Biosensor-assisted evolution for high-level production of 4-hydroxyphenylacetic acid in Escherichia coli.

4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, and biochemicals, and requires sustainable production to meet increasing demand. Here, we use a 4HPAA biosensor to overcome the difficulty of conventional library screening in identification of preferred mutants. Strains with higher 4HPAA production and tolerance are successfully obtained by atmospheric and room temperature plasma (ARTP) mutagenesis coupled with adaptive laboratory evolution using this biosensor. Genome shuffling integrates preferred properties in the strain GS-2-4, which produces 25.42 g/L 4HPAA. Chromosomal mutations of the strain GS-2-4 are identified by whole genome sequencing. Through comprehensive analysis and experimental validation, important genes, pathways and regulations are revealed. The best gene combination in inverse engineering, acrD-aroG, increases 4HPAA production of strain GS-2-4 by 37% further. These results emphasize precursor supply and stress resistance are keys to efficient 4HPAA biosynthesis. Our work shows the power of biosensor-assisted screening of mutants from libraries. The methods developed here can be easily adapted to construct cell factories for the production of other aromatic chemicals. Our work also provides many valuable target genes to build cell factories for efficient 4HPAA production in the future.

Long-term outcome after perineural injection with 5% dextrose for carpal tunnel syndrome: a retrospective follow-up study.

OBJECTIVE: Perineural injection therapy with 5% dextrose water is progressively becoming a mainstream method for treating carpal tunnel syndrome. However, its long-term outcome is still unknown. Hence, the purpose of this retrospective study was to investigate the long-term outcome after perineural injection therapy using 5% dextrose water. METHODS: A total of 185 patients diagnosed with carpel tunnel syndrome at least 1 year post-therapy were enrolled. All the patients underwent ultrasound-guided perineural injection therapy using 10 ml of 5% dextrose water at the outpatient department. In a structured telephone interview, the patients were asked about the outcome post-therapy compared with pre-injection. A symptom relief ≥50% indicated effective outcome, and a symptom relief <50% was indicative of a poor outcome. RESULTS: In total, 88.6% patients reported an effective outcome, and 11.4% rated the outcome as poor, after a mean of 2.2 injections with a mean of 1-3 years' post-injection follow-up. The outcome was significantly related with severity level, and the patients that reported a poor outcome had a significantly higher incidence of severe grade compared with those who reported an effective outcome (52.4% vs 31.7%, P = 0.03). Patients with mild, moderate and severe grades, respectively, required an average of 1.7 (0.1), 2.4 (0.2) and 2.6 (0.3) injections to reach an effective outcome (P = 0.006) (severe vs mild, P = 0.008; moderate vs mild, P = 0.062). CONCLUSION: Perineural injection therapy is a novel approach for treatment of carpal tunnel syndrome with safe and outstanding long-term effects.

The Efficacy of Hyaluronic Acid for Carpal Tunnel Syndrome: A Randomized Double-Blind Clinical Trial.

OBJECTIVES: To investigate the effect of hyaluronic acid (HA) in patients diagnosed with mild or moderate carpal tunnel syndrome (CTS). DESIGN: A prospective randomized, double-blinded control study with 6 months of follow-up. SETTING: Rehabilitation outpatient clinic of one single medical center. SUBJECTS: Thirty-five participants with mild or moderate CTS. METHODS: Participants were enrolled and randomly assigned to HA or control groups. The HA group received one ultrasound-guided perineural injection of 2.5 mL HA while the control group received 2.5 mL normal saline injection through in-plane, long-axis approach to separate the median nerve from the flexor retinaculum via nerve hydrodissection. Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) scores were the primary outcome, while secondary outcomes included the numeric rating scale (NRS), electrophysiological domains, and the cross-sectional area of the median nerve. The assessment was conducted prior to injection and during the second week and 1-, 3-, and 6-months post-injection. RESULTS: Thirty-two patients (17 wrists in HA group and 15 wrists in control group) completed the study. Compared with the control group, the HA group did not show significantly superior outcomes, except in BCTQ and NRS at the second week post-injection (all P < .0125). CONCLUSIONS: A single ultrasound guided perineural HA injection may have short-term therapeutic efficacy for mild or moderate CTS; however, the 2-weeks superior efficacy was not beneficial for chronic neuropathy. Further studies with larger sample sizes are required to verify its therapeutic efficacy.

