Isoliquiritigenin Suppresses Breast Tumor Development by Enhancing Host Antitumor Immunity.

Isoliquiritigen (ISL), a constituent of licorice, has been shown to possess antitumorigenic effects in diverse cancer types. In this study, we observed that ISL suppressed breast tumor development significantly more effectively in immunocompetent mice than in immunocompromised ones. In exploring the cause of such a discrepancy, we detected robust tumor infiltration of CD8[Formula: see text] T lymphocytes in mice treated with ISL, not seen in tumors derived from vehicle-treated mice. Moreover, we found a dramatic reduction in PD-L1 in both experimental breast tumors and cultured breast cancer cells upon ISL treatment. In further experiments, we showed that ISL selectively elevated miR-200c in breast cancer and confirmed that PD-L1 mRNA is the target of miR-200c in both murine and human breast cancer cells. ISL suppression of PD-L1 was functionally linked to miR-200c/ZEB1/2 because (1) ISL diminished ZEB1/2; (2) knockdown of ZEB1/2 led to the disappearance of PD-L1; and (3) miR-200c antagomiR disabled ISL to reduce PD-L1. We found evidence that ISL reduced the level of PD-L1 by simultaneously intercepting the ERK and Src signaling pathways. In agreement with clinical finding that PD-L1 antibodies enhance efficacy of taxane-based therapy, we showed that ISL improved the tumoricidal effects of paclitaxel in an orthopedic murine breast tumor model. This study demonstrates that ISL-led tumor suppression acts through the augmentation of host antitumor immunity.

Elevational and temporal patterns of pollination success in distylous and homostylous buckwheats (Fagopyrum) in the Hengduan Mountains.

Reproductive strategies of sexually dimorphic plants vary in response to the environment. Here, we ask whether the sexual systems of Fagopyrum species (i.e., selfing homostylous and out-crossing distylous) represent distinct adaptive strategies to increase reproductive success in changing alpine environments. To answer this question, we determined how spatial and temporal factors (e.g., elevation and peak flowering time) affect reproductive success (i.e., stigmatic pollen load) in nine wild Fagopyrum species (seven distylous and two homostylous) among 28 populations along an elevation gradient of 1299-3315 m in the Hengduan Mountains, southwestern China. We also observed pollinators and conducted hundreds of hand pollinations to investigate inter/intra-morph compatibility, self-compatibility and pollen limitation in four Fagopyrum species (two distylous and two homostylous). We found that Fagopyrum species at higher elevation generally had bigger flowers and more stigmatic pollen loads; late-flowering individuals had smaller flowers and lower pollen deposition. Stigmatic pollen deposition was more variable in distylous species than in homostylous species. Although seed set was not pollen-limited in all species, we found that fruit set was much lower in distylous species, which rely on frequent pollinator visits, than in homostylous species capable of autonomous self-pollination. Our findings that pollination success increases at high elevations and decreases during the flowering season suggest that distylous and homostylous species have spatially and temporally distinct reproductive strategies related to environment-dependent pollinator activity.

Mecapegfilgrastim for the prophylaxis of chemotherapy-induced neutropenia in locally advanced nasopharyngeal carcinoma: A prospective phase II clinical study.

BACKGROUND: Induction chemotherapy of docetaxel plus cisplatin (TP) is myelosuppressive, leading to severe neutropenia and febrile neutropenia (FN). Herein, we aimed to investigate the efficacy and safety of mecapegfilgrastim in the prevention of neutropenia in patients with locally advanced nasopharyngeal carcinoma who received the TP regimen. METHODS: A total of 30 treatment-naive patients with locally advanced nasopharyngeal carcinoma were included in this study. Mecapegfilgrastim 6 mg was injected 24-48 h after the completion of induction chemotherapy with the TP regimen. RESULTS: The incidence of grade ≥3 neutropenia during the three induction chemotherapy cycles was 6.7% (95% CI, 0.8%-22.1%). In the first cycle of chemotherapy, the incidence of grade ≥3 neutropenia was 3.3% (95% CI, 0.1%-17.2%). No FN or antibiotic usage was reported. All 30 patients completed the induction chemotherapy cycles. CONCLUSION: Mecapegfilgrastim effectively reduced the incidence of chemotherapy-induced neutropenia and FN in patients with locally advanced nasopharyngeal carcinoma.

