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The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (∼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation.One-sentence summaryCorynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.

Pleurotus ostreatus is a crucial commercial mushroom widely cultivated for diverse uses. Scientists have worked on breeding disease-resistant and high-yielding varieties to secure food supply. Studies on the molecular genetic mechanism of growth and development can provide valuable information to facilitate crop breeding programs by genetic engineering. Aegerolysins are pore-forming proteins widely distributed in both prokaryotes and eukaryotes, which are reported to have haemolytic activity and be involved in the early stages of fructification. The present study aimed to explore biological function of a differential expressed aegerolysin gene PriA in P. ostreatus. The expression level of PriA gene was higher in primordium and fruiting body than that in mycelium. The PriA expression in overexpression (OE) and RNAi interference (RNAi) strains was detected by qRT-PCR. The RNAi strains grew at slightly slower rates and advanced producing yellow pigments than the wild type, while OE strains showed no prominent phenotypic characteristics. Furthermore, Pseudomonas tolaasii infection assays showed that the PriA OE strains could enhance mycelia and caps resistance to P. tolaasii. These data demonstrate PriA from P. ostreatus play an essential role in mycelial development and increase antagonism against P. tolaasii. Our study provides some reference information on interactions between edible fungi and pathogenic bacteria and offers a new resistance-conferring gene for breeding. [ABSTRACT FROM AUTHOR]

Since genetic mutations during brain development play a significant role in the genesis of epilepsy, and such genetically determined epilepsies are the most difficult to treat, there is a need to study the mechanisms of epilepsy development with deletions of various transcription factors. We utilized heterozygous mice (Sip1wt/fl) with a neuronal deletion of the transcription factor Sip1 (Smad interacting protein 1) in the cerebral cortex. These mice are characterized by cognitive impairment and are prone to epilepsy. It is known that the brain-derived neurotrophic factor (BDNF) has a neuroprotective effect in various neurodegenerative diseases. Therefore, we created and applied an adeno-associated construct carrying the BDNF sequence selectively in neurons. Using in vitro and in vivo research models, we were able to identify a key gen, the disruption of whose expression accompanies the deletion of Sip1 and contributes to hyperexcitation of neurons in the cerebral cortex. Overexpression of BDNF in cortical neurons eliminated epileptiform activity in neurons obtained from heterozygous Sip1 mice in a magnesium-free model of epileptiform activity (in vitro). Using PCR analysis, it was possible to identify correlations in the expression profile of genes encoding key proteins responsible for neurotransmission and neuronal survival. The effects of BDNF overexpression on the expression profiles of these genes were also revealed. Using BDNF overexpression in cortical neurons of heterozygous Sip1 mice, it was possible to achieve 100% survival in the pilocarpine model of epilepsy. At the level of gene expression in the cerebral cortex, patterns were established that may be involved in the protection of brain cells from epileptic seizures and the restoration of cognitive functions in mice with Sip1 deletion. [ABSTRACT FROM AUTHOR]

Although enhanced fibroblast growth factor (FGF) signaling has been demonstrated to be crucial in many cases of syndromic cleft palate caused by tongue malposition in humans, animal models that recapitulate this phenotype are limited, and the precise mechanisms remain elusive. Mutations in FGF9 with the effect of either loss- or gain-of-function effects have been identified to be associated with cleft palate in humans. Here, we generated a mouse model with a transgenic Fgf9 allele specifically activated in cranial neural crest cells, aiming to elucidate the gain-of-function effects of Fgf9 in palatogenesis. We observed cleft palate with 100% penetrance in mutant mice. Further analysis demonstrated that no inherent defects in the morphogenic competence of palatal shelves could be found, but a passively lifted tongue prevented the elevation of palatal shelves, leading to the cleft palate. This tongue malposition was induced by posterior spatial confinement that was exerted by temporomandibular joint (TMJ) dysplasia characterized by a reduction in Sox9+ progenitors within the condyle and a structural decrease in the posterior dimension of the lower jaw. Our findings highlight the critical role of excessive FGF signaling in disrupting spatial coordination during palate development and suggest a potential association between palatal shelf elevation and early TMJ development. [ABSTRACT FROM AUTHOR]

Plant size is a critical component of agricultural productivity as larger plants produce more biomass. To identify genes related to plant size, we grouped C4 grasses into small and large and used OrthoFinder to find orthologous genes present in large but absent in small grasses. Three such genes were identified from sorghum (Sorghum bicolor [L.] Moench) by phylogenomic approach, and they encode nitrate transporter (Sobic.007G213200), oxysterol binding protein (SbRio.01G578800) and thioredoxin reductase (SbRio.05G168300), respectively. Overexpression of all three genes driven by the maize ubiquitin promoter in Setaria viridis (L.) Beauv. indicates that they all affected plant size as measured by plant height and tiller number. Both nitrate transporter and oxysterol binding protein increased plant height and tiller number, and thioredoxin reductase significantly decreased tiller number but had minimal effect on plant height. In rice (Oryza sativa L.), all three constructs reduced plant height significantly. The only commonality between the transgenic species was that nitrate transporter and oxysterol binding protein increased tiller number in both S. viridis and rice. Overall, we have demonstrated that phytogenomic approach can be used to identify genes responsible for large plant size in the grasses. [ABSTRACT FROM AUTHOR]

Plants produce secondary metabolites that serve various functions, including defense against biotic and abiotic stimuli. Many of these secondary metabolites possess valuable applications in diverse fields, including medicine, cosmetic, agriculture, and food and beverage industries, exhibiting their importance in both plant biology and various human needs. Small RNAs (sRNA), such as microRNA (miRNA) and small interfering RNA (siRNA), have been shown to play significant roles in regulating the metabolic pathways post-transcriptionally by targeting specific key genes and transcription factors, thus offering a promising tool for enhancing plant secondary metabolite biosynthesis. In this review, we summarize current approaches for manipulating sRNAs to regulate secondary metabolite biosynthesis in plants. We provide an overview of the latest research strategies for sRNA manipulation across diverse plant species, including the identification of potential sRNAs involved in secondary metabolite biosynthesis in non-model plants. We also highlight the potential future research directions, focusing on the manipulation of sRNAs to produce high-value compounds with applications in pharmaceuticals, nutraceuticals, agriculture, cosmetics, and other industries. By exploring these advanced techniques, we aim to unlock new potentials for biotechnological applications, contributing to the production of high-value plant-derived products. [ABSTRACT FROM AUTHOR]

Drought and salt stress are two important environmental factors that significantly restrict plant growth and reproduction. Malate dehydrogenase is essential to life as it is engaged in numerous physiological processes in cells, particularly those related to abiotic stress reactions. However, a complete understanding of MDH family members in kenaf is not clear yet. In this study, subcellular localization analysis and a yeast transcriptional activation assay revealed that HcMDH1 was localized in chloroplasts but had no transcriptional activation activity. When exposed to salt or drought stress, yeast cells expressing the HcMDH1 gene exhibit an increased survival rate. Overexpression of HcMDH1 in Arabidopsis increased seed germination rate and root growth when transgenic lines were exposed to varying concentrations of mannitol and NaCl. Subsequent physiological studies revealed that transgenic lines had higher concentrations of soluble carbohydrates, proline, and chlorophyll and lower concentrations of malondialdehyde (MDA) and reactive oxygen species (ROS). Furthermore, inhibiting HcMDH1 in kenaf using virus-induced gene silencing (VIGS) decreased salt and drought tolerance due to elevated ROS and MDA levels. In these silenced lines, the expression of six essential genes engaged in stress-resistance and photosynthesis, namely HcGAPDH, HcGLYK, HcFBA, HcFBPase, HcPGA, and HcLSD, is significantly altered under salt and drought stress. In summary, HcMDH1 is a complex and positive regulatory gene that plays a key role in regulating chlorophyll content, antioxidant enzyme activity and osmotic regulation under salt and drought stress, which may have implications for kenaf transgenic breeding. Highlights: HcMDH1 is located in the chloroplasts and could be induced by salt and drought stresses. Expression of the HcMDH1 in yeast improved its survival rate under salt and drought conditions. HcMDH1 can improve salt and drought tolerance ability by changing antioxidant activity. Silencing HcMDH1 reduced salt and drought tolerance by altering the expression of some stress-responsive genes. [ABSTRACT FROM AUTHOR]

