Mitochondrial One-Carbon Pathway Supports Cytosolic Folate Integrity in Cancer Cells.

Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.

The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

An atlas of substrate specificities for the human serine/threonine kinome.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

Ultrapotent influenza hemagglutinin fusion inhibitors developed through SuFEx-enabled high-throughput medicinal chemistry.

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.

Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools.

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

Advances in Nonreactive Polymer Compatibilizers for Commodity Polyolefin Blends.

Recycling mixed polyolefin plastics is a significant challenge due to the limitations in sorting and degraded mechanical properties of blends. Nonreactive compatibilization by adding a small amount of polymeric additive is a widespread approach to restoring the performance and value of recycled plastics. Over the past several decades, synthetic advances have enabled access to low-cost copolymers and precision architectures for deepening the understanding of compatibilization mechanisms in semicrystalline polyolefins. This review covers the design parameters of a polymeric compatibilizer, the testing of blends, the synthetic methods of producing economically viable additives, and surveys the literature of blends of compatibilized HDPE, LLDPE, LDPE, and iPP. From this, readers should gain a comprehension of the polymer mechanics, synthesis, and macromolecular engineering of processable polyolefin blends, along with the field's future directions.

Ferroelastically protected reversible orthorhombic to monoclinic-like phase transition in ZrO2 nanocrystals.

Robust ferroelectricity in nanoscale fluorite oxide-based thin films enables promising applications in silicon-compatible non-volatile memories and logic devices. However, the polar orthorhombic (O) phase of fluorite oxides is a metastable phase that is prone to transforming into the ground-state non-polar monoclinic (M) phase, leading to macroscopic ferroelectric degradation. Here we investigate the reversibility of the O-M phase transition in ZrO2 nanocrystals via in situ visualization of the martensitic transformation at the atomic scale. We reveal that the reversible shear deformation pathway from the O phase to the monoclinic-like (M') state, a compressive-strained M phase, is protected by 90° ferroelectric-ferroelastic switching. Nevertheless, as the M' state gradually accumulates localized strain, a critical tensile strain can pin the ferroelastic domain, resulting in an irreversible M'-M strain relaxation and the loss of ferroelectricity. These findings demonstrate the key role of ferroelastic switching in the reversibility of phase transition and also provide a tensile-strain threshold for stabilizing the metastable ferroelectric phase in fluorite oxide thin films.

Concepts Relevant for the Kinetic Analysis of Reversible Reaction Systems.

The net rate of a reversible chemical reaction is the difference between unidirectional rates of traversal along forward and reverse reaction paths. In a multistep reaction sequence, the forward and reverse trajectories, in general, are not the microscopic reverse of one another; rather, each unidirectional route is comprised of distinct rate-controlling steps, intermediates, and transition states. Consequently, traditional descriptors of rate (e.g., reaction orders) do not reflect intrinsic kinetic information but instead conflate unidirectional contributions determined by (i) the microscopic occurrence of forward/reverse reactions (i.e., unidirectional kinetics) and (ii) the reversibility of reaction (i.e., nonequilibrium thermodynamics). This review aims to provide a comprehensive resource of analytical and conceptual tools which deconvolute the contributions of reaction kinetics and thermodynamics to disambiguate unidirectional reaction trajectories and precisely identify rate- and reversibility-controlling molecular species and steps in reversible reaction systems. The extrication of mechanistic and kinetic information from bidirectional reactions is accomplished through equation-based formalisms (e.g., De Donder relations) grounded in principles of thermodynamics and interpreted in the context of theories of chemical kinetics developed in the past 25 years. The aggregate of mathematical formalisms detailed herein is general to thermochemical and electrochemical reactions and encapsulates a diverse body of scientific literature encompassing chemical physics, thermodynamics, chemical kinetics, catalysis, and kinetic modeling.

BCLAF1 binds SPOP to stabilize PD-L1 and promotes the development and immune escape of hepatocellular carcinoma.

