Uncovering the Power of HSCCC: Separation of Diastereomeric and Regioisomeric Styrylpyrones from the Stem Bark of Goniothalamus leiocarpus.

The plant Goniothalamus leiocarpus of the Annonaceae family is used as an alternative medicine in tropical regions. Applying high-speed counter current chromatography (HSCCC), eight new bioactive styrylpyrone isomers, including 6R,7S,8R,2'S-goniolactone B (1), 6S,7S,8S,2'S-goniolactone B (2), 6R,7R,8R,2'S-goniolactone B (3), 6R,7S,8S,2'S-goniolactone C (4), 6R,7S,8R,2'S-goniolactone C (5), 6S,7R,8S,2'S-goniolactone C (6), and two positional isomers, 6R,7R,8R,2'S-goniolactone G (7) and 6S,7R,8R,2'S-goniolactone G (8), were isolated from a chloroform fraction (2.1 g) of G. leiocarpus, which had a prominent spot by TLC analysis. The structures of the new compounds were elucidated by MS, NMR, IR, and UV spectra, and their absolute configurations were determined by Mosher's method, ECD, and X-ray diffraction analysis. The isolates are characteristic components found in plants of the genus Goniothalamus and consist of two structural moieties: a styrylpyrone and a dihydroflavone unit. The isolation of the eight new compounds demonstrates the effectiveness of HSCCC in separating the isomers of natural styrylpyrone. In a bioactivity assessment, compounds 1 and 6 exhibited cytotoxic effects against the human colon carcinoma cell lines LS513 and SW620 with IC50 values ranging from 1.6 to 3.9 µM. Compounds 1, 2, 7, and 8 showed significant synergistic activity against antibiotic-resistant Staphylococcus aureus strains.

Evolution of the WRKY Family in Angiosperms and Functional Diversity under Environmental Stress.

The transcription factor is an essential factor for regulating the responses of plants to external stimuli. The WRKY protein is a superfamily of plant transcription factors involved in response to various stresses (e.g., cold, heat, salt, drought, ions, pathogens, and insects). During angiosperm evolution, the number and function of WRKY transcription factors constantly change. After suffering from long-term environmental battering, plants of different evolutionary statuses ultimately retained different numbers of WRKY family members. The WRKY family of proteins is generally divided into three large categories of angiosperms, owing to their conserved domain and three-dimensional structures. The WRKY transcription factors mediate plant adaptation to various environments via participating in various biological pathways, such as ROS (reactive oxygen species) and hormone signaling pathways, further regulating plant enzyme systems, stomatal closure, and leaf shrinkage physiological responses. This article analyzed the evolution of the WRKY family in angiosperms and its functions in responding to various external environments, especially the function and evolution in Magnoliaceae plants. It helps to gain a deeper understanding of the evolution and functional diversity of the WRKY family and provides theoretical and experimental references for studying the molecular mechanisms of environmental stress.

Dynamical interplay between superconductivity and pseudogap in cuprates as revealed by terahertz third-harmonic generation spectroscopy.

We report on nonlinear terahertz third-harmonic generation (THG) measurements on YBa2Cu3O6+x thin films. Different from conventional superconductors, the THG signal starts to appear in the normal state, which is consistent with the crossover temperature T* of pseudogap over broad doping levels. Upon lowering the temperature, the THG signal shows an anomaly just below Tc in the optimally doped sample. Notably, we observe a beat pattern directly in the measured real-time waveform of the THG signal. We elaborate that the Higgs mode, which develops below Tc, couples to the mode already developed below T*, resulting in an energy level splitting. However, this coupling effect is not evident in underdoped samples. We explore different potential explanations for the observed phenomena. Our research offers valuable insight into the interplay between superconductivity and pseudogap.

Annonaceous Acetogenins Synergistically Inhibit Hepatocellular Carcinoma with Sorafenib.