Simple Summary: Canine lung adenocarcinomas are malignant tumors generally treated by surgical excision. When inoperable, chemotherapy can be performed with limited benefit. For this reason, the use of targeted drugs can be attempted, as has already happened in human medicine. In this retrospective study, 19 canine lung adenocarcinomas were investigated with three different methods to evaluate the expression of HER2. This transmembrane receptor for the human epidermal growth factor is often altered in human epithelial tumors, for which many targeted drugs have recently been produced. Using immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing, we found 3 cases out of 19 with protein overexpression, 2 cases with amplification, and only 1 case with a specific type of mutation (V659E), probably sensitive to tyrosine kinase inhibitory drugs. Due to the similar HER2 molecular changes in dogs and humans, this study provides preliminary information regarding the possible future use of targeted therapies in canine pulmonary adenocarcinomas. Recently, human epidermal growth factor receptor 2 (HER2) has emerged as a therapeutic target of interest for non-small-cell lung cancer in humans. The role of HER2 in canine pulmonary adenocarcinomas is poorly documented. To address this gap, this study employed three methodologies: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS) to investigate the protein expression, gene amplification, and mutation of HER2 in 19 canine primary pulmonary adenocarcinomas. By IHC, 3 out of 19 cases were overexpressed 3+, 6 were 2+, and 10 were negative. With FISH, 2 cases were amplified (12.5%), 3 were inadequate for the analyses, and the others were non-amplified. With NGS, seven cases were inadequate. All other cases were wild-type, except for one IHC 3+ case, which was amplified with FISH and with a specific mutation already described in human pulmonary adenocarcinoma, V659E. This mutation is probably sensitive to tyrosine kinase inhibitory drugs. These results are similar to those in human medicine and to the few data in the literature on canine lung carcinomas; the presence of 12.5% of amplified cases in dogs lays the foundation for future targeted drugs against HER2 alterations. [ABSTRACT FROM AUTHOR]

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the deamination of phenylalanine, which is the initial step in the biosynthesis of phenylpropanoids. It serves as a crucial enzyme that facilitates the transfer of carbon from primary to secondary metabolism in plants. Duckweed is regarded as a promising chassis plant in synthetic biology research and application, due to its being rich in secondary metabolites and other advantages. The genes encoding PAL in Spirodela polyrhiza (L.) Schleid, the giant duckweed, were investigated in this study. Three SpPAL genes (SpPAL1–SpPAL3) were identified and cloned. All of them were successfully expressed in E. coli, and their recombinant proteins all showed PAL activity. In addition, SpPAL1 and SpPAL2 proteins could also utilize tyrosine as substrate, although the activity was low. A qRT-PCR analysis demonstrated that the expression of SpPAL3 was most pronounced in young fronds. It was found that the expression of SpPAL1 and SpPAL3 was significantly induced by MeJA treatment. Overexpression of SpPAL3 in Lemna turionifera inhibited the growth of fronds and adventitious roots in the transgenic plants, indicating the importance of SpPAL3 in duckweed besides its involvement in the secondary metabolism. [ABSTRACT FROM AUTHOR]

Simple Summary: The xanthophyll cycle plays an important role in plants' responses to abiotic stress. Zeaxanthin epoxidase (ZEP) contributes to the xanthophyll cycle and the biosynthesis of abscisic acid (ABA) and carotenoids. The biological function of ZEP has been extensively studied in higher plants but has not been reported in Ulva prolifera. In this study, the relative content of xanthophylls was determined in U. prolifera. We also cloned and analyzed a ZEP gene from U. prolifera, namely UpZEP. The expression and biological function of the UpZEP gene were examined. The results of this study provide a theoretical basis for understanding the high-salt resistance of U. prolifera. The xanthophyll cycle is a photoprotective mechanism in plants and algae, which protects the photosynthetic system from excess light damage under abiotic stress. Zeaxanthin is considered to play a pivotal role in this process. In this study, the relative content of xanthophylls was determined using HPLC-MS/MS in Ulva prolifera exposed to different salinities. The results showed that high-salt stress significantly increased the relative content of xanthophylls and led to the accumulation of zeaxanthin. It was speculated that the accumulated zeaxanthin may contribute to the response of U. prolifera to high-salt stress. Zeaxanthin epoxidase (ZEP) is a key enzyme in the xanthophyll cycle and is also involved in the synthesis of abscisic acid and carotenoids. In order to explore the biological function of ZEP, a ZEP gene was cloned and identified from U. prolifera. The CDS of UpZEP is 1122 bp and encodes 373 amino acids. Phylogenetic analysis showed that UpZEP clusters within a clade of green algae. The results of qRT-PCR showed that high-salt stress induced the expression of UpZEP. In addition, heterologous overexpression of the UpZEP gene in yeast and Chlamydomonas reinhardtii improved the salt tolerance of transgenic organisms. In conclusion, the UpZEP gene may be involved in the response of U. prolifera to high-salt stress and can improve the high-salt tolerance of transgenic organisms. [ABSTRACT FROM AUTHOR]

Burkholderia is the second largest source of natural product bacteria after Actinomyces and can produce many secondary metabolites including pyrrolnitrin (PRN). Natural products of microbial origin are usually found in trace amounts, so in metabolic engineering, promoter engineering is often used to regulate gene expression to increase yield. In this study, an endogenous strong promoter was identified based on RNA-seq to overexpress biosynthetic genes to increase the production of PRN. By analyzing the transcriptomic data of the antagonistic bacterium Burkholderia sp. JP2-270 in three different development periods, we screened 50 endogenous promoters with high transcriptional activity, nine of which were verified by an obvious fluorescent signal via fluorescence observation. Then, combined with RT-qPCR analysis, Php, the promoter of a hypothetical protein, was found to be significantly expressed in all three periods. In order to increase the suitability of endogenous promoters, the promoter Php was shortened at different lengths, and the results show that a sequence length of 173 bp was necessary for its activity. Moreover, this promoter was used to overexpress the PRN biosynthesis genes (prnA, prnB, prnC and prnD) in JP2-270, resulting in a successful increase in gene expression levels by 40–80 times. Only the overexpression of the prnB gene successfully increased PRN production to 1.46 times that of the wild type. Overall, the endogenous strong promoters screened in this study can improve gene expression and increase the production of secondary metabolites in JP2-270 and other strains. [ABSTRACT FROM AUTHOR]

The significant reduction in cassava (Manihot esculenta Crantz) yields attributed to cassava bacterial blight (CBB) constitutes an urgent matter demanding prompt attention. The current study centered on the MebHLH149 transcription factor, which is acknowledged to be reactive to CBB and exhibits augmented expression levels, as indicated by laboratory transcriptome data. Our exploration, encompassing Xanthomonas phaseoli pv. manihotis strain CHN01 (Xpm CHN01) and hormone stress, disclosed that the MebHLH149 gene interacts with the pathogen at the early stage of infection. Furthermore, the MebHLH149 gene has been discovered to be responsive to the plant hormones abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), intimating a potential role in the signaling pathways mediated by these hormones. An analysis of the protein's subcellular localization suggested that MebHLH149 is predominantly located within the nucleus. Through virus-induced gene silencing (VIGS) in cassava, we discovered that MebHLH149-silenced plants manifested higher disease susceptibility, less ROS accumulation, and significantly larger leaf spot areas compared to control plants. The proteins MePRE5 and MePRE6, which are predicted to interact with MebHLH149, demonstrated complementary downregulation and upregulation patterns in response to silencing and overexpression of the MebHLH149 gene. This implies a potential interaction between MebHLH149 and these proteins. Both MePRE5 and MePRE6 genes are involved in the initial immune response to CBB. Notably, MebHLH149 was identified as a protein that physically interacts with MePRE5 and MePRE6. Based on these findings, it is hypothesized that the MebHLH149 gene likely functions as a positive regulator in the defense mechanisms of cassava against CBB. [ABSTRACT FROM AUTHOR]