Comparison of short- and long-axis nerve hydrodissection for carpal tunnel syndrome: A prospective randomized, single-blind trial.

Background: This study is to compare the efficacy of short-axis hydrodissection with long-axis hydrodissection for patients with mild-to-moderate carpal tunnel syndrome (CTS). Methods: Forty-seven patients with mild-to-moderate CTS were enrolled in a prospective, randomized, single-blinded, controlled trial (6 months follow-up). With ultrasound guidance, patients in both groups (short-axis or long-axis groups) were injected with normal saline (5 mL per session). Assessments were performed before and 2 weeks after the injection, as well as at 1, 3, and 6 months post-intervention. The primary outcome measure was the Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) score and secondary outcomes included the cross-sectional area of the median nerve and electrophysiological studies. Results: Forty-four patients (21 wrists in the short-axis group and 23 wrists in the long-axis group) completed the study. Compared with the baseline, both groups showed improved BCTQ and cross-sectional area at all follow-up assessments (p<0.05). The short-axis group was not more effective except significant improvements in BCTQ-severity and BCTQ-function 1 month post-injection compared to the long-axis group (p = 0.031 and p = 0.023, respectively). Conclusions: Both short- and long-axis hydrodissection were effective for patients with mild-to-moderate CTS and the short-axis approach was not more effective than long-axis injection. Further studies with larger sample sizes, multiple injections, and larger injection volume are encouraged in the future.

One-Year Efficacy of Platelet-Rich Plasma for Moderate-to-Severe Carpal Tunnel Syndrome: A Prospective, Randomized, Double-Blind, Controlled Trial.

OBJECTIVE: To assess the therapeutic effect of platelet-rich plasma (PRP) for moderate-to-severe carpal tunnel syndrome (CTS). DESIGN: A prospective, randomized, double-blinded, controlled trial (1-year follow-up). SETTING: Outpatient of local medical center settings. PARTICIPANTS: Patients (N=26) who were diagnosed with bilateral moderate-to-severe CTS (total 52 wrists) were included. For each patient, one wrist was randomized into either the PRP or control group and the contralateral wrist of the same patient was allocated to another group. Twenty-four patients were included in the final data analysis. INTERVENTIONS: The wrists in the PRP group received a single ultrasound-guided dose of PRP injection (3.5mL), and the control group received a single ultrasound-guided injection with normal saline (3.5mL). MAIN OUTCOME MEASURES: The Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) scores were used as the primary outcome. Secondary outcomes encompassed the cross-sectional area of the median nerve and electrophysiological study. Assessments were conducted prior to injection and 1, 3, 6, and 12 months postinjection. RESULTS: Compared to the control group, the PRP group exhibited significant improvements in BCTQ severity scores at all time points, BCTQ functional scores at the sixth month, and cross-sectional area at the 12th month postinjection (P<.0125). CONCLUSIONS: A single dose of ultrasound-guided perineural PRP injection can provide therapeutic effect for 1 year postinjection.

Outcome predictors of platelet-rich plasma injection for moderate carpal tunnel syndrome.