Interaction between programmed death-1 (PD-1) ligand 1 (PD-L1) on tumor cells and PD-1 on T cells allows tumor cells to evade T cell-mediated immune surveillance. Strategies targeting PD-1/PD-L1 have shown clinical benefits in a variety of cancers. However, limited response rates in hepatocellular carcinoma (HCC) have prompted us to investigate the molecular regulation of PD-L1. Here, we identify B cell lymphoma-2-associated transcription factor 1 (BCLAF1) as a key PD-L1 regulator in HCC. Specifically, BCLAF1 interacts with SPOP, an E3 ligase that mediates the ubiquitination and degradation of PD-L1, thereby competitively inhibiting SPOP-PD-L1 interaction and subsequent ubiquitination and degradation of PD-L1. Furthermore, we determined an SPOP-binding consensus (SBC) motif mediating the BCLAF1-SPOP interaction on BCLAF1 protein and mutation of BCLAF1-SBC motif disrupts the regulation of the SPOP-PD-L1 axis. In addition, BCLAF1 expression was positively correlated with PD-L1 expression and negatively correlated with biomarkers of T cell activation, including CD3 and CD8, as well as with the level of immune cell infiltration in HCC tissues. Besides, BCLAF1 depletion leads to a significant reduction of PD-L1 expression in vitro, and this reduction of PD-L1 promoted T cell-mediated cytotoxicity. Notably, overexpression of BCLAF1 sensitized tumor cells to checkpoint therapy in an in vitro HCC cells-Jurkat cells co-culture model, whereas BCLAF1-SBC mutant decreased tumor cell sensitivity to checkpoint therapy, suggesting that BCLAF1 and its SBC motif serve as a novel therapeutic target for enhancing anti-tumor immunity in HCC.

Cryo-EM structure of the fully assembled Elongator complex.

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.

The multi-subunit Elongator complex mediates the addition of a carboxymethyl group to wobble uridines in eukaryotic tRNAs. This tRNA modification is crucial to preserve the integrity of cellular proteomes and to protects us against severe neurodegenerative diseases. Elongator is organized in two distinct modules (i) the larger Elp123 subcomplex that binds and modifies the suitable tRNA substrate and (ii) the smaller Elp456 subcomplex that assists the release of the modified tRNA. The presented cryo-EM structures of Elongator show that the assemblies are very dynamic and undergo conformational rearrangements at consecutive steps of the process. Last but not least, the study provides a detailed reaction scheme and shows that the architecture of Elongator is highly conserved from yeast to mammals.

Boosting the reaction kinetics in aprotic lithium-carbon dioxide batteries with unconventional phase metal nanomaterials.

Given the high energy density and eco-friendly characteristics, lithium-carbon dioxide (Li-CO2) batteries have been considered to be a next-generation energy technology to promote carbon neutral and space exploration. However, Li-CO2 batteries suffer from sluggish reaction kinetics, causing large overpotential and poor energy efficiency. Here, we observe enhanced reaction kinetics in aprotic Li-CO2 batteries with unconventional phase 4H/face-centered cubic (fcc) iridium (Ir) nanostructures grown on gold template. Significantly, 4H/fcc Ir exhibits superior electrochemical performance over fcc Ir in facilitating the round-trip reaction kinetics of Li+-mediated CO2 reduction and evolution, achieving a low charge plateau below 3.61 V and high energy efficiency of 83.8%. Ex situ/in situ studies and theoretical calculations reveal that the boosted reaction kinetics arises from the highly reversible generation of amorphous/low-crystalline discharge products on 4H/fcc Ir via the Ir-O coupling. The demonstration of flexible Li-CO2 pouch cells with 4H/fcc Ir suggests the feasibility of using unconventional phase nanomaterials in practical scenarios.

New Insight into Bulk Structural Degradation of High-Voltage LiCoO2 at 4.55 V.

The aggravated mechanical and structural degradation of layered oxide cathode materials upon high-voltage charging invariably causes fast capacity fading, but the underlying degradation mechanisms remain elusive. Here we report a new type of mechanical degradation through the formation of a kink band in a Mg and Ti co-doped LiCoO2 cathode charged to 4.55 V (vs Li/Li+). The local stress accommodated by the kink band can impede crack propagation, improving the structural integrity in a highly delithiated state. Additionally, machine-learning-aided atomic-resolution imaging reveals that the formation of kink bands is often accompanied by the transformation from the O3 to O1 phase, which is energetically favorable as demonstrated by first-principles calculations. Our results provide new insights into the mechanical degradation mechanism of high-voltage LiCoO2 and the coupling between electrochemically triggered mechanical failures and structural transition, which may provide valuable guidance for enhancing the electrochemical performance of high-voltage layered oxide cathode materials for lithium-ion batteries.

The diverse structural modes of tRNA binding and recognition.

tRNAs are short noncoding RNAs responsible for decoding mRNA codon triplets, delivering correct amino acids to the ribosome, and mediating polypeptide chain formation. Due to their key roles during translation, tRNAs have a highly conserved shape and large sets of tRNAs are present in all living organisms. Regardless of sequence variability, all tRNAs fold into a relatively rigid three-dimensional L-shaped structure. The conserved tertiary organization of canonical tRNA arises through the formation of two orthogonal helices, consisting of the acceptor and anticodon domains. Both elements fold independently to stabilize the overall structure of tRNAs through intramolecular interactions between the D- and T-arm. During tRNA maturation, different modifying enzymes posttranscriptionally attach chemical groups to specific nucleotides, which not only affect translation elongation rates but also restrict local folding processes and confer local flexibility when required. The characteristic structural features of tRNAs are also employed by various maturation factors and modification enzymes to assure the selection, recognition, and positioning of specific sites within the substrate tRNAs. The cellular functional repertoire of tRNAs continues to extend well beyond their role in translation, partly, due to the expanding pool of tRNA-derived fragments. Here, we aim to summarize the most recent developments in the field to understand how three-dimensional structure affects the canonical and noncanonical functions of tRNA.

