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Many pathological conditions are a result of intragenic epistasis; however, there are ambiguities in current epistasis models. Herein, the new Mutation Interaction Spectrum model defines a discrete outcome, named a Mutation Interaction, for each double point mutation in a gene and its component single mutations. The model is a universal genetic model of all types of mutation interactions and their functional outcomes and is derived from digital logic, commonly used in electrical engineering. Mutation interactions are normally classified as positive and negative epistasis. The model logics unifies common genetic relationships into one model, normalizing biological nomenclature, and disambiguates them with the 16 possible logic-based interactions. The model was tested by assaying transcriptional activity induced by HIV-1 Tat protein, for a random sampling of 3429 double mutations and all 1615 single mutations. All possible types of logic were observed for the Tat mutation interactions., Competing Interests: Declaration of competing interest The authors declare that CJM and MRS are employees of Heligenics and JIR is a Director of Heligenics. The authors have no other potential conflicts of interest to disclose. All authors reviewed the final version of the manuscript and agreed with its publication., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Globally, the continuous spread and evolution of SARS-CoV-2, along with its variants, profoundly impact human well-being, health, security, and the growth of socio-economic. In the field of development of drugs against COVID-19, the main protease (M pro ) is a critical target as it plays a core role in the lifecycle of SARS-CoV-2. Bofutrelvir acts as a potent inhibitor of SARS-CoV-2 M pro , demonstrating high efficacy and broad-spectrum antiviral activity. Compared to therapies that require pharmacokinetic boosters, such as ritonavir, the monotherapy approach of Bofutrelvir reduces the risk of potential drug interactions, making it suitable for a wider patient population. However, further studies on the potency and mechanism of inhibition of Bofutrelvir against the M pro of COVID-19 and its variants, together with other coronaviruses, are needed to prepare for the possibility of a possible re-emerging threat from an analogous virus in the future. Here, we reveal the effective inhibition of Bofutrelvir against the M pro of SARS-CoV-2, SARS-CoV, and HCoV-229E through FRET and crystallographic analysis. Furthermore, the inhibitory mechanisms of Bofutrelvir against two SARS-CoV-2 M pro mutants (G15S and K90R) were also elucidated through FRET and crystallographic studies. Through detailed analysis and comparison of these crystal structures, we identified crucial structural determinants of inhibition and elucidated the binding mode of Bofutrelvir to M pro s from different coronaviruses. These findings are hopeful to accelerate the development of safer and more potent inhibitors against the M pro of coronavirus, and to provide important references for the prevention and treatment of similar viruses that may emerge in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

After successfully performing Agrobacterium- mediated CRISPR-Cas9-based gene editing in plants, isolation of the Cas9 T-DNA is essential for the stable inheritance of induced mutations. Here, we report a simple technique that allows the isolation of Cas9 -free mutants, eliminating the need for outcrossing or other intricate methods. This method is based on the ability of Basta-sensitive Arabidopsis thaliana seedlings, which generally perish, to recover and grow once transplanted to Basta-free growth media. By growing gene-edited heterozygous populations of single-locus insertion Basta-resistant plants on Basta selection media, plants lacking the Cas9 T-DNA can be identified. These pale-looking plants lacking Cas9 are then rescued on media lacking the Basta to recover Cas9 -free plants. The ability of seedlings to recover from Basta selection was also studied in camelina, canola, and wheat. All three crops showed different recovery rates, with wheat demonstrating the highest recovery once transplanted from Basta to normal growth media. In summary, our findings demonstrate that by harnessing the recovery capability of Basta-sensitive seedlings, we can effectively identify and rescue plants lacking the Cas9 T-DNA, enabling the isolation of Cas9-free mutants in Arabidopsis and potentially extending to other crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ahmed, Hulbert, Xin and Neff.)

Mutations play a pivotal role in shaping the trajectory and outcomes of a species evolution and domestication. Maize (Zea mays) has been a major staple crop and model for genetic research for more than 100 yr. With the arrival of site-directed mutagenesis and genome editing (GE) driven by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), maize mutational research is once again in the spotlight. If we combine the powerful physiological and genetic characteristics of maize with the already available and ever increasing toolbox of CRISPR-Cas, prospects for its future trait engineering are very promising. This review aimed to give an overview of the progression and learnings of maize screening studies analyzing forward genetics, natural variation and reverse genetics to focus on recent GE approaches. We will highlight how each strategy and resource has contributed to our understanding of maize natural and induced trait variability and how this information could be used to design the next generation of mutational screenings., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

The voltage-dependent potassium channels (Kv channels) show several different types of inactivation. N-type inactivation is a fast inactivating mechanism, which is essentially an open pore blockade by the amino-terminal structure of the channel itself or the auxiliary subunit. There are several functionally discriminatable slow inactivation (C-type, P-type, U-type), the mechanism of which is supposed to include rearrangement of the pore region. In some Kv1 channels, the actual inactivation is brought about by coupling of N-type and C-type inactivation (N-C coupling). In the present study, we focused on the N-C coupling of the Aplysia Kv1 channel (AKv1). AKv1 shows a robust N-type inactivation, but its recovery is almost thoroughly from C-type inactivated state owing to the efficient N-C coupling. In the I8Q mutant of AKv1, we found that the inactivation as well as its recovery showed two kinetic components apparently correspond to N-type and C-type inactivation. Also, the cumulative inactivation which depends on N-type mechanism in AKv1 was hindered in I8Q, suggesting that N-type inactivation of I8Q is less stable. We also found that Zn 2 + specifically accelerates C-type inactivation of AKv1 and that H382 in the pore turret is involved in the Zn 2 + binding. Because the region around Ile 8 (I8) in AKv1 has been suggested to be involved in the pre-block binding of the amino-terminal structure, our results strengthen a hypothesis that the stability of the pre-block state is important for stable N-type inactivation as well as the N-C coupling in the Kv1 channel inactivation., (© 2024. The Author(s).)

The lignan secoisolariciresinol (SECO) diglucoside (SDG) is a phytoestrogen with diverse effects. LuUGT74S1 glucosylates SECO to SDG, whereby only small amounts of the monoglucoside SMG are formed intermediately, which exhibit increased activity. To identify critical amino acids that are important for enzymatic activity and the SMG/SDG ratio, 3D structural modeling and docking, as well as site-directed mutation studies, were performed. Enzyme assays with ten mutants revealed that four of them had identical kinetic data to LuUGT74S1, while three showed reduced and one increased catalytic efficiency k cat . S82F and E189L substitutions resulted in the complete absence of activity. A17 and Q136 are crucial for the conversion of SMG to SDG as A17S and Q136F mutants exhibited the highest SMG/SDG ratios of 0.7 and 0.4. Kinetic analyses show that diglucosylation is an essentially irreversible reaction, while monoglycosylation is kinetically favored. The results lay the foundation for the biotechnological production of SMG.K m . S82F and E189L substitutions resulted in the complete absence of activity. A17 and Q136 are crucial for the conversion of SMG to SDG as A17S and Q136F mutants exhibited the highest SMG/SDG ratios of 0.7 and 0.4. Kinetic analyses show that diglucosylation is an essentially irreversible reaction, while monoglycosylation is kinetically favored. The results lay the foundation for the biotechnological production of SMG.