Inborn errors of immunity and its clinical significance in children with lymphoma in China: a single-center study

Abstract Objective: To investigate the incidence, clinical and genetic characteristics of pediatric lymphoma patients of China with inborn errors of immunity (IEI)-related gene mutations, which have not been fully studied. Method: From Jan. 2020 to Mar. 2023, IEI-related genetic mutations were retrospectively explored in 108 children with lymphomas admitted to Beijing Children's Hospital by NGS. Genetic rule and clinical characteristics as well as treatment outcomes were compared between patients with or without IEI-related gene mutations. Results: A total of 17 patients (15.7 %) harbored IEI-associated mutations, including 4 cases with X-linked lymphoproliferative syndrome (XLP), 3 cases had mutations in tumor necrosis factor receptor superfamily 13B (TNFRSF13B), 2 cases with Activated p110 syndrome (APDS). Patients with IEI all had alteration of immunocompetence with decreased levels of immunoglobulin and lymphocyte subsets. Recurrent infection existed in 41.2 % of patients. The 18-month event-free survival (EFS) and the overall response rate (ORR) of patients with IEI are significantly lower than those without IEI (33.86% vs. 73.26 %, p = 0.011; 52.94% vs. 87.91 %, p = 0.002, respectively). In addition, patients with IEI had a higher progression disease (PD) rate of 23.5 % than those without IEI of 4.4% (p = 0.006). Conclusion: The present study demonstrated that IEI-associated lymphomas were much more common than originally appreciated in pediatric lymphomas, and those were insensitive to treatment and more likely to progress or relapse. The genomic analysis and a thorough review of the medical history of IEI can be used to distinguish them from pediatric lymphomas without IEI, which are beneficial for the early diagnosis and direct intervention.

Remote ischemic conditioning prevents ischemic cerebrovascular events in children with moyamoya disease: a randomized controlled trial.

BACKGROUND: Moyamoya disease (MMD) is a significant cause of childhood stroke and transient ischemic attacks (TIAs). This study aimed to assess the safety and efficacy of remote ischemic conditioning (RIC) in children with MMD. METHODS: In a single-center pilot study, 46 MMD patients aged 4 to 14 years, with no history of reconstructive surgery, were randomly assigned to receive either RIC or sham RIC treatment twice daily for a year. The primary outcome measured was the cumulative incidence of major adverse cerebrovascular events (MACEs). Secondary outcomes included ischemic stroke, recurrent TIA, hemorrhagic stroke, revascularization rates, and clinical improvement assessed using the patient global impression of change (PGIC) scale during follow-up. RIC-related adverse events were also recorded, and cerebral hemodynamics were evaluated using transcranial Doppler. RESULTS: All 46 patients completed the final follow-up (23 each in the RIC and sham RIC groups). No severe adverse events associated with RIC were observed. Kaplan-Meier analysis indicated a significant reduction in MACEs frequency after RIC treatment [log-rank test (Mantel-Cox), P = 0.021]. At 3-year follow-up, two (4.35%) patients had an ischemic stroke, four (8.70%) experienced TIAs, and two (4.35%) underwent revascularization as the qualifying MACEs. The clinical improvement rate in the RIC group was higher than the sham RIC group on the PGIC scale (65.2% vs. 26.1%, P < 0.01). No statistical difference in cerebral hemodynamics post-treatment was observed. CONCLUSIONS: RIC is a safe and effective adjunct therapy for asymptomatic children with MMD. This was largely due to the reduced incidence of ischemic cerebrovascular events.

Au24Cd Nanoenzyme Coating for Enhancing Electrochemical Sensing Performance of Metal Wire Microelectrodes.

