Search Results (33)

The development of novel antimicrobial peptides (AMPs) with broad-spectrum activity represents a promising strategy to overcome multidrug resistance in pathogenic bacteria. In this study, we investigated the antimicrobial activity of three designed peptides—R44K^S*, V31K^S*, and R23F^S*—engineered to incorporate an amyloidogenic fragment from the S1 protein of Staphylococcus aureus and one or two cell-penetrating peptide (CPP) fragments to enhance cellular uptake. The antimicrobial efficacy of these peptides and their combinations was assessed against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Bacillus cereus. The results demonstrated that all three peptides exhibited significant antibacterial activity in a concentration-dependent manner, with R44K^S* being the most potent. Peptide combinations, particularly V31K^S*/R23F^S* and R44K^S*/V31K^S*, showed enhanced inhibitory effects and reduced minimum inhibitory concentrations (MICs), suggesting synergistic or additive interactions. Fractional inhibitory concentration index (FICI) analysis confirmed that most combinations exhibited synergy or additive effects. These findings highlight the potential of CPP-modified peptides as antimicrobial agents and underscore the importance of optimizing peptide combinations for therapeutic applications. Full article

As integrated circuit technology continues to advance, clock tree synthesis has become increasingly significant in the design of ultra-large-scale integrated circuits. Traditional clock tree synthesis methods often face challenges such as insufficient computational resources and buffer fan-out limitations when dealing with ultra-large-scale clock trees. To address this issue, this paper proposes an improved clock tree synthesis algorithm called incomplete balanced KSR (IB-KSR). Building upon the KSR algorithm, this proposed algorithm efficiently reduces the consumption of computational resources and constrains the fan-out of each buffer by incorporating incomplete minimum spanning tree (IMST) technology and a clustering strategy grounded in Balanced Split. In experiments, the IB-KSR algorithm was compared with the GSR algorithm. The results indicated that IB-KSR reduced the global skew of the clock tree by 43.4% and decreased the average latency by 34.3%. Furthermore, during program execution, IB-KSR maintained low computational resource consumption. Full article

Kluyveromyces marxianus is an emerging yeast cell host for diverse products, but multiple-gene expression in K. marxianus faces challenges due to limited current knowledge of cis-regulatory elements and insertion loci. Our previous study transferred an alien Saccharomyces cerevisiae chromosome I (R1) into K. marxianus, resulting in the creation of the monochromosomal hybrid yeast KS-R1. All R1 genes were actively transcribed, providing a series of loci with varying transcriptional activities. Here, we explore the use of R1 as a novel platform for stable, multi-gene integration and expression. By deleting three essential K. marxianus genes while complementing their functions with orthologs on R1, we achieved stable propagation of R1 in the absence of selective pressure. We characterized several loci on R1 that exhibit stable transcriptional activities under various conditions. GFP inserted in place of genes at six such loci demonstrated varying expression levels. Strains with GFP at two loci exhibited significantly higher expression than those with GFP at a single locus. Furthermore, we replaced five R1 genes with disulfide bond formation genes from Pichia pastoris at distinct loci, resulting in the active expression of all five genes and significantly enhanced production of heterologous glucoamylases BadGLA and TeGlaA. Our findings demonstrate that alien chromosomes offer a stable and versatile platform for the coordinated expression of multiple heterologous genes, serving as valuable tools for metabolic engineering and synthetic biology. Full article

Conventional biochemical methods for studying cellular signaling cascades have relied on destructive cell disruption. In contrast, the live cell imaging of fluorescent-tagged transfected proteins offers a non-invasive approach to understanding signal transduction events. One strategy involves monitoring the phosphorylation-dependent shuttling of a fluorescent-labeled kinase between the nucleus and cytoplasm using nuclear localization, export signals, or both. In this paper, we introduce a simple method to visualize intracellular signal transduction in live cells by exploring the translocation properties of PKC from the cytoplasm to the membrane. We fused bait protein to PKC, allowing the bait (RFP-labeled) and target (GFP-labeled) proteins to co-translocate from the cytoplasm to the membrane. However, in non-interacting protein pairs, only the bait protein was translocated to the plasma membrane. To verify our approach, we examined the Raf–MEK–ERK signaling cascade (ERK pathway). We successfully visualized direct Raf1/MEK2 interaction and the KSR1-containing ternary complex (Raf1/MEK2/KSR1). However, the interaction between MEK and ERK was dependent on the presence of the KSR1 scaffold protein under our experimental conditions. Full article

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein–protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins. Full article