Humans and other primates lack the ability to synthesize the essential nutrient, Vitamin C, which is derived exclusively from the diet. Crucial for effective vitamin C uptake are the Na + dependent Vitamin C transporters, SVCT1 and SVCT2, members of the nucleobase ascorbate transporter (NAT) family. SVCT1 and 2 actively transport the reduced form of Vitamin C, ascorbic acid, into key tissues. The recent structure of the mouse SVCT1 revealed the molecular basis of substrate binding and that, like the other structurally characterised members of the NAT family, it exists as a closely associated dimer. SVCT1 is likely to function via the elevator mechanism with the core domain of each protomer able to bind substrate and move through the membrane carrying the substrate across the membrane. Here we explored the function of a range of variants of the human SVCT1, revealing a range of residues involved in substrate selection and binding, and confirming the importance of the C-terminus in membrane localisation. Furthermore, using a dominant negative mutant we show that the dimer is essential for transport function, as previously seen in the fungal homologue, UapA. In addition, we show that a localisation deficient C-terminal truncation of SVCT1 blocks correct localisation of co-expressed, associated wildtype SVCT1. These results clearly show the importance of the dimer in both correct SVCT1 trafficking and transport activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Interactions between plants and microorganisms are pivotal for plant growth and productivity. Several plant molecular mechanisms that shape these microbial communities have been identified. However, the importance of nitric oxide (NO) produced by plants for the associated microbiota remains elusive. Using Arabidopsis thaliana isogenic mutants overproducing NO (nox1, NO overexpression) or down-producing NO (i.e. nia1nia2 impaired in the expression of both nitrate reductases NR1/NIA1 and NR2/NIA2; the 35s::GSNOR1 line overexpressing nitrosoglutathione reductase (GSNOR) and 35s::AHB1 line overexpressing haemoglobin 1 (AHB1)), we investigated how altered NO homeostasis affects microbial communities in the rhizosphere and in the roots, soil microbial activity and soil metabolites. We show that the rhizosphere microbiome was affected by the mutant genotypes, with the nox1 and nia1nia2 mutants causing opposite shifts in bacterial and fungal communities compared with the wild-type (WT) Col-0 in the rhizosphere and roots, respectively. These mutants also exhibited distinctive soil metabolite profiles than those from the other genotypes while soil microbial activity did not differ between the mutants and the WT Col-0. Our findings support our hypothesis that changes in NO production by plants can influence the plant microbiome composition with differential effects between fungal and bacterial communities., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Background: The emergence and spread of multidrug-resistant Staphylococcus aureus strains demonstrates the urgent need for new antimicrobials. Xanthorrhizol, a plant-derived sesquiterpenoid compound, has a rapid killing effect on methicillin-susceptible strains and methicillin-resistant strains of S. aureus achieving the complete killing of staphylococcal cells within 2 min using 64 μg/mL xanthorrhizol. However, the mechanism of its action is not yet fully understood., Methods: The S. aureus cells treated with xanthorrhizol were studied using optical diffraction tomography. Activity of xanthorrhizol against the wild-type and mscL null mutant of S. aureus ATCC 29213 strain was evaluated in the time-kill assay. Molecular docking was conducted to predict the binding of xanthorrhizol to the SaMscL protein., Results: Xanthorrhizol treatment of S. aureus cells revealed a decrease in cell volume, dry weight, and refractive index (RI), indicating efflux of the cell cytoplasm, which is consistent with the spontaneous activation of the mechanosensitive MscL channel. S. aureus ATCC 29213Δ mscL was significantly more resistant to xanthorrhizol than was the wild-type strain. Xanthorrhizol had an enhanced inhibitory effect on the growth and viability of exponentially growing S. aureus ATCC 29213Δ mscL cells overexpressing the SaMscL protein and led to a noticeable decrease in their viability in the stationary growth phase. The amino acid residues F5, V14, M23, A79, and V84 were predicted to be the residues of the binding pocket for xanthorrhizol. We also showed that xanthorrhizol increased the efflux of solutes such as K + and glutamate from S. aureus ATCC 29213Δ mscL cells overexpressing SaMscL. Xanthorrhizol enhanced the antibacterial activity of the antibiotic dihydrostreptomycin, which targets the MscL protein., Conclusion: Our findings indicate that xanthorrhizol targets the SaMscL protein in S. aureus cells and may have important implications for the development of a safe antimicrobial agent., Competing Interests: EM and J-GP were employed by GenoFocus Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mordukhova, Kim, Jin, No and Pan.)

The lack of effective treatment options for an increasing number of cancer cases highlights the need for new anticancer therapeutic strategies. Immunotherapy mediated by Salmonella enterica Typhimurium is a promising anticancer treatment. Candidate strains for anticancer therapy must be attenuated while retaining their antitumor activity. Here, we investigated the attenuation and antitumor efficacy of two S. enterica Typhimurium mutants, Δ tolRA and Δ ihfABpmi , in a murine melanoma model. Results showed high attenuation of Δ tolRA in the Galleria mellonella model, and invasion and survival in tumor cells. However, it showed weak antitumor effects in vitro and in vivo . Contrastingly, lower attenuation of the attenuated Δ ihfABpmi strain resulted in regression of tumor mass in all mice, approximately 6 days after the first treatment. The therapeutic response induced by Δ ihfABpmi was accompanied with macrophage accumulation of antitumor phenotype (M1) and significant increase in the mRNAs of proinflammatory mediators (TNF-α, IL-6, and iNOS) and an apoptosis inducer (Bax). Our findings indicate that the attenuated Δ ihfABpmi exerts its antitumor activity by inducing macrophage infiltration or reprogramming the immunosuppressed tumor microenvironment to an activated state, suggesting that attenuated S. enterica Typhimurium strains based on nucleoid-associated protein genes deletion could be immunotherapeutic against cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pérez Jorge, Gontijo, Silva, Goes, Jaimes-Florez, Coser, Rocha, Giorgio and Brocchi.)