Sorafenib was first approved as the standard treatment for advanced hepatocellular carcinoma (HCC). Despite providing an advantage in terms of patient survival, sorafenib has shown poor clinical efficacy and severe side effects after long-term treatment. Thus, combination treatment is a potential way to increase the effectiveness and reduce the dose-limiting toxicity of sorafenib. Extracts of the seeds of Annona montana have shown synergistic antitumor activity with sorafenib, and seven annonaceous acetogenins, including three new acetogenins, muricin P (2), muricin Q (3), and muricin R (4), were isolated from the extracts by bioguided fractionation and showed synergy with sorafenib. The structures of these compounds were determined using spectroscopic and chemical methods. Annonacin (1) and muricin P (2), which reduced intracellular ATP levels and promoted apoptosis, exhibited synergistic cytotoxicity with sorafenib in vitro. In vivo, annonacin (1) displayed synergistic antitumor activity by promoting tumor cell apoptosis. Moreover, the potential mechanism of annonacin (1) was predicted by transcriptomic analysis, which suggested that SLC33A1 is a potential target in HCC. Annonacin (1) might be a novel candidate for combination therapy with sorafenib against advanced HCC.

Pharmacokinetic study of five components of Fuzheng Huayu tablets in healthy human plasma.

Natural medicines play a crucial role in clinical drug applications, serving as a primary traditional Chinese medicine for the clinical treatment of liver fibrosis. Understanding the in vivo metabolic process of the Fuzheng Huayu (FZHY) formula is essential for delving into its material basis and mechanism. In recent years, there has been a growing body of research focused on the mechanisms and component analysis of FZHY. This study aimed to examine the pharmacokinetics of FZHY in healthy volunteers following oral administration. Blood samples were collected at designated time intervals after the oral intake of 9.6-g FZHY tablets. A UHPLC-Q/Exactive method was developed to assess the plasma concentrations of five components post-FZHY ingestion. The peak time for all components occurred within 10 min. The peak concentration (Cmax ) values for amygdalin, schisandrin, and schisandrin A ranged from 3.47 to 28.80 ng/mL, with corresponding AUC(0-t) values ranging from 10.63 to 103.20 ng h/mL. For schisandrin B and prunasin, Cmax values were in the range of 86.52 to 229.10 ng/mL, and their AUC(0-t) values ranged from 375.26 to 1875.54 ng h/mL, indicating significant exposure within the body. These findings demonstrate that the developed method enables rapid and accurate detection and quantification of the five components in FZHY, offering a valuable reference for its clinical study.

Revealing the frequency-dependent oscillations in the nonlinear terahertz response induced by the Josephson current.

Nonlinear responses of superconductors to intense terahertz radiation has been an active research frontier. Using terahertz pump-terahertz probe spectroscopy, we investigate the c-axis nonlinear optical response of a high-temperature superconducting cuprate. After excitation by a single-cycle terahertz pump pulse, the reflectivity of the probe pulse oscillates as the pump-probe delay is varied. Interestingly, the oscillatory central frequency scales linearly with the probe frequency, a fact widely overlooked in pump-probe experiments. By theoretically solving the nonlinear optical reflection problem on the interface, we show that our observation is well explained by the Josephson-type third-order nonlinear electrodynamics, together with the emission coefficient from inside the material into free space. The latter results in a strong enhancement of the emitted signal whose physical frequency is around the Josephson plasma edge. Our result offers a benchmark for and new insights into strong-field terahertz spectroscopy of related quantum materials.

Potential effects of biomaterials on macrophage function and their signalling pathways.

