Breast cancer is a leading cause of mortality and morbidity among females worldwide. As part of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2023, we provided an updated comprehensive assessment of the epidemiological trends, disease burden, and risk factors associated with breast cancer globally, regionally, and nationally from 1990 to 2023. Breast cancer incidence, mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs) were estimated by age and sex for 204 countries and territories from 1990 to 2023. Mortality estimates were generated using GBD Cause of Death Ensemble models, leveraging data from population-based cancer registration systems, vital registration systems, and verbal autopsies. Mortality-to-incidence ratios were calculated to derive both mortality and incidence estimates. Prevalence was calculated by combining incidence and modelled survival estimates. YLLs were established by multiplying age-specific deaths with the GBD standard life expectancy at the age of death. YLDs were estimated by applying disability weights to prevalence estimates. The sum of YLLs and YLDs equalled the number of DALYs. Breast cancer burden attributable to seven risk factors was examined through the comparative risk assessment framework. The GBD forecasting framework was used to forecast breast cancer incidence and mortality from 2024 to 2050. Age-standardised rates were calculated for each metric using the GBD 2023 world standard population. In 2023, there were an estimated 2·30 million (95% uncertainty interval [UI] 2·01 to 2·61) breast cancer incident cases, 764 000 deaths (672 000 to 854 000), and 24·1 million (21·3 to 27·5) DALYs among females globally. In the World Bank low-income group, where a low age-standardised incidence rate (ASIR) was estimated (44·2 per 100 000 person-years [31·2 to 58·4]), the age-standardised mortality rate (ASMR) was the highest (24·1 per 100 000 [16·8 to 31·9]). The highest ASIR was in the high-income group (75·7 per 100 000 [67·1 to 84·0]), and the lowest ASMR was in the upper-middle-income group (11·2 per 100 000 [10·2 to 12·3]). Between 1990 and 2023, the ASIR in the low-income group increased by 147·2% (38·1 to 271·7), compared with a 1·2% (-11·5 to 17·2) change in the high-income group. The ASMR decreased in the high-income group, changing by -29·9% (-33·6 to -25·9), but increased by 99·3% (12·5 to 202·9) in the low-income group. The increase in age-standardised DALY rates followed that of ASMRs. Risk factors such as dietary risks, tobacco use, and high fasting plasma glucose contributed to 28·3% (16·6 to 38·9) of breast cancer DALYs in 2023. The risk factors with a decrease in attributable DALYs between 1990 and 2023 were high alcohol use and tobacco. By 2050, the global incident cases of breast cancer among females were forecast to reach 3·56 million (2·29 to 4·83), with 1·37 million (0·841 to 2·02) deaths. The stable incidence and declining mortality rates of female breast cancer in high-income nations reflect success in screening, diagnosis, and treatment. In contrast, the concurrent rise in incidence and mortality in other regions signals health system deficits. Without effective interventions, many countries will fall short of the WHO Global Breast Cancer Initiative's ambitious target of achieving an annual reduction of 2·5% in age-standardised mortality rates by 2040. The mounting breast cancer burden, disproportionately affecting some of the world's most vulnerable populations, will further exacerbate health inequalities across the globe without decisive immediate action. Gates Foundation, St Jude Children's Research Hospital.
Chicken primordial germ cells (cPGCs) hold great potential for genetic modification and germ cell research in chickens. In this study, we evaluated the cellular characteristics of three cPGC lines: cPGC-1, cPGC-2, and cPGC-3. cPGC-1 and cPGC-2 were derived from male chickens, whereas cPGC-3 was derived from a female chicken. We analyzed and compared cell proliferation rates, marker gene expression, and gonadal colonization abilities. Three different cell culture temperatures were assessed (37 °C, 39 °C, and 41 °C) and proliferation rates were highest for all cPGC lines at 39 °C. Additionally, cPGC-1 demonstrated a higher proliferation rate than cPGC-2. No significant differences were observed between cPGC-1 and cPGC-2 with regard to the expression of germ cell and pluripotency marker genes (Cvh, Dazl, Pou5f3, and Nanog). To assess changes in cellular characteristics before and after genetic modification, we performed a green fluorescent protein (GFP) gene knock-in using the CRISPR/Cas9 system, followed by site-specific integration of the scFv-Fc gene using the Cre-loxP system. Transplantation experiments revealed that cPGC-2/GFP exhibited higher gonadal colonization efficiency than cPGC-1/GFP. This study demonstrates differences in cellular characteristics among established cPGC lines and highlights the impact of genetic modification on cPGC function. Our findings emphasize the importance of selecting appropriate cell lines and optimizing culture conditions based on cPGC traits to achieve efficient and reproducible production of transgenic chickens. These insights will aid in the conservation of poultry genetic resources and the advancement of transgenic chicken production for both research and industrial applications.