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The current study was carried out at Nuclear Institute of Agriculture, Tandojam to assess ten mungbean mutants together with one check cultivar in two separate cropping seasons. The findings revealed that all mutants, with the exception of the branches per plant, had significantly different examined traits. By characterizing ten variables, including plant height (cm), number of branches/plant, number of seeds per pod, grain yield per plot (g/plot), grain weight per plant, pod length (cm), pods per plant, days to flowering, above ground biological weight per plot (g/plot) and days to maturity, the results could considerably differentiate between low and high producing mutants. Discriminant analysis was used to choose high-yielding genotypes. The discriminant score demonstrated a significant canonical correlation of 0.994** and could account for 98.8 % of differences in mungbean production. According to the results of discriminant function analysis, the most significant features are pod length, days to flowering, plant height and above ground biological weight. The highest discriminant scores were displayed by the genotypes AEM66, AEM27, AEM25 and AEM14, identified as high yielding mutants. The low yielding mungbean mutants, designated Viz, AEM20, AEM30, AEM35, AEM-96, AEM29, AEM40 and AEM32 are those that exhibit the lowest values of the discriminant score. Mungbean cultivation is more successful in the summer than it is in the spring., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Muhammad Ali reports administrative support and equipment, drugs, or supplies were provided by Nuclear Institute of Agriculture, Tandojam, SIndh, Pakistan., (© 2024 The Author(s).)

Introduction: The emergence of SARS-CoV-2 triggered a global health emergency, causing > 7 million deaths thus far. Limited early knowledge spurred swift research, treatment, and vaccine developments. Implementation of public health measures such as, lockdowns and social distancing, disrupted economies and strained healthcare. Viral mutations highlighted the need for flexible strategies and strong public health infrastructure, with global collaboration crucial for pandemic control., Areas Covered: (i) Revisiting diagnostic strategies, (ii) adapting to the evolving challenge of the virus, (iii) vaccines against new variants, (iv) vaccine hesitancy in the light of the evolving disease, (v) treatment strategies, (vi) hospital preparedness for changing clinical needs, (vii) global cooperation and data sharing, (viii) economic implications, and (ix) education and awareness- keeping communities informed., Expert Opinion: The COVID-19 crisis forced unprecedented adaptation, emphasizing public health readiness, global unity, and scientific advancement. Key lessons highlight the importance of adaptability and resilience against uncertainties. As the pandemic evolves into a 'new normal,' ongoing vigilance, improved understanding, and available vaccines and treatments equip us for future challenges. Priorities now include proactive pandemic strategies, early warnings, supported healthcare, public education, and addressing societal disparities for better health resilience and sustainability.

The Ca 2+ /calmodulin (CaM)-dependent protein kinase II (CaMKII) is a ubiquitous mediator of cellular Ca 2+ signals with both enzymatic and structural functions. Here, we briefly introduce the complex regulation of CaMKII and then provide a comprehensive overview of the expanding toolbox to study CaMKII. Beyond a variety of distinct mutants, these tools now include optical methods for measurement and manipulation, with the latter including light-induced inhibition, stimulation, and sequestration. Perhaps most importantly, there are now three mechanistically distinct classes of specific CaMKII inhibitors, and their combined use enables the interrogation of CaMKII functions in a manner that is powerful and sophisticated yet also accessible. This review aims to provide guidelines for the interpretation of the results obtained with these tools, with careful consideration of their direct and indirect effects., Competing Interests: Declaration of interests K.U.B. is co-founder and board member of Neurexis Therapeutics, a company that seeks to develop a CaMKII inhibitor into a therapeutic drug for cerebral ischemia., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vaccination is the most effective means of preventing and controlling porcine epidemic diarrhea (PED). Conventional vaccines developed from porcine epidemic diarrhea virus (PEDV) GI-a subtypes (CV777 and SM98) have played a vital role in preventing classical PED. However, with the emergence of PEDV mutants in 2010, conventional PEDV GI-a subtype-targeting vaccines no longer provide adequate protection against PEDV GII mutants, thereby making novel-type PED vaccine development an urgent concern to be addressed. Novel vaccines, including nucleic acid vaccines, genetically engineered subunit vaccines, and live vector vaccines, are associated with several advantages, such as high safety and stability, clear targeting, high yield, low cost, and convenient usage. These vaccines can be combined with corresponding ELISA kits to differentiate infected from vaccinated animals, which is beneficial for disease confirmation. This review provides a detailed overview of the recent advancements in PED vaccines, emphasizing on the research and application evaluation of novel PED vaccines. It also considers the future directions and challenges in advancing these vaccines to widespread use in clinics., Competing Interests: Conflict of interest statement Neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides, which are advantageous for plant immunity, animal nutrition and health. However, thermophilic endo-chitinases are scarce and the transformation from exo- to endo-activity of chitinases is still a challenging problem. In this study, to enhance the endo-activity of the thermophilic chitinase Chi304, we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses. Four effective single-point mutants were identified among 28 designed mutations. The ratio of (GlcNAc) 3 to (GlcNAc) 2 quantity (DP 3/2 ) in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89, 1.65, 1.24, and 1.38 times that of Chi304, respectively. When combining to double-point mutants, the DP 3/2 proportions produced by F79A/W140R, F79A/M264L, F79A/W272R, and M264L/W272R were 2.06, 1.67, 1.82, and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity. When applied to produce chitooligosaccharides (DP ≥ 3), F79A/W140R accumulated the most (GlcNAc) 4 , while M264L/W272R was the best to produce (GlcNAc) 3 , which was 2.28 times that of Chi304. The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate. Overall, this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides., (© 2024. The Author(s).)

We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants., (© 2024 Wiley Periodicals LLC.)

Plastid-encoded RNA polymerase (PEP) forms a multisubunit complex in operating chloroplasts, where PEP subunits and a sigma factor are tightly associated with 12 additional nuclear-encoded proteins. Mutants with disrupted genes encoding PEP-associated proteins (PAPs) provide unique tools for deciphering mutual relationships among phytohormones. A block of chloroplast biogenesis in Arabidopsis pap mutants specifying highly altered metabolism in white tissues induced dramatic fluctuations in the content of major phytohormones and their metabolic genes, whereas hormone signaling circuits mostly remained functional. Reprogramming of the expression of biosynthetic and metabolic genes contributed to a greatly increased content of salicylic acid (SA) and a concomitant decrease in 1-aminocyclopropane-1-carboxylic acid (ACC) and oxophytodienoic acid (OPDA), precursors of ethylene and jasmonic acid, respectively, in parallel to reduced levels of abscisic acid (ABA). The lack of differences in the free levels of indole-3-acetic acid (IAA) between the pap mutants and wild-type plants was accompanied by fluctuations in the contents of IAA precursors and conjugated forms as well as multilayered changes in the expression of IAA metabolic genes. Along with cytokinin (CK) overproduction, all of these compensatory changes aim to balance plant growth and defense systems to ensure viability under highly modulated conditions.

