Efficacy comparison of fruquintinib, regorafenib monotherapy or plus programmed death-1 inhibitors for microsatellite stable metastatic colorectal cancer.

BACKGROUND: Regorafenib (R) and fruquintinib (F) are the standard third-line regimens for colorectal cancer (CRC) according to the National Comprehensive Cancer Network guidelines, but both have limited efficacy. Several phase 2 trials have indicated that R or F combined with immune checkpoint inhibitors can reverse immunosuppression and achieve promising efficacy for microsatellite stable or proficient mismatch repair (MSS/pMMR) CRC. Due to the lack of studies comparing the efficacy between F, R, F plus programmed death-1 (PD-1) inhibitor, and R plus PD-1 inhibitors (RP), it is still unclear whether the combination therapy is more effective than monotherapy. AIM: To provide critical evidence for selecting the appropriate drugs for MSS/pMMR metastatic CRC (mCRC) patients in clinical practice. METHODS: A total of 2639 CRC patients were enrolled from January 2018 to September 2022 in our hospital, and 313 MSS/pMMR mCRC patients were finally included. RESULTS: A total of 313 eligible patients were divided into F (n = 70), R (n = 67), F plus PD-1 inhibitor (FP) (n = 95) and RP (n = 81) groups. The key clinical characteristics were well balanced among the groups. The median progression-free survival (PFS) of the F, R, FP, and RP groups was 3.5 months, 3.6 months, 4.9 months, and 3.0 months, respectively. The median overall survival (OS) was 14.6 months, 15.7 months, 16.7 months, and 14.1 months. The FP regimen had an improved disease control rate (DCR) (P = 0.044) and 6-month PFS (P = 0.014) and exhibited a better trend in PFS (P = 0.057) compared with F, and it was also significantly better in PFS than RP (P = 0.030). RP did not confer a significant survival benefit; instead, the R group had a trend toward greater benefit with OS (P = 0.080) compared with RP. No significant differences were observed between the R and F groups in PFS or OS (P > 0.05). CONCLUSION: FP is superior to F in achieving 6-month PFS and DCR, while RP is not better than R. FP has an improved PFS and 6-month PFS compared with RP, but F and R had similar clinical efficacy. Therefore, FP may be a highly promising strategy in the treatment of MSS/pMMR mCRC.

Asymmetric catalytic synthesis of chiral covalent organic framework composite (S)-DTP-COF@SiO2 for HPLC enantioseparations by normal-phase and reversed-phase chromatographic modes.

A novel CCOF core-shell composite material (S)-DTP-COF@SiO2 was prepared via asymmetric catalytic and in situ growth strategy. The prepared (S)-DTP-COF@SiO2 was utilized as separation medium for HPLC enantioseparation using normal-phase and reversed-phase chromatographic modes, which displays excellent chiral separation performance for alcohols, esters, ketones, and epoxides, etc. Compared with chiral commercial chromatographic columns (Chiralpak AD-H and Chiralcel OD-H columns) and some previously reported chiral CCOF@SiO2 (CC-MP CCTF@SiO2 and MDI-ß-CD-modified COF@SiO2)-packed columns, there are 4, 3, 13, and 15 tested racemic compounds that could not be resolved on the Chiralpak AD-H column, Chiralcel OD-H column, CC-MP CCTF@SiO2 column, and MDI-ß-CD-modified COF@SiO2 column, respectively, which indicates that the resolution effect of (S)-DTP-COF@SiO2-packed column can be complementary to the other ones. The effects of the analyte mass, column temperature, and mobile phase composition on the enantiomeric separation were investigated. The chiral column exhibits good reproducibility after multiple consecutive injections. The RSDs (n = 5) of the peak area and retention time were less than 1.5% for repetitive separation of 2-methoxy-2-phenylethanol and 1-phenyl-1-pentanol. The chiral core-shell composite (S)-DTP-COF@SiO2 exhibited good enantiomeric separation performance, which not only demonstrates its potential as a novel CSP material in HPLC but also expands the range of applications for chiral COFs.

Subcomponent self-assembly construction of tetrahedral cage FeII4L4 for high-resolution gas chromatographic separation.