Recently, sake aged for several years to decades, called long-term aged sake, has attracted some attention. Aged liquors also exist in other countries, such as Chinese rice wine (Huangjiu) and fortified wines (Sherry, Madeira, Port). To understand the characteristics of long-term aged sake among the typical aged liquors worldwide, we analyzed and compared the flavor components and sensory characteristics of aged sake, Huangjiu, and fortified wines. Accordingly, flavor component analysis revealed a common trend toward increases in sotolon, aldehydes, polysulfides, ethyl esters, and decreases in acetate esters with age. However, the increases in aldehydes and polysulfides were more significant in sake and Huangjiu, whereas the increases in ethyl esters were more significant in fortified wines. Moreover, the concentrations and variances of acetate esters in sake were highest among the five liquor categories. Sensory evaluation revealed that sake and Huangjiu presented stronger sulfur aroma and bitter taste, whereas fortified wines presented stronger dried fruit, honey aroma and sweet taste. Furthermore, sake samples showed a larger variance in the intensity of caramel, burnt, and soy sauce aroma than did the other liquor categories. These results indicate that the compositional and sensory characteristics of the aged liquors can be grouped into sake/Huangjiu and fortified wines and that sake is featured by extensive variations in the levels of age-related components and sensory attributes.
Tobacco fermentation is a crucial process for improving tobacco quality by reducing undesirable substances and enhancing aroma characteristics. In this study, an enzyme-microbe combined fermentation strategy was established and evaluated. Cytobacillus oceanisediminis C4 combined with amylase pretreatment was applied in the fermentation of tobacco powder (TP) to improve the quality of tobacco, yielding an enhanced sensory score of 84.50, compared with 82.00 in the untreated TP. Compared with the control, the reducing sugar content increased significantly, whereas starch and protein contents decreased markedly after combined fermentation. Furthermore, total aromatic components of TP increased by 34.77 %, along with noticeable changes in TP surface structure. Bacterial community structure analysis revealed a noteworthy shift at the genus level, with the relative abundance of Bacillus and Pseudomonas escalating from 0.30 % and 1.15 % to 74.67 % and 4.40 %, respectively. Additionally, fungal community structure analysis revealed that the relative abundance of Monascus surged from 1.69 % to 87.59 %. Metabolomic analysis demonstrated that fermentation reprogrammed the metabolic profile toward flavor-active and aroma-precursor compounds, characterized by increased levels of amino acids and phenolic acids and decreased levels of lipids and flavonoids. These coordinated changes contributed to enhanced aroma, reduced irritation, and overall improvement in tobacco quality. Overall, this study establishes an effective enzyme-microbe combined fermentation strategy, demonstrates its applicability for improving heat-not-burn (HnB) tobacco products, and provides multi-omics insights into the underlying mechanisms of quality enhancement.
Strawberry cultivars have recently been diversified, and breeding objectives generally focus on production characteristics, as well as those to attract consumers. Strawberry cultivar selection and breeding rely heavily on human sensory evaluation. However, methods relying solely on sensory evaluation face challenges, such as being influenced by the subjectivity and physical condition of the panelists. Therefore, if compounds correlated with sensory profiles can be identified through instrumental analysis, combining this with sensory evaluation could enable more robust evaluation and improve the selection of new strawberry cultivars. Previous studies have investigated the relationship between strawberry metabolites and sensory profiles but have mainly focused on volatiles, certain sugars, and organic acids. However, some non-volatile compounds also influence flavor. Therefore, investigation of the correlations between non-volatile compounds and sensory profiles based on comprehensive analytical data is valuable. The objective of this study was to investigate the compounds correlated with the sensory profiles of strawberries. Sensory evaluations and gas chromatography/mass spectrometry (GC/MS)-based compound analyses were performed on five strawberry cultivars. Orthogonal partial least squares (OPLS) regression analysis was then performed using the compound data as explanatory variables and the sensory evaluation scores as response variables. Models capable of accurately predicting sensory profiles were constructed, and several non-volatile and volatile compounds showing high correlations were identified. This study confirmed the correlations between the sensory profiles of strawberries and volatile as well as non-volatile compounds, including amino and organic acids, providing useful insights into the breeding of new strawberry cultivars.