INTRODUCTION: Platelet-rich plasma (PRP) injection is effective for mild-to-moderate carpal tunnel syndrome (CTS), and physicians have been using PRP injections to treat CTS. However, the predictive factors of PRP injections have not been evaluated. This retrospective study sought to identify the predictive factors of PRP injections in patients with moderate CTS. METHODS: Seventy-one patients with moderate CTS receiving single PRP injections were enrolled. The outcomes at the third- and sixth-month postinjection visits were categorised into good and poor groups according to the following: (1) good outcome, with visual analogue scale (VAS) score decrease ≧50% and (2) poor outcome, with VAS score decrease <50% of preinjection scores. Significant variables between groups were entered into a binary logistic regression to determine the predictive factors. RESULTS: The baseline body weight (BW), distal motor latency (DML), sensory nerve conduction velocity (SNCV), and cross-sectional area (CSA) of the median nerve were significantly different between the groups in the third month. The odds ratios (ORs) of all features were significant, except for SNCV (BW, OR: 0.911; P = .016; DML, OR: 0.383; P = .028; CSA, OR: 0.694; P = .003), and they remained significant in the sixth month (BW, OR: 0.909; P = .004; DML, OR: 0.530; P = .011; CSA, OR: 0.828; P = .032). CONCLUSION: Lower BW, DML, and CSA values of the median nerve predict better outcomes after perineural injection of PRP for moderate CTS at the 3- and 6-month follow-ups.

Contrast-enhanced ultrasound-guided celiac plexus neurolysis in patients with upper abdominal cancer pain: initial experience.

OBJECTIVES: The purpose of this study was to determine the efficacy and safety of contrast-enhanced ultrasound (CEUS)-guided celiac plexus neurolysis (CPN) in patients with upper abdominal cancer pain. METHODS: Thirty-five patients with upper abdominal cancers tortured by intractable upper abdominal pain underwent CEUS-guided CPN with ethanol. The pain alleviation and opioid intake were observed and evaluated during a 3-month follow-up after CPN. The dispersion of alcohol around the aorta was evaluated on 3D-CEUS. Complications were assessed during CPN and at follow-up. RESULTS: All of the 35 patients' CPN was successfully achieved. Pain relief was observed in 28 (80%), 20 (57.1%), 27 (77.1%), 20 (57.1%), and 10 (29.4%) patients immediately, 1 day, 1 month, 2 months, and 3 months after CPN, respectively. The agent dispersion around the aorta on CEUS images of 28 patients who showed pain relief was at least 90° of the circumference around the aorta. The median duration of pain alleviation was 2.7 months (95% confidence interval [CI], 2.5-2.9). Less than half of the patients had minor complications including irritant pain at the puncture site (8 of 35; 22.9%), diarrhea (4 of 35; 11.4%), nausea and vomiting (3 of 35; 8.6%), and post-procedural hypotension (1 of 35; 2.9%). CONCLUSIONS: CEUS-guided CPN is a safe and effective method to alleviate refractory upper abdominal pain in patients with upper abdominal cancers. CEUS image allows the visualization of puncture path and observation of drug dispersion. The pain relief is relevant to the dispersion of neurolytic agent around the aorta. KEY POINTS: • CEUS-guided celiac plexus neurolysis (CPN) is feasible and easy. • It allows direct visualization of the diffusion of the neurolytic agent in the retroperitoneal anatomic space. • CEUS-guided CPN improves safety of CPN by clearly delineating the needle path.

Perineural Dextrose and Corticosteroid Injections for Ulnar Neuropathy at the Elbow: A Randomized Double-blind Trial.

OBJECTIVE: To assess the effects of perineural corticosteroid and 5% dextrose water (D5W) injections in patients with mild to moderate ulnar neuropathy at the elbow (UNE). DESIGN: Prospective, randomized, double-blind, controlled trial (6-month follow-up). SETTING: Outpatients of local medical center settings. PARTICIPANTS: Patients (N=36) with mild to moderate UNE were randomized, and 33 participants were included in the final data analysis. INTERVENTIONS: Patients were administered a single perineural injection with 5 mL D5W and 3 mL corticosteroid (triamcinolone acetonide, 10mg/mL) mixed with 2 mL normal saline under ultrasound guidance in the dextrose and steroid groups, respectively. MAIN OUTCOME MEASURES: The visual analog scale digital pain or paresthesia/dysesthesia score was the primary outcome. The secondary outcomes were the Disabilities of the Arm, Shoulder, and Hand questionnaire, motor nerve conduction velocity, and cross-sectional area (CSA) of the ulnar nerve. The measurement assessment was conducted before and 1, 3, 4, and 6 months after injection. RESULTS: Thirty-three patients completed the study. Both injections were found to be equally effective at most measurement points, although the dextrose group experienced larger reductions in symptom severity and CSA of the ulnar nerve from the third month onward. CONCLUSIONS: We suggest D5W as a more suitable injectate for perineural injection in patients with UNE.