Metal organic cages (MOCs), as an emerging discrete supramolecular compounds, have received widespread attention in separation, biomedicine, gas capture, catalysis, and molecular recognition due to their porosity, adjustability and stability. Herein, we present a new chiral MOC FeII4L4 coated capillary column prepared for gas chromatographic (GC) separation of different types of organic compounds, including n-alkanes, n-alcohols, alkylbenzenes, isomers, especially for racemic compounds. There are 20 different kinds of racemates (e.g., alcohols, ethers, epoxides, esters, alkenes, and aldehydes) were well resolved on the FeII4L4 chiral column and a maximum resolution value for 1-phenyl-1-propanol reaches 6.17. The FeII4L4 coated column exhibited high column efficiency (3100 plates m-1 for n-dodecane) and good enantiomeric resolution complementary to that of a commercial ß-DEX 120 column and the previously reported chiral MOC [Fe4L6] (ClO4)8 coated column. The relative standard deviation (RSDs) of the peak area and retention time of glycidol and nitrotoluene were below 1.2 %. This study reveals that chiral MOCs have good application prospects in chromatographic separation.

Effect of Goji Berry extract on cell viability of Lactiplantibacillus plantarum M5 microcapsules during in vitro gastrointestinal digestion.

Lactiplantibacillus plantarum M5 and Goji Berry extract were co-microencapsulated to maintain the activity of cells during gastrointestinal digestion, and the mechanism by which they could maintain high activity was investigated. The results showed that the microcapsules with 3% Goji Berry extract(A-GE-3) had the largest encapsulation efficiency(EE) of 92.41 ± 0.58%. SEM showed that the structure of A-GE-3 microcapsules were smoother and denser. Cell viability in A-GE-3 microcapsules remained at 7.17 log10 CFU/g after gastrointestinal digestion. Meanwhile, during the gastrointestinal digestion with 3% Goji Berry extract, cell membrane damage detected by fluorescent probes propidium iodide(PI) and 1.1-N-phenylnaphthylamine(NPN) was significantly reduced; the contents of arginine, glutamic acid and oleic acid in cell membrane were increased, which helped to maintain the dynamic balance of intracellular pH and regulated cell membrane fluidity in response to gastrointestinal environment. This study demonstrated the potential of Goji Berry extract as a probiotic protector in gastrointestinal digestion.

Large Flux Supply of NAD(H) under Aerobic Conditions by Candida glycerinogenes.

NAD is a redox coenzyme and is the center of energy metabolism. In metabolic engineering modifications, an insufficient NAD(H) supply often limits the accumulation of target products. In this study, Candida glycerinogenes was found to be able to supply NAD(H) in large fluxes, up to 7.6 times more than Saccharomyces cerevisiae in aerobic fermentation. Aerobic fermentation in a medium without amino nitrogen sources demonstrated that C. glycerinogenes NAD synthesis was not dependent on NAD precursors in the medium. Inhibition by antisense RNA and the detection of transcript levels indicated that the main NAD supply pathway is the de novo biosynthesis pathway. It was further demonstrated that NAD(H) supply was unaffected by changes in metabolic flow through C. glycerinogenes ΔGPD aerobic fermentation (80 g/L ethanol). In conclusion, the ability of C. glycerinogenes to supply NAD(H) in large fluxes provides a new approach to solving the NAD(H) supply problem in synthetic biology.

Effective Management of Polycythemia Vera With Ropeginterferon Alfa-2b Treatment.

