Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa.

Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity. Given that two C-10 modified camptothecin derivatives, topotecan and irinotecan, have been approved as potent anticancer agents, there is a critical need for methods to access other aromatic ring-functionalized congeners (e.g., C-9, C-10, etc.). However, contemporary methods for chemical oxidation are generally harsh and low-yielding when applied to the camptothecin scaffold, thereby limiting the development of modified derivatives. Reported herein, we have identified four tailoring enzymes responsible for C-9 modifications of camptothecin from Nothapodytes tomentosa, via metabolomic and transcriptomic analysis. These consist of a cytochrome P450 (NtCPT9H) which catalyzes the regioselective oxidation of camptothecin to 9-hydroxycamptothecin, as well as two methyltransferases (NtOMT1/2, converting 9-hydroxycamptothecin to 9-methoxycamptothecin), and a uridine diphosphate-glycosyltransferase (NtUGT5, decorating 9-hydroxycamptothecin to 9-ß-D-glucosyloxycamptothecin). Importantly, the critical residues that contribute to the specific catalytic activity of NtCPT9H have been elucidated through molecular docking and mutagenesis experiments. This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering. This will hasten the discovery of novel C-9 modified camptothecin derivatives, with profound implications for pharmaceutical manufacture.

O-Alkylazoxymycins A-F, Naturally Occurring Azoxy-Aromatic Compounds from Streptomyces sp. Py50.

Six new azoxy-aromatic compounds (o-alkylazoxymycins A-F, 1-6) and two new nitrogen-bearing phenylvaleric/phenylheptanoic acid derivatives (o-alkylphemycins A and B, 7 and 8) were isolated from Streptomyces sp. Py50. Their structures were elucidated based on HRESIMS, NMR, UV spectroscopic analyses, and X-ray crystallographic data. O-Alkylazoxymycins A-F (1-6) are the first natural examples of azoxy compounds with the azoxy bond attached to the ortho-position of the phenylheptanoic acid or phenylvaleric acid moiety. Compounds 1, 5, and 6 were active against Epidermophyton floccosum with MIC50 values ranging from 10.1 to 51.2 µM. A plausible biosynthetic pathway of 2 and 3 was proposed.

Phytochemical Analysis of Nothapodytes tomentosa and Distribution and Content of Camptothecin and its Analogues in Four Plants.

Camptothecin (CPT) and its derivatives have attracted worldwide attention because of their notable anticancer activity. However, the growing demand for CPT in the global pharmaceutical industry has caused a severe shortage of CPT-producing plant resources. In this study, phytochemical analysis of Nothapodytes tomentosa results in the isolation and identification of CPT (13: ) and 16 analogues (1:  - 12, 14:  - 17: ), including a new (1: ) and five known (9, 10, 12, 15: , and 17: ) CPT analogues with an open E-ring. In view of the potential anticancer activity of CPT analogues with an open E-ring, the fragmentation pathways and mass spectra profiles of these six CPT analogues (1, 9, 10, 12, 15: , and 17: ) are investigated, providing a reference for the rapid detection of these compounds in other plants. Furthermore, based on the fragmentation patterns of CPT (13: ) and known analogues (2:  - 8, 11, 14, 16, 18:  - 26: ), the distribution and content of these compounds in different tissues of N. tomentosa, N. nimmoniana, Camptotheca acuminata, and Ophiorrhiza japonica are further studied. Our findings not only provide an alternative plant resource for further expanding the development and utilization of CPT and its analogues, but also lay a foundation for improving the utilization of known CPT-producing plant resources.

Discovery and Engineering of the Cocaine Biosynthetic Pathway.

Cocaine, the archetypal tropane alkaloid from the plant genus Erythroxylum, has recently been used clinically as a topical anesthesia of the mucous membranes. Despite this, the key biosynthetic step of the requisite tropane skeleton (methylecgonone) from the identified intermediate 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (MPOA) has remained, until this point, unknown. Herein, we identify two missing enzymes (EnCYP81AN15 and EnMT4) necessary for the biosynthesis of the tropane skeleton in cocaine by transient expression of the candidate genes in Nicotiana benthamiana. Cytochrome P450 EnCYP81AN15 was observed to selectively mediate the oxidative cyclization of S-MPOA to yield the unstable intermediate ecgonone, which was then methylated to form optically active methylecgonone by methyltransferase EnMT4 in Erythroxylum novogranatense. The establishment of this pathway corrects the long-standing (but incorrect) biosynthetic hypothesis of MPOA methylation first and oxidative cyclization second. Notably, the de novo reconstruction of cocaine was realized in N. benthamiana with the two newly identified genes, as well as four already known ones. This study not only reports a near-complete biosynthetic pathway of cocaine and provides new insights into the metabolic networks of tropane alkaloids (cocaine and hyoscyamine) in plants but also enables the heterologous synthesis of tropane alkaloids in other (micro)organisms, entailing significant implications for pharmaceutical production.

