Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation.

Extracellular vesicles (EVs) play important roles in cell-cell communication and pathological development. Cargo profiling for the EVs present in clinical specimens can provide valuable insights into their functions and help discover effective EV-based markers for diagnostic and therapeutic purposes. However, the highly abundant and complex matrix components pose significant challenges for specific identification of low-abundance EV cargos. Herein, we combine asymmetrical flow field-flow fractionation (AF4) with large-volume sample stacking and capillary electrophoresis (LVSS/CE), to attain EVs with high purity for downstream protein profiling. This hyphenated system first separates the EVs from the contamination of smaller serum proteins by AF4, and second resolves the EVs from the coeluted, nonvesicular matrix components by CE following LVSS. The optimal LVSS condition permits the injection of 10-fold more EVs into CE compared to the nonstacking condition without compromising separation resolution. Collection and downstream analysis of the highly pure EVs after CE separation were demonstrated in the present work. The high EV purity yields a much-improved labeling efficiency when detected by fluorescent antibodies compared to those collected from the one-dimension separation of AF4, and permits the identification of more EV-specific cargos by LC-MS/MS compared to those isolated by ultracentrifugation (UC), the exoEasy Maxi Kit, and AF4. Our results strongly support that AF4-LVSS/CE can improve EV isolation and cargo analysis, opening up new opportunities for understanding EV functions and their applications in the biomedical fields.

Low-Dose Exposure of WS2 Nanosheets Induces Differential Apoptosis in Lung Epithelial Cells.

Escalating the production and application of tungsten disulfide (WS2) nanosheets inevitably increases environmental human exposure and warrants the necessity of studies to elucidate their biological impacts. Herein, we assessed the toxicity of WS2 nanosheets and focused on the impacts of low doses (≤10 µg/mL) on normal (BEAS-2B) and tumorigenic (A549) lung epithelial cells. The low doses, which approximate real-world exposures, were found to induce cell apoptosis, while doses ≥ 50 µg/mL cause necrosis. Focused studies on low-dose exposure to WS2 nanosheets revealed more details of the impacts on both cell lines, including reduction of cell metabolic activity, induction of lipid peroxidation in cell membranes, and uncoupling of mitochondrial oxidative phosphorylation that led to the loss of ATP production. These phenomena, along with the expression situations of a few key proteins involved in apoptosis, point toward the occurrence of mitochondria-dependent apoptotic signaling in exposed cells. Substantial differences in responses to WS2 exposure between normal and tumorigenic lung epithelial cells were noticed as well. Specifically, BEAS-2B cells experienced more adverse effects and took up more nanosheets than A549 cells. Our results highlight the importance of dose and cell model selection in the assessment of nanotoxicity. By using doses consistent with real-world exposures and comparing normal and diseased cells, we can gain knowledge to guide the development of safety precautions for mitigating the adverse impacts of nanomaterial exposure on human health.

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

Biological Impacts of Reduced Graphene Oxide Affected by Protein Corona Formation.

Applications of reduced graphene oxide (rGO) in many different areas have been gradually increasing owing to its unique physicochemical characteristics, demanding more understanding of their biological impacts. Herein, we assessed the toxicological effects of rGO in mammary epithelial cells. Because the as-synthesized rGO was dissolved in sodium cholate to maintain a stable aqueous dispersion, we hypothesize that changing the cholate concentration in the dispersion may alter the surface property of rGO and subsequently affect its cellular toxicity. Thus, four types of rGO were prepared and compared: rGO dispersed in 4 and 2 mg/mL sodium cholate, labeled as rGO and concentrated-rGO (c-rGO), respectively, and rGO and c-rGO coated with a protein corona through 1 h incubation in culture media, correspondingly named pro-rGO and pro-c-rGO. Notably, c-rGO and pro-c-rGO exhibited higher toxicity than rGO and pro-rGO and also caused higher reactive oxygen species production, more lipid membrane peroxidation, and more significant disruption of mitochondrial-based ATP synthesis. In all toxicological assessments, pro-c-rGO induced more severe adverse impacts than c-rGO. Further examination of the material surface, protein adsorption, and cellular uptake showed that the surface of c-rGO was coated with a lower content of surfactant and adsorbed more proteins, which may result in the higher cellular uptake observed with pro-c-rGO than pro-rGO. Several proteins involved in cellular redox mediation were also more enriched in pro-c-rGO. These results support the strong correlation between dispersant coating and corona formation and their subsequent cellular impacts. Future studies in this direction could reveal a deeper understanding of the correlation and the specific cellular pathways involved and help gain knowledge on how the toxicity of rGO could be modulated through surface modification, guiding the sustainable applications of rGO.