Background: Polycythemia vera (PV) is a myeloproliferative neoplasm. Ropeginterferon alfa-2b is a new-generation polyethylene glycol-conjugated proline-interferon. It is approved for the treatment of PV at a starting dose of 100 µg (50 µg for patients receiving hydroxyurea (HU)) and dose titrations up to 500 µg by 50 µg increments. The study was aimed at assessing its efficacy and safety at a higher starting dose and simpler intra-patient dose escalation. Methods: Forty-nine patients with PV having HU intolerance from major hospitals in China were treated biweekly with an initial dose of 250 µg, followed by 350 µg and 500 µg thereafter if tolerated. Complete hematological response (CHR) was assessed every 12 weeks based on the European LeukemiaNet criteria. The primary endpoint was the CHR rate at week 24. The secondary endpoints included CHR rates at weeks 12, 36 and 52, changes of JAK2V617F allelic burden, time to first CHR, and safety assessments. Results: The CHR rates were 61.2%, 69.4% and 71.4% at weeks 24, 36, and 52, respectively. Mean allele burden of the driver mutation JAK2V617F declined from 58.5% at baseline to 30.1% at 52 weeks. Both CHR and JAK2V617F allele burden reduction showed consistent increases over the 52 weeks of the treatment. Twenty-nine patients (63.0%) achieved partial molecular response (PMR) and two achieved complete molecular response (CMR). The time to CHR was rapid and median time was 5.6 months according to central lab results. The CHRs were durable and median CHR duration time was not reached at week 52. Mean spleen index reduced from 55.6 cm2 at baseline to 50.2 cm2 at week 52. Adverse events (AEs) were mostly mild or moderate. Most common AEs were reversible alanine aminotransferase and aspartate aminotransferase increases, which were not associated with significant elevations in bilirubin levels or jaundice. There were no grade 4 or 5 AEs. Grade 3 AEs were reversible and manageable. Only one AE led to discontinuation. No incidence of thromboembolic events was observed. Conclusion: The 250-350-500 µg dosing regimen was well tolerated and effectively induced CHR and MR and managed spleen size increase. Our findings demonstrate that ropeginterferon alfa-2b at this dosing regimen can provide an effective management of PV and support using this dosing regimen as a treatment option.

Enhancing the catalytic efficiency of M32 carboxypeptidase by semi-rational design and its applications in food taste improvement.

BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization. RESULTS: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components. CONCLUSION: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.

Development of a co-culture system for green production of caffeic acid from sugarcane bagasse hydrolysate.

Caffeic acid (CA) is a phenolic acid compound widely used in pharmaceutical and food applications. However, the efficient synthesis of CA is usually limited by the resources of individual microbial platforms. Here, a cross-kingdom microbial consortium was developed to synthesize CA from sugarcane bagasse hydrolysate using Escherichia coli and Candida glycerinogenes as chassis. In the upstream E. coli module, shikimate accumulation was improved by intensifying the shikimate synthesis pathway and blocking shikimate metabolism to provide precursors for the downstream CA synthesis module. In the downstream C. glycerinogenes module, conversion of p-coumaric acid to CA was improved by increasing the supply of the cytoplasmic cofactor FAD(H2). Further, overexpression of ABC transporter-related genes promoted efflux of CA and enhanced strain resistance to CA, significantly increasing CA titer from 103.8 mg/L to 346.5 mg/L. Subsequently, optimization of the inoculation ratio of strains SA-Ec4 and CA-Cg27 in this cross-kingdom microbial consortium resulted in an increase in CA titer to 871.9 mg/L, which was 151.6% higher compared to the monoculture strain CA-Cg27. Ultimately, 2311.6 and 1943.2 mg/L of CA were obtained by optimization of the co-culture system in a 5 L bioreactor using mixed sugar and sugarcane bagasse hydrolysate, respectively, with 17.2-fold and 14.6-fold enhancement compared to the starting strain. The cross-kingdom microbial consortium developed in this study provides a reference for the production of other aromatic compounds from inexpensive raw materials.

Progress in the development of piezoelectric biomaterials for tissue remodeling.

Piezoelectric biomaterials have demonstrated significant potential in the past few decades to heal damaged tissue and restore cellular functionalities. Herein, we discuss the role of bioelectricity in tissue remodeling and explore ways to mimic such tissue-like properties in synthetic biomaterials. In the past decade, biomedical engineers have adopted emerging functional biomaterials-based tissue engineering approaches using innovative bioelectronic stimulation protocols based on dynamic stimuli to direct cellular activation, proliferation, and differentiation on engineered biomaterial constructs. The primary focus of this review is to discuss the concepts of piezoelectric energy harvesting, piezoelectric materials, and their application in soft (skin and neural) and hard (dental and bone) tissue regeneration. While discussing the prospective applications as an engineered tissue, an important distinction has been made between piezoceramics, piezopolymers, and their composites. The superiority of piezopolymers over piezoceramics to circumvent issues such as stiffness mismatch, biocompatibility, and biodegradability are highlighted. We aim to provide a comprehensive review of the field and identify opportunities for the future to develop clinically relevant and state-of-the-art biomaterials for personalized and remote health care.