The kinase suppressor of Ras 2 (KSR2) gene is associated with monogenic obesity, and loss-of-function variants in KSR2 have been identified in individuals with severe early-onset obesity. This study investigated KSR2 variants in 9 pediatric patients with severe early-onset obesity in Qatar using whole genome sequencing among a cohort of 240 individuals. We focused on KSR2 variants with a minor allele frequency (MAF) below 1% and a Combined Annotation Dependent Depletion (CADD) score above 13 to identify potential causative variants. Our analysis identified four KSR2 variants: one intronic (c.1765-8G>A) and three missense variants (c.1057G>A, c.1673G>A, and c.923T>C) in nine patients. The intronic variant c.1765-8G>A was the most frequent (seen in six individuals) and had a CADD score of 21.10, suggesting possible pathogenicity. This variant showed a significantly higher allele frequency in the Qatari population compared to the Genome Aggregation Database (gnomAD), indicating a possible founder effect. Molecular modeling of the missense variants revealed structural changes in the protein structure. The study concludes that these four KSR2 variants are associated with monogenic obesity, with an autosomal dominant inheritance pattern. The c.1765-8G>A variant’s prevalence in Qatar underscores its importance in genetic screening for severe obesity. This research advances the understanding of genetic factors in severe early-onset obesity and may inform better management strategies. Full article

Cotton is highly sensitive to potassium, and Xinjiang, China’s leading cotton-producing region, faces a severe challenge due to reduced soil potassium availability. Biofertilizers, particularly potassium-solubilizing rhizobacteria (KSR), convert insoluble potassium into plant-usable forms, offering a sustainable solution for evergreen agriculture. This study isolated and characterized KSR from cotton, elucidated their potassium solubilization mechanisms, and evaluated the effects of inoculating KSR strains on cotton seedlings. Twenty-three KSR strains were isolated from cotton rhizosphere soil using modified Aleksandrov medium. Their solubilizing capacities were assessed in a liquid medium. Strain A10 exhibited the highest potassium solubilization capacity (21.8 ppm) by secreting organic acids such as lactic, citric, acetic, and succinic acid, lowering the pH and facilitating potassium release. A growth curve analysis and potassium solubilization tests of A10 under alkali stress showed its vigorous growth and maintained solubilization ability at pH 8–9, with significant inhibition at pH 10. Furthermore, 16S rRNA sequencing identified strain A10 as Pseudomonas aeruginosa. Greenhouse pot experiments showed that inoculating cotton plants with strain A10 significantly increased plant height and promoted root growth. This inoculation also enhanced dry biomass accumulation in both the aerial parts and root systems of the plants, while reducing the root–shoot ratio. These results suggest that Pseudomonas aeruginosa A10 has potential as a biofertilizer, offering a new strategy for sustainable agriculture. Full article

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells. Full article

In vitro spermatogenesis (IVS) has important applications including fertility preservation of prepubertal cancer patients; however, thus far, IVS has only been achieved using mouse models. To study the effects of growth factors on the maintenance of testicular tissue integrity, germ cell numbers, and potential induction of IVS using a porcine model, we cultured small testicular fragments (~2 mg) from 1-wk-old piglets under six different media conditions (DMEM + 10%KSR alone or supplemented with GDNF, bFGF, SCF, EGF, or a combination of all) for 8 weeks. Overall, tissues supplemented with GDNF and bFGF had the greatest seminiferous tubule integrity and least number of apoptotic cells. GDNF-supplemented tissues had the greatest number of gonocytes per tubule, followed by bFGF-supplemented tissues. There was evidence of gradual Sertoli cell maturation in all groups. Moreover, histological examination and the expression of c-KIT (a marker of differentiating spermatogonia and spermatocytes) and STRA8 (a marker of the pre/meiotic stage germ cells) confirmed the induction of IVS in all groups. However, GDNF- and bFGF-supplemented tissue cultures had greater numbers of seminiferous tubules with spermatocytes compared to other groups. In conclusion, overall, GDNF and bFGF supplementation better maintained the tissue integrity and gonocyte numbers and induced IVS in cultured testicular tissues. Full article

Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation. Full article

Silver nanoparticles have gained considerable interest in recent decades due to their antimicrobial activity and are used in water disinfection, wound healing, food packaging, and plant protection. This study tested the potential of silver nanoparticles synthesized using the neem (Azadirachta indica) leaf extract against Alternaria solani causes early blight disease in tomato plants. The pathogen was isolated from infected tomato plants and identified using morphological and molecular features. The results showed significant variation among isolates. Isolates, Shk-1 and Ksr-1 were highly pathogenic, causing up to 80% disease incidence. The potential of silver nanoparticles against each isolate was determined using different concentrations of silver nanoparticles. During in vitro and in vivo experiments, the growth inhibition rate of the pathogen was 70–100% at 50 ppm. Lower concentrations of silver nanoparticles (5 and 10 ppm) increased phenolics, PO, PPO, and PAL production by more than 50% as compared to the untreated control. These defensive mechanisms clearly demonstrate the fungicidal potential of AgNPs and recommend their utilization in different crop protection programs. Full article