Microbial fermentation is widely used in the production of food, pharmaceuticals, and bioenergy. Proper monitoring of the fermentation process is essential to ensure consistent product quality and yield. Although the indicators of fermentation progress vary among systems, they are generally evaluated by quantifying the major metabolites in the fermentation broth. However, these measurements rely on offline analyses involving time-consuming sampling and pretreatment, which hinder the real-time detection of process deviations. In this study, volatile organic compounds (VOCs) in the fermentation gas during beer fermentation were analyzed to develop a non-invasive method for the prediction of the current state of sugar, organic acid, and ethanol concentrations in the fermentation broth at each sampling point. VOCs emitted during fermentation serve as valuable indicators reflecting the metabolic state of the yeast. Beer brewing was adopted as a model system to validate VOC monitoring, because it involves sequential sugar consumption, organic acid and ethanol formation, and abundant VOC generation. We constructed multivariate regression models using the VOC profiles obtained from gas chromatography-mass spectrometry (GC-MS) analysis using Tenax TA. Parallel sampling of the fermentation gas and broth followed by orthogonal partial least squares (OPLS) regression yielded highly accurate models for predicting key fermentation indicators at corresponding time points. Furans and aldehydes abundant at early stages showed inverse correlations with higher alcohols and esters produced later, indicating distinct metabolic transitions. This study demonstrated the feasibility of applying VOC profiles for the quantitative prediction of the current fermentation progress and highlights the potential of this approach as a novel non-invasive monitoring method linking aroma chemistry and process control.
Phthalate esters, widely used as plasticizers in plastic manufacturing, are known for their endocrine-disrupting effects. γ-Oryzanol derivatives, functional lipids composed of sterol and ferulic acid esters, possess diverse biological activities. In this study, we cloned and heterologously expressed a lipase-encoding gene (lipO745) from Aspergillus oryzae RIB40, deleting a 23-amino-acid N-terminal signal peptide, in Pichia pastoris using the pPICZαC vector. The resulting recombinant enzyme (rLipO745Δ23) was successfully secreted as an active extracellular protein. The purified recombinant lipase exhibited an apparent molecular mass of approximately 65 kDa, as determined using SDS-PAGE. Substrate specificity assays using p-nitrophenyl (pNP) esters (C2-C16) revealed that pNP butyrate (pNP-C4) was hydrolyzed most efficiently, with a specific activity of 369.2 ± 6.6 nmol·mL-1 mg-1. rLipO745Δ23 catalyzed the conversion of dibutyl phthalate (DBP) to monobutyl phthalate (MBP) without further degradation to phthalic acid, and exhibited an activity of 7.24 ± 0.61 nmol·mL-1 mg-1 toward DBP. The kinetic parameters (Km and kcat) were 0.66 ± 0.1 mM and 37.8 ± 6.4 s-1, respectively, for pNP-C4 substrate, and 0.40 ± 0.05 mM and 1.30 ± 0.23 s-1, respectively, for DBP. Moreover, rLipO745Δ23 showed detectable hydrolytic activity toward γ-oryzanol, including cycloartenyl ferulate, a conjugate of ferulic acid and triterpene alcohol. Notably, β-sitosterol, a major hydrolysate of phytosterol type γ-oryzanol, did not inhibit enzymatic activity. These findings highlight the potential of rLipO745Δ23 as a versatile biocatalyst for the enzymatic degradation of phthalates and γ-oryzanol derivatives in environmental and industrial applications.