Potentides: New Cysteine-Rich Peptides with Unusual Disulfide Connectivity from Potentilla anserina.

Cysteine-rich peptides (CRPs), which are disulfide-constrained peptides with 3 to 5 disulfide bonds and molecular weights of 2 to 6 kDa, are generally hyperstable and resistant to thermal, chemical, and enzymatic degradation. Herein, the discovery and characterization of a novel suite of CRPs, collectively named potentides pA1-pA16 from the root of the medicinal herb Potentilla anserina L, are described. Through a combination of proteomic and transcriptomic methods, it is shown that 35-residue potentide pA3, which is the most abundant member of potentides, exhibits high stability against heat, acidic, and proteolytic degradation. Transcriptomic analysis revealed that potentide precursor sequences contained four tandem repeats in the mature domain, which is the first report on tandem repeats being found in the Rosaceae family. Disulfide mapping showed that potentide pA3 displayed a novel disulfide connectivity of C1-C3, C2-C6, and C4-C5; a cystine motif that has not been reported in plant CRPs. Transcriptomic data mining and a neighbor-joining clustering analysis revealed 56 potentide homologues and their distribution in the families of Rosaceae and Ranunculaceae in angiosperm. Altogether, these results reveal a new plant CRP structure with an unusual cystine connectivity. Additionally, this study expands the families and structure diversity of CRPs as potentially active peptide pharmaceuticals.

Herpes zoster increased risk of neuralgic amyotrophy: a retrospective, population-based matched cohort study.

Although neuralgic amyotrophy (NA) has occasionally been reported to be associated with reactivated herpes zoster, their associated risk remains unknown. The aim of this study was to assess the risk of developing NA following preceding herpes zoster. The authors used the National Health Insurance Research Database of Taiwan to select 41,548 patients with newly diagnosed herpes zoster during the period 2000 to 2010 and randomly extracted 166,192 matched control subjects. All participants in the study and control groups were followed for 3 months after the diagnosis to identify those who developed NA. Cox proportional hazards regression analyses were performed to evaluate the subsequent risk of NA. Twenty-one subjects from the group with herpes zoster (0.05%) developed NA over the 3-month period and 46 from the group without herpes zoster (0.03%). The patients with herpes zoster had a higher risk of developing NA (adjusted hazard ratio = 1.408, 95% confidence interval = 1.013-2.319, P = 0.030). In the patients with herpes zoster, female sex, age ≥ 65, hepatitis E virus (HEV), and having had a recent infectious event including pneumonia and influenza were risk factors for developing NA (adjusted HR 2.746, 1.998, 2.735, 2.016, and 1.718, respectively, all P < 0.05). Patients with herpes zoster attack have a higher risk of developing NA over a 3-month period after diagnosis, especially those who are female, age ≥ 65, HEV, or have experienced a recent infectious event or pneumonia and influenza.

Randomized double-blinded clinical trial of 5% dextrose versus triamcinolone injection for carpal tunnel syndrome patients.