This article analyzes the complete corpus of live-action X-Men movies for their depictions of genetics and otherness. The researchers watched and qualitatively coded all thirteen movies produced by 20th Century Fox that take place in the same shared cinematic universe, beginning with X-Men (2000) and ending with The New Mutants (2020). The X-Men movies are unusual summer blockbusters since they explore genetic topics through their central characters, mutants, who are genetically different from their non-mutant peers. Mutants in the films evoke a plurality of analogies, such as mutant-as-Black and mutant-as-queer. These intersecting metaphors build upon a core of genetic difference to create a versatile but limited picture of prejudice, solidarity, and otherness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Grimsted, Krizner, Porter and Clayton.)

In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis requires DNA acquisition and its stable insertion into the chromosome by allelic exchange. In this chapter, we described two techniques for the preparation of genomic constructs (cloning and overlapping extension PCR) and for DNA uptake (electroporation and transformation). The protocols are accompanied with examples. All described protocols were successful on our hands with the reference S. suis strain P1/7., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To provide an updated summary of recent advances in the application of gamma irradiation to elicit secondary metabolism and for induction of mutations in plant cell and organ cultures for the production of industrially important specialized metabolites (SMs)., Conclusions: Research on the application of gamma radiation with plants has contributed a lot to microbial decontamination of seeds, and the promotion of physiological processes such as seed germination, seedling vigor, plant growth, and development. Various studies have demonstrated the influence of gamma rays on the morphology, physiology, and biochemistry of plants. Recent research efforts have also shown that low-dose gamma (5-100 Gy) irradiation can be utilized as an expedient solution to alleviate the deleterious effect of abiotic stresses and to obtain better yields of plants. Inducing mutagenesis using gamma irradiation has also evolved as a better option for inducing genetic variability in crops, vegetables, medicinal and ornamentals for their genetic improvement. Plant SMs are gaining increasing importance as pharmaceutical, therapeutic, cosmetic, and agricultural products. Plant cell, tissue, and organ cultures represent an attractive alternative to conventional methods of procuring useful SMs. Among the varied approaches the elicitor-induced in vitro culture techniques are considered an efficient tool for studying and improving the production of SMs. This review focuses on the utilization of low-dose gamma irradiation in the production of high-value SMs such as phenolics, terpenoids, and alkaloids. Furthermore, we present varied successful examples of gamma-ray-induced mutations in the production of SMs.

Purpose: Bagasse, the residue left after extracting juice from sugarcane stalks, is rich in lignocellulosic biomass. The lignin present in this plant biomass is the key factor that hinders the efficient extraction of ethanol from the bagasse. In the current study, γ-irradiated sugarcane mutants were evaluated for variation in lignin content and its corresponding caffeic acid-O-methyl transferase ( COMT ) gene., Materials and Methods: The acetyl bromide method was used to estimate lignin content in sugarcane mutants. PCR-based cloning of the COMT gene was performed in low lignin mutants as well as control plants in E. coli (strain DH5α) to understand the mechanism of variation at the molecular level. The Sanger sequencing for cloned gene was performed to check variation in gene sequence., Results: In comparison to the control (21.5%), the mutant plants' lignin content ranged from 13 to 28%. The Sanger sequencing revealed approximately the same length of the gene from mutants as well as a control plant. In comparison to the reference gene, the mutated gene showed SNPs and indels in different regions, which may have an impact on lignin content., Conclusions: Therefore, γ-irradiated mutagenesis is an acceptable approach to develop novel mutants of sugarcane with low lignin content to enhance bioethanol production from waste material using bioprocess technology.

Purpose: This manuscript aimed for the generation of γ-irradiation derived mutants of potato genotype PAU/RR-1501 possessing desirable processing traits., Materials and Methods: Nodal cuttings from virus-free explants were established on basal MS medium and irradiated with different doses (0, 5, 10 and 20 Gy) of γ-irradiation. The 5 and 10 Gy treated plantlets were multiplied and used for micro-tuber induction. Harvested micro-tubers were planted in pots for the selection and evaluation of mutants in M1V2 generation., Results: Four weeks post-treatment, plantlets (5 Gy) showed enhanced growth as compared to the control while 20 Gy treatment exhibited completely ceased shoot growth. The highest number and weight of mini-tubers per plant was recorded for 10 Gy followed by 5 Gy treatment as compared to control. The γ-irradiation treatments caused changes in the skin color and shape of M1V2 tubers., Conclusion: Under the 5 Gy treatment 49.9% of clones produced exhibited cream and 8.53% brown skin color. Nine putative mutants were identified in genotype PAU/RR-1501 exhibiting promising processing traits.

Metacaspases are cysteine proteases belonging to the CD clan of the C14 family. They possess important characteristics, such as specificity for cleavage after basic residues (Arg/Lys) and dependence on calcium ions to exert their catalytic activity. They are defined by the presence of a large subunit (p20) and a small subunit (p10) and are classified into types I, II, and III. Type I metacaspases have a characteristic pro-domain at the N-terminal of the enzyme, preceding a region rich in glutamine and asparagine. In the yeast Saccharomyces cerevisiae, a type I metacaspase is found. This organism encodes a single metacaspase that participates in the process of programmed cell death by apoptosis. The study focuses on cloning, expressing, and mutating Saccharomyces cerevisiae metacaspase (ScMCA-Ia). Mutations in Cys 155 and Cys 276 were introduced to investigate autoprocessing mechanisms. Results revealed that Cys 155 plays a crucial role in autoprocessing, initiating a conformational change that activates ScMCA-Ia. Comparative analysis with TbMCA-IIa highlighted the significance of the N-terminal region in substrate access to the active site. The study proposes a two-step processing mechanism for type I metacaspases, where an initial processing step generates the active form, followed by a distinct intermolecular processing step. This provides new insights into ScMCA-Ia's activation and function. The findings hold potential implications for understanding cellular processes regulated by metacaspases. Overall, this research significantly advances knowledge in metacaspase biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

SUMMARYRecBCD enzyme is a multi-functional protein that initiates the major pathway of homologous genetic recombination and DNA double-strand break repair in Escherichia coli . It is also required for high cell viability and aids proper DNA replication. This 330-kDa, three-subunit enzyme is one of the fastest, most processive helicases known and contains a potent nuclease controlled by Chi sites, hotspots of recombination, in DNA. RecBCD undergoes major changes in activity and conformation when, during DNA unwinding, it encounters Chi (5'-GCTGGTGG-3') and nicks DNA nearby. Here, we discuss the multitude of mutations in each subunit that affect one or another activity of RecBCD and its control by Chi. These mutants have given deep insights into how the multiple activities of this complex enzyme are coordinated and how it acts in living cells. Similar studies could help reveal how other complex enzymes are controlled by inter-subunit interactions and conformational changes., Competing Interests: The authors declare no conflict of interest.