It is difficult to evaluate the pre-symptomatic state of mental disorders and prevent its onset. Since stress could be a trigger of mental disorders, it may be helpful to identify stress-responsive biomarkers (stress markers) for the evaluation of stress levels. We have so far performed omics analyses of the rat brain and peripheral blood after various kinds of stress and have found numerous factors that respond to stress. In this study, we investigated the effects of relatively moderate stress on these factors in the rat to identify stress marker candidates. Adult male Wistar rats underwent water immersion stress for 12 h, 24 h, or 48 h. Stress caused weight loss and elevated serum corticosterone levels, and alterations regarded as anxiety and/or fear-like behaviors. Reverse-transcription PCR and Western blot analyses revealed significant alterations in the expressions of hippocampal genes and proteins by the stress for no longer than 24 h, such as mitogen-activated protein kinase phosphatase 1 (MKP-1), CCAAT/enhancer-binding protein delta (CEBPD), small ubiquitin-like modifier proteins 1/sentrin-specific peptidase 5 (SENP5), matrix metalloproteinase-8 (MMP-8), kinase suppressor of Ras 1 (KSR1), and MKP-1, MMP-8, nerve growth factor receptor (NGFR). Similar alterations were observed in three genes (MKP-1, CEBPD, MMP-8) in the peripheral blood. The present results strongly suggest that these factors may serve as stress markers. The correlation of these factors in the blood and brain may enable the evaluation of stress-induced changes in the brain by blood analysis, which will contribute to preventing the onset of mental disorders. Full article

Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum—FBS—or knockout serum replacement—KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks. Full article

In nematode Caenorhabditis elegans, exposure to polystyrene nanoparticles (PS-NPs) at predicted environmental concentrations can cause induction of transgenerational toxicity. However, the underlying mechanisms for toxicity formation of PS-NP in the offspring remain largely unknown. In this study, based on high-throughput sequencing, Ephrin ligand EFN-3 was identified as a target of KSR-1/2 (two kinase suppressors of Ras) in the germline during the control of transgenerational PS-NP toxicity. At parental generation (P0-G), exposure to 0.1–10 μg/L PS-NP caused the increase in expression of germline efn-3, and this increase in germline efn-3 expression could be further detected in the offspring, such as F1-G and F2-G. Germline RNAi of efn-3 caused a resistance to transgenerational PS-NP toxicity, suggesting that the activation of germline EFN-3 at P0-G mediated transgenerational PS-NP toxicity. In the offspring, Ephrin receptor VAB-1 was further activated by the increased EFN-3 caused by PS-NP exposure at P0-G, and RNAi of vab-1 also resulted in resistance to transgenerational PS-NP toxicity. VAB-1 acted in both the neurons and the germline to control toxicity of PS-NP in the offspring. In the neurons, VAB-1 regulated PS-NP toxicity by suppressing expressions of DBL-1, JNK-1, MPK-1, and GLB-10. In the germline, VAB-1 regulated PS-NP toxicity by increasing NDK-1 and LIN-23 expressions and decreasing EGL-1 expression. Therefore, germline Ephrin ligand EFN-3 and its receptor VAB-1 acted together to mediate the formation of transgenerational PS-NP toxicity. Our data highlight the important role of activation in germline Ephrin signals in mediating transgenerational toxicity of nanoplastics at predicted environmental concentrations in organisms. Full article

This study utilizes LA-ICP-MS-determined minor and trace element contents of megacrystic blocky K-feldspar to reveal the chemical variability and fractionation degree of albite-spodumene and barren feldspar pegmatites of the Kolmozero lithium deposit in the Kola region, Russia. K-feldspar from albite-spodumene pegmatite is represented by two generations: early microcline-I and late microcline-II. Rb, Cs, Li, and Tl are the most typical impurity elements in K-feldspar that replace K in its crystal lattice. Microcline-II differs from microcline-I: (i) relatively high contents of Rb (6520 and 4490 ppm, respectively), Cs (146 and 91 ppm), and Li (86 and 68 ppm), Tl (34 and 28 ppm); and (ii) low contents of Ba (13 and 29 ppm), Sr (8 and 24 ppm), and Pb (14 and 26 ppm). K-feldspar from feldspar pegmatites of the Kolmozero pegmatite field differs from those in the Kolmozero Li deposit in (i) low contents of Rb, Cs, Li, Tl, and an orthoclase component; and (ii) high contents of Sr, Ba, Pb, and an albite component. K/Sr, K/Ba, Rb/Ba, and Rb/Sr element ratios increase, while K/Rb, K/Cs, K/Tl, and K/Li element ratios decrease in K-feldspar, from feldspar pegmatites to albite-spodumene pegmatites. These trends reflect different fractionation degrees of pegmatite evolution. The implications of the detected trace element variations in K-feldspar are discussed in respect of tracing the rare element enrichments in pegmatite systems. A model is proposed for the formation of the Kolmozero pegmatites by differentiation from a hypothetical parental granite, rather than by anatexis of the host rock. Full article