OBJECTIVE: Perineural injection with 5% dextrose (D5W) is a novel strategy in the treatment of carpal tunnel syndrome (CTS). In contrast, perineural injection with corticosteroid has been used for decades for treating CTS, but possible neurotoxicity has been a major concern. No studies investigating the comparative effects have been published so far. The authors performed a prospective, randomized, double-blinded, head-to-head comparative trial to compare these two approaches for patients having mild-to-moderate CTS. METHODS: Fifty-four participants with mild-to-moderate CTS were randomly divided into dextrose and steroid groups. The patients were administered 1 session of perineural injection with 5ml D5W (dextrose group) or 3ml triamcinolone acetonide mixed with 2ml normal saline (steroid group), under ultrasound guidance. A visual analog scale was assigned to assess the primary outcome. The secondary outcomes were assessed using the Boston Carpal Tunnel Syndrome Questionnaire, cross-sectional area of the median nerve, and electrophysiological studies. The assessment was performed prior to injection and 1, 3, 4, and 6 months postinjection. RESULTS: All patients (27 wrists per group) completed the study. Compared with the steroid group, the dextrose group exhibited a significant reduction in pain and disability through the 4th to the 6th month (p < 0.01). INTERPRETATION: Our study demonstrates that perineural injection of D5W is more beneficial than that of corticosteroid in patients with mild-to-moderate CTS at 4 to 6 months postinjection. Ann Neurol 2018;84:601-610.

Nerve hydrodissection for carpal tunnel syndrome: A prospective, randomized, double-blind, controlled trial.

INTRODUCTION: In this study we explored the efficacy of nerve hydrodissection for mild-to-moderate carpal tunnel syndrome (CTS). METHODS: Thirty-four participants were randomly assigned to an intervention group or a control group. One 5-ml dose of normal saline was injected into the intracarpal and subcutaneous regions in subjects of both groups, respectively. The primary outcome measure was the Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) score. Secondary outcomes were cross-sectional area of the median nerve and electrophysiological studies. Assessments were performed before the injection and at 1, 2, 3, and 6 months postintervention. RESULTS: Compared with the control group, the intervention group showed significantly greater improvement at the second and third posttreatment months according to BCTQ severity score and at all time-points for cross-sectional area of the median nerve (P < 0.01). DISCUSSION: Our study demonstrates the therapeutic effects of nerve hydrodissection for mild-to-moderate CTS. Muscle Nerve 59:174-180, 2019.

Metabolic evolution and a comparative omics analysis of Corynebacterium glutamicum for putrescine production.

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor L-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.

The Applications of Promoter-gene-Engineered Biosensors.

A promoter is a small region of a DNA sequence that responds to various transcription factors, which initiates a particular gene expression. The promoter-engineered biosensor can activate or repress gene expression through a transcription factor recognizing specific molecules, such as polyamine, sugars, lactams, amino acids, organic acids, or a redox molecule; however, there are few reported applications of promoter-enhanced biosensors. This review paper highlights the strategies of construction of promoter gene-engineered biosensors with human and bacteria genetic promoter arrays with regard to high-throughput screening (HTS) molecular drugs, the study of the membrane protein's localization and nucleocytoplasmic shuttling mechanism of regulating factors, enzyme activity, detection of the toxicity of intermediate chemicals, and probing bacteria density to improve value-added product titer. These biosensors' sensitivity and specificity can be further improved by the proposed approaches of Mn2+ and Mg2+ added random e error-prone PCR that is a technique used to generate randomized genomic libraries and site-directed mutagenesis approach, which is applied for the construction of bacteria's "mutant library". This is expected to establish a flexible HTS platform (biosensor array) to large-scale screen transcription factor-acting drugs, reduce the toxicity of intermediate compounds, and construct a gene-dynamic regulatory system in "push and pull" mode, in order to effectively regulate the valuable medicinal product production. These proposed novel promoter-engineered biosensors aiding in synthetic genetic circuit construction will maximize the efficiency of the bio-synthesis of medicinal compounds, which will greatly promote the development of microbial metabolic engineering and biomedical science.

Preparation of isoquercitrin and rhamnose from readily accessible rutin by a highly specific recombinant α-L-rhamnosidase (r-Rha1).