Lin28A and Lin28B are paralogous RNA‐binding proteins that play fundamental roles in development and cancer by regulating the microRNA family of tumor suppressor Let‐7. Although Lin28A and Lin28B share some functional similarities with Let‐7 inhibitors, they also have distinct expression patterns and biological functions. Increasing evidence indicates that Lin28A and Lin28B differentially impact cancer stem cell properties, epithelial‐mesenchymal transition, metabolic reprogramming, and other hallmarks of cancer. Therefore, it is important to understand the overexpression of Lin28A and Lin28B paralogs in specific cancer contexts. In this review, we summarize the main similarities and differences between Lin28A and Lin28B, their implications in different cellular processes, and their role in different types of cancer. In addition, we provide evidence of other specific targets of each lin28 paralog, as well as the lncRNAs and miRNAs that promote or inhibit its expression, and how this impacts cancer development and progression. [ABSTRACT FROM AUTHOR]

BACKGROUND: SMAD family member 4 (SMAD4) can promote bone remodeling in osteoporotic rats, but it is unclear whether SMAD4 interferes with the expression of iron metabolism related proteins in osteoporotic rats. OBJECTIVE: To explore the effect of SMAD4 overexpression on the expression of iron metabolism related proteins in osteoporotic rats. METHODS: Rats were randomized into sham group, ovariectomy group, transfection control group and SMAD4 overexpression group. Animal models of osteoporosis were established in the latter three groups by ovariectomy, and only adipose tissue was removed in the sham group. One week later, adenovirus was injected into the femoral bone marrow cavity. SMAD4 overexpression group and transfection control group were injected with adenovirus overexpressing SMAD4 gene and control empty virus, respectively. Index detection was performed at 1 month after injection. Micro-CT, hematoxylin-eosin staining and tartrate resistant acid phosphatase staining were used to detect bone formation and bone resorption in osteoporotic rats. ELISA was used to detect serum ferritin and hepcidin levels. Immunohistochemical staining was used to detect alkaline phosphatase, osteocalcin, receptor activator for nuclear factor-κB ligand and tartrate resistant acid phosphatase levels in femoral tissue. RT-qPCR was used to detect SMAD4, hepcidin, divalent metal transporter 1, transferrin receptor1 and ferroportin1 mRNA levels in femoral tissue. Western blot was used to detect SMAD4, alkaline phosphatase, osteocalcin, osteoprotegerin, receptor activator for nuclear factor-κB ligand, tartrate resistant acid phosphatase, β-Crosslaps, hepcidin, divalent metal transporter 1, transferrin receptor 1, and ferroportin 1 protein levels. RESULTS AND CONCLUSION: In the sham group, bone trabeculae in femur tissue were intact, and almost no osteoclasts were found. In the ovariectomy and transfection control groups, the bone trabeculae were sparse and a large number of osteoclasts were present. In the SMAD4 overexpression group, the number of bone trabeculae was increased and the number of osteoclasts was decreased. Compared with the sham group, the ovariectomy group showed a significant reduction in the protein expression of SMAD4, alkaline phosphatase, osteocalcin, and osteoprotegerin in femoral tissue and hepcidin levels in serum and femoral tissue, while receptor activator for nuclear factor-κB ligand, tartrate resistant acid phosphatase, β-Crosslaps protein levels, divalent metal transporter 1, transferrin receptor1, ferroportin1 mRNA and protein levels were significantly increased (P < 0.05). Compared with the transfection control group, the SMAD4 overexpression showed a significant increase in SMAD4, alkaline phosphatase, osteocalcin, and osteoprotegerin protein levels in femoral tissue and hepcidin levels in serum and femoral tissue, while the expressions of receptor activator for nuclear factor-κB ligand, tartrate resistant acid phosphatase, β-Crosslaps protein levels, divalent metal transporter 1, transferrin receptor1, and ferroportin 1 at mRNA and protein levels were significantly decreased (P < 0.05). To conclude, overexpression of SMAD4 promotes bone remodeling in osteoporotic rats by interfering with the expression of iron metabolism related proteins. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the probiogenomic features of artisanal bacteriocin-producing Enterococcus faecium BGPAS1-3 and the use of the improved pMALc5HisEk expression vector for overexpressing class II bacteriocins and the application of purified bacteriocin 31 in a milk model as a preservative against L. monocytogenes. The BGPAS1-3 strain was isolated from traditional fresh soft cheese manufactured in households on a small scale in rural locations surrounding Pale Mountain City in Bosnia and Herzegovina. The whole-genome sequencing approach and bioinformatics analyses revealed that the strain BGPAS1-3 was non-pathogenic to humans. The presence of bacteriocin operons suggested the ability of the isolate to suppress the growth of pathogens. Coding regions for three maturated bacteriocins (bacteriocin 31, bacteriocin 32, and enterocin P) produced by BGPAS1-3 were amplified and expressed in Escherichia coli ER2523 using the pMALc5HisEk system. All three bacteriocins were successfully overexpressed and purified after enterokinase cleavage but showed different antimicrobial activity. Bacteriocin 31 showed significantly stronger antimicrobial activity compared with bacteriocin 32. It was the only one that proved to be suitable for use as a food preservative against L. monocytogenes in a milk model. [ABSTRACT FROM AUTHOR]

Tomato Solanum lycopersicum is an important economic fruit, and widely consumed for its high nutritional quality. Our previous study found that AtERF022, a member of Arabidopsisthaliana ethylene response factors (ERFs) group III, acted a vital role in Arabidopsis growth and development. However, whether or how endogenous AtERF022 confers tomato fruit quality is still elusive. To explore the role of AtERF022 gene in tomato fruit ripening and nutritional quality, we generated AtERF022-overexpressing tomato line by transgenic and plant tissue culture technology. Our results showed that the higher expression of AtERF022 was found in tomato flowers and fruits, especially in orange ripening stage of fruits. Compared to wild-type, AtERF022-overexpressing tomato exhibited morphological growth advantages and elevated fruit ripening and nutritional quality. Further experiments and transcriptome sequencing showed that overexpression of AtERF022 enhanced the expression of genes involved in ethylene biosynthesis, cell wall metabolism, chlorophyll degradation, carotenoids and flavonoids biosynthesis, and thereby promoting fruit ripening, β-carotene, flavonoids, vitamin C and total phenols accumulation. Together, our results provide genetic evidence that AtERF022 regulates tomato fruit ripening and nutritional quality. [ABSTRACT FROM AUTHOR]

Phosphatidylethanolamine-binding proteins (PEBPs) play a crucial role in the growth and development of various organisms. Due to the low sequence similarity compared to plants, humans, and animals, the study of pebp genes in fungi has not received significant attention. The redifferentiation of fruiting bodies is exceedingly rare in fungal development. Hitherto, only a few studies have identified the Capebp2 gene as being associated with this phenomenon in Cyclocybe aegerita. Thus, exploring the role of pebp genes in fruiting body development is imperative. In the present study, four Capebp genes (Capebp1, Capebp3, Capebp4, and Capebp5) were cloned from the AC0007 strain of C. aegerita based on genome sequencing and gene prediction. The findings indicate that the pebp family, in C. aegerita, comprises a total of five genes. Moreover, the sequence similarity was low across the five CAPEBP protein sequences in C. aegerita, and only a few conserved sequences, such as HRY and RHF, were identical. Expression analyses revealed that, similarly to Capebp2, the four Capebp genes exhibit significantly higher expression levels in the fruiting bodies than in the mycelium. Furthermore, overexpressed and RNA interference Capebp1 or Capebp5 transformants were analyzed. The results demonstrate that overexpression of Capebp1 or Capebp5 could induce the regeneration of the lamella or fruiting body, whereas the knockdown of Capebp1 or Capebp5 could lead to the accelerated aging of fruiting bodies. These findings highlight a significant role of Capebp genes in the generation of C. aegerita fruiting bodies and provide a foundation for further exploration into their involvement in basidiomycete growth and development. [ABSTRACT FROM AUTHOR]

Plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) proteins play critical roles in plant development and stress responses; however, their functions in chrysanthemum (Chrysanthemum morifolium) have not been well-studied. In this study, we isolated and characterized the chrysanthemum TCP transcription factor family gene CmTCP13, a homolog of AtTCP13. This gene encoded a protein harboring a conserved basic helix–loop–helix motif, and its expression was induced by salinity stress in chrysanthemum plants. Subcellular localization experiments showed that CmTCP13 localized in the nucleus. Sequence analysis revealed the presence of multiple stress- and hormone-responsive cis-elements in the promoter region of CmTCP13. The heterologous expression of CmTCP13 in Arabidopsis plants enhanced their tolerance to salinity stress. Under salinity stress, CmTCP13 transgenic plants exhibited enhanced germination, root length, seedling growth, and chlorophyll content and reduced relative electrical conductivity compared with those exhibited by wild-type (WT) plants. Moreover, the expression levels of stress-related genes, including AtSOS3, AtP5CS2, AtRD22, AtRD29A, and AtDREB2A, were upregulated in CmTCP13 transgenic plants than in WT plants under salt stress. Taken together, our results demonstrate that CmTCP13 is a critical regulator of salt stress tolerance in plants. [ABSTRACT FROM AUTHOR]

Taraxacum kok-saghyz (TKS) is a natural rubber (NR)-producing plant and a model plant for studying the biosynthesis of NR. Analyzing and studying the biosynthetic mechanism of NR is an important way to cultivate high-yield rubber TKS varieties. JAZ proteins, which belong to the Jasmonate ZIM domain family, function as negative regulators in the jasmonic acid (JA) signal transduction pathway. MYC2 is typically regarded as a regulatory factor for the target genes of JAZ proteins; JAZ proteins indirectly influence the gene expression regulated by MYC2 by modulating its activity. Theoretically, JAZ is expected to participate in growth, development, and responses to environmental cues related to rubber and biomass accumulation in TKS, all of which rely on the interaction between JAZ and MYC2. In this study, we identified 11 TkJAZs through homology searching of the TKS genomes and bioinformatics analyses. Subcellular localization, Y2H, and BiFC analysis demonstrate that TkJAZs and TkMYC2 are localized in the nucleus, with all TkJAZs and TkMYC2 showing nuclear colocalization interactions. Overexpression of TkMYC2 in TKS inhibited leaf development, promoted root growth, and simultaneously increased NR production. RNA-seq and qRT-PCR analysis revealed that the TkSRPP/REF genes exhibit varying degrees of upregulation compared to the wild type, upregulating the TkREF1 gene by 3.7-fold, suggesting that TkMYC2 regulates the synthesis of NR by modulating the TkSRPP/REF genes. [ABSTRACT FROM AUTHOR]

Watermelon (Citrullus lanatus) holds global significance as a fruit with high economic and nutritional value. Exploring the regulatory network of watermelon male reproductive development is crucial for developing male sterile materials and facilitating cross-breeding. Despite its importance, there is a lack of research on the regulation mechanism of male reproductive development in watermelon. In this study, we identified that ClESR2, a VIIIb subclass member in the APETALA2/Ethylene Responsive Factor (AP2/ERF) superfamily, was a key factor in pollen development. RNA in situ hybridization confirmed significant ClESR2 expression in the tapetum and pollen during the later stage of anther development. The pollens of transgenic plants showed major defects in morphology and vitality at the late development stage. The RNA-seq and protein interaction assay confirmed that ClESR2 regulates pollen morphology and fertility by interacting with key genes involved in pollen development at both transcriptional and protein levels. These suggest that Enhancer of Shoot Regeneration 2 (ESR2) plays an important role in pollen maturation and vitality. This study helps understand the male reproductive development of watermelon, providing a theoretical foundation for developing male sterile materials.

Abstract Drought and salt stress are two important environmental factors that significantly restrict plant growth and reproduction. Malate dehydrogenase is essential to life as it is engaged in numerous physiological processes in cells, particularly those related to abiotic stress reactions. However, a complete understanding of MDH family members in kenaf is not clear yet. In this study, subcellular localization analysis and a yeast transcriptional activation assay revealed that HcMDH1 was localized in chloroplasts but had no transcriptional activation activity. When exposed to salt or drought stress, yeast cells expressing the HcMDH1 gene exhibit an increased survival rate. Overexpression of HcMDH1 in Arabidopsis increased seed germination rate and root growth when transgenic lines were exposed to varying concentrations of mannitol and NaCl. Subsequent physiological studies revealed that transgenic lines had higher concentrations of soluble carbohydrates, proline, and chlorophyll and lower concentrations of malondialdehyde (MDA) and reactive oxygen species (ROS). Furthermore, inhibiting HcMDH1 in kenaf using virus-induced gene silencing (VIGS) decreased salt and drought tolerance due to elevated ROS and MDA levels. In these silenced lines, the expression of six essential genes engaged in stress-resistance and photosynthesis, namely HcGAPDH, HcGLYK, HcFBA, HcFBPase, HcPGA, and HcLSD, is significantly altered under salt and drought stress. In summary, HcMDH1 is a complex and positive regulatory gene that plays a key role in regulating chlorophyll content, antioxidant enzyme activity and osmotic regulation under salt and drought stress, which may have implications for kenaf transgenic breeding. Graphical Abstract

BACKGROUND: Previous studies have shown that puerarin can inhibit the differentiation of osteoclasts, and the expression of Notch signaling pathway-related proteins such as Notch1, HES1, and Jagged1 is decreased. However, the specific mechanism of the Notch1 signaling pathway for the inhibition of osteoclast differentiation by puerarin is not clear. OBJECTIVE: To explore the effect of Notch signaling pathway on puerarin inhibiting the differentiation of mouse macrophage Raw264.7 into osteoclasts. METHODS: Raw264.7 cells were divided into seven groups for intervention culture. Blank control group was cultured in high-sugar DMEM medium; the osteoclast induction group was cultured in osteoclast induction medium; the puerarin intervention group was cultured with 50 μmol/L puerarin at the same time of osteoclast induction; Notch1 siRNA control group, Notch1 siRNA group, Notch1 overexpression control group and Notch1 overexpression group were transfected with Notch1 siRNA control sequence, Notch1 siRNA, Notch1 overexpression control plasmid and Notch1 overexpression plasmid, respectively, and then cultured with osteoclast induction medium and puerarin. The number and size of osteoclasts were observed by tartrate-resistant acid phosphatase staining, the skeleton formation of osteoclasts was observed by F-actin staining, and the gene expression level of osteoclast formation markers was detected by RT-PCR. RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining results showed that puerarin intervention could inhibit the generation of osteoclasts, Notch1 silencing could further reduce the number of osteoclasts, while the number of osteoclasts in the osteoclast-induced group increased significantly after Notch1 overexpression. The results of F-actin showed that Raw264.7 cells could form a well-defined F-actin ring after osteoclast induction. Puerarin intervention would inhibit the formation of cytoskeleton, and Notch1 silencing could aggravate the inhibitory effect of cytoskeleton formation, while Notch1 overexpression could alleviate this inhibitory effect of puerarin. RT-PCR results showed that puerarin could inhibit the mRNA expression levels of tartrateresistant acid phosphatase, Cathepsin K and c-Fos, the expression of the above-mentioned three factors decreased significantly after Notch1 gene silencing, and Notch1 overexpression could upregulate the expression of these factors. These finding indicate that puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Human serum albumin (HSA) is the most abundant protein in plasma, playing crucial roles in regulating osmotic pressure and maintaining protein homeostasis. It is widely applied in the clinical treatment of various diseases. HSA can be purified from plasma or produced using recombinant DNA technology. Due to the improved efficiency and reduced costs, a growing body of research has focused on enhancing albumin production through bacterial strain overexpression. However, there have been few studies on the effect of albumin on the characteristics of the overexpressing‐strain itself, particularly stress resistance. In this study, we utilized Lactiplantibacillus plantarum (L. plantarum) AR113 as the expression host and successfully constructed the albumin overexpression strain AR113‐pLLY01 through gene editing technology. The successful expression of albumin was achieved and subsequently compared with the wild‐type strain AR113‐pIB184. RESULTS: The results demonstrated that the survival rate of AR113‐pLLY01 was also significantly better than that of AR113‐pIB184 after lyophilization. In addition, AR113‐pLLY01 exhibited a significantly better protective effect than AR113‐pIB184 at pH 3, indicating that albumin possesses a certain tolerance to acidic stress. At bile salt concentrations higher than 0.03%, both strains showed limited growth, but at a concentration of 0.02%, AR113‐pLLY01 had a significant protective effect. CONCLUSION: This study suggest that albumin can improve strain tolerance, which has significant implications for future applications. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in Δ creA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (bgl1). Interestingly, bgl1 expression in Δ creA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in A. aculeatus. [ABSTRACT FROM AUTHOR]