Efficacy and safety of anlotinib plus anti-PD-1 agents in patients with refractory advanced biliary tract cancers.

BACKGROUND: Anlotinib has demonstrated promising anti-tumor efficacy in various solid tumors. Additionally, there is evidence suggesting that immune therapy can enhance the systemic responses of anlotinib. This study aimed to assess the effectiveness and safety of combining anlotinib with PD-1 inhibitors compared to fluoropyrimidine-based chemotherapy as a second-line treatment option for advanced biliary tract cancers (BTCs). METHODS: A total of 242 patients with BTCs were screened at the First Affiliated Hospital of Zhengzhou University from October 2015 to October 2022. Among them, 78 patients who received either anlotinib plus PD-1 inhibitors (AP) or fluoropyrimidine-based chemotherapy (FB) as second-line treatment were included in the study. The primary endpoint was progression-free survival (PFS), and the secondary endpoints included objective response rate (ORR), disease control rate (DCR), overall survival (OS), safety, and predictive tumor biomarkers. RESULTS: Among the 78 patients with BTCs, 39 patients received AP, while 39 patients were administered FB. The ORR in the AP group was 20.5%, compared to 5.1% in the FB group. The DCR was 87.2% in the AP group and 66.7% in the FB group. The AP group demonstrated significantly better ORR and DCR compared to the FB group (p = 0.042, p = 0.032). The median PFS and OS in the AP group were 7.9 months (95% CI: 4.35-11.45) and 13.9 months (95% CI: 5.39-22.41), respectively. In the FB group, the median PFS and OS were 4.1 months (95% CI: 3.17-5.03) and 13.2 months (95% CI: 8.72-17.68), respectively. The AP group exhibited significantly better median PFS than the FB group (p = 0.027). In the subgroup analysis, patients without liver metastasis had a much longer PFS in the AP group compared to the FB group (14.3 vs. 5.5 months, p = 0.016). Similarly, patients with CEA ≤ 5 µg/L also demonstrated a longer PFS in the AP group compared to the FB group (8.7 vs. 3.9 months, p = 0.008). CONCLUSIONS: The combination of anlotinib and PD-1 inhibitors demonstrated a promising clinical effect compared to fluoropyrimidine-based chemotherapy in the second-line treatment of refractory advanced BTCs. Liver metastases and CEA levels may serve as predictive factors for identifying patients who may benefit from AP therapy.

An S-locus F-box protein as pollen S determinant targets non-self S-RNase underlying self-incompatibility in Citrus.

Self-incompatibility (SI) is a crucial mechanism that prevents self-fertilization and inbreeding in flowering plants. Citrus exhibits SI regulated by a polymorphic S-locus containing an S-RNase gene and multiple S-locus F-box (SLF) genes. It has been documented that S-RNase functions as the pistil S determinant, but there is no direct evidence that the SLF genes closely linked with S-RNase function as pollen S determinants in Citrus. This study assembled the genomes of two pummelo (Citrus grandis) plants, obtained three novel complete and well-annotated S-haplotypes, and isolated 36 SLF or SLF-like alleles on the S-loci. Phylogenetic analysis of 138 SLFs revealed that the SLF genes were classified into 12 types, including six types with divergent or missing alleles. Furthermore, transformation experiments verified that the conserved S6-SLF7a protein can lead to the transition of SI to self-compatibility by recognizing non-self S8-RNase in 'Mini-Citrus' plants (S7S8 and S8S29, Fortunella hindsii), a model plant for citrus gene function studies. In vitro assays demonstrated interactions between SLFs of different S haplotypes and the Skp1-Cullin1-F-box subunit CgSSK1 protein. This study provides direct evidence that SLF controls the pollen function in Citrus, demonstrating its role in the 'non-self recognition' SI system.

Spatiotemporal Regulation and Transport Engineering for Sustainable Production of Geraniol in Candida glycerinogenes.