Dopamine (DA), ascorbic acid (AA), and uric acid (UA) are crucial neurochemicals, and their abnormal levels are involved in various neurological disorders. While electrodes for their detection have been developed, achieving the sensitivity required for in vivo applications remains a challenge. In this study, we proposed a synthetic Au24Cd nanoenzyme (ACNE) that significantly enhanced the electrochemical performance of metal electrodes. ACNE-modified electrodes demonstrated a remarkable 10-fold reduction in impedance compared to silver microelectrodes. Furthermore, we validated their excellent electrocatalytic activity and sensitivity using five electrochemical detection methods, including cyclic voltammetry, differential pulse voltammetry, square-wave pulse voltammetry, normal pulse voltammetry, and linear scanning voltammetry. Importantly, the stability of gold microelectrodes (Au MEs) modified with ACNEs was significantly improved, exhibiting a 30-fold enhancement compared to Au MEs. This improved performance suggests that ACNE functionalization holds great promise for developing micro-biosensors with enhanced sensitivity and stability for detecting small molecules.

Advances in Multifunctional Electronic Catheters for Precise and Intelligent Diagnosis and Therapy in Minimally Invasive Surgery.

The advent of catheter-based minimally invasive surgical instruments has provided an effective means of diagnosing and treating human disease. However, conventional medical catheter devices are limited in functionalities, hindering their ability to gather tissue information or perform precise treatment during surgery. Recently, electronic catheters have integrated various sensing and therapeutic technologies through micro/nanoelectronics, expanding their capabilities. As micro/nanoelectronic devices become more miniaturized, flexible, and stable, electronic surgical catheters are evolving from simple tools to multiplexed sensing and theranostics for surgical applications. The review on multifunctional electronic surgical catheters is lacking and thus is not conducive to the reader's comprehensive understanding of the development trend in this field. This review covers the advances in multifunctional electronic catheters for precise and intelligent diagnosis and therapy in minimally invasive surgery. It starts with the summary of clinical minimally invasive surgical instruments, followed by the background of current clinical catheter devices for sensing and therapeutic applications. Next, intelligent electronic catheters with integrated electronic components are reviewed in terms of electronic catheters for diagnosis, therapy, and multifunctional applications. It highlights the present status and development potential of catheter-based minimally invasive surgical devices, while also illustrating several significant challenges that remain to be overcome.

Identification of a Novel 16.8Kb Deletion of the α-Globin Gene Cluster by Third-Generation Sequencing.

α-thalassemia major (α-TM) often causes Hb Bart's (c4) hydrops fetalis and severe obstetric complications in the mother. Step-wise screening for couples at risk of having offspring(s) affected by α-TM is the efficient prevention method but some rare genotypes of thalassemia cannot be detected. A 32-year-old male with low HbA2 (2.4%) and mild anemia was performed real-time PCR-based multicolor melting curve analysis (MMCA) because his wife was -SEA deletion carrier. The result of multiplex ligation-dependent probe amplification (MLPA) suggested the existence of -SEA deletion in the proband. A novel deletion of the α-globin gene cluster was found using self-designed MLPA probes combined with longer PCR, which was further accurately described to be 16.8Kb (hg38, Chr16:1,65,236-1,82,113) deletion by the third-generation sequencing. A fragment ranging from 1,53,226 to 1,54,538(GRch38/hg38) was identified which suggested the existence of the homologous recombination event. The third-generation sequencing is accurate and efficient in obtaining accurate information for complex structural variations.

Customizing carrier screening in the Chinese population: Insights from a 334-gene panel.

OBJECTIVE: This study aimed to evaluate the yield and applicability of expanded carrier screening and propose carrier rate screening thresholds suitable for the Chinese population by comparing the current screening panel with the American College of Medical Genetics and Genomics recommended panel of 113 genes. METHODS: Using targeted next-generation sequencing, a customized panel with 334 genes was performed on 2168 individuals without clinical phenotypes for expanded carrier screening purpose. Variant interpretation followed the American College of Medical Genetics and Genomics guidelines. Carrier rates were calculated for each identified variant and each gene. At-risk couple rates were also assessed. The yield of expanded carrier screening was evaluated through calculating cumulative carrier rate. RESULTS: Overall, 65.87% of the individuals were found to be carriers of at least 1 disease causing variants. The overall at-risk couple rate was 11.76%, of which the GJB2:c.109G > A related at-risk couple rate was 5.78%. The cumulative carrier rate of 334-panel was 65.53%. When screened genes with gene carrier rate ≥1/1000, the expanded carrier screening can cover over 90% of the cumulative carrier rate and at-risk couples. A total of 86 genes overlapped with American College of Medical Genetics and Genomics Tier-3 genes and were attributed to the cumulative carrier rate of 47.33%. CONCLUSION: Expanded carrier screening using the 334-gene panel showed high screening efficiency. A threshold of gene carrier rate ≥1/1000 is recommended for selecting carrier screening genes in the Chinese Han population. This study highlights the importance of customizing screening panels based on the ACMG Tier-3 genes in conjunction with population-specific carrier frequencies to improve the accuracy and effectiveness of expanded carrier screening.