Tuberculosis, a disease caused by the bacterium Mycobacterium tuberculosis, is a major public health concern. Innate and adaptive immunity provide robust defense against pathogens. However, M. tuberculosis, which co-evolved with humans, has acquired many mechanisms to evade the immune response and ensure its intracellular existence and long-term survival within the host. Moreover, emerging evidence suggests that this secretive bacterium can alter expression of regulatory noncoding RNAs (including microRNAs), leading to dysregulation of biological processes underlying tuberculosis pathogenesis. For example, miR-222-3p has been shown to regulate the functional reprogramming of macrophages and is involved in the regulation of host innate immunity. Previously, we demonstrated the important role of miR-222-3p as a biological marker of tuberculosis activity. To confirm their biological targets and understand their role in the pathogenesis of tuberculosis, many research groups are working to establish functional relationships between miRNA expression under different conditions and their actual biological action using molecular biology and bioinformatics methods. In the present study, we demonstrated the effect of miR-222-3p overexpression on several functions of human macrophages of monocytic origin activated with M. tuberculosis antigens in in vitro culture. Specifically, we found that miR-222-3p overexpression significantly decreased IL-6 and IFNγ expression and increased IL-1β and cxcl10 expression in cultures of uninfected macrophages. Infected macrophages overexpressing miR-222-3p were characterized by increased NF-κB and IL-6 expression, as were infected macrophages without transfection. Another important finding was that miR-222-3p overexpression caused a small but significant increase in reactive nitrogen species production by infected macrophages, but did not affect their bacteriostatic activity against M. tuberculosis. Elucidating the functions of different microRNAs in regulating different pathogenic pathways in TB may lead to discovering new therapeutic targets. The detailed study of microRNAs that regulate immune-associated pathways will be useful for the design of miRNA mimetic molecules, either as inhibitors or as activators. Immune effects induced by miRNA drugs are currently a major challenge for miRNA therapeutics.

Abstract Background Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application. Results In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1′,4′-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1′,4′-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1′,4′-trans-ABA-diol synthesis. To further increase the yield of 1′,4′-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1′,4′-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1′,4′-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain. Conclusions We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1′,4′-trans-ABA-diol. By overexpressing the crucial genes involved in the 1′,4′-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1′,4′-trans-ABA-diol.

Plant basic helix-loop-helix (bHLH) transcription factors (TFs) play central roles in various abiotic stresses. However, its role in plant cold resistance is largely unknown. Previously, we characterised CaNAC035 in pepper, which positively regulates tolerance to cold, salt and drought stresses tolerance. Here, we identified CabHLH035, a CaNAC035-interacting protein in pepper. To explore its functions in cold stress tolerance, we silenced the gene in pepper via virus-induced gene silencing (VIGS) and overexpressed the gene in Arabidopsis. The results showed that CabHLH035 expression was induced by cold treatment, and silencing of CabHLH035 decreased cold stress tolerance. Conversely, overexpression of CabHLH035 in Arabidopsis increased cold stress tolerance. To investigate homologs genes of C-repeat binding factor (CBF) pathway proteins and reactive oxygen species (ROS) marker gene expression blocking by CabHLH035, we performed yeast one-hybrid (Y1H), dual luciferase and electrophoretic mobility shift assay experiments. The results showed that CabHLH035 bound to the region upstream of the CaCBF1A and CaAPX promoters. Additionally, CaCBF1A bound to the CaDHN4 promoter. Taken together, our results showed that CabHLH035 plays a crucial role in cold stress tolerance and its potential as a target for breeding cold-resistant crops. The findings provide a basis for studying the functions and regulatory network of cold stress tolerance in pepper.

Breast infection is the primary etiology of mastitis in dairy cows, leading to a reduction in the quality of dairy products and resulting in substantial economic losses for animal husbandry. Although antibiotic treatment can eliminate the pathogenic microorganisms that induce mastitis, it cannot repair the inflammatory damage of mammary epithelial cells and blood milk barrier. Mas1 is a G protein-coupled receptor, and its role in lipopolysaccharide (LPS) -induced inflammatory injury to mammary epithelial cells has not been studied. LPS treatment of EpH4 EV cells led to a significant downregulation of Mas1 transcript levels, which attracted our great interest, suggesting that Mas1 may be an important target for the treatment of mastitis. Therefore, this study intends to verify the role of Mas1 in the inflammatory injury of EpH4 EV cells by gene overexpression technology and gene silencing technology. The findings demonstrated that the overexpression of the Mas1 gene effectively reversed the activation of the nuclear factor-κB/mitogen-activated protein kinase (NF-κB/MAPK) signaling pathways induced by LPS, while also suppressing the upregulation of pro-inflammatory mediators. Furthermore, overexpression of the Mas1 gene reversed the downregulation of zonula occludens 1 (ZO-1), Occludin, and Claudin-3 caused by LPS, suggesting that Mas1 could promote to repair the blood-milk barrier. However, the silencing of the Mas1 gene using siRNA resulted in a contrasting effect. These results indicated that Mas1 alleviated the inflammatory injury of mammary epithelial cells induced by LPS. [ABSTRACT FROM AUTHOR]

AbstractMicroalgae and cyanobacteria are a rich source of carotenoids that are well known for their potent bioactivities, including antioxidant, anti-cancer, anti-proliferative, anti-inflammatory, and anti-obesity properties. Recently, many interests have also been focused on the biological activities of these microalgae/cyanobacteria-derived carotenoids, such as fucoxanthin and β-carotene potential to be the salutary nutraceuticals, on treating or preventing human common diseases (e.g., cancers). This is due to their special chemical structures that demonstrate unique bioactive functions, in which the biologically active discrepancies might attribute to the different spatial configurations of their molecules. In addition, their abundance and bioaccessibilities make them more popularly applied in food and pharmaceutical industries, as compared to the macroalgal/fungal-derived ones. This review is focused on the recent studies on the bioactivities of fucoxanthin and some carotenoids derived from microalgae and cyanobacteria in relationship with human health and diseases, with emphasis on their potential applications as natural antioxidants. Various biotechnological approaches employed to induce the production of these specific carotenoids from the culture of microalgae/cyanobacteria are also critically reviewed. These well-developed and emerging biotechnologies present promise to be applied in food and pharmaceutical industries to facilitate the efficient manufacture of the bioactive carotenoid products derived from microalgae and cyanobacteria. [ABSTRACT FROM AUTHOR]