Tropane alkaloid biosynthesis: a centennial review.

Covering: 1917 to 2020Tropane alkaloids (TAs) are a remarkable class of plant secondary metabolites, which are characterized by an 8-azabicyclo[3.2.1]octane (nortropane) ring. Members of this class, such as hyoscyamine, scopolamine, and cocaine, are well known for their long history as poisons, hallucinogens, and anaesthetic agents. Since the structure of the tropane ring system was first elucidated in 1901, organic chemists and biochemists have been interested in how these mysterious tropane alkaloids are assembled in vitro and in vivo. However, it was only in 2020 that the complete biosynthetic route of hyoscyamine and scopolamine was clarified, and their de novo production in yeast was also achieved. The aim of this review is to present the innovative ideas and results in exploring the story of tropane alkaloid biosynthesis in plants from 1917 to 2020. This review also highlights that Robinson's classic synthesis of tropinone, which is one hundred years old, is biomimetic, and underscores the importance of total synthesis in the study of natural product biosynthesis.

Bisaspochalasins D and E: Two Heterocycle-Fused Cytochalasan Homodimers from an Endophytic Aspergillus flavipes.

Two heterocycle-fused cytochalasan homodimers, bisaspochalasins D (1) and E (2), were isolated from an endophytic Aspergillus flavipes. Their chemical structures were elucidated using a combination of HRESIMS, NMR, theoretical calculations, and crystallographic techniques. Bisaspochalasin D (1) is dimerized by the first reported naturally occurring triple heterobridged 3,8-dioxa-6-azabicyclo[3.2.1]octane framework, while bisaspochalasin E (2) employs a pyrrole ring as the linking moiety. Possible dimerization mechanisms of bisaspochalasins D and E were proposed. The bioassay screening revealed that bisaspochalasin D showed cytotoxic activities against five cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) with IC50 values ranging from 4.45 to 22.99 µM. Additionally, bisaspochalasin D exhibited neurotrophic activities in a PC12 cell-based assay. At a concentration of 10 µM, bisaspochalasin D can promote neurite growth by inducing a differentiation rate of 12.52% for PC12 cells.

Functional genomics analysis reveals two novel genes required for littorine biosynthesis.

Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood. Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS). UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added. This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

Antibacterial Pentacyclic Polyketides from a Soil-Derived Streptomyces.

Nine new pentacyclic polyketides, fasamycins G-K (1-5) and formicamycins N-Q (6-9), along with 10 known analogues (10-19), were isolated from a rhizospheric soil-derived Streptomyces sp. KIB-1414. Their structures and absolute configurations were elucidated by interpretation of NMR and HRMS data and comparisons of CD data. The compounds were active against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Bacillus subtilis, and Escherichia coli strains, with MIC values ranging from 0.20 to 50.00 µg/mL.

Benwamycins A-G, Trialkyl-Substituted Benzene Derivatives from a Soil-Derived Streptomyces.

Seven new trialkyl-substituted benzene derivatives named benwamycins A-G (1-7), together with three known congeners, 8-10, were isolated from culture broth of the soil-derived Streptomyces sp. KIB-H1471. Their structures were elucidated by using 1D and 2D NMR analyses in combination with HRESIMS data. The absolute configurations of 1-9 were determined by chemical conversion and comparison of circular dichroism spectra and confirmed for 1 by single-crystal X-ray crystallography. Compounds 6 and 7 have a unique γ-pyrone-like ring on one side chain. Compounds 2 and 6 inhibited human T cell proliferation with IC50 values of 14.3 and 12.5 µM, respectively, without obvious cytotoxicity for naïve human T cells. Compounds 3 and 6 could weakly enhance insulin-stimulated glucose uptake.

Carbon molecular sieve based micro-matrix-solid-phase dispersion for the extraction of polyphenols in pomegranate peel by UHPLC-Q-TOF/MS.