Penpulimab, an anti-PD-1 antibody, for heavily pretreated metastatic nasopharyngeal carcinoma: a single-arm phase II study.

Penpulimab is an anti-programmed cell death-1 (PD-1) IgG1 antibody with no Fc gamma receptor (FcγR) binding activity, and thus theoretically reduced immune-related adverse events (irAEs) while maintaining efficacy. This single-arm, phase II trial conducted across 20 tertiary care centers in China enrolled adult patients with metastatic nasopharyngeal carcinoma (NPC) who had failed two or more lines of previous systemic chemotherapy. Patients received 200-mg penpulimab intravenously every 2 weeks (4 weeks per cycle) until disease progression or intolerable toxicities. The primary endpoint was objective response rate (ORR) per RECIST (version 1.1), as assessed by an independent radiological review committee. The secondary endpoints included progression-free survival (PFS) and overall survival (OS). One hundred thirty patients were enrolled and 125 were efficacy evaluable. At the data cutoff date (September 28, 2022), 1 patient achieved complete response and 34 patients attained partial response. The ORR was 28.0% (95% CI 20.3-36.7%). The response was durable, with 66.8% still in response at 9 months. Thirty-three patients (26.4%) were still on treatment. The median PFS and OS were 3.6 months (95% CI = 1.9-7.3 months) and 22.8 months (95% CI = 17.1 months to not reached), respectively. Ten (7.6%) patients experienced grade 3 or higher irAEs. Penpulimab has promising anti-tumor activities and acceptable toxicities in heavily pretreated metastatic NPC patients, supporting further clinical development as third-line treatment of metastatic NPC.

Multichannel microneedle dry electrode patches for minimally invasive transdermal recording of electrophysiological signals.

The collection of multiple-channel electrophysiological signals enables a comprehensive understanding of the spatial distribution and temporal features of electrophysiological activities. This approach can help to distinguish the traits and patterns of different ailments to enhance diagnostic accuracy. Microneedle array electrodes, which can penetrate skin without pain, can lessen the impedance between the electrodes and skin; however, current microneedle methods are limited to single channels and cannot achieve multichannel collection in small areas. Here, a multichannel (32 channels) microneedle dry electrode patch device was developed via a dimensionality reduction fabrication and integration approach and supported by a self-developed circuit system to record weak electrophysiological signals, including electroencephalography (EEG), electrocardiogram (ECG), and electromyography (EMG) signals. The microneedles reduced the electrode-skin contact impedance by penetrating the nonconducting stratum corneum in a painless way. The multichannel microneedle array (MMA) enabled painless transdermal recording of multichannel electrophysiological signals from the subcutaneous space, with high temporal and spatial resolution, reaching the level of a single microneedle in terms of signal precision. The MMA demonstrated the detection of the spatial distribution of ECG, EMG and EEG signals in live rabbit models, and the microneedle electrode (MNE) achieved better signal quality in the transcutaneous detection of EEG signals than did the conventional flat dry electrode array. This work offers a promising opportunity to develop advanced tools for neural interface technology and electrophysiological recording.

Fabrication of Multiple-Channel Electrochemical Microneedle Electrode Array via Separated Functionalization and Assembly Method.