Geraniol is an attractive natural monoterpene with significant industrial and commercial value in the fields of pharmaceuticals, condiments, cosmetics, and bioenergy. The biosynthesis of monoterpenes suffers from the availability of key intermediates and enzyme-to-substrate accessibility. Here, we addressed these challenges in Candida glycerinogenes by a plasma membrane-anchoring strategy and achieved sustainable biosynthesis of geraniol using bagasse hydrolysate as substrate. On this basis, a remarkable 2.4-fold improvement in geraniol titer was achieved by combining spatial and temporal modulation strategies. In addition, enhanced geraniol transport and modulation of membrane lipid-associated metabolism effectively promoted the exocytosis of toxic monoterpenes, significantly improved the resistance of the engineered strain to monoterpenes and improved the growth of the strains, resulting in geraniol yield up to 1207.4 mg L-1 at shake flask level. Finally, 1835.2 mg L-1 geraniol was obtained in a 5 L bioreactor using undetoxified bagasse hydrolysate. Overall, our study has provided valuable insights into the plasma membrane engineering of C. glycerinogenes for the sustainable and green production of valuable compounds.

Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette.

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C. glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C. glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C. glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.

Crosstalk between colorectal CSCs and immune cells in tumorigenesis, and strategies for targeting colorectal CSCs.

Cancer immunotherapy has emerged as a promising strategy in the treatment of colorectal cancer, and relapse after tumor immunotherapy has attracted increasing attention. Cancer stem cells (CSCs), a small subset of tumor cells with self-renewal and differentiation capacities, are resistant to traditional therapies such as radiotherapy and chemotherapy. Recently, CSCs have been proven to be the cells driving tumor relapse after immunotherapy. However, the mutual interactions between CSCs and cancer niche immune cells are largely uncharacterized. In this review, we focus on colorectal CSCs, CSC-immune cell interactions and CSC-based immunotherapy. Colorectal CSCs are characterized by robust expression of surface markers such as CD44, CD133 and Lgr5; hyperactivation of stemness-related signaling pathways, such as the Wnt/ß-catenin, Hippo/Yap1, Jak/Stat and Notch pathways; and disordered epigenetic modifications, including DNA methylation, histone modification, chromatin remodeling, and noncoding RNA action. Moreover, colorectal CSCs express abnormal levels of immune-related genes such as MHC and immune checkpoint molecules and mutually interact with cancer niche cells in multiple tumorigenesis-related processes, including tumor initiation, maintenance, metastasis and drug resistance. To date, many therapies targeting CSCs have been evaluated, including monoclonal antibodies, antibody‒drug conjugates, bispecific antibodies, tumor vaccines adoptive cell therapy, and small molecule inhibitors. With the development of CSC-/niche-targeting technology, as well as the integration of multidisciplinary studies, novel therapies that eliminate CSCs and reverse their immunosuppressive microenvironment are expected to be developed for the treatment of solid tumors, including colorectal cancer.

Glycerol Production from Undetoxified Lignocellulose Hydrolysate by a Multiresistant Engineered Candida glycerinogenes.

Glycerol is an important platform compound with multidisciplinary applications, and glycerol production using low-cost sugar cane bagasse hydrolysate is promising. Candida glycerinogenes, an industrial yeast strain known for its high glycerol production capability, has been found to thrive in bagasse hydrolysate obtained through a simple treatment without detoxification. The engineered C. glycerinogenes exhibited significant resistance to furfural, acetic acid, and 3,4-dimethylbenzaldehyde within undetoxified hydrolysates. To further enhance glycerol production, genetic modifications were made to Candida glycerinogenes to enhance the utilization of xylose. Fermentation of undetoxified bagasse hydrolysate by CgS45 resulted in a glycerol titer of 40.3 g/L and a yield of 40.4%. This process required only 1 kg of bagasse to produce 93.5 g of glycerol. This is the first report of glycerol production using lignocellulose, which presents a new way for environmentally friendly industrial production of glycerol.

Efficacy, safety and genomic analysis of SCT200, an anti-EGFR monoclonal antibody, in patients with fluorouracil, irinotecan and oxaliplatin refractory RAS and BRAF wild-type metastatic colorectal cancer: a phase â ¡ study.