The world's increasing demand for petrochemical, pharmaceutical, and nutraceutical products necessitates the development of new strategies for producing these high-value chemicals. The depletion of the natural fossil reserves and environmental pollution associated with their procurement further compel us to find sustainable, greener, and cost-effective alternatives. In light of this, a shift has been witnessed in deriving these products from fossil-based sources or chemical synthesis to biomanufacturing (production using living systems). However, to fully utilize the potential of biomanufacturing, novel tools and strategies that can function in bacteria, archaea, and eukaryotes, regulate multi-enzyme pathways, offer precise and conditional gene regulation, and possess versatility are highly required. This review presents a comprehensive summary of the latest gene modulation tools and strategies used by metabolic engineers, along with a mechanistic overview and their applications. In addition, we presented the tools that have the potential to be used for pathway optimization but are still less explored. Within this context, we categorized these tools based on their molecular level of gene regulation, i.e., at and beyond the central dogma. We believe that a deeper understanding of the design, development, and application of these tools would be beneficial for metabolic engineers to reprogram biosynthetic pathways by adopting system-specific approaches, as a single strategy cannot be applied to all systems. Lastly, we discussed the challenges and future prospects of developing these gene regulatory tools to further advance the biomanufacturing field.
The correction (editing) of mutated genes at the DNA level is expected to cure gene-inherited diseases and cancers. A 5'-tailed duplex (TD) with an approximately 80-base editor strand (E-strand) plus a 35-base assistant strand (A-strand) was developed for gene editing without artificial nucleases. The E-strand has the normal (or desired) sequence and the A-strand hybridizes to the 3'-region of the E-strand. In this study, the polarity-dependency of gene editing by TDs was examined. The sense and antisense E-strands for eight transcribed target genes, including the WRN (Werner syndrome) gene, were designed, and the target plasmid DNAs were co-transfected with the TDs into human U2OS cells. Most TDs with the antisense E-strand corrected the targets more efficiently than those with the sense E-strand. However, transcription had only a slight effect on gene correction efficiency. These results suggested that the TDs containing the antisense E-strand are more useful editing tools than the sense TDs.
The development of conventional antibiotics is being severely challenged by the rise of bacterial resistance and the obstacle of biofilm-associated infections. Given their enhanced permeability, nano micelles have emerged as a widely utilized platform for the delivery of hydrophobic drugs. In this study, a novel material named BS-12-PEG-OH (PB12) was synthesized, and an intelligent nanoplatform was successfully developed through thin film dispersion method by co assembling vitamin E polyethylene glycol succinate (TPGS) with PB12 into hybrid micelles via thin film dispersion method, thereby enabling the encapsulation of Honokiol (designated as HK@PB12/TPGS Ms). The hybrid micelle can effectively penetrate the extracellular polymeric substance barrier of the biofilm. Upon reaching the acidic microenvironment, it responsively releases dodecyl dimethyl betaine (BS-12) and honokiol (HK), thus eliminating the biofilm structure and killing the embedded bacteria. Under acidic conditions, HK@PB12/TPGS Ms achieved a substantial bacterial reduction of 9.60 log10 CFU/ml. Furthermore, under the acidic condition of the biofilm microenvironment, they effectively cleared 83.32 % of the biofilm and reduced the embedded S. aureus by 3.75 log10 CFU/ml. The results indicate that the HK@PB12/TPGS Ms could effectively penetrate Staphylococcus aureus biofilms, disrupt their structure, and eliminate the bacteria inside the biofilm, hence preventing new infections and providing a novel therapeutic strategy for combating stubborn biofilm-associated infections.
Sub-Saharan Africa (SSA) is experiencing an epidemiological transition where non-communicable diseases are becoming the leading cause of disability and mortality alongside infectious diseases such as HIV/AIDS. Multimorbidity, the coexistence of two or more long-term conditions, is increasing in SSA. However, the cost of managing multimorbidity is largely unknown. This study aimed to estimate the economic cost of public outpatient primary care for adults with multimorbidity (HIV, hypertension, and/or diabetes, and their associated conditions: cardiovascular disease, and TB) in rural South Africa. This study used a cross-sectional, retrospective cost-of-illness approach to estimate the direct and indirect costs of multimorbidity management in Bushbuckridge, Mpumalanga, in 2022. Data were synthesized from patient-level data from eight public primary healthcare facilities within the Agincourt study site-a rapidly transitioning rural South African setting. Additionally, government reports and an existing study on transport costs and productivity losses conducted within the Agincourt study site were used to estimate the costs of managing patients in the primary care facilities. Results showed that patients with multimorbidity had higher average economic costs per patient compared to those with single conditions. Overall, patients with multimorbidity increase costs above the baseline of a patient with a single condition (R4 900/annum) by between 42% and 83%. Patients with multimorbidity also incur slightly higher costs associated with accessing primary care services compared to those with a single condition. However, our model shows that the additive cost of managing multiple conditions in separate consultations is higher than managing all conditions in one visit. This shows that managing patients within an integrated care model seems to have a cost-limiting effect. However, treatment guidelines for managing multimorbidity in South Africa should be developed to ensure standardized care.