A rapid, simple, and efficient sample extraction method based on micro-matrix-solid-phase dispersion (micro-MSPD) was applied to the extraction of polyphenols from pomegranate peel. Five target analytes were determined by ultra-HPLC coupled with Q-TOF/MS. Carbon molecular sieve (CMS) was firstly used as dispersant to improve extraction efficiency in micro-MSPD. The major micro-MSPD parameters, such as type of dispersant, amount of dispersant, grinding time, and the type and the volume of elution solvents, were studied and optimized. Under optimized conditions, 26 mg of pomegranate peel was dispersed with 32.5 mg of CMS, the grinding time was selected as 90 s, the dispersed sample was eluted with 100 µL of methanol. Results showed that the proposed method was of good linearity for concentrations of analytes against their peak areas (coefficient of determination r2 > 0.990), the LOD was as low as 3.2 ng/mL, and the spiking recoveries were between 88.1 and 106%. Satisfactory results were obtained for the extraction of gallic acid, punicalagin A, punicalagin B, catechin, and ellagic acid from pomegranate peel sample, which demonstrated nice reliability and high sensitivity of this approach.

Lorneic Acid Analogues from an Endophytic Actinomycete.

Our natural products discovery program utilizes endophytic actinomycetes associated with plants and employs biological assays and HPLC-based metabolite profiles as the preliminary screen to identify strains of interest, followed by large-scale fermentation and isolation, leading to new and/or bioactive natural products. Six new trialkyl-substituted aromatic acids, namely, lorneic acids E-J (1-6), together with two known analogues (7 and 8), were isolated and identified from the culture extract of Streptomyces sp. KIB-H1289, an endophytic actinomycete obtained from the inner tissue of the bark of Betula mandshurica Nakai. The structures were characterized by interpretation of their spectroscopic data, mainly 1D and 2D NMR. Among them, compound 5 contains a unique disulfide bond that is presumably derived from N-acetylcysteine. All isolated metabolites were evaluated for their inhibitory activity on tyrosinase.

Evodiamine Suppresses ABCG2 Mediated Drug Resistance by Inhibiting p50/p65 NF-κB Pathway in Colorectal Cancer.

Evodiamine (Evo), extracted from the Chinese herbal medicine Evodia rutaecarpa, has cytotoxic effects on different types of human cancer cells. However, its effects on drug resistance and their molecular mechanism and therapeutic target in colorectal cancer are not well understood. In the present study, we observed that Evo inhibited cell growth and induced apoptosis in adose-and time-dependent manner in HCT-116/L-OHP cells. Moreover, Evo treatment reduced Rhodamine 123 accumulation and ATPase activity in HCT-116/L-OHP cells, indicating that Evo decreased the efflux function in HCT-116/L-OHP cells. Interestingly, phosphorylation of NF-κB pathway, particularly p50/p65, was also inhibited by Evo treatment. Furthermore the effect of Evo in reversing drug resistance and suppressing phosphorylation of NF-κB pathway were attenuated after treatment with the NF-κB activator (LPS). Additionally, Evo inhibited the tumor growth in a colorectal MDR cancer xenograft model and down regulated p-NF-κB level in vivo. Our study provided the first direct evidence that Evo can attenuate multidrug resistance by blocking p-NF-κB signaling pathway in human colorectal cancer. Evo could be a potential candidate for cancer chemotherapy.

Aerosol optical depth under "clear" sky conditions derived from sea surface reflection of lidar signals.

There are considerable demands for accurate atmospheric correction of satellite observations of the sea surface or subsurface signal. Surface and sub-surface reflection under "clear" atmospheric conditions can be used to study atmospheric correction for the simplest possible situation. Here "clear" sky means a cloud-free atmosphere with sufficiently small aerosol particles. The "clear" aerosol concept is defined according to the spectral dependence of the scattering cross section on particle size. A 5-year combined CALIPSO and AMSR-E data set was used to derive the aerosol optical depth (AOD) from the lidar signal reflected from the sea surface. Compared with the traditional lidar-retrieved AOD, which relies on lidar backscattering measurements and an assumed lidar ratio, the AOD retrieved through the surface reflectance method depends on both scattering and absorption because it is based on two-way attenuation of the lidar signal transmitted to and then reflected from the surface. The results show that the clear sky AOD derived from the surface signal agrees with the clear sky AOD available in the CALIPSO level 2 database in the westerly wind belt located in the southern hemisphere, but yields significantly higher aerosol loadings in the tropics and in the northern hemisphere.