BACKGROUND: Limited therapeutic options are available for metastatic colorectal cancer (mCRC) patients after failure of first- and second-line therapies, representing an unmet medical need for novel therapies. METHODS: This is an open-label, single arm, multicenter, phase Ⅱ study aiming to perform the efficacy, safety and genomic analysis of SCT200, a noval fully humanized IgG1 anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in patients with fluorouracil, irinotecan and oxaliplatin refractory RAS and BRAF wild-type mCRC. SCT200 (6 mg/kg) was given weekly for the first six weeks, followed by a higher dose of 8 mg/kg every two weeks until disease progression or unacceptable toxicity. Primary endpoint was independent review committee (IRC)-assessed objective response rate (ORR) and secondary endpoints included ORR in patients with left-sided tumor, disease control rate (DCR), duration of response (DoR), time to response (TTR), progression-free survival (PFS), overall survival (OS) and safety. FINDINGS: From February 12, 2018 to December 1, 2019, a total of 110 patients aged between 26 and 77 years (median: 55; interquartile range [IQR]: 47-63) with fluorouracil, oxaliplatin, and irinotecan refractory RAS and BRAF wild-type mCRC were enrolled from 22 hospitals in China. As the data cut-off date on May 15, 2020, the IRC-assessed ORR and DCR was 31% (34/110, 95% confidence interval [CI] 22-40%) and 75% (82/110, 95% CI 65-82%), respectively. Thirty one percent (34/110) patients achieved confirmed partial response (PR). The median PFS and median OS were 5.1 months (95% CI 3.4-5.2) and 16.2 months (95% CI 11.1-not available [NA]), respectively. The most common ≥ grade 3 treatment-related adverse events (TRAEs) were hypomagnesemia (17%, 19/110) and acneiform dermatitis (11%, 12/110). No deaths occurred. Genomic analysis suggested positive association between MYC amplification and patients' response (P = 0.0058). RAS/RAF mutation and MET amplification were the most frequently detected resistance mechanisms. Patients with high circulating tumor DNA (ctDNA) at baseline or without ctDNA clearance at the 7th week after the first dose of SCT200 administration before receiving SCT200 had worse PFS and OS. INTERPRETATION: SCT200 exhibited promising clinical efficacy and manageable safety profiles in RAS and BRAF wild-type mCRC patients progressed on fluorouracil, irinotecan and oxaliplatin treatment. The baseline ctDNA and ctDNA clearance status at the 7th week after the first dose of SCT200 administration before receiving SCT200 could be a potential prognostic biomarker for RAS and BRAF wild-type mCRC patients with SCT200 therapy. FUNDING: This study was sponsored by Sinocelltech Ltd., Beijing, China and partly supported by the National Science and Technology Major Project for Key New Drug Development (2019ZX09732001-006, 2017ZX09304015).

Regulatory mechanisms and cell membrane properties of Candida glycerinogenes differ under 2-phenylethanol addition or fermentation conditions.

The biosynthesis of 2-phenylethanol (2-PE) at high yields and titers is often limited by its toxicity. In this study, we describe the molecular mechanisms of 2-PE tolerance in the multi-stress tolerant industrial yeast, Candida glycerinogenes. They were different under 2-PE addition or fermentation conditions. After extracellular addition of 2-PE, C. glycerinogenes cells became rounder and bigger, which reduced specific surface area. However, during 2-PE fermentation C. glycerinogenes cells were smaller, which increased specific surface area. Other differences in the tolerance mechanisms were studied by analyzing the composition and molecular parameters of the cell membrane. Extracellular 2-PE stress resulted in down-regulation of transcriptional expression of unsaturated fatty acid synthesis genes. This raised the proportion of saturated fatty acids in the cell membrane, which increased rigidity of the cell membrane and reduced 2-PE entry to the cell. However, intracellular 2-PE stress resulted in up-regulation of transcriptional expression of unsaturated fatty acid synthesis genes, and increased the proportion of unsaturated fatty acids in the cell membrane; this in turn enhanced flexibility of the cell membrane which accelerated efflux of 2-PE. These contrasting mechanisms are mediated by transcriptional factors Hog1 and Swi5. Under 2-PE addition, C. glycerinogenes activated Hog1 and repressed Swi5 to upregulate erg5 and erg4 expression, which increased cell membrane rigidity and resisted 2-PE import. During 2-PE fermentation, C. glycerinogenes activated Hog1 and repressed Swi5 to upregulate 2-PE transporter proteins cdr1 and Acyl-CoA desaturase 1 ole1 to increase 2-PE export, thus reducing 2-PE intracellular toxicity. The results provide new insights into 2-PE tolerance mechanisms at the cell membrane level and suggest a novel strategy to improve 2-PE production by engineering anti-stress genes.