A robust heterologous expression system was developed in Pseudomonas putida KT2440 for the production of recombinant xanthine oxidase (XOD) from Cellulosimicrobium sp. TH20. To alleviate metabolic burden and enable inducer-free expression, a constitutive expression plasmid, pPegP119, was constructed by replacement of the pUCP18 origin with the low-copy-number pSC101 origin. Evaluation of the expression products from two gene clusters indicated that the complete xodCBA cluster yielded significantly higher activity (CsXodCBA, 5.6 U/mL) compared to the truncated xodBA cluster (CsXodBA, 2.1 U/mL), directly supporting the role of XodC in enzyme maturation. This role was further substantiated biochemically: purified CsXodCBA was found to contain approximately 2.6 times more molybdenum than CsXodBA and exhibited a substantially higher specific activity (28.1 U/mg versus 15.3 U/mg). Despite the genetic differences, the final assembled forms of CsXodCBA and CsXodBA were identical, consisting solely of the XODA (106 kDa) and XODB (30 kDa) subunits. Biochemical characterization of purified CsXodCBA demonstrated optimal activity at pH 7.0 and 50 °C, with kinetic parameters of Km = 85 ± 4 μM and Vmax = 20.6 ± 1.3 μM min-1. Enzyme activity was potently inhibited by Cu2+, Hg2+, and Zn2+. Thus, an efficient and cost-effective platform for the production of active XOD has been established through this work.
Methylotrophic yeasts are promising hosts for bioproduction from methanol, but their growth is significantly reduced in the presence of methanol. In this study, we investigated metabolic bottlenecks in Komagataella phaffii, Ogataea polymorpha, and Candida boidinii during methanol assimilation through comparative metabolome analysis. We found a common decrease in α-ketoglutarate-derived amino acids and tricarboxylic acid (TCA) cycle intermediates in all three species during methanol assimilation, suggesting a metabolic bottleneck around the TCA cycle entry. Additionally, K. phaffii and O. polymorpha accumulate trehalose in the presence of methanol. ΔΔG analysis suggested reduced activity of pyruvate kinase and TCA cycle entry enzymes as potential rate-limiting steps. Biosynthesis of TCA cycle-derived amino acids, including Glu, Arg, and Pro, is a limiting factor because supplementation with these amino acids significantly enhanced growth rates and final cell density for all three species. Furthermore, disruption of the trehalose biosynthesis gene (TPS2) in K. phaffii further improved growth, indicating that trehalose accumulation negatively affects methanol-based growth. Amino acid supplementation with TPS2 disruption in K. phaffii resulted in a 1.2-fold increase in the specific growth rate and a 2.73-fold increase in final cell density. These findings provide insights into the metabolic limitations of methylotrophic yeast growth in the presence of methanol and offer strategies for improving bioproduction efficiency.
Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are membrane-bound vesicles secreted by cells. They play essential roles in intercellular communications and are involved in numerous physiological processes. Given their functional importance, EVs have emerged as promising tools for diagnosing and treating various diseases. In this study, we focused on the utility of EVs and explored their application in the analysis of contact-dependent cell-cell interactions, which are essential for the control of cell differentiation and induction of immune responses. Although several methods have been developed to evaluate these interactions, they often require complex procedures and advanced optimization, limiting their broad applicability. To overcome these limitations, we developed a novel method utilizing EVs to present membrane proteins in their native conformations. Our strategy involved producing fluorescently labeled EVs with target antigens and quantitatively assessing their binding to target cells via flow cytometry. Using fluorescently labeled EVs presenting with either an N-terminal pro-brain natriuretic peptide or interleukin-2 receptor, we successfully detected specific interactions with corresponding hybridoma B cell receptors. This simpler method requires no advanced optimization and effectively analyzes cell-cell interactions under physiological conditions in a high-throughput and quantitative manner. Our findings highlight the potential of this EV-based system as a valuable tool for studying membrane protein-mediated cell-cell interactions in bioscience research.