Isoquercitrin has superior in vivo bioactivities with respect to its primary glycoside rutin. Its conventional preparation was ineffective, with large chemical consumption and many by-products. Rhamnose, a high value-added monosaccharide, is usually separated from acid hydrolytes of rutin. This study aimed to establish a novel enzymatic hydrolysis-based approach for their preparation. α-L-rhamnosidase was expressed in Pichia pastoris GS115 and applied to enzymolysis of rutin. Then, one-factor-at-a-time optimisation of hydrolysis conditions was performed. Two compounds were produced in 0.02 M HAc-NaAc buffer (pH4.50) containing α-L-rhamnosidase/rutin (1:4, w/w) at 60 °C. Consequently, 20.0 g/L rutin was completely hydrolysed in 2 hrs, and isoquercitrin was obtained after purification by HPD-100 resin. Additionally, rhamnose was enriched by decolorisation and crystallisation. MD simulation analysis suggested that rutin was catalysed on the hydrophobic surface of r-Rha1 with van-der-Waals force being main driving force. This strategy is an efficient approach for preparation of isoquercitrin and rhamnose.

Purification and immobilization of ß-glucosidase using surface modified mesoporous silica Santa Barbara Amorphous 15 for eco-friendly preparation of sagittatoside A.

Functionalized mesoporous materials have become a promising carrier for enzyme immobilization. In this study, Santa Barbara Amorphous 15 (SBA-15) was modified by N-aminoethyl-γ-aminopropyl trimethoxy (R). R-SBA-15 was employed to purify and immobilize recombinant ß-glucosidase from Terrabacter ginsenosidimutans (BgpA) in one step for the first time. Optimum pH of the constructed R-SBA-15@BgpA were 7.0, and it has 20 â„ƒ higher optimal temperature than free enzyme. Relative activity of R-SBA-15@BgpA still retained > 70% at 42 â„ƒ after 8-h incubation. The investigation on organic reagent resistance revealed that the immobilized enzyme can maintain strong stability in 15% DMSO. In leaching test and evaluation of storage stability, only trace amount of protein was detected in buffer of the immobilized enzyme after storage at 4 â„ƒ for 33 days, and the immobilized BgpA still maintained > 50% relative activity. It also demonstrated good reusability, with 76.1% relative activity remaining after fourteen successive enzymatic hydrolyses of epimedin A to sagittatoside A. The newly proposed strategy is an effective approach for the purification and immobilization of BgpA concurrently. In addition, R-SBA-15@BgpA was demonstrated to have high efficiency and stability in this application, suggesting its great feasibility and potential to produce bioactive compounds such as secondary glycosides or aglycones from natural products.

IBI310 (anti-CTLA-4 antibody) monotherapy or in combination with sintilimab in advanced melanoma or urothelial carcinoma.

IBI310 is a recombinant fully human IgG1 antibody against cytotoxic T lymphocyte antigen 4. This study was conducted to evaluate IBI310 monotherapy or combination therapy with sintilimab in the patients with advanced melanoma or urothelial carcinoma (UC). Patients in phase 1a received IBI310 at 0.3/1/2/3 mg/kg intravenously (IV) every 3 weeks (Q3W) following the accelerated titration and 3 + 3 escalation design. Patients in phase 1b received IBI310 (1/2/3 mg/kg IV, Q3W) plus sintilimab (200 mg IV, Q3W) for four cycles, followed by sintilimab maintenance therapy. The phase 1b expansion of IBI310 plus sintilimab was performed in patients with advanced melanoma or UC. Overall, 53 patients were enrolled, including 10 patients with melanoma in phase 1a, 34 with melanoma, and 9 with UC in phase 1b. Overall, 94.3% of patients (50/53) experienced at least one treatment-related adverse event (TRAE) with most being grade 1-2; 26.4% of patients (14/53) experienced grade 3 or higher TRAEs. In phase 1a, the disease control rate (DCR) was 50.0% (95% confidence interval [CI], 18.7%-81.3%). In phase 1b, the objective response rate (ORR) and DCR were 17.6% (95% CI, 6.8%-34.5%) and 44.1% (95% CI, 27.2%-62.1%), respectively, for melanoma, and were 22.2% (95% CI, 2.8%-60.0%) and 66.7% (95% CI, 29.9%-92.5%), respectively, for UC. IBI310 monotherapy or combination therapy with sintilimab was well tolerated with favorable antitumor activity across patients with advanced melanoma and UC.