Solid-Phase Synthesis of Well-Defined Multiblock Copolymers by Atom Transfer Radical Polymerization.

Solid-phase polymer synthesis, historically rooted in peptide synthesis, has evolved into a powerful method for achieving sequence-controlled macromolecules. This study explores solid-phase polymer synthesis by covalently immobilizing growing polymer chains onto a poly(ethylene glycol) (PEG)-based resin, known as ChemMatrix (CM) resin. In contrast to traditional hydrophobic supports, CM resin's amphiphilic properties enable swelling in both polar and nonpolar solvents, simplifying filtration, washing, and drying processes. Combining atom transfer radical polymerization (ATRP) with solid-phase techniques allowed for the grafting of well-defined block copolymers in high yields. This approach is attractive for sequence-controlled polymer synthesis, successfully synthesizing di-, tri-, tetra-, and penta-block copolymers with excellent control over the molecular weight and dispersity. The study also delves into the limitations of achieving high molecular weights due to confinement within resin pores. Moreover, the versatility of the method is demonstrated through its applicability to various monomers in organic and aqueous media. This straightforward approach offers a rapid route to developing tailored block copolymers with unique structures and functionalities.

Evolution of genome and immunogenome in esophageal squamous cell carcinomas driven by neoadjuvant chemoradiotherapy.

Neoadjuvant chemoradiotherapy (NCRT) followed by surgery is a standard treatment for locally advanced esophageal squamous cell carcinomas (ESCCs). However, the evolution of genome and immunogenome in ESCCs driven by NCRT remains incompletely elucidated. We performed whole-exome sequencing of 51 ESCC tumors collected before and after NCRT, 36 of which were subjected to transcriptome sequencing. Clonal analysis identified clonal extinction in 13 ESCC patients wherein all pre-NCRT clones disappeared after NCRT, and clonal persistence in 9 patients wherein clones endured following NCRT. The clone-persistent patients showed higher pre-NCRT genomic intratumoral heterogeneity and worse prognosis than the clone-extinct ones. In contrast to the clone-extinct patients, the clone-persistent patients demonstrated a high proportion of subclonal neoantigens within pre-treatment specimens. Transcriptome analysis revealed increased immune infiltrations and up-regulated immune-related pathways after NCRT, especially in the clone-extinct patients. The number of T cell receptor-neoantigen interactions was higher in the clone-extinct patients than in the clone-persistent ones. The decrease in T cell repertoire evenness positively correlated to the decreased number of clonal neoantigens after NCRT, especially in the clone-extinct patients. In conclusion, we identified two prognosis-related clonal dynamic modes driven by NCRT in ESCCs. This study extended our knowledge of the ESCC genome and immunogenome evolutions driven by NCRT.

RTL1/PEG11 imprinted in human and mouse brain mediates anxiety-like and social behaviors and regulates neuronal excitability in the locus coeruleus.

RTL1/PEG11, which has been associated with anxiety disorders, is a retrotransposon-derived imprinted gene in the placenta. However, imprinting patterns and functions of RTL1 in the brain have not been well-investigated. We found Rtl1 was paternally, but not maternally, expressed in brain stem, thalamus, and hypothalamus of mice, and imprinting status of RTL1 was maintained in human brain. Paternal Rtl1 knockout (Rtl1m+/p-) mice had higher neonatal death rates due to impaired suckling, and low body weights beginning on embryonic day 16.5. High paternal expression of Rtl1 was detected in the locus coeruleus (LC) and Rtl1m+/p- mice showed an increased delay in time of onset for action potentials and inward currents with decreased neuronal excitability of LC neurons. Importantly, Rtl1m+/p- mice exhibited behaviors associated with anxiety, depression, fear-related learning and memory, social dominance, and low locomotor activity. Taken together, our findings demonstrate RTL1 is imprinted in brain, mediates emotional and social behaviors, and regulates excitability in LC neurons.

Using the Diabetes Care System for a County-Wide Hepatitis C Elimination: An Integrated Community-Based Shared Care Model in Taiwan.