Wheat is one of the most important food crops globally, and understanding the regulation of grain size is crucial for wheat breeding to achieve a higher grain yield. MicroRNAs (miRNAs) play vital roles in plant growth and development. However, the miRNA-mediated mechanism underlying grain size regulation remains largely elusive in wheat. Here, we report the characterization and functional validation of a miRNA, TamiR397a, associated with grain size regulation in wheat. The function of three TaMIR397 homoeologs was determined through histochemical β-glucuronidase-dependent assay. MiRNA expression was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the function of TamiR397a was validated through its transgenic overexpression and repression in wheat. It was found that TaMIR397-6A and TaMIR397-6B encode active TamiR397a. The expression profiling indicated that TamiR397a was differentially expressed in various tissues and gradually up-regulated during grain filling. The inhibition of TamiR397a perturbed grain development, leading to a decrease in grain size and weight. Conversely, the overexpression of TamiR397a resulted in increased grain size and weight by accelerating the grain filling process. Transcriptome analysis revealed that TamiR397a regulates a set of genes involved in hormone response, desiccation tolerance, regulation of cellular senescence, seed dormancy, and seed maturation biological processes, which are important for grain development. Among the down-regulated genes in the grains of the TamiR397a-overexpressing transgenic plants, 11 putative targets of the miRNA were identified. Taken together, our results demonstrate that TamiR397a is a positive regulator of grain size and weight, offering potential targets for breeding wheat with an increased grain yield. [ABSTRACT FROM AUTHOR]

Key message: Overexpressing the copper transporter LbCOPT1 leads to a notable increase in the abundance of mycorrhizal arbuscules that suggests the potential application of LbCOPT1 in breeding programs aimed at enhancing symbiotic nutrient uptake in Lycium barbarum L. [ABSTRACT FROM AUTHOR]

Photoperiod is a pivotal regulatory factor in the flowering of Camellia oleifera Abel. (C. oleifera). GIGANTEA (GI) serves as a pivotal regulator, not only orchestrating the intricate circadian rhythm but also governing photoperiod-dependent flowering. In order to explore the function of GI in C. oleifera (CoGI), we obtained a CoGI gene-coding sequence and analyzed a CoGI protein sequence using bioinformatics. Furthermore, we conducted a spatiotemporal expression analysis of CoGI. And a yeast two-hybridization assay was used to screen the interacting proteins of CoGI. Evolutionary analysis revealed high conservation of the CoGI protein, which clustered with the GI protein from Camellia sinensis (CsGI) on a common evolutionary branch. The expression of CoGI was different in each part, and a tissue expression analysis revealed that the relative expression level of the CoGI gene is highest in the leaves of C. oleifera, while it is at its lowest in the seed coats. Transgenic Arabidopsis thaliana (Arabidopsis) overexpressing CoGI exhibited early flowering under long-day conditions. In addition, the yeast two-hybrid library screening revealed interactions between seven C. oleifera proteins and CoGI: CoACR9, CoLAO, CoDExH12-like, CoIT1K-like, CoUPF0481, CoIDM3, and CoAt4g27190-like. The findings demonstrated that CoGI is crucial to C. oleifera's flowering. [ABSTRACT FROM AUTHOR]

Melatonin (Mel) is highly conserved and stress regulator molecule in plants and regulates several key physiological mechanisms relating to growth and development under abiotic stress conditions. Given that, abiotic stress conditions significantly reduce plant growth, thus it is imperative to improve abiotic stress tolerance in plants. This mini-review specifically dissects the role of genes relating to Mel-biosynthesis and metabolism in regulating abiotic stress adaptive responses. Literature analysis showed that the overexpression of different genes relating to Mel-biosynthesis or Mel metabolism regulate abiotic stress tolerance by regulating ROS production and activation of antioxidant, ionic homeostasis, hormonal regulation, and activation of stress responsive genes. Nonetheless, the regulation of such stress adaptive responses is highly plant species-specific, abiotic stress type, and Mel-related genes. The role of phytomelatonin receptor 1-Mel receptor in plants, in regulating abiotic stress tolerance has also been briefly discussed. Therefore, we entrusted that this article improved our understanding the molecular role of Mel in regulating abiotic stress adaptive responses. [ABSTRACT FROM AUTHOR]

Background: Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application. Results: In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1′,4′-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1′,4′-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1′,4′-trans-ABA-diol synthesis. To further increase the yield of 1′,4′-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1′,4′-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1′,4′-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain. Conclusions: We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1′,4′-trans-ABA-diol. By overexpressing the crucial genes involved in the 1′,4′-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1′,4′-trans-ABA-diol. [ABSTRACT FROM AUTHOR]

Proteasome 26S Subunit, Non-ATPase 9 (psmd9) plays an important role in the balance of protamine and the stability of the nucleolar structure during spermatogenesis. In this study, we cloned the psmd9 of Cynoglossus semilaevis and analyzed its expression pattern. psmd9 was identified on the Z chromosome of C. semilaevis, which is considered an interesting candidate gene for spermatogenesis. qRT-PCR and FISH experiments showed that the psmd9 gene was significantly highly expressed in the testes. It is worth noting that the expression level of psmd9 in male fish testes is significantly higher than that in pseudomales. In order to further explore the role of psmd9 in spermatogenesis, a male testicular cell line was used as the experimental material. The results of the psmd9-RNAi and overexpression experiments showed that psmd9 had a synergistic effect with spermatogenesis-related genes dnd1, cfap69, dnah3 and dnajb13, but had an antagonistic effect with ccne2. Our findings offer a scientific foundation for comprehending the role of psmd9 in the spermatogenesis regulatory network of C. semilaevis. [ABSTRACT FROM AUTHOR]

Introduction: Chemokine-like factor 1 (CKLF1) is a chemokine that is overexpressed in several diseases. Our previous findings revealed a significant increase in CKLF1 expression in the ischemic brain, suggesting its potential as a therapeutic target for ischemic stroke. Methods: In this study, we examined the expression dynamics of CKLF1 in both in vivo and in vitro models of ischemic cardiac injury. Myocardial infarction (MI) was induced in vivo by ligation of the left anterior descending artery (LAD) of the rat heart. The levels of CKLF1, Creatine Kinase MB Isoenzyme (CK-MB), and Lactate dehydrogenase (LDH) in the serum were detected using Enzyme-linked immunosorbent assay (ELISA). The expression of CKLF1 in the infarcted area was detected by immunohistochemistry, immunofluorescence, quantitative PCR (qPCR), and Western blotting (WB). H9C2 and AC16 cardiomyocytes cultured in vitro were subjected to oxygen and glucose deprivation (OGD). LDH was used to detect cell damage, and CKLF1 expression was detected by qPCR and WB. Results: CKLF1 mRNA and protein expression were significantly increased in h9c2 cells at 1.5 h and in AC16 cells at 4 h after OGD. The serum CK-MB in rats increased significantly on the first day after infarction, while the LDH concentration increased significantly on the third day after infarction. CKLF1 blood levels significantly increased on the first day following MI in rats. CKLF1 expression notably increased in the infarct area on days 1, 3, and 7 post-MI. In MI tissue, CKLF1 colocalizes with cardiomyocytes, macrophages, and neutrophils. Conclusion: CKLF1 was substantially expressed during myocardial ischemia injury both in vivo and in vitro and was colocalized with macrophages and neutrophils, indicating that CKLF1 is expected to be a biomarker and a drug target for the treatment of myocardial infarction. [ABSTRACT FROM AUTHOR]

The last decade has been highlighted by the increased use of next-generation DNA sequencing technology to identify novel human disease genes. A critical downstream part of this process is assigning function to a candidate gene variant. Functional studies in Drosophila melanogaster, the common fruit fly, have made a prominent contribution in annotating variant impact in an in vivo system. The use of patient-derived knock-in flies or rescue-based, "humanization", approaches are novel and valuable strategies in variant testing but have been recently widely reviewed. An often-overlooked strategy for determining variant impact has been GAL4/upstream activation sequence-mediated tissue-defined overexpression in Drosophila. This mini-review will summarize the recent contribution of ectopic overexpression of human reference and variant cDNA in Drosophila to assess variant function, interpret the consequence of the variant, and in some cases infer biological mechanisms. [ABSTRACT FROM AUTHOR]

Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi. [ABSTRACT FROM AUTHOR]