[Effects of Couplet Medicines (Astragalus Membranaceus and Jiaozhen) on Intestinal Barrier in Postoperative Colorectal Cancer Patients].

OBJECTIVE: To observe the effect of the couplet medicines (Astragalus Membranaceus and Jiaozhen) on intestinal barrier functions of postoperative colorectal cancer patients. METHODS: Totally 90 inpatients with confirmed colorectal cancer by pathological diagnosis were recruited as subjects in this study. They were assigned to the Chinese medicine group (CM, treated with Astragalus Membranaceus and Jiaozhen), the Western medicine group (WM, treated with glutamine), and the blank control group (treated with normal saline) according to random digit table, 30 in each group. The treatment course consisted of eight days. Levels of blood D-lactic acid, diamine oxidase (DAO), urinary lactulose/mannitol ratio (L/M), ET, TNF-alpha, and postoperative recovery time of bowel sound were observed before surgery and after surgery. The effect of the couplet medicines (Astragalus Membranaceus and Jiaozhen) on intestinal barrier functions of postoperative colorectal cancer patients were comprehensively assessed by taking blood D-lactic acid levels, DAO levels, urinary L/M as main potency indices; ET and TNF-alpha, recovery time of bowel sound as the secondary potency indices. RESULTS: CM showed similar effect with that of WM in improving blood D-lactic acid levels and DAO levels, and urinary L/M ratio, with no statistical difference between them (P > 0.05). But they showed better effect than that of the blank control group (P < 0.05). Levels of ET and TNF-alpha were decreased more in the CM group than in the WM group (P < 0.05). The recovery time of bowel sound was shorter in the CM group than in the WM group (P < 0.05, P < 0.01). Levels of ET and TNF-alpha were decreased more in the WM group than in the blank control group (P < 0.05). There was no statistical difference in the recovery time of bowel sound between the WM group and the blank control group (P > 0.05). CONCLUSIONS: The couplet medicines (Astragalus Membranaceus and Jiaozhen) had obvious protection for intestinal barrier dysfunction of postoperative colorectal cancer patients, showing similar efficacy to that of WM. It was even superior to glutamine in restoring bowel functions, reducing toxin absorption, and lowering levels of pro-inflammatory factors.

Growth attenuation under saline stress is mediated by the heterotrimeric G protein complex.

BACKGROUND: Plant growth is plastic, able to rapidly adjust to fluctuation in environmental conditions such as drought and salinity. Due to long-term irrigation use in agricultural systems, soil salinity is increasing; consequently crop yield is adversely affected. It is known that salt tolerance is a quantitative trait supported by genes affecting ion homeostasis, ion transport, ion compartmentalization and ion selectivity. Less is known about pathways connecting NaCl and cell proliferation and cell death. Plant growth and cell proliferation is, in part, controlled by the concerted activity of the heterotrimeric G-protein complex with glucose. Prompted by the abundance of stress-related, functional annotations of genes encoding proteins that interact with core components of the Arabidopsis heterotrimeric G protein complex (AtRGS1, AtGPA1, AGB1, and AGG), we tested the hypothesis that G proteins modulate plant growth under salt stress. RESULTS: Na+ activates G signaling as quantitated by internalization of Arabidopsis Regulator of G Signaling protein 1 (AtRGS1). Despite being components of a singular signaling complex loss of the Gß subunit (agb1-2 mutant) conferred accelerated senescence and aborted development in the presence of Na+, whereas loss of AtRGS1 (rgs1-2 mutant) conferred Na+ tolerance evident as less attenuated shoot growth and senescence. Site-directed changes in the Gα and Gßγ protein-protein interface were made to disrupt the interaction between the Gα and Gßγ subunits in order to elevate free activated Gα subunit and free Gßγ dimer at the plasma membrane. These mutations conferred sodium tolerance. Glucose in the growth media improved the survival under salt stress in Col but not in agb1-2 or rgs1-2 mutants. CONCLUSIONS: These results demonstrate a direct role for G-protein signaling in the plant growth response to salt stress. The contrasting phenotypes of agb1-2 and rgs1-2 mutants suggest that G-proteins balance growth and death under salt stress. The phenotypes of the loss-of-function mutations prompted the model that during salt stress, G activation promotes growth and attenuates senescence probably by releasing ER stress.

Chromosome level genome assembly of endangered medicinal plant Anisodus tanguticus.