Regulation of Cell Division in Streptococci: Comparing with the Model Rods.

Streptococcus is a genus of oval-shaped bacteria that act as both commensals and pathogens. Streptococcal infections are relevant to high morbidity and huge socioeconomic costs, with drug resistant strains becoming an increasing threat. Cell division plays an essential role during streptococcal colonization and infection, rendering it an ideal target for antibiotics. Substantial progress has been made to uncover the molecular biology and cellular processes of cell division, favoring the target strategies. This review discusses recent advances in our understanding of streptococcal cell division and its regulatory mechanisms regarding the conserved proteins, by comparing with model rods. Peptidoglycan synthesis that involved in septum formation and the maintenance of the unique oval shape have been spatiotemporally controlled in concert with the pace of division. With newly available tools of genetic and cytological study, streptococci will become an additional model bacterial system for cytokinesis and novel therapeutic agents that target cell division.

Benzothiadiazole Versus Thiophene: Influence of the Auxiliary Acceptor on the Photovoltaic Properties of Donor-Acceptor-Based Copolymers.

Two donor-acceptor (D-A) type conjugated copolymers, P1 and P2, are designed and synthesized. A classical benzothiadiazole acceptor is used to replace a thiophene unit in the polymer chain of P1 to obtain P2 terpolymer. Compared with P1, P2 exhibits broader absorption spectra, higher absorption coefficient, deeper lowest unoccupied molecular orbital level, and a relatively lower band gap. As a result, the P2-based solar cell exhibits a high power conversion efficiency (PCE) of 6.60%, with a short-circuit current (J sc ) of 12.43 mA cm-2 , and a fill factor (FF) of 73.1%, which are higher than those of the P1-based device with a PCE of 4.70%, a J sc of 9.43 mA cm-2 , and an FF of 61.6%.

Serotype distribution and antibiotic resistance of Streptococcus pneumoniae isolates from 17 Chinese cities from 2011 to 2016.

BACKGROUND: Streptococcus pneumoniae, the leading pathogen of bacterial infections in infants and the elderly, is responsible for pneumococcal diseases with severe morbidity and mortality. Emergence of drug-resistant strains presented new challenges for treatment and prevention. Vaccination has proven to be an effective means of preventing pneumococcal infection worldwide. Detailed epidemiological information of antibiotic susceptibilities and serotype distribution will be of great help to the management of pneumococcal infections. METHODS: A total of 881 S. pneumoniae isolates were collected from patients at 23 teaching hospitals in 17 different cities from 2011 to 2016. The main specimen types included sputum, blood, broncho-alveolar lavage fluid, pharyngeal swabs, and cerebrospinal fluid. Minimum inhibitory concentrations (MICs) were determined using the agar dilution method. Capsular serotypes were identified using latex agglutination and quellung reaction test. Molecular epidemiology was investigated using multilocus sequence typing. RESULTS: S. pneumoniae isolates were highly resistant to macrolides, tetracycline, and trimethoprim/sulfamethoxazole. The rate of resistance to penicillin was 51.6% (oral breakpoint). However, levofloxacin and moxifloxacin maintained excellent antimicrobial activity and all of the isolated strains were susceptible to vancomycin. Twenty-two serotypes were identified among the 881 isolates. Prevalent serotypes were 19F (25.7%), 19A (14.0%), 15 (6.8%), 6B (3.6%), 6A (3.0%), and 17 (2.8%). The overall vaccine coverage rates for 7- and 13-valent pneumococcal conjugate vaccines were 37.5% and 58.3%, respectively. Vaccine coverage rates in young children and economically underdeveloped regions were higher than those in older adults and developed regions. Vaccine-covered serotypes demonstrated higher resistance compared with uncovered serotypes. Molecular epidemiological typing demonstrated that S. pneumoniae showed significant clonal dissemination and that ST271 (120, 28.3%), ST320 (73, 17.2%) and ST81 (27, 6.6%) were the major STs. CONCLUSIONS: High resistance to clinical routine antibiotics was observed for all 881 S. pneumoniae strains. Drug resistance varied among different serotypes and age groups. Prevalent serotypes among the isolates were 19F, 19A, 15, 6B, 6A, and 17. Community-acquired strains should also be included in future studies to gain a better understanding of the prevalence and resistance of S. pneumoniae in China.