The use of biomaterials in biomedicine and healthcare has increased in recent years. Macrophages are the primary immune cells that induce inflammation and tissue repair after implantation of biomaterials. Given that macrophages exhibit high heterogeneity and plasticity, the influence of biomaterials on macrophage phenotype should be considered a crucial evaluation criterion during the development of novel biomaterials. This review provides a comprehensive summary of the physicochemical, biological, and dynamic characteristics of biomaterials that drive the regulation of immune responses in macrophages. The mechanisms involved in the interaction between macrophages and biomaterials, including endocytosis, receptors, signalling pathways, integrins, inflammasomes and long non-coding RNAs, are summarised in this review. In addition, research prospects of the interaction between macrophages and biomaterials are discussed. An in-depth understanding of mechanisms underlying the spatiotemporal changes in macrophage phenotype induced by biomaterials and their impact on macrophage polarization can facilitate the identification and development of novel biomaterials with superior performance. These biomaterials may be used for tissue repair and regeneration, vaccine or drug delivery and immunotherapy.

Natural Product BO-1 as an Inner Responsive Molecule Inhibits Antimicrobial-Resistant Staphylococcus aureus via Synergism.

Multidrug-resistant Staphylococcus aureus, a Gram-positive bacterium that causes several difficult-to-treat human infections, is a considerable threat to global healthcare. We hypothesize that there exist inner responsive molecules (IRMs) which can function synergistically with antibiotics to restore the sensitivity of resistant bacteria to existing antibiotics without inducing new antibiotic resistance. An investigation of the extracts of the Chinese medicinal herb Piper betle L. led to the isolation of six benzoate esters, BO-1-BO-6. Among these, BO-1 as a distinct IRM displayed considerable synergism by potentiating antibacterial activity against five antibiotic-resistant S. aureus strains. Mechanistic studies demonstrated that BO-1 acted as a suppressing drug resistance IRM via inhibiting efflux activity. A combination of BO-1 with ciprofloxacin significantly inhibited resistance to this antibiotic and reversed its resistance in the S. aureus strain. Furthermore, BO-1 effectively enhanced the activity of ciprofloxacin against the efflux fluoroquinolone-resistant S. aureus strain SA1199B that caused infection in two animal models and significantly decreased the inflammatory factors IL-6 and C-reactive protein of the infected mice, thereby showing the practice utility of this approach.

[Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae].

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Metal-Organic Frameworks-Based Optical Nanosensors for Analytical and Bioanalytical Applications.

Metal-organic frameworks (MOFs)-based optical nanoprobes for luminescence and surface-enhanced Raman spectroscopy (SERS) applications have been receiving tremendous attention. Every element in the MOF structure, including the metal nodes, the organic linkers, and the guest molecules, can be used as a source to build single/multi-emission signals for the intended analytical purposes. For SERS applications, the MOF can not only be used directly as a SERS substrate, but can also improve the stability and reproducibility of the metal-based substrates. Additionally, the porosity and large specific surface area give MOF a sieving effect and target molecule enrichment ability, both of which are helpful for improving detection selectivity and sensitivity. This mini-review summarizes the advances of MOF-based optical detection methods, including luminescence and SERS, and also provides perspectives on future efforts.

Antiliver Fibrosis Formula of Fuzheng Huayu Alleviates Inflammatory Response.

Fuzheng Huayu's (FZHY) formula ameliorated liver fibrosis in clinical and experimental practice. Based on the close link between fibrosis and inflammation, its anti-inflammatory effect and related mechanisms were explored in this present study. With the aid of the inflammatory macrophage model, FZHY significantly blocked nitrite accumulation without observable cytotoxicity due to its suppression of inducible nitric oxide synthase (iNOS) gene and protein expressions in a concentration-depended manner. Proinflammatory mediators including IL-6, CD86, and CD40 were also restrained by FZHY. Interestingly, FZHY induced anti-inflammatory mediators heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) expressions simultaneously. Downregulation of iNOS and miR-155 and upregulation of PPAR-γ were also observed in CCl4-induced liver fibrosis mice upon FZHY administration. Mechanically, FZHY strikingly eliminated the phosphorylation of STAT1 and MAPK. Taken together, FZYH regulated the balance of proinflammatory and anti-inflammatory mediators partially via modulating STAT1/MAPK pathways and the miR-155/PPAR-γ axis.

Controllable growth of wafer-scale monolayer transition metal dichalcogenides ternary alloys with tunable band gap.