[Dynamic regulation using a quorum-sensing circuit enhances the production of tyrosol by Escherichia coli].

Tyrosol is a natural polyphenolic product that is widely used in chemical, pharmaceutical and food industries. Currently, the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield. Therefore, the phenylpyruvate decarboxylase mutant ARO10F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression, and the optimal ARO10F138L/D218G-L-YahK produced 1.09 g/L tyrosol in shake flasks. In order to further improve tyrosol production, feaB, a key gene in the competing pathway of 4-hydroxyphenylacetic acid, was knocked out, and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15% compared to that of the control. To overcome the low cell density in tyrosol fermentation, the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway, so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition. Using this strategy, the yield of tyrosol was increased to 1.74 g/L, a 33.82% increase. In a 2 L fermenter, the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD600 of 42.88. Compared with those in the engineered strain TRF5 statically regulated by induced expression, the yield was increased by 38.58% and the OD600 was enhanced by 43.62%. The combination of blocking the competing pathway using gene knockout technology, and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol. This study may facilitate the biosynthesis of other chemicals with high toxicity.

Multidisciplinary strategies to enhance therapeutic effects of flavonoids from Epimedii Folium: Integration of herbal medicine, enzyme engineering, and nanotechnology.

Flavonoids such as baohuoside I and icaritin are the major active compounds in Epimedii Folium (EF) and possess excellent therapeutic effects on various diseases. Encouragingly, in 2022, icaritin soft capsules were approved to reach the market for the treatment of hepatocellular carcinoma (HCC) by National Medical Products Administration (NMPA) of China. Moreover, recent studies demonstrate that icaritin can serve as immune-modulating agent to exert anti-tumor effects. Nonetheless, both production efficiency and clinical applications of epimedium flavonoids have been restrained because of their low content, poor bioavailability, and unfavorable in vivo delivery efficiency. Recently, various strategies, including enzyme engineering and nanotechnology, have been developed to increase productivity and activity, improve delivery efficiency, and enhance therapeutic effects of epimedium flavonoids. In this review, the structure-activity relationship of epimedium flavonoids is described. Then, enzymatic engineering strategies for increasing the productivity of highly active baohuoside I and icaritin are discussed. The nanomedicines for overcoming in vivo delivery barriers and improving therapeutic effects of various diseases are summarized. Finally, the challenges and an outlook on clinical translation of epimedium flavonoids are proposed.

A pH-sensitive intelligent packaging film harnessing Dioscorea zingiberensis starch and anthocyanin for meat freshness monitoring.

Abundant starch was isolated from Dioscorea zingiberensis C.H. Wright, a novel and underutilized industrial crop resource. In this study, an intelligent packaging film able to indicate food freshness was developed and characterized. D. zingiberensis starch (DZS) was bleached first, and its particle size, total starch content, amylose content, and gelatinization temperature were then measured. Butterfly pea (Clitoria ternatea Linn.) flowers were selected as the source of polyphenols, which rendered the prepared film intelligent and progressively blue-violet. SEM and FT-IR analyses showed the homogeneous dispersion of butterfly pea flower extract (BPE) in the film. The BPE-loaded film showed improved flexibility and resistance to UV and oxidation while maintaining sufficient mechanical strength and physical properties. Moreover, the film underwent a distinguishable color change from red to blue-violet and finally to green-yellow with increasing pH from 2 to 13. Similar color alteration also occurred when the film was exposed to ammonia. When the film was used to monitor the freshness of chicken stored at room temperature, it exhibited an obvious color change, implying its deterioration. Therefore, the newly developed BPE-DZS film, which was produced from readily accessible natural substances, can serve as an intelligent packaging material, indicating food freshness and prolonging shelf life.