Real-time monitoring of physiological indicators inside the body is pivotal for contemporary diagnostics and treatments. Implantable electrodes can not only track specific biomarkers but also facilitate therapeutic interventions. By modifying biometric components, implantable electrodes enable in situ metabolite detection in living tissues, notably beneficial in invasive glucose monitoring, which effectively alleviates the self-blood-glucose-managing burden for patients. However, the development of implantable electrochemical electrodes, especially multi-channel sensing devices, still faces challenges: (1) The complexity of direct preparation hinders functionalized or multi-parameter sensing on a small scale. (2) The fine structure of individual electrodes results in low spatial resolution for sensor functionalization. (3) There is limited conductivity due to simple device structures and weakly conductive electrode materials (such as silicon or polymers). To address these challenges, we developed multiple-channel electrochemical microneedle electrode arrays (MCEMEAs) via a separated functionalization and assembly process. Two-dimensional microneedle (2dMN)-based and one-dimensional microneedle (1dMN)-based electrodes were prepared by laser patterning, which were then modified as sensing electrodes by electrochemical deposition and glucose oxidase decoration to achieve separated functionalization and reduce mutual interference. The electrodes were then assembled into 2dMN- and 1dMN-based multi-channel electrochemical arrays (MCEAs), respectively, to avoid damaging functionalized coatings. In vitro and in vivo results demonstrated that the as-prepared MCEAs exhibit excellent transdermal capability, detection sensitivity, selectivity, and reproducibility, which was capable of real-time, in situ glucose concentration monitoring.

Concurrent nimotuzumab and intensity-modulated radiotherapy for elderly patients with locally advanced nasopharyngeal carcinoma.

Because of the common physical condition, reduced organ function, and comorbidities, elderly patients with nasopharyngeal carcinoma (NPC) are often underrepresented in clinical trials. The optimal treatment of elderly patients with locally advanced NPC remains unclear. The purpose of this study was to evaluate the efficacy of concurrent nimotuzumab combined with intensity-modulated radiotherapy (IMRT) in elderly patients with locally advanced NPC. We conducted a single-arm, phase II trial for elderly patients with stage III-IVA NPC (according to UICC-American Joint Committee on Cancer TNM classification, 8th edition). All patients received concurrent nimotuzumab (200 mg/week, 1 week prior to IMRT) combined with IMRT. The primary end-point was complete response (CR) rate. The secondary end-points were survival, safety, and geriatric assessment. Between March 13, 2017 and November 12, 2018, 30 patients were enrolled. In total, 20 (66.7%) patients achieved CR, and objective response was observed in 30 (100.0%) patients 1 month after radiotherapy. The median follow-up time was 56.05 months (25th-75th percentile, 53.45-64.56 months). The 5-year locoregional relapse-free survival, distant metastasis-free survival, cancer-specific survival, disease-free survival, and overall survival were 89.4%, 86.4%, 85.9%, 76.5%, and 78.8%, respectively. Grade 3 mucositis occurred in 10 (33%) patients and grade 3 pneumonia in 3 (10%) patients. Concurrent nimotuzumab combined with IMRT is effective and well-tolerated for elderly patients with locally advanced NPC.

Impact of DNA ligase inhibition on the nick sealing of polß nucleotide insertion products at the downstream steps of base excision repair pathway.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) ß at the downstream steps. We previously demonstrated that a functional interplay between polß and BER ligases is critical for efficient repair, and polß mismatch or oxidized nucleotide insertions confound final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polß+/+ cells more than polß-/- null cells. We showed less efficient ligation after polß nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polß inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polß, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.

Long-term efficacy analysis of radiotherapy and local management of metastases in patients with newly diagnosed oligometastatic nasopharyngeal carcinoma: A prospective, single-arm, single-center clinical study.

PURPOSE: We conducted a single-center, single-arm study (NCT03129412) to prospectively analyze the long-term outcomes of newly diagnosed patients with oligometastatic nasopharyngeal carcinoma (NPC) who received radical radiotherapy and local treatment of metastases. PATIENTS AND METHODS: Patients who reached disease controll after platinum-based palliative chemotherapy continued to receive radical radiotherapy for the nasopharyngeal region and neck. Appropriate local treatments were selected to treat the metastatic lesions. The primary endpoint of this study was overall survival (OS) and the secondary endpoint was progression-free survival (PFS). RESULTS: Fifty-one patients were included in the final analysis. During a median follow-up of 60 months, the median OS and PFS were 53.87 and 24.23 months, respectively. The 1-year, 3-year, and 5-year PFS and OS rates were 76.5 %, 38.1 %, and 31.8 % and 98 %, 75.4 %, 45.6 %, respectively. Both single and multivariate analysis indicated that maintenance therapy after radiotherapy could significantly increase PFS (36.43 vs. 16.1 months, P = 0.005). The OS of patients with single organ metastasis was significantly better than that of patients with double organ metastasis (P = 0.001). In addition, the number of metastatic organs also significantly affected PFS in the multifactor analysis. CONCLUSION: Patients with newly diagnosed oligometastatic NPC can achieve long-term survival after receiving radical radiotherapy to the primary site and local treatment for metastases.