Fungal pigments such as melanin and carotenoids are distinctive markers of animal and plant pathogenic fungi as well as their environmental relatives. These complex pigments play important roles in pathogenicity and stress tolerance while also being useful as biomarkers. Accordingly, it is important to be able to identify in situ the pigments in black fungi, a group of clinical and environmental importance. In this study, wild-type and genetically modified strains of Knufia petricola A95 and wild fungal cells attached to ancient rock were investigated for their spectroscopic and microscopic Raman features and morphological appearance. Knockout mutants of melanin synthesis genes pks1 (polyketide synthase), sdh1 (scytalone dehydratase), and both pks1 and the carotenoid synthesis gene phd1 (phytoene desaturase) were studied We applied two different Raman microscopes using two lasers, with 633 nm and 488 nm wavelengths. We analyzed and compared Raman spectra between the measured reference substances and the mutant and wild-type strains. In the wild strain WT:A95, the peaks close to melanin peals were found at 1353 cm -1 and 1611 cm -1 . There are no characteristic melanin peaks at 1580-1600 cm -1 and around 1350 cm -1 at the spectrum of the Δpks1/Δphd1 mutant and the Δsdh1 mutant. The Δpks1 mutant spectrum has the peaks at the beta-carotene v2 C-C in-plane stretch at 1155 cm -1 and v3 C-CH3 deformation at 1005 cm -1 . The peaks of carotenoids and melanin were found in all mutants and the wild strain, except the Δpks1/Δphd1 mutant. Raman spectra allow for discrimination between the various pigments. Hence, interactions between natural fungal melanin, as well as other protective pigments, and complex environmental matrices can be characterized on a range of spatial and temporal scales., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Maria Dittrich reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Maria Dittrich reports financial support was provided by Canadian Foundation for Innovation. Maria Dittrich reports financial support was provided by BAM Federal Institute for Materials Research and Testing., (Copyright © 2023 Elsevier B.V. All rights reserved.)

NAC family gene - SNAC1 (Stress-responsive NAC1) is responsive to drought, salt, cold stress, and ABA. It acts as a regulator in mediating tolerance to abiotic stress through different pathways. Abiotic stress, among them drought and salinity, are adverse factors for plant growth and crop productivity. SNAC1 was an object of high interest according to the effect of improved drought and salt tolerance when overexpressed in different plant species such as rice, wheat, barley, cotton, maize, banana, or oat. SNAC1 functions by regulating the expression of genes that contain the NAC Recognized Sequence (NACRS) within their promoter region. This gene is induced by drought, specifically in guard cells. Its downstream targets have been identified. The role of SNAC1 in molecular and physiological responses during abiotic stress has been proposed, but this knowledge still needs to be expanded. Here, we describe recent advances in understanding the action of SNAC1 in adapting plants to abiotic stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Aldehyde dehydrogenase (ALDH), a common oxidoreductase in organisms, is an aldehyde scavenger involved in various metabolic processes. However, its function in different pathogenic fungi remains unknown. Fusarium graminearum causes Fusarium head blight in cereals, which reduces grain yield and quality and is an important global food security problem. To elucidate the pathogenic mechanism of F. graminearum , seven genes encoding ALDH were knocked out and then studied for their function. Single deletions of seven ALDH genes caused a decrease in spore production and weakened the pathogenicity. Furthermore, these deletions altered susceptibility to various abiotic stresses. FGSG_04194 is associated with a number of functions, including mycelial growth and development, stress sensitivity, pathogenicity, toxin production, and energy metabolism. FGSG_00139 and FGSG_11482 are involved in sporulation, pathogenicity, and SDH activity, while the other five genes are multifunctional. Notably, we found that FGSG_04194 has an inhibitory impact on ALDH activity, whereas FGSG_00979 has a positive impact. RNA sequencing and subcellular location analysis revealed that FGSG_04194 is responsible for biological process regulation, including glucose and lipid metabolism. Our results suggest that ALDH contributes to growth, stress responses, pathogenicity, deoxynivalenol synthesis, and mitochondrial energy metabolism in F. graminearum . Finally, ALDH presents a potential target and theoretical basis for fungicide development.

The ease and throughput of single-cell genomics have steadily improved, and its current trajectory suggests that surveying single-cell populations will become routine. We discuss the merger of quantitative genetics with single-cell genomics and emphasize how this synergizes with advantages intrinsic to plants. Single-cell population genomics provides increased detection resolution when mapping variants that control molecular traits, including gene expression or chromatin accessibility. Additionally, single-cell population genomics reveals the cell types in which variants act and, when combined with organism-level phenotype measurements, unveils which cellular contexts impact higher-order traits. Emerging technologies, notably multiomics, can facilitate the measurement of both genetic changes and genomic traits in single cells, enabling single-cell genetic experiments. The implementation of single-cell genetics will advance the investigation of the genetic architecture of complex molecular traits and provide new experimental paradigms to study eukaryotic genetics.

The effects of high-intensity light on the pigment content, photosynthetic rate, and fluorescence parameters of photosystem II in high-pigment tomato mutants ( hp 3005) and low-pigment mutants ( lp 3617) were investigated. This study also evaluated the dry weight percentage of low molecular weight antioxidant capacity, expression patterns of some photoreceptor-regulated genes, and structural aspects of leaf mesophyll cells. The 3005 mutant displayed increased levels of photosynthetic pigments and anthocyanins, whereas the 3617 mutant demonstrated a heightened content of ultraviolet-absorbing pigments. The photosynthetic rate, photosystem II activity, antioxidant capacity, and carotenoid content were most pronounced in the high-pigment mutant after 72 h exposure to intense light. This mutant also exhibited an increase in leaf thickness and water content when exposed to high-intensity light, suggesting superior physiological adaptability and reduced photoinhibition. Our findings indicate that the enhanced adaptability of the high-pigment mutant might be attributed to increased flavonoid and carotenoid contents, leading to augmented expression of key genes associated with pigment synthesis and light regulation.