Sago is a high-carbohydrate, naturally gluten-free product derived from tropical palm trees, serving as an essential staple food in many regions of Southeast Asia and the Pacific Islands. The various processing methods used to produce sago starch can influence its overall quality, including its flavor. To date, a comprehensive analysis of its flavor quality across different processing methods has not yet been conducted. This study aimed to characterize the flavor quality of sago starch by combining physicochemical, metabolomic, and sensory analyses. Sago starch samples produced using traditional, semi-mechanized, and modern methods were collected and analyzed. Principal component and heatmap analyses revealed that traditional processing resulted in lower sensory and physicochemical quality, characterized by higher off-flavor compounds, particularly organic acids, likely due to uncontrolled microbial activity. In contrast, modern processing yielded higher levels of sugars such as sucrose and fructose, associated with desirable flavor, while semi-mechanized processing produced intermediate flavor profiles, possibly due to partial fermentation. Partial least squares regression analysis identified potential key metabolites related to flavor deterioration in sago starch, including octanoic acid, 3-methylbutyric acid, and hexanal. These findings can support improvements in sago starch processing to enhance flavor quality and guide quality control strategies in the industry.
Sake yeast Kyokai no. 11 (K11) is an ethanol-tolerant mutant of Kyokai no. 7 (K7) and produces a higher ethanol concentration in the sake mash than K7. A previous study revealed that stress-induced genes under the control of STRE elements were upregulated in K11. To elucidate the causal mutation responsible for ethanol tolerance, we compared the genome sequences of the ethanol-tolerant mutants (K11 and K7AT2, a newly isolated ethanol-tolerant mutant of K7) with that of their parental strain, K7. We identified a shared loss of heterozygosity region in the left arm of chromosome X in both mutants. We focused on CYR1 in this region, as it encodes adenylate cyclase, which negatively regulates expression of STRE-regulated genes through the upregulation of protein kinase A. Nucleotide 2066 of CYR1 was changed from G/A (amino acids Arg/His) in K7 to A/A (amino acids His/His) in K11 and K7AT2. When the plasmid containing CYR12066G was introduced into K11 or K7AT2, the stress tolerance of the transformants decreased to the level of K7, whereas the introduction of CYR12066A had a minimal effect. Consistently, disruption of the CYR12066G allele in K7 increased stress tolerance, whereas disruption of CYR12066A decreased stress tolerance. Furthermore, when CYR1 in a laboratory haploid strain was disrupted and either the CYR12042A or CYR12042G allele of S288C (corresponding to K7CYR12066) was introduced into the disruptant, the transformants with CYR12042A showed higher stress tolerance than those with CYR12042G. We concluded that the CYR1G2066A mutation was responsible for ethanol tolerance in K11.
Harnessing solar energy more efficiently and sustainably remains a key challenge in advancing renewable, bio-based production systems. Artificial photosynthesis tends to mimic natural photosynthesis by using catalytic systems and semiconductor assemblies to capture light and convert H2O and CO2 into energy-rich fuels such as H2 or hydrocarbons, whereas biophotovoltaics utilize living organisms or biological components (such as photosystems, chloroplasts, microalgae, or bacteria) integrated with electrodes for solar-to-electrical conversion. The review paper provides novel insights into exploring the integration of artificial photosynthesis and biophotovoltaics, discussing how their amalgamation can enhance and solidify solar-to-chemical and solar-to-electrical energy conversion. It emphasizes the crucial role of biocatalysts, such as microalgae, cyanobacteria, and bacteria, which can operate within these biohybrid systems. It also discusses the advanced strategies for enhancing biocatalyst efficiency, including genetic engineering, boosting carotenoid biosynthesis for better photoprotection and energy transfer, and integrating machine learning and Internet of Things to optimize the performance of microorganisms. In addition, the potential applications of artificial photosynthesis systems and biophotovoltaics are outlined, including biorefineries, biohydrogen production, chemical synthesis, and sustainable biofuel and food production.