The tuning of band gap is very important for the application of two-dimensional (2D) materials in optoelectronic devices. Alloying of 2D transition metal dichalcogenides (TMDCs) is an important way to tune the wide band gap. In this study, we report a multi-step vapor deposition method to grow monolayer TMDC ternary alloy films with wafer scale, including Mo1-xWxS2, Mo1-xWxSe2and MoS2xSe2(1-x), which are accurately controllable in the elemental proportion (xis from 0 to 1). The band gap of the three 2D ternary alloy materials are continuously tuned for the whole range of metal and chalcogen compositions. The metal compositions are controlled by the as-deposited thickness. Raman, photoluminescence, elemental maps and TEM show the high spatial homogeneity in the compositions and optical properties across the whole wafer. The band gap can be continuously tuned from 1.86 to 1.99 eV for Mo1-xWxS2, 1.56 to 1.65 eV for Mo1-xWxSe2, 1.56 to 1.86 eV for MoS2xSe2(1-x). Electrical transport measurements indicate that Mo1-xWxS2and MoS2xSe2(1-x)monolayers shown-type semiconductor behaviors, and the carrier types of Mo1-xWxSe2can be tuned asn-type, bipolar andp-type. Moreover, this control process can be easily generalized to other 2D alloy films, even to quaternary or multi-element alloy materials. Our study presents a promising route for the preparation of large-scale homogeneous monolayer TMDC alloys and the application for future functional devices.

Age-dependent modulation of oleoresin production in the stem of Sindora glabra.

Plants produce specialized metabolites in various organs which serve important functions in defense and development. However, the molecular regulatory mechanisms of oleoresin production in stems from broadleaved tree species are not fully understood. To determine whether endogenous developmental cues play a role in the regulation of oleoresin biosynthesis in tree stems, anatomy, multi-omics and molecular experiments were utilized to investigate the change of secretory structures, chemical profiles and gene expression in different ontogenetic stages of Sindora glabra tree, which accumulates copious amount of sesquiterpene-rich oleoresin in stems. The size of secretory canals and the concentration of five sesquiterpenes in Sindora stems exhibited obvious increase with plant age, from 0.5- to 20-year-old plants. Moreover, α-copaene and ß-copaene were found to be stem-specific sesquiterpenes. Metabolomic analysis revealed that salicylic acid highly accumulated in mature stems, but the content of triterpenes was greatly decreased. The expression of three repressors AUX/IAA, DELLA and JAZ involved in hormone signaling transduction pathways was significantly downregulated in stems of 10- and 20-year-old plants. Two key genes SgTPS3 and SgTPS5 were identified, whose expression was highly correlated with the accumulation patterns of specific sesquiterpenes and their enzymatic products were consistent with the chemical profiles in the stem. The promoters of three SgTPSs exhibiting high activity were isolated. Furthermore, we demonstrated that SgSPL15 directly interacts with SgTPS3 and SgTPS5 promoters and activates SgTPS5 expression but SgSPL15 inhibits SgTPS3 expression. In addition, SgSPL15 enhanced sesquiterpene levels by upregulating AtTPSs expression in Arabidopsis. These results suggested that sesquiterpene biosynthesis in S. glabra stem was dependent on the regulation of endogenous hormones as well as plant age, and SgSPL15 might act as a buffering factor to regulate sesquiterpene biosynthesis by targeting SgTPS genes.

In vitro and vivo study of tranilast protects from acute respiratory distress syndrome and early pulmonary fibrosis induced by smoke inhalation.

