs). Cholesterol plays a pivotal role in this process, which is essential for the infectivity of many enveloped viruses. The interaction between MFPs and cholesterol is often facilitated by specific amino acid motifs known as cholesterol recognition/interaction amino acid consensus (CRAC) motifs and reverse CARC motifs. In a previous study, we demonstrated that CRAC1 and CRAC2 in GP64 are required for Bombyx mori nucleopolyhedrovirus (BmNPV) infection. This study further investigates the role of CARC in the GP64 protein of BmNPV, revealing their complex interaction with cholesterol and the influence of signal peptide (SP) retention on viral infectivity. We identified six putative CARC motifs in GP64 and generated mutants to assess their function. Our findings show that CARC1, CARC2, CARC3, and CARC4 are indispensable for viral fusion and infection when the SP is retained, whereas only CARC2 and CARC3 remain essential after SP cleavage. In contrast, CARC1 and CARC4 are necessary for viral infection through a cholesterol-independent mechanism resulting from double mutations in the CRAC1 and CRAC2 motifs of GP64. These insights not only deepen our understanding of BmNPV GP64-mediated fusion but also highlight potential antiviral targets, underscoring the adaptability and resilience of viral fusion mechanisms.IMPORTANCEUnderstanding viral membrane fusion mechanisms is crucial for developing antiviral strategies. This study provides novel insights into the intricate roles of CARC and CRAC motifs in the GP64 protein of BmNPV, particularly their interaction with cholesterol and the influence of signal peptide retention. The discovery that certain CARC motifs are essential for cholesterol-dependent fusion, whereas others function in a cholesterol-independent context advances our understanding of viral fusion processes. These findings emphasize the potential of targeting CARC motifs for therapeutic interventions and underline the importance of cholesterol interactions in viral infections. This research not only deepens our understanding of BmNPV fusion mechanisms but also has broader implications for other enveloped viruses.

From lock and key to molecular diplomacy: understanding pollen recognition and discrimination in brassicaceae.

Hybridization barriers in Brassicaceae play a pivotal role in governing reproductive success and maintaining speciation. In this perspective, we highlight recent advances revealing the intricate molecular mechanisms and the interplay among key players governing these barriers. Recent studies have shed light on the molecular mechanisms that govern hybridization barriers in Brassicaceae. The interplay between pollen coat proteins, stigmatic receptors, and signaling peptides plays a crucial role in determining the success of pollination. At the core of this system, autocrine stigmatic RALF peptides (sRALF) maintain the stigmatic barrier by activating the FERONIA (FER) and ANJEA (ANJ) receptor complex, triggering the RAC/ROP-RBOHD pathway and subsequent reactive oxygen species (ROS) production. It is now established that incompatible pollen rejection is mediated by two parallel pathways: the FER-RAC/ROP-RBOHD pathway, which generates ROS, and the ARC1-mediated pathway, which degrades compatible factors required for pollen growth. Conversely, compatible pollen overcomes the stigmatic barrier through the action of pollen coat proteins (PCP-B) and paracrine pollen-derived RALF peptides (pRALF), which compete with autocrine sRALF for receptor binding, enabling successful pollen hydration and tube penetration. The "lock-and-key" mechanism involving sRALF and pRALF provides species-specific recognition of compatible pollen. These findings offer valuable insights into the molecular basis of hybridization barriers and open new possibilities for overcoming these barriers in interspecific and intergeneric crosses within Brassicaceae, with potential applications in plant breeding and crop improvement. Future research should focus on elucidating the evolutionary dynamics of these signaling pathways and exploring their manipulation for crop breeding purposes.

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the structure, the biosynthetic pathway, and the mode of action of a RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the bacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear nonheme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are rare examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide allowing them to be exported by the ubiquitous Sec pathway. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by complexing excess metal ions. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in bacteria.

Evaluation of a New Tandem Mass Spectrometry Method for Sickle Cell Disease Newborn Screening

In France, sickle cell disease newborn screening (SCD NBS) has been targeted to at-risk regions since 1984, but generalization to the whole population will be implemented from November 2024. Although tandem mass spectrometry (MS/MS) is already used for the NBS of several inherited metabolic diseases, its application for SCD NBS has not been widely adopted worldwide. The aim of this study was to evaluate a dedicated MS/MS kit (Targeted MS/MS Hemo, ZenTech, LaCAR Company, Liege, Belgium) for SCD NBS and to compare the results obtained with those from an NBS reference center using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and cation-exchange high-performance liquid chromatography (CE-HPLC, Variant NBS, Biorad Laboratories, Inc., Hercules, CA, USA) as confirmatory method. The MS/MS Hemo kit was used according to the manufacturer’s instructions and performed on a Waters Xevo TQ-D (Waters Corporation, USA). The software provided by the manufacturer was used for the calculation and analysis of peptide signal ratios. Among the 1333 samples, the results of 1324 samples were consistent with the HPLC and/or MALDI-TOF results (1263 FA, 50 FAS, 7 FAC, 1 FAO-Arab, and 3 FS). All the discordant results (one FAS on MS/MS vs. FA in CE-HPLC, one FA on MS/MS vs. FAS in CE-HPLC, seven FS on MS/MS vs. FAS in CE-HPLC) were corrected after modifying the peptide signal ratios thresholds, allowing the MS/MS Hemo kit to achieve near-100% sensitivity and specificity for SCD NBS. In conclusion, the MS/MS Hemo kit appears to be an effective method for SCD NBS, particularly for laboratories already equipped with MS/MS technology. However, these results should be confirmed in a larger cohort including a greater number of positive samples for SCD.

Comparative Secretory Efficiency of Two Chitosanase Signal Peptides from Bacillus subtilis in Escherichia coli.

The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1mM and 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1mM IPTG induction for 3days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region. This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency. Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E. coli.