The prolonged use of current anti-inflammatory therapies has significant limitations for chronic inflammation management, requiring safer alternatives. In this study, we investigated the anti-inflammatory effect of methylxanthine derivative, pentoxifylline. MTT assay was used for the determination of the cytotoxicity of pentoxifylline. Lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells were used for the determination of the anti-inflammatory effect of pentoxifylline using Griess assay, qRT-PCR, Western blot, gene reporter assay and cell migration assay. Pentoxifylline exhibited a high level of safe therapeutic window with no cytotoxicity up to 20 μM in the MTT assay on RAW 264.7 cells. Pentoxifylline demonstrated concentration and time-dependent anti-inflammatory effects with significantly (p < 0.05) reduced NO production at 10 and 20 μM concentrations. Gene expression revealed significant downregulation of inflammatory mediator genes: Nos2, Tnfα, and Il1β (p < 0.05). Protein analysis confirmed these effects with reductions in iNOS, TNFα, IL1β, and IL6 (p < 0.05). Pentoxifylline caused a significant reduction (p < 0.05) of transcriptional activity of NF-κB and AP-1. Further, a significant reduction of nuclear translocation of NF-κB by pentoxifylline confirmed the transcriptional inhibition. Additionally, in the scratch assay, pentoxifylline exhibited a significant reduction (p < 0.05) in macrophage migration, supporting its role in inhibiting inflammation progression. These comprehensive multi-target anti-inflammatory effects of pentoxifylline support its promising potential for use as a therapeutic candidate for inflammatory diseases.
Rapid and precise detection of small-molecule metabolites is crucial for optimizing the bioproduction processes. Cell-free systems (CFSs) offer an ideal platform for developing such biosensors due to their speed and suitability for automation. However, transcription factor (TF)-based biosensors, which are key elements for metabolite sensing, suffer from a severe bottleneck in in vitro environments. Their performance is often compromised due to the absence of nucleoid-associated proteins and different DNA topology compared to in vivo conditions. Here, we present a systematic framework for rationally engineering high-performance TF biosensors optimized for CFSs through integrated control of TF availability and promoter sequence. Using an itaconate (ITA)-responsive biosensor regulated by the LysR-type transcriptional regulator ItcR as a model system, we demonstrate that modulating TF supply and redesigning promoter elements substantially enhance sensitivity and dynamic range. Promoter dissection revealed that upstream sequences that function normally in vivo interfered with regulated transcription in CFSs, and that truncation to remove this region restored inducible behavior. Subsequent fine-tuning of the -35 and -10 motifs enhanced RNA polymerase recruitment and regulator interaction, resulting in 19-fold higher maximum signal, a 3.3-fold lower detection limit (0.003 g/L ITA), and a steeper dose-response curve (Hill slope increase from 2.7 to 34.7). The same promoter engineering strategy also improved a 3-hydroxypropionate-responsive biosensor, demonstrating its generality across distinct TF-promoter systems. Collectively, this framework establishes a rational, modular approach for constructing high-performance, topology-aware biosensors in CFSs, directly enabling high-throughput screening and automated biofoundry integration for synthetic biology and metabolic engineering applications.
Glucose dehydrogenases (GDHs) are oxidoreductases that catalyze the oxidation of d-glucose to glucono-δ-lactone and are widely used in industrial applications such as biosensors, bioelectrodes, and biocatalytic cofactor regeneration. In particular, NAD(P)+-dependent GDHs are frequently employed as NAD(P)H-regenerating enzymes, with extensive efforts devoted to improving their robustness. BmGDHM6, an engineered variant of the Bacillus megaterium GDH with enhanced chemical and organic solvent tolerance, was developed as a potent cofactor-regeneration enzyme. In parallel, enzyme condensation via liquid-liquid phase separation has attracted increasing attention as a potential mechanism for organizing enzyme-catalyzed reactions, and short peptides capable of promoting condensate formation. However, efficient screening and evaluation of such peptide tags using conventional cell-based expression systems have remained challenging. This study established a peptide tag screening strategy using the PURE system, a reconstituted cell-free protein synthesis platform, and applied it to BmGDHM6 as a model enzyme. A small library of metabolic enzymes transiently assembling (META) body-forming signal (METAfos) tags and intrinsically disordered region (IDR)-derived peptide tags was evaluated with respect to expression, solubility, and assembly behavior within a defined in vitro environment. From this library, a short peptide tag, K7G3, 10-amino-acids-long, was identified that conferred droplet-like assembly-forming properties on BmGDHM6 under specified crowding conditions. These results demonstrated that the PURE system provided a rapid and controllable platform for screening peptide tags and down-selection of candidates that modulate enzyme assembly behavior in vitro.