3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) enzyme plays an important role in the biosynthesis of diterpene lactones such as andrographolide, a specialized metabolite that is widely used as a therapeutic agent. This study aimed to enhance the content of andrographolide through the overexpression of 3-hydroxy-3-methylglutaryl coenzyme-A reductase-1 (HMGR1) in A. paniculata. HMGR1 was constructed into the pBI121 expression vector controlled by the cauliflower mosaic virus (CaMV 35S) promoter. The pBI121-HMGR1 construct was transformed to A. paniculata through Agrobacteriummediated transformation followed by plant regeneration to obtain transgenic plants. Polymerase chain reaction (PCR) analysis was conducted to validate the transgenic plants, quantitative reverse transcription PCR (RT-qPCR) was used for the HMGR1 expression analysis, and high-performance liquid chromatography was employed to quantify the andrographolide content of the transgenic plants. PCR analysis on the transgenic plantlets indicated the presence of a 2,262 bp fragment that corresponded to HMGR1, thus confirming the integration of HMGR1 into the A. paniculata genome. The RT-qPCR results showed a 52.5-fold greater HMGR1 expression in the transgenic plant under the CaMV 35S promoter compared to the normal plant. As the expression level of HMGR1 increased, the amount of andrographolide was enhanced by up to 2.6 times in the transgenic plant compared to the control plant. This study demonstrates that the overexpression of HMGR1 driven by CaMV 35S can enhance the production of andrographolide in A. paniculata. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Acinetobacter baumannii poses a significant threat to public health globally, especially due to its ability to produce multiple carbapenemases, leading to treatment challenges. This study aimed to investigate the antibiotic resistance pattern of carbapenem-resistant A. baumannii isolates collected from different clinical settings in North East India, focusing on their genotypic and phenotypic resistance profiles. A total of 172 multidrug-resistant A. baumannii isolates were collected and subjected to antibiotic susceptibility test using the Kirby–Bauer disk diffusion method. Various phenotypic tests were performed to detect extended-spectrum β-lactamase (ESBL), metallo-β-lactamase (MBL), class C AmpC β-lactamase (AmpC), and carbapenem hydrolyzing class D β-lactamase (CHDL) production among the isolates. Overexpression of carbapenemase and cephalosporinase genes was detected among the isolates through both phenotypic and genotypic investigation. The antibiotic resistance profile of the isolates revealed that all were multidrug-resistant; 25% were extensively drug-resistant, 9.30% were pan-drug-resistant, whereas 91.27% were resistant to carbapenems. In the genotypic investigation, 80.81% of isolates were reported harbouring at least one metallo-β-lactamase encoding gene, with blaNDM being the most prevalent at 70.34%, followed by blaIMP at 51.16% of isolates. Regarding class D carbapenemases, blaOXA-51 and blaOXA-23 genes were detected in all the tested isolates, while blaOXA-24,blaOXA-48, and blaOXA-58 were found in 15.11%, 6.97%, and 1.74% isolates respectively. Further analysis showed that 31.97% of isolates co-harboured ESBL, MBL, AmpC, and CHDL genes, while 31.39% of isolates co-harboured ESBL, MBL, and CHDL genes with or without ISAba1 leading to extensively drug-resistant or pan drug-resistant phenotypes. This study highlights the complex genetic profile and antimicrobial-resistant pattern of the isolates circulating in North East India, emphasizing the urgent need for effective infection control measures and the development of alternative treatment strategies to combat these challenging pathogens. [ABSTRACT FROM AUTHOR]

The suppressor of cytokine signaling 3 (SOCS3) is a key signaling molecule that regulates milk synthesis in dairy livestock. However, the molecular mechanism by which SOCS3 regulates lipid synthesis in goat milk remains unclear. This study aimed to screen for key downstream genes associated with lipid synthesis regulated by SOCS3 in goat mammary epithelial cells (GMECs) using RNA sequencing (RNA-seq). Goat SOCS3 overexpression vector (PC-SOCS3) and negative control (PCDNA3.1) were transfected into GMECs. Total RNA from cells after SOCS3 overexpression was used for RNA-seq, followed by differentially expressed gene (DEG) analysis, functional enrichment analysis, and network prediction. SOCS3 overexpression significantly inhibited the synthesis of triacylglycerol, total cholesterol, non-esterified fatty acids, and accumulated lipid droplets. In total, 430 DEGs were identified, including 226 downregulated and 204 upregulated genes, following SOCS3 overexpression. Functional annotation revealed that the DEGs were mainly associated with lipid metabolism, cell proliferation, and apoptosis. We found that the lipid synthesis-related genes, STAT2 and FOXO6, were downregulated. In addition, the proliferation-related genes BCL2, MMP11, and MMP13 were upregulated, and the apoptosis-related gene CD40 was downregulated. In conclusion, six DEGs were identified as key regulators of milk lipid synthesis following SOCS3 overexpression in GMECs. Our results provide new candidate genes and insights into the molecular mechanisms involved in milk lipid synthesis regulated by SOCS3 in goats. [ABSTRACT FROM AUTHOR]

In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different poly-Ub (UBQ) gene overexpression plants of Arabidopsis thaliana. One is transformed with hexa-6His-UBQ (designated 6HU), driven by the cauliflower mosaic virus 35S promoter, while the other expresses hexa-6His-TEV-UBQ (designated 6HTU), driven by the endogenous promoter of UBQ10. We discovered that 6HU and 6HTU had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both 6HU and 6HTU. Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of UBQ expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply. [ABSTRACT FROM AUTHOR]

Salt stress is one of the major factors limiting plant growth and productivity. Many studies have shown that serine hydroxymethyltransferase (SHMT) gene play an important role in growth, development and stress response in plants. However, to date, there have been few studies on whether SHMT3 can enhance salt tolerance in plants. Therefore, the effects of overexpression or silencing of CsSHMT3 gene on cucumber seedling growth under salt stress were investigated in this study. The results showed that overexpression of CsSHMT3 gene in cucumber seedlings resulted in a significant increase in chlorophyll content, photosynthetic rate and proline (Pro) content, and antioxidant enzyme activity under salt stress condition; whereas the content of malondialdehyde (MDA), superoxide anion (H2O2), hydrogen peroxide (O2·−) and relative conductivity were significantly decreased when CsSHMT3 gene was overexpressed. However, the content of chlorophyll and Pro, photosynthetic rate, and antioxidant enzyme activity of the silenced CsSHMT3 gene lines under salt stress were significantly reduced, while MDA, H2O2, O2·− content and relative conductivity showed higher level in the silenced CsSHMT3 gene lines. It was further found that the expression of stress-related genes SOD, CAT, SOS1, SOS2, NHX, and HKT was significantly up-regulated by overexpressing CsSHMT3 gene in cucumber seedlings; while stress-related gene expression showed significant decrease in silenced CsSHMT3 gene seedlings under salt stress. This suggests that overexpression of CsSHMT3 gene increased the salt tolerance of cucumber seedlings, while silencing of CsSHMT3 gene decreased the salt tolerance. In conclusion, CsSHMT3 gene might positively regulate salt stress tolerance in cucumber and be involved in regulating antioxidant activity, osmotic adjustment, and photosynthesis under salt stress. Key message: CsSHMT3 gene may positively regulate the expression of osmotic system, photosynthesis, antioxidant system and stress-related genes in cucumber. [ABSTRACT FROM AUTHOR]

Several studies suggest that transgene expression in lettuce may be unstable, possibly as a result of epigenetic silencing of the cauliflower mosaic virus 35 S RNA promoter (35SCaMV). To solve this problem, the ACT2 promoter from Arabidopsis thaliana (pAtACT2) and the novel SlAVP1 promoter from Solanum lycopersicum (pSlAVP1) were evaluated to express the gus reporter gene in lettuce plants. Three generations of genetically modified plants were evaluated using histochemical and fluorometric GUS assays. The results demonstrated that the GUS activity of plants expressing gus under the control of the pAtACT2 and pSlAVP1 promoters was higher and more stable than that of plants expressing the gus gene under the control of the 35SCaMV promoter. It was observed in different mature and immature tissues and organs, such as seeds, cotyledons, leaves, stems, roots and flowers. This demonstrated that the alternative promoters, pAtACT2 and pSlAVP1, have the potential to direct constitutive expression in lettuce leaf tissues and achieve more stable transgene expression. In addition, the study also explored the reactivation of transgene expression in 35SCaMV plants treated with 5-azacytidine, a compound known to affect DNA methylation and potentially reverse epigenetic silencing. The results indicated that transgene expression in 35SCaMV plants could be reactivated with 5-azacytidine treatment. These results suggest that the alternative promoters pAtACT2 and pSlAVP1 are superior to the conventional 35SCaMV promoter for achieving more stable and elevated levels of transgene expression in lettuce.Key message: Two alternative promoters (from the actin 2 and AVP1 genes) were evaluated for lettuce genetic engineering. Transgenic plants presented stable and consistent transgene expression for at least three generations. [ABSTRACT FROM AUTHOR]