Anisodus tanguticus is a medicinal herb that belongs to the Anisodus genus of the Solanaceae family. This endangered herb is mainly distributed in Qinghai-Tibet Plateau. In this study, we combined the Illumina short-read, Nanopore long-read and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to de novo assemble the A. tanguticus genome. A high-quality chromosomal-level genome assembly was obtained with a genome size of 1.26 Gb and a contig N50 of 25.07 Mb. Of the draft genome sequences, 97.47% were anchored to 24 pseudochromosomes with a scaffold N50 of 51.28 Mb. In addition, 842.14 Mb of transposable elements occupying 66.70% of the genome assembly were identified and 44,252 protein-coding genes were predicted. The genome assembly of A. tanguticus will provide genetic repertoire to understand the adaptation strategy of Anisodus species in the plateau, which will further promote the conservation of endangered A. tanguticus resources.

Four unreported aporphine alkaloids with antifungal activities from Artabotrys hexapetalus.

In this study, the extract from Artabotrys hexapetalus showed strong antifungal activity against phytopathogenic fungi in vitro. Four unreported aporphine alkaloids, hexapetalusine A-D (1-4), were isolated from stems and roots of Artabotrys hexapetalus (L.f.) Bhandari, along with six known aporphine alkaloids (5-10). Their chemical structures were elucidated by extensive spectroscopic analysis. The absolute configurations of 1-3 were determined using single-crystal X-ray diffractions and ECD calculations. Hexapetalusine A-C (1-3) were special amidic isomers. Additionally, all isolated compounds were evaluated for their antifungal activity against four phytopathogenic fungi in vitro. Hexapetalusine D (4) exhibited weak antifungal activity against Curvularia lunata. Liriodenine (5) displayed significant antifungal activity against Fusarium proliferatum and Fusarium oxysporum f. sp. vasinfectum, which is obviously better than positive control nystatin, suggesting that it had great potential to be developed into an effective and eco-friendly fungicide.

Efficacy and safety of Tianqi Pingchan Granule, a compound Chinese herbal medicine, for levodopa-induced dyskinesia in Parkinson's disease: A randomized double-blind placebo-controlled trial.

BACKGROUND: Patients with Parkinson's disease (PD) undergoing long-term levodopa therapy are prone to develop levodopa-induced dyskinesia (LID). Amantadine is the main drug recommended for the treatment of LID by current guidelines, but it is far from meeting clinical needs. Tianqi Pingchan Granule (TPG), a compound Chinese herbal medicine, has been developed to relieve symptom of LID. OBJECTIVE: This randomized controlled trial evaluated the efficacy and safety of the combination of TPG and amantadine for LID. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: This is a randomized double-blind placebo-controlled trial, conducted from January 2020 to August 2021 at 6 sites in Jiangsu, Zhejiang and Shanghai, China. One hundred PD patients with ≥ 0.5 h of LID were randomly assigned to either the TPG plus amantadine group (TPG group) or the placebo plus amantadine group (placebo group), and treated for a period of 12 weeks. To ensure unbiased results, all study participants, investigators and sponsors were unaware of group allocations. Additionally, the data analysts remained blinded until the analysis was finalized. MAIN OUTCOME MEASURES: The primary outcome was assessed using the Unified Dyskinesia Rating Scale (UDysRS) (Range 0-104). The key secondary end point was improvement of motor and non-motor symptoms. Safety analyses included all enrolled patients. RESULTS: One hundred patients were enrolled and randomized into the two treatment groups. The changes in UDysRS at week 12 were -11.02 for the TPG group and -4.19 for the placebo group (treatment difference -6.83 [-10.53 to -3.12]; P = 0.0004). Adverse events were reported for 2 of 50 patients (4.0%) in each of the groups. CONCLUSION: This study indicated that a 12-week treatment of amantadine plus TPG effectively reduced UDysRS scores and was well tolerated, demonstrating the efficacy and safety of TPG for the treatment of LID in PD. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04173832. PLEASE CITE THIS ARTICLE AS: Zhang Y, Zhu XB, Zhao Y, Cui GY, Li WT, Yuan CX, Huang JP, Wan Y, Wu N, Song L, Zhao JH, Liang Y, Xu CY, Liu MJ, Gao C, Chen XX, Liu ZG. Efficacy and safety of Tianqi Pingchan Granule, a compound Chinese herbal medicine, for levodopa-induced dyskinesia in Parkinson's disease: A randomized double-blind placebo-controlled trial. J Integr Med. 2024; Epub ahead of print.