Flavonoids, of which the major groups are flavones, flavonols, and anthocyanins, confer a variety of colors on plants. Bud sports with variation of floral colors occur occasionally during chrysanthemum cultivation. Although it has been reported that methylation at the promoter of CmMYB6 was related to anthocyanin contents, the regulatory networks of flavonoid biosynthesis still remain largely unknown in mutation of chrysanthemum. We compared phenotypes, pigment composition and transcriptomes in two chrysanthemum cultivars, 'Anastasia Dark Green' and 'Anastasia Pink', and regenerated bud sports of these cultivars with altered floral colors. Increased anthocyanins turned the 'Anastasia Dark Green' mutant red, while decreased anthocyanins turned the 'Anastasia Pink' mutant white. Moreover, total flavonoids were reduced in both mutants. Multiple flavonoid biosynthetic genes and regulatory genes encoding MYBs and bHLHs transcription factors were differentially expressed in pairwise comparisons of transcriptomes in 'Anastasia Dark Green' or 'Anastasia Pink' and their mutants at different flowering stages. Among these regulatory genes, the expression patterns of CmMYB6 and CmbHLH2 correlated to changes of anthocyanin contents, and down-regulation of CmMYB11 correlated to decreased total flavonoid contents in two mutants. CmMYB11 was shown to directly activate the promoter activities of CmCHS2, CmCHI, CmDFR, CmANS, CmFNS, and CmFLS. Furthermore, overexpression of CmMYB11 increased both flavonols and anthocyanins in tobacco petals. Our work provides new insights into regulatory networks involved in flavonoid biosynthesis and coloration in chrysanthemum., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Countless individuals have fallen victim to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and have generated antibodies, reducing the risk of secondary infection in the short term. However, with the emergence of mutated strains, the probability of subsequent infections remains high. Consequently, the demand for simple and accessible methods for distinguishing between different variants is soaring. Although monitoring viral gene sequencing is an effective approach for differentiating between various types of SARS-CoV-2 variants, it may not be easily accessible to the general public. In this article, we provide an overview of the reported techniques that use combined approaches and adaptable testing methods that use editable recognition receptors for simultaneous detection and distinction of current and emerging SARS-CoV-2 variants. These techniques employ straightforward detection strategies, including tests capable of simultaneously identifying and differentiating between different variants. Furthermore, we recommend advancing the development of uncomplicated protocols for distinguishing between current and emerging variants. Additionally, we propose further development of facile protocols for the differentiation of existing and emerging variants., (© 2023. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

Coxsackievirus A10 (CV-A10) is a prevailing causative agent of hand-foot-mouth disease, necessitating the isolation and adaptation of appropriate strains in cells allowed for human vaccine development. In this study, amino acid sequences of CV-A10 strains with different cell tropism on RD and Vero cells were compared. Various amino acids on the structural and non-structural proteins related to cell tropism were identified. The reverse genetic systems of several CV-A10 strains with RD + /Vero - and RD + /Vero + cell tropism were developed, and a set of CV-A10 recombinants were produced. The binding, entry, uncoating, and proliferation steps in the life cycle of these viruses were evaluated. P1 replacement of CV-A10 strains with different cell tropism revealed the pivotal role of the structural proteins in cell tropism. Further, seven amino acid substitutions in VP2 and VP1 were introduced to further investigate their roles played in cell tropism. These mutations cooperated in the growth of CV-A10 in Vero cells. Particularly, the valine to isoleucine mutation at the position VP1-236 (V1236I) was found to significantly restrict viral uncoating in Vero cells. Co-immunoprecipitation assays showed that the release of viral RNA from the KREMEN1 receptor-binding virions was restricted in r0195-V1236I compared with the parental strain r0195 (a RD + /Vero + strain). Overall, this study highlights the dominant effect of structural proteins in CV-A10 adaption in Vero cells and the importance of V1236 in viral uncoating, providing a foundation for the mechanism study of CV-A10 cell tropism, and facilitating the development of vaccine candidates.

To investigate the role of ethylene (ET) in abiotic stress tolerance in petunia cv. 'Mirage Rose', petunia plants in which the ET biosynthesis gene 1-aminocyclopropane-1-carboxylic acid oxidase 4 (ACO4) was knocked out (phaco4 mutants) and wild-type (WT) plants were exposed to heat and drought conditions. Loss of function of ACO4 significantly delayed leaf senescence and chlorosis under heat and drought stress by maintaining the SPAD values and the relative water content, indicating a greater stress tolerance of phaco4 mutants than that of WT plants. This tolerance was related to the lower ET and reactive oxygen species levels in the mutants than in WT plants. Furthermore, the stress-induced expression of genes related to ET signal transduction, antioxidant and proline activities, heat response, and biosynthesis of abscisic acid was higher in the mutants than in WT plants, indicating a greater stress tolerance in the former than in the latter. These results demonstrate the deleterious effects of stress-induced ET on plant growth and provide a better physiological and molecular understanding of the role of stress ET in the abiotic stress response of petunia. Because the loss of function of ACO4 in petunia improved stress tolerance, we suggest that ACO4 plays a vital role in stress-induced leaf senescence and acts as a negative regulator of abiotic stress tolerance., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Bacterial infections in the lungs of persons with cystic fibrosis are typically composed of multispecies biofilm-like communities, which modulate clinically relevant phenotypes that cannot be explained in the context of a single species culture. Most analyses to date provide a picture of the transcriptional responses of individual pathogens; however, there is relatively little data describing the transcriptional landscape of clinically relevant multispecies communities. Harnessing a previously described cystic fibrosis-relevant, polymicrobial community model consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica , we performed an RNA-Seq analysis on the biofilm population to elucidate the transcriptional profiles of the community grown in artificial sputum medium (ASM) as compared to growth in monoculture, without mucin, and in fresh medium supplemented with tobramycin. We provide evidence that, although the transcriptional profile of P. aeruginosa is community agnostic, the transcriptomes of S. aureus and S. sanguinis are community aware. Furthermore, P. aeruginosa and P. melaninogenica are transcriptionally sensitive to the presence of mucin in ASM, whereas S. aureus and S. sanguinis largely do not alter their transcriptional profiles in the presence of mucin when grown in a community. Only P. aeruginosa shows a robust response to tobramycin. Genetic studies of mutants altered in community-specific growth provide complementary data regarding how these microbes adapt to a community context. IMPORTANCE Polymicrobial infections constitute the majority of infections in the cystic fibrosis (CF) airway, but their study has largely been neglected in a laboratory setting. Our lab previously reported a polymicrobial community that can help explain clinical outcomes in the lungs of persons with CF. Here, we obtained transcriptional profiles of the community versus monocultures to provide transcriptional information about how this model community responds to CF-related growth conditions and perturbations. Genetic studies provide complementary functional outputs to assess how the microbes adapt to life in a community.