INTRODUCTION: Despite the serious risks of diabetes with hepatitis C virus (HCV) infection, this preventable comorbidity is rarely a priority for HCV elimination. We aim to examine how a shared care model could eliminate HCV in patients with diabetes (PwD) in primary care. METHODS: There were 27 community-based Diabetes Health Promotion Institutes in each township/city of Changhua, Taiwan. PwD from these institutes from January 2018 to December 2020 were enrolled. HCV screening and treatment were integrated into diabetes structured care through collaboration between diabetes care and HCV care teams. Outcome measures included HCV care continuum indicators. Township/city variation in HCV infection prevalence and care cascades were also examined. RESULTS: Of the 10,684 eligible PwD, 9,984 (93.4%) underwent HCV screening, revealing a 6.18% (n = 617) anti-HCV seroprevalence. Among the 597 eligible seropositive individuals, 507 (84.9%) completed the RNA test, obtaining 71.8% positives. Treatment was initiated by 327 (89.8%) of 364 viremic patients, and 315 (86.5%) completed it, resulting in a final cure rate of 79.4% (n = 289). Overall, with the introduction of antivirals in this cohort, the prevalence of viremic HCV infection dropped from 4.44% to 1.34%, yielding a 69.70% (95% credible interval 63.64%-77.03%) absolute reduction. DISCUSSION: Although HCV prevalence varied, the care cascades achieved consistent results across townships/cities. We have further successfully implemented the model in county-wide hospital-based diabetes clinics, eventually treating 89.6% of the total PwD. A collaborative effort between diabetes care and HCV elimination enhanced the testing and treatment in PwD through an innovative shared care model.

Heterogeneous Integration of Memristive and Piezoresistive MDMO-PPV-Based Copolymers in Nociceptive Transmission with Fast and Slow Pain for an Artificial Pain-Perceptual System.

Nociceptive pain perception is a remarkable capability of organisms to be aware of environmental changes and avoid injury, which can be accomplished by specialized pain receptors known as nociceptors with 4 vital properties including threshold, no adaptation, relaxation, and sensitization. Bioinspired systems designed using artificial devices are investigated to imitate the efficacy and functionality of nociceptive transmission. Here, an artificial pain-perceptual system (APPS) with a homogeneous material and heterogeneous integration is proposed to emulate the behavior of fast and slow pain in nociceptive transmission. Retention-differentiated poly[2-methoxy-5-(3,7-dimethyoctyoxyl)-1,4-phenylenevinylene] (MDMO-PPV) memristors with film thicknesses of 160 and 80 nm are manufactured and adopted as A-δ and C nerve fibers of nociceptor conduits, respectively. Additionally, a nociceptor mimic, the ruthenium nanoparticles (Ru-NPs)-doped MDMO-PPV piezoresistive pressure sensor, is fabricated with a noxiously stimulated threshold of 150 kPa. Under the application of pricking and dull noxious stimuli, the current flows predominantly through the memristor to mimic the behavior of fast and slow pain, respectively, in nociceptive transmission with postsynaptic potentiation properties, which is analogous to biological pain perception. The proposed APPS can provide potential advancements in establishing the nervous system, thus enabling the successful development of next-generation neurorobotics, neuroprosthetics, and precision medicine.

Using rDNA ITS2 barcoding to identify kratom (Mitragyna speciosa) from the genus Mitragyna and Neolamarckia cadamba.

This study collected 80 samples of suspected kratom plant powder. A polymerase chain reaction sequence analysis was conducted using two sets of DNA barcode primers for plant ribosomal (r)DNA internal transcribed spacers (ITSs), namely, ITS3/ITS4 and ITS-p3/ITS-u4. Among the 80 samples, 40 were analyzed using the ITS3/ITS4 primer pair, and then DNA sequences were subjected to a National Center for Biotechnology Information-Basic Local Alignment Search Tool (NCBI-BLAST) comparison. Results showed that 29 samples had a 100% match (364/364) with Mitragyna speciosa (kratom), and 6 samples had a 99.73% match (363/364) with M. speciosa, whereas 5 samples had disordered and unreadable sequences. The 5 unreadable samples and an additional 40 suspected kratom samples were then analyzed using the ITS-p3/ITS-u4 primer pair, followed by an NCBI-BLAST comparison. Among these, 32 samples had a 100% match (404/404) with M. speciosa, and 11 samples had a 99.75% match (403/404) with M. speciosa. Among the samples with sequences matching M. speciosa, three distinct types were observed (no variance/404, 287M/404, and 287A/404). One sample had a 99.51% match (404/406) with Neolamarckia cadamba, and another sample had a sequencing length of 305 bp, with 25 positions showing mixed base pairs, indicating a mixture of different species. Analysis of the mixed base pair pattern suggested a possible mixture of M. speciosa and N. cadamba. Actually, M. speciosa and N. cadamba have very similar external morphologies. This indicates that the ITS-p3/ITS-u4 primer pair is effective in distinguishing mixtures of M. speciosa and N. cadamba and is thus more suitable than ITS3/ITS4 for identifying and analyzing samples of suspected kratom plant powder.