Construction of Triboelectric Series and Chirality Detection of Amino Acids Using Triboelectric Nanogenerator.

Triboelectrification necessitates a frictional interaction between two materials, and their contact electrification is characteristically based on the polarity variance in the triboelectric series. Utilizing this fundamental advantage of the triboelectric phenomenon, different materials can be identified according to their contact electrification capability. Herein, an in-depth analysis of the amino acids present in the stratum corneum of human skin is performed and these are quantified regarding triboelectric polarization. The principal focus of this study lies in analyzing and identifying the amino acids present in copious amounts in the stratum corneum to explain their positive behavior during the contact electrification process. Thus, an augmented triboelectric series of amino acids with quantified triboelectric charging polarity by scrutinizing the transfer charge, work function, and atomic percentage is presented. Furthermore, the chirality of aspartic acid as it is most susceptible to racemization with clear consequences on the human skin is detected. The study is expected to accelerate research exploiting triboelectrification and provide valuable information on the surface properties and biological activities of these important biomolecules.

Fine-Tuned Gene Expression Elements from Hybrid Promoter Libraries in Pichia pastoris.

As a desirable microbial cell factory, Pichia pastoris has garnered extensive utilization in metabolic engineering. Nevertheless, the lack of fine-tuned gene expression components has significantly constrained the potential scope of applications. Here, a gradient strength promoter library was constructed by random hybridization and high-throughput screening. The hybrid promoter, phy47, performed best with 2.93-fold higher GFP expression levels than GAP. The broad applicability of the novel hybrid promoter variants in biotechnological production was further validated in the biosynthesis of pinene and rHuPH20 with higher titers. The upstream regulatory sequences (UASE and URSD) were identified and applied to promoters GAP and ENO1, resulting in a 34 and 43% increase and an 18 and 37% decrease in the expression level, respectively. Yeast one-hybrid analysis showed that transcription factor HAP2 activates the hybrid promoter through a direct interaction with the crucial regulatory region UASH. Furthermore, a short segment of tunable activation sequence (20 bp) was also screened, and artificial promoters were constructed in tandem with the addition of regulatory sequence, resulting in a 61% expansion of the expression range. This study provides a molecular tool and regulatory elements for further synthetic biology research in P. pastoris.

Guanidinium-Functionalized Polymer Dielectrics for Triboelectric Bacterial Detection.

Development of rapid detection strategies that target potentially pathogenic bacteria has gained increasing attention due to the increasing awareness for better health and safety. In this study, we evaluate an intrinsically antimicrobial polymer, 2Gdm, which is a poly(norbornene)-based functional polymer featuring guanidinium groups as side chains, for bacterial detection by the means of triboelectric nanogenerators (TENGs) and triboelectric nanosensors (TENSs). Attachment of bacteria to the sensing layer is anticipated to alter the overall triboelectric properties of the underlying polymer layer. The positively charged guanidinium functional groups can interact with the negatively charged phospholipid bilayer of bacteria and lead to bacterial death, which can then be detected by optical microscopy, X-ray photoelectron microscopy, and more advanced self-powered sensing techniques such as TENGs and TENSs. The double bonds present along the poly(norbornene) backbone allow for thermally induced cross-linking to obtain X-2Gdm and thus rendering materials remain stable in water. By monitoring the change in voltage output after immersion in various concentrations of Gram-negative Escherichia coli (E. coli) and Gram-positive Streptococcus pneumoniae (S. pneumoniae), we have demonstrated the utility of X-2Gdm as a new polymer dielectric for autonomous bacterial detection. As the bacterial concentration increases, the amount of adsorbed bacteria also increases, resulting in a decrease in the surface potential of the X-2Gdm thin film; this reduction in surface potential can cause a decrease in the triboelectric output for both TENGs and TENSs, which serves as a key working mechanism for facile bacterial detection. TENG and TENS systems are capable of detecting E. coli and S. pneumoniae within a range of 4 × 105 to 4 × 108 CFU/mL with a limit of detection of 106 CFU/mL. This report highlights the promising prospects of employing TENGs and TENSs as innovative sensing technologies for rapid bacterial detection by leveraging the electrostatic interactions between bacterial cell membranes and cationic groups present on polymer surfaces.