Dominant-negative mutations can help to investigate the biological mechanisms and to understand the selective pressures for multifunctional proteins. However, most studies have focused on recessive mutant effects that occur in the absence of a second functional gene copy, which overlooks the fact that most eukaryotic genomes contain more than one copy of many genes. We have identified dominant effects on yeast growth rate among all possible point mutations in ubiquitin expressed alongside a wild-type allele. Our results reveal more than 400 dominant-negative mutations, indicating that dominant-negative effects make a sizable contribution to selection acting on ubiquitin. Cellular and biochemical analyses of individual ubiquitin variants show that dominant-negative effects are explained by varied accumulation of polyubiquitinated cellular proteins and/or defects in conjugation of ubiquitin variants to ubiquitin ligases. Our approach to identify dominant-negative mutations is general and can be applied to other proteins of interest., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Peter K. Vogt reports a relationship with Umbra Therapeutics that includes: consulting or advisory., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Stress-induced alterations vary with the species of plants, the intensity and duration of the exposure, and stressors availability in nature or soil. Purine catabolism acts as an inherent defensive mechanism against various abiotic stresses and plays a pivotal role in the stress acclimatisation of plants. The intermediate metabolite of purine catabolism, allantoin, compensates for soil nitrogen deficiency due to the low carbon/nitrogen ratio, thereby maintaining nitrogen homeostasis and supporting plant growth and development. Allantoin accounts for 90% of the total nitrogenous compound in legumes, while it contributes only 15% in non-leguminous plants. Moreover, studies on a variety of plant species have reported the differential accumulation of allantoin in response to abiotic stresses, endowing allantoin as a stress modulator. Allantoin functions as signalling molecule to stimulate stress-responsive genes ( P5CS ; pyrroline-5-carboxylase synthase) and ROS (reactive oxygen species) scavenging enzymes (antioxidant). Moreover, it regulates cross-talk between the abscisic acid and jasmonic acid pathway, and maintains ion homeostasis by increasing the accumulation of putrescine and/or spermine, consequently enhancing the tolerance against stress conditions. Further, key enzymes of purine catabolism (xanthine dehydrogenase and allantoinase) have also been explored by constructing various knockdown/knockout mutant lines to decipher their impact on ROS-mediated oxidative injury in plants. Thus, it is established that allantoin serves as a regulatory signalling metabolite in stress protection, and therefore a lower accumulation of allantoin also reduces plant stress tolerance mechanisms. This review gives an account of metabolic regulation and the possible contribution of allantoin as a photo protectant, osmoprotectant, and nitrogen recycler to reduce abiotic-stress-induced impacts on plants.

The presence of so-called anti-nutritional factors can reduce the bioavailability of nutrients following consumption of seeds which are otherwise an excellent source of proteins, carbohydrates and micronutrients. Among the proteins associated with negative effects on quality in pea (Pisum sativum L.) seeds are lectin, pea albumin 2 (PA2) and trypsin inhibitors (TI). Here we have investigated the impact of these proteins on protein digestibility and amino acid availability, using naturally occurring and derived mutant lines of pea lacking these proteins. The mutations were stacked to generate a triple mutant which was compared with a wild-type progenitor and a line lacking the major seed trypsin inhibitors alone. In vitro digestions following the INFOGEST protocol revealed significant differences in the degree of hydrolysis, protein profile and apparent amino acid availability among the pea variants. Proteins resistant to digestion were identified by MALDI-TOF mass spectrometry and amino acid profiles of digested samples determined. The results indicate that pea seeds lacking certain proteins can be used in the development of novel foods which have improved protein digestibility, and without negative impact on seed protein concentration or yield., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Proso millet ( Panicummiliaceum L.) is a short-duration C 4 crop that is drought tolerant and nutritionally rich and can grow well in marginal lands. Though the crop has many climate-resilient traits like tolerance to drought and heat, its yield is lower than that of common cereals like rice, wheat, and maize. Being an underutilized crop, the molecular resources in the crop are limited. The main aim of the present study was to develop and characterize contrasting mutants for yield and generate functional genomic information for the trait in proso millet. Gamma irradiation-induced mutant population was screened to identify high-yielding mutants, which were evaluated up to M 4 generation. One mutant with a dense panicle and high yield (ATL_hy) and one with a lax panicle and low yield (ATL_ly) along with the wild type were sequenced using the genotyping-by-sequencing approach. The variants detected as single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) were annotated against the reference genome of proso millet. Bioinformatic analyses using the National Center for Biotechnology Information (NCBI) and UniProt databases were performed to elucidate genetic information related to the SNP variations. A total of 25,901, 30,335, and 31,488 SNPs, respectively, were detected in the wild type, ATL_hy mutants, and ATL_ly mutants. The total number of functional SNPs identified in high-yielding and low-yielding mutants was 84 and 171, respectively. Two functional SNPs in the high-yielding mutant (ATL_ hy ) and one in the low-yielding mutant (ATL_ ly ) corresponded to the gene coding for "E3 ubiquitin-protein ligase UPL7". Pathway mapping of the functional SNPs identified that two SNPs in ATL_ly were involved in the starch biosynthetic pathway coding for the starch synthase enzyme. This information can be further used in identifying genes responsible for various metabolic processes in proso millet and in designing useful genetic markers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Francis, Rajasekaran, Rajagopalan, Bakya, Muthurajan, Kumar, Alagarswamy, Krishnamoorthy and Thiyagarajan.)

Real-time PCR methods were developed to quantify the frequency of SDHC-H134R and SDHB-H277Y mutants associated with succinate dehydrogenase inhibitors (SDHI) resistance in Alternaria populations from pistachio. The linearity of the standard curves demonstrated the applicability in the quantification of the assays. The accuracy and reliability of the qPCR protocols to determine the frequency of mutants in real samples were corroborated. Orchards visibly affected by Alternaria late blight were sampled. The frequency of mutants was determined using the qPCR assays, while the frequency of resistant phenotypes was determined using a single discriminatory dose. The statistical analysis showed that the frequencies of the mutation SDHC-H134R determined with the qPCR assay were highly correlated with those estimated with the conventional method. The survey also evidenced that resistance to boscalid is still widespread in California. Results also indicated the possible contribution of other mutations to SDHI resistance. Our results confirmed the prevalence of SDHC-H134R mutants and the occurrence of mutation SDHB-H277Y at low frequencies. The real-time PCR methods developed in this study were able to detect differences in the frequencies of resistant mutants caused by the use of chemical fungicides. Finally, the effects of two fungicide programs on the frequency of mutants resistant to SDHI and quinone outside inhibitors fungicides were studied using qPCR assays. The experiments demonstrated that the use of anilinopyrimidine and demethylation inhibitors fungicides in the same program reduced the frequency of these mutations in Alternaria populations. The qPCR methods developed and used in this study can be used to track resistance levels in the pistachio orchards on a large scale., Competing Interests: The author(s) declare no conflict of interest.