Pepper, which is a widely cultivated important vegetable, is sensitive to salt stress, and the continuous intensification of soil salinization has affected pepper production worldwide. However, genes confer to salt tolerance are rarely been cloned in pepper. Since the REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylase that plays a crucial regulatory role in plants in response to various abiotic stresses, including salt stress. We cloned a ROS1 gene in pepper, named CaROS1 (LOC107843637). Bioinformatic analysis showed that the CaROS1 protein contains the HhH-GPD glycosylase and RRM_DME domains. qRT-PCR analyses showed that the CaROS1 was highly expressed in young and mature fruits of pepper and rapidly induced by salt stress. Functional characterization of the CaROS1 was performed by gene silencing in pepper and overexpressing in tobacco, revealed that the CaROS1 positively regulates salt tolerance ability. More detailly, CaROS1-silenced pepper were more sensitive to salt stress, and their ROS levels, relative conductivity, and malondialdehyde content were significantly higher in leaves than those of the control plants. Besides, CaROS1-overexpressing tobacco plants were more tolerant to salt stress, with a higher relative water content, total chlorophyll content, and antioxidant enzyme activity in leaves compared to those of WT plants during salt stress. These results revealed the CaROS1 dose play a role in salt stress response, providing the theoretical basis for salt tolerance genetic engineering breeding in pepper. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is one of the most aggressive, heterogeneous, and fatal types of human cancer; therefore, more effective therapeutic drugs are urgently needed. Human epidermal growth factor receptor 2 (HER2) overexpression and amplification have been identified as a cornerstone in this pathology. The aim of this review is to identify HER2 membrane overexpression in relation to pancreatic cancer pathways that can be used in order to develop a targeted therapy. After searching the keywords, 174 articles were found during a time span of 10 years, between 2013 and 2023, but only twelve scientific papers were qualified for this investigation. The new era of biomolecular research found a significant relationship between HER2 overexpression and pancreatic cancer cells in 25–30% of cases. The variables are dependent on tumor-derived cells, with differences in receptor overexpression between PDAC (pancreatic ductal adenocarcinoma), BTC (biliary tract cancer), ampullary carcinoma, and PNETs (pancreatic neuroendocrine tumors). HER2 overexpression is frequently encountered in human pancreatic carcinoma cell lines, and the ERBB family is one of the targets in the near future of therapy, with good results in phase I, II, and III studies evaluating downregulation and tumor downstaging, respectively. [ABSTRACT FROM AUTHOR]

Fucosyltransferases (Fut) regulate the fucosylation process associated with tumorogenesis in different cancer types. Ascitic fluid (AF) from patients diagnosed with advanced stage of epithelial ovarian cancer (EOC) is considered as a dynamic tumor microenvironment associated with poor prognosis. Previous studies from our laboratory showed increased fucosylation in SKOV‐3 and OVCAR‐3, cancer‐derived cell lines, when these cells were incubated with AFs derived from patients diagnosed with EOC. In the present work we studied three fucosyltransferases (Fut 2, Fut 4, and Fut 8) in SKOV‐3, OVCAR‐3 and CAOV‐3 cell lines in combination with five different AFs from patients diagnosed with this disease, confirming that all tested AFs increased fucosylation. Then, we demonstrate that mRNAs of these three enzymes were overexpressed in the three cell lines under treatment with AFs. SKOV‐3 showed the higher overexpression of Fut 2, Fut 4, and Fut 8 in comparison with the control condition. We further confirmed, in the SKOV‐3 cell line, by endpoint PCR, WB, and confocal microscopy, that the three enzymes were overexpressed, being Fut 4 the most overexpressed enzyme compared to Fut 2 and Fut 8. These enzymes were concentrated in vesicular structures with a homogeneous distribution pattern throughout the cytoplasm. Moreover, we found that among the three enzymes, only Fut 4 was located inside the nuclei. The nuclear location of Fut 4 was confirmed for the three cell lines. These results allow to propose Fut 2, Fut 4, and Fut 8 as potential targets for EOC treatment or as diagnostic tools for this disease. [ABSTRACT FROM AUTHOR]

The resilience of plants to concurrent abiotic stresses—such as drought, salinity, extreme temperatures, heavy metals, and elevated CO2 levels—is paramount in the era of climate change. Reactive oxygen species (ROS), traditionally perceived as mere byproducts of metabolic processes, serve a dual role: as crucial signaling molecules that facilitate plant adaptation and as deleterious agents causing cellular damage when excessively accumulated. In this review, we highlighted the intricate equilibrium that plants maintain through both enzymatic and non-enzymatic antioxidant defenses to mitigate ROS-mediated oxidative stress, emphasizing the sophisticated strategies plants deploy to counteract a spectrum of combined abiotic stresses. Some plant species, however, exhibit insufficient enhancement of their intrinsic antioxidant defenses to counterbalance stress-induced ROS accumulation and consequent oxidative damage. Consequently, we explored the pivotal role of diverse signaling molecules in further strengthening antioxidant defenses, offering profound insights into bolstering plant resilience. Furthermore, the advent of genetic engineering technologies unveils novel avenues for crop improvement, with the strategic overexpression of antioxidant genes such as SOD, APX, CAT, GPX, DHAR, GR, and GST showing immense potential in fortifying plants against oxidative challenges imposed by multiple abiotic stresses. Future perspectives entail deepening our understanding of the molecular mechanisms governing ROS generation and scavenging, investigating the synergistic effects of co-expressing antioxidant genes, and elucidating the interactions between endogenous plant hormones and exogenously applied signaling molecules. We advocate for integrative research methodologies, combining field experiments, controlled environmental studies, and computational modeling, to bridge the gap between laboratory discoveries and practical agricultural applications.

High glycogen in oysters contributes to their flavor, quality, and hardiness, making it a crucial target trait for breeding purposes. Until now, the upstream regulatory mechanism for glycogen synthesis has remained unclear. Protein phosphatase-1 regulatory subunit 3 (PPP1R3) is a member of a family that directs the protein phosphatase-1 (PP1) to glycogen, resulting in the activation of glycogen synthase (GS) and the inactivation of glycogen phosphorylase (GP). However, their functions in molluscan species have remained unclear until now. In our study, we verified the function of the PP1α/PPP1R3D pathways in the glycogen metabolism process in Crassostrea gigas. Initially, we conducted a phylogenetic analysis and identified two PP1 and PPP1R3 members in molluscan species, emphasizing copy number variations in eukaryotic animals. Additionally, we identified distinct substitutions of conserved motifs specific to bivalves, Cephalopoda, or Gastropods, revealing functional diversity in PP1 and PPP1R3. Our primary focus was on PP1α/PPP1R3D in the oyster C.gigas, detected in the muscle glycogen fraction of bivalve species, with mRNA expression showing a positive correlation with glycogen content. By manipulating CgPPP1R3D expression in oysters through adenovirus-mediated overexpression or siRNA-mediated downregulation, we established a direct positive correlation between CgPPP1R3D expression levels and glycogen content. Colocation analysis confirmed protein interactions between CgPP1α and CgPPP1R3D. Furthermore, to elucidate the correlation between gene expression and glycogen accumulation, we conducted an association analysis and identified a cluster of significant SNP loci upstream of CgPPP1R3D significantly related to glycogen content. The high polymorphism observed may be linked to differential expressions of CgPPP1R3D in oyster individuals with high and low glycogen levels. These findings serve as a crucial reference for enhancing glycogen traits in molluscan species.