BACKGROUND: Tranilast (N-[3＇,4＇-dimethoxycinnamoyl]-anthranilic acid) is an analog of a tryptophan metabolite. It was identified with anti-inflammatory and antifibrotic activities, and used in the treatment of a variety of diseases, such as anti - allergy, bronchial asthma, and hypertrophic scars. As a drug with few adverse reactions, tranilast has attracted great attention, but its application is limited due to the uncertainty of dosages and mechanisms. In this study, the protection effects of different doses of tranilast on smoke inhalation mediated lung injury on rats, and on the damage of three kinds of lung cells in vitro were investigated. METHOD: In vivo, Sprague-Dawley rats were randomly divided into sham group, smoke group (rats were exposed to pine sawdust smoke three times, each time for 5 min), different doses of tranilast treatment group (doses were 100 mg/kg, 200 mg/kg and 300 mg/kg, ip.) and placebo group. After 1, 3 and 7 days, pulmonary function, pathologic injury by HE staining, cytokines and oxidative stress level by kits were determined. At 7days, lung fibrosis was assessed by Masson's trichrome staining and the level of hydroxyproline (HYP). In vitro, three kinds of lung cells from normal rats were isolated: type II alveolar epithelial cells (AT-II), pulmonary microvascular endothelial cells (PMVECs) and pulmonary fibroblasts (PFs). To investigate the potential effects of tranilast on cell proliferation, cell cycle and cytokine production of three kinds of lung cells exposed to smoke. RESULTS: Compared with smoke group and placebo group, tranilast treatment significantly reduced histopathological changes (such as pulmonary hemorrhage, edema and inflammatory cell infiltration, etc.), significantly reduced histopathological score (p < 0.05), increased arterial oxygen partial pressure, and decreased the levels of IL-1ß, TNF-α, TGF-ß1 (p < 0.05), oxidative stress and the expression of nuclear transcription factor κB (NF-κB) smoke exposed rats (p < 0.01). In particular, the effect of 200 mg/kg dose was more prominent. In vitro, smoke induced AT-II and PMVECs apoptosis, improved PFs proliferation (p < 0.01), activity of SOD and decreased the content of MDA (p < 0.01). However, tranilast seems to be turning this trend well. The inflammatory factor IL-11ß, TNF-α and TGF-ß1, and the expression of NF-κB were significantly lower in the tranilast treatment than in the smoke group (p < 0.01). CONCLUSION: This study indicates that tranilast had a protective effect on acute respiratory distress syndrome and early pulmonary fibrosis of rats in vivo. In addition, tranilast promotes proliferation of AT-II and PMVECs but inhibits PFs proliferation, down-regulates secretion of inflammatory cytokines and alleviates oxidative stress of AT-II, PMVECs and PFs after smoke stimuli in vitro.

[Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae].

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Tuning the morphology of 2D transition metal chalcogenides via oxidizing conditions.

Two-dimensional transition metal chalcogenides (TMCs) are emerging as an intriguing platform to realize nascent properties in condensed matter physics, materials science and device engineering. Controllable growing of TMCs becomes increasingly important, especially for the layer number, doping, and morphology. Here, we successfully tune the morphology of MoS2, MoSe2, WS2and WSe2, from homogenous films to individual single crystalline grains only via changing the oxidizing growth conditions. The oxidization degrees are determined by the oxygen that adsorbed on substrates and the oxygen concentrations in reaction gas together. We find the homogenous films are easily formed under the reductive conditions, triangular grains prefer the weak oxidizing conditions, and medium oxidizing conditions bring in dendritic grains with higher oxygen doping and inhomogenous photoluminescence intensities from edge to interior regions shown in the dendritic grains. These growth rules under different oxidizing conditions are easily generalized to other TMCs, which also show potential for growing specific TMCs with designed oxygen doping levels.

[Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae].

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Simultaneous determination of eight analytes of Fuzheng Huayu recipe in beagle dog plasma by UHPLC-Q/exactive Orbitrap HRMS and its application to toxicokinetics.