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Semiochemical-based management strategies are important for controlling bark beetles, such as invasive Red Turpentine Beetle (Denroctonus valens), the causal agent for mass mortality of pine trees (Pinus spp.) in China. It has been previously shown that the pheromone verbenone regulates the attack density of this beetle in a dose-dependent manner and that the gut bacteria of D. valens are involved in verbenone production. However, molecular functional verification of the role of gut bacteria in the pheromone production of D. valens is still lacking. To better understand the molecular function of gut bacterial verbenone production, we chose a facultative anaerobic gut bacterium (Enterobacter xiangfangensis) of D. valens based on its strong ability to convert cis-verbenol to verbenone, as shown in our previous study, and investigated its transcriptomics in the presence or absence of cis-verbenol under anaerobic conditions (simulating the anoxic environment in the beetle's gut). Based on this transcriptome analysis, aldehyde dehydrogenase (ALDH1) was identified as a putative key gene responsible for verbenone production and was knocked-down by homologous recombination to obtain a mutant E. xiangfangensis strain. Our results show that these mutants had significantly decreased the ability to convert the monoterpene precursor to verbenone compared with the wild-type bacteria, indicating that ALDH1 is primarily responsible for verbenone conversion for this bacterium species. These findings provide further mechanistic evidence of bacterially mediated pheromone production by D. valens, add new perspective for functional studies of gut bacteria in general, and may aid the development of new gene silencing-based pest management strategies., (© 2022 Institute of Zoology, Chinese Academy of Sciences.)

Introduction: Identifying rice ( Oryza sativa ) germplasm with improved efficiency of primary metabolism is of utmost importance in order to increase yields. One such approach can be attained through screening genetically diverse populations under altered environmental conditions. Growth or treatment under low carbon dioxide (CO 2 ) concentrations can be used as a means of revealing altered leaf photorespiration, respiration and other metabolic variants., Methods: We developed a pipeline for very high throughput treatment of gamma- and ethyl methanesulfonate- (EMS) induced mutant populations of IR64 rice seedlings at very low CO 2 for 7 days. 1050 seedlings per batch at 5 th leaf stage were exposed to 60 ppm CO 2 for the first day and 30 ppm for the remaining three days. Following this, putative candidates were identified by measuring chlorophyll depletion using SPAD. Screening results showed a distinct difference between the mutants and the WTs., Results and Discussion: The mean chlorophyll loss in WTs ranged from 65% to 11% respectively, whereas in the mutant lines chlorophyll loss ranged from 0 to 100%, suggesting considerable phenotypic variation. Rice mutants with a reduced chlorophyll reduction (<10%) were identified as 'Chlorophyll retention mutants' (CRMs) under low CO 2 stress. In total, 1909 mutant lines (14,000 seedlings) were screened for chlorophyll content under 30 ppm CO 2, with 26 lines selected for detailed screening. These 26 putative candidates were self-seeded to produce an M 5 generation, used to determine the genetic control of the altered response to low CO 2. Gas exchange of light and CO 2 response revealed that there were significant variations among photosynthetic properties in two selected rice mutants. The CO 2 compensation points in the absence of photorespiration and leaf respiration rates were lower than the WTs and anatomical analyses showed that CRM 29 had improved mesophyll cell area. We propose that this approach is useful for generating new material for breeding rice with improved primary metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mubarak, Burgess, Pyke, Quick and Murchie.)

Carbon ion beam (CIB) irradiation is a powerful way to create mutations in animals, plants, and microbes. Research on the mutagenic effects and molecular mechanisms of radiation is an important and multidisciplinary issue. However, the effect of carbon ion radiation on cotton is uncertain. In this study, five different upland cotton varieties and five CIB doses were used to identify the suitable irradiation dose for cotton. Three mutagenized progeny cotton lines from the wild-type Ji172 were re-sequenced. The effect of half-lethal dose on mutation induction indicated that 200 Gy with LET max of 226.9 KeV/μm was the most effective heavy-ion dose for upland cotton and a total of 2,959-4,049 single-base substitutions (SBSs) and 610-947 insertion-deletion polymorphisms (InDels) were identified among the three mutants by resequencing. The ratio of transition to transversion in the three mutants ranged from 2.16 to 2.24. Among transversion events, G:C>C:G was significantly less common than three other types of mutations (A:T>C:G, A:T>T:A, and G:C>T:A). The proportions of six types of mutations were very similar in each mutant. The distributions of identified SBSs and InDels were similar with unevenly distributed across the genome and chromosomes. Some chromosomes had significantly more SBSs than others, and there were "hotspot" mutation regions at the ends of chromosomes. Overall, our study revealed a profile of cotton mutations caused by CIB irradiation, and these data could provide valuable information for cotton mutation breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Zhao, Geng, Geng, Dou, Liu, An, Zhang and Wang.)

In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an overcompensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites., Competing Interests: Conflicts of interest None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Genetics Society of America.)

Two-component signaling systems (TCSs) are finely regulated mechanisms by which bacteria adapt to environmental conditions by modifying the expression of target genes. In bacterial pathogenesis, TCSs play important roles in modulating adhesion to mucosal surfaces, resistance to antibiotics, and metabolic adaptation. In the context of urinary tract infections (UTI), one of the most common types infections causing significant health problems worldwide, uropathogens use TCSs for adaptation, survival, and establishment of pathogenicity. For example, uropathogens can exploit TCSs to survive inside bladder epithelial cells, sense osmolar variations in urine, promote their ascension along the urinary tract or even produce lytic enzymes resulting in exfoliation of the urothelium. Despite the usefulness of studying the function of TCSs in in vitro experimental models, it is of primary necessity to study bacterial gene regulation also in the context of host niches, each displaying its own biological, chemical, and physical features. In light of this, the aim of this review is to provide a concise description of several bacterial TCSs, whose activity has been described in mouse models of UTI.

Research on enzymes for lignocellulose biomass degradation has progressively increased in recent years due to the interest in taking advantage of this natural resource. Among these enzymes are the lytic polysaccharide monooxygenases (LPMOs) that oxidatively depolymerize crystalline cellulose using a reactive oxygen species generated in a reduced mono‑copper active site. The copper site comprises of a highly conserved histidine-brace, providing three equatorial nitrogen ligands, whereas less conserved residues close to the copper contribute to shaping and confining the site. The catalytic copper site is exposed to the solvent and to the crystalline substrates, and as so, the influence of the copper environment on LPMO properties, including the redox potential, is of great interest. In the current work, a direct electrochemical study of an LPMO (ScLPMO10C) was conducted allowing to retrieve kinetic and thermodynamic data associated with the redox transition in the catalytic centre. Moreover, two residues that do not bind to the copper but shape the copper sites were mutated, and the properties of the mutants were compared with those of the wild-type enzyme. The direct electrochemical studies, using cyclic voltammetry, yielded redox potentials in the +200 mV range, well in line with LPMO redox potentials determined by other methods. Interestingly, while the mutations hardly affected the formal redox potential of the enzyme, they drastically affected the reactivity of the copper site and enzyme functionality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)