Ultra-low frequency magnetic energy focusing for highly effective wireless powering of deep-tissue implantable electronic devices.

The limited lifespan of batteries is a challenge in the application of implantable electronic devices. Existing wireless power technologies such as ultrasound, near-infrared light and magnetic fields cannot charge devices implanted in deep tissues, resulting in energy attenuation through tissues and thermal generation. Herein, an ultra-low frequency magnetic energy focusing (ULFMEF) methodology was developed for the highly effective wireless powering of deep-tissue implantable devices. A portable transmitter was used to output the low-frequency magnetic field (<50 Hz), which remotely drives the synchronous rotation of a magnetic core integrated within the pellet-like implantable device, generating an internal rotating magnetic field to induce wireless electricity on the coupled coils of the device. The ULFMEF can achieve energy transfer across thick tissues (up to 20 cm) with excellent transferred power (4-15 mW) and non-heat effects in tissues, which is remarkably superior to existing wireless powering technologies. The ULFMEF is demonstrated to wirelessly power implantable micro-LED devices for optogenetic neuromodulation, and wirelessly charged an implantable battery for programmable electrical stimulation on the sciatic nerve. It also bypassed thick and tough protective shells to power the implanted devices. The ULFMEF thus offers a highly advanced methodology for the generation of wireless powered biodevices.

Respective evolution of soil and biochar on competitive adsorption mechanisms for Cd(II), Ni(II), and Cu(II) after 2-year natural ageing.

To reveal the respective evolution of soil and biochar on competitive heavy metal adsorption mechanisms after natural ageing, three soils and two biochars were tested in this study. The soil-biochar interlayer samples were buried in the field for 0.5, 1, and 2 years, for which competitive adsorption characteristics and mechanisms of soils and biochars in four systems (Cd, Cd+Ni, Cd+Cu, and Cd+Ni+Cu) were investigated. Results showed that physicochemical properties, adsorption capacity and mechanisms of soils and biochars all changed the most in the first 0.5 years. The properties and adsorption capacity of biochars gradually weakened with the ageing time, meanwhile, those of soils gradually enhanced. After co-ageing with acidic soil for 0.5 years, the Cd(II) adsorption capacity of modified biochar decreased by 86.59% in the ternary system; meanwhile, that of acidic soil increased by 65.52%. The contributions of mineral mechanisms decreased significantly, while non-mineral mechanisms were slightly affected by ageing. This study highlighted that when using biochar to remediate heavy metal-contaminated soils, biochar should be applied at least half a year in advance before planting crops so that biochar can fully contact and react with the soil.

High toxin concentration in pollen may deter collection by bees in butterfly-pollinated Rhododendron molle.

BACKGROUNDS AND AIMS: The hypothesis that plants evolve features that protect accessible pollen from consumption by flower visitors remains poorly understood. METHODS: To explore potential chemical defenses against pollen consumption, we examined the pollinator assemblage, foraging behaviour, visitation frequency and pollen transfer efficiency in Rhododendron molle, a highly toxic shrub containing Rhodojaponin III. Nutrient (protein and lipid) and toxic components in pollen and other tissues were measured. KEY RESULTS: Overall in the five populations, floral visits by butterflies and bumblebees were relatively more frequent than visits by honeybees. All foraged for nectar but not pollen. Butterflies did not differ from bumblebees in the amount of pollen removed per visit, but deposited more pollen per visit. Pollination experiments indicated that R. molle was self-compatible, but both fruit and seed production were pollen limited. Our analysis indicated that the pollen was not protein-poor and had a higher concentration of the toxic compound Rhodojaponin III than petals and leaves, which compound was undetectable in nectar. CONCLUSION: Pollen toxicity in Rhododendron flowers may discourage pollen robbers (bees) from taking the freely accessible pollen grains, while the toxin-free nectar rewards effective pollinators, promoting pollen transfer. This preliminary study supports the hypothesis that chemical defense in pollen would be likely to evolve in species without physical protection from pollinivores.