Fuzheng Huayu recipe (FZHY) is a Chinese patent medicine for the treatment of liver fibrosis. This study aimed to investigate the toxicokinetics of FZHY in beagle dogs after oral administration. Blood samples were collected on days 1, 15 and 28 after oral gavage of FZHY dosages of 400 or 1,200 mg/kg body weight once a day. A UHPLC-Q-Orbitrap method was developed and validated to simultaneously determine and quantify eight components of FZHY in beagle dog plasma. The times to peak concentration for eight components were18-120 min. The peak concentrations (Cmax ) of amygdalin, genistein, daidzein and 3,4-dihydroxybenzaldehyde were 1.43-43.50 ng/ml, the areas under the concentration-time curve (AUC(0-t) ) were 2.45-6,098.25 ng min/ml, and the apparent volumes of distribution (Vd ) were 0.05-131.23 × 104 ml/kg. The values of Cmax of prunasin, schisantherin A, schisandrin A and schisandrin were 7.35-1,450.73 ng/ml, the values of AUC(0-t) were 3,642.30-330,388.65 ng min/ml, and the values of Vd were 11.15-1,087.18 × 104 ml/kg. No obvious accumulation of the eight compounds was observed in beagle dogs. The results showed that the method is rapid, accurate and sensitive, and is suitable for detecting the eight analytes of FZHY. This study provides an important basis for the assessment of FZHY safety.

Universal Nanoparticle Counting Platform for Tetraplexed Biomarkers by Integrating Immunorecognition and Nucleic Acid Hybridization in One Assay.

The development of a simple and universal strategy for simultaneous quantification of proteins and nucleic acid biomarkers in one assay is valuable, particularly for disease diagnosis and pathogenesis studies. Herein, a universal and amplification-free quantum dot-doped nanoparticle counting platform was developed by integrating immunorecognition and nucleic acid hybridization in one assay. The assay can be performed at room temperature, which is friendly for routine analysis. Multiplexed biomarkers associated with Alzheimer's disease (AD) including proteins and nucleic acids were detected. For simultaneous detection of tetraplex biomarkers, the assay for amyloid ß 1-42 (Aß42), tau protein, miR-146a, and miR-138 presented limit of detection values of 250 pg/mL, 55.7 pg/mL, 52.5 pM, and 0.62 pM, respectively. By spiking all the above four biomarkers in one artificial cerebrospinal fluid sample, the recoveries were found to be 94.7-117.2%. Using tau protein as the model, four measurements in 88 days presented a coefficient of variance of 7.5%. The proposed platform for the multiplexed assay of proteins and nucleic acids presents the universality, reasonable sensitivity, and repeatability, which may open a new door for early diagnosis and pathogenesis research for AD and other diseases.

Engineering yeast subcellular compartments for increased production of the lipophilic natural products ginsenosides.

Eukaryotic yeasts have a variety of subcellular compartments and are ideal platform strains for the construction of complex heterologous natural product biosynthesis pathways. Improving the synthesis efficiency of microbial cell factories through the utilization and modification of subcellular compartments by synthetic biology has good application prospects. Here, we used the yeast PLN1 protein to target the normally endoplasmic reticulum (ER)-localized cytochrome P450 enzyme protopanaxadiol (PPD) synthase (PPDS) to lipid droplets (LDs), which are the storage organelles of the PPDS substrate dammarenediol-II (DD). The efficiency of converting DD to PPD was significantly increased by 394%, and the conversion rate of DD increased from 17.4% to 86.0%. Furthermore, increasing the volume of LDs can significantly enhance the production of DD and its derivatives, but the change in the ratio of the volume and surface area of LDs decreased the conversion efficiency of DD to PPD. Additionally, the biosynthetic pathways of the PPD-type saponin ginsenoside compound K (CK) was reconstituted in a PPD-producing chassis strain, and CK production reached 21.8 mg/L/OD, 4.4-fold higher compared to the native ER-expression strategy. Next, we enhanced the expression of the Pn3-29 gene module to further reduce the accumulation of PPD and increase the production of CK to 41.3 mg/L/OD. Finally, the CK titer of the resulting strain reached 5 g/L in 5 L fed-batch fermentations. This study provides a new strategy for engineering yeast to produce complex natural products.