[Bactericidal effects and smear layer removal of Er:YAG-laser radiation against Enterococcus faecalis in root canals].

OBJECTIVE: To evaluate the cleaning ability of Er:YAG laser against Enterococcus faecalis in root canals.
 Methods: The single-rooted human teeth were sterilized and inoculated with Enterococcus faecalis and were randomly assigned into 2 groups. A group of teeth was irrigated with saline during root canal preparation while another group with NaClO. After mechanical preparation, the two groups were randomly divided into 3 subgroups according to the different treatments: Samples, laser radiation, and calcium hydroxide intracanal medication for 7 days. Bactericical effects were compared among groups. Root canal walls and dental tubules were observed under scanning electron microscope.
 Results: Er:YAG laser was the most efficient way for anti-bacteria among the groups (P<0.05), but there was no significant difference between the 2 laser groups (P>0.05). Meanwhile the smear laser was efficiently removed by laser compared with other treatments, and the laser could open the dentinal tubules.
 Conclusion: Er:YAG laser can be effectively used for root canal disinfection without NaClO and Ca(OH)2.

Colocalization of protein and microRNA markers reveals unique extracellular vesicle subpopulations for early cancer detection.

Extracellular vesicles (EVs) play important roles in cell-cell communication but are highly heterogeneous, and each vesicle has dimensions smaller than 200 nm with very limited amounts of cargos encapsulated. The technique of NanOstirBar (NOB)-EnabLed Single Particle Analysis (NOBEL-SPA) reported in the present work permits rapid inspection of single EV with high confidence by confocal fluorescence microscopy, thus enables colocalization assessment for selected protein and microRNA (miRNA) markers in the EVs produced by various cell lines, or present in clinical sera samples. EV subpopulations marked by the colocalization of unique protein and miRNA combinations were discovered to be able to detect early-stage (stage I or II) breast cancer (BC). NOBEL-SPA can be adapted to analyze other types of cargo molecules or other small submicron biological particles. Study of the sorting of specific cargos to heterogeneous vesicles under different physiological conditions can help discover distinct vesicle subpopulations valuable in clinical examination and therapeutics development and gain better understanding of their biogenesis.

Fine Structure of the Mouthparts of Three Tomicus Beetles Co-Infecting Pinus yunnanensis in Southwestern China with Some Functional Comments.

Tomicus yunnanensis, T. brevipilosus, and T. minor are the most economically significant pests of Pinus yunnanensis in Southwestern China. Chemical and physical factors play critical roles in diverse biological activities. Here, we describe the fine structure of the adult mouthparts of these three Tomicus species using scanning and transmission electron microscopy. We identified three types of mandibular shapes, which determine their biomechanical properties, their ability to process food, and their preferred foraging locations on tree trunks. Eleven types of sensilla were discernible, including sensilla basiconica (Sb.1-2), sensilla twig basiconica (Stb.1-3), sensilla coeloconica (Sco), sensilla chaetica (Sch.1-2), sensilla trichoidea (Str.1-2), and sensilla digitiformia (Sdi). Each basiconic sensillum occurs on the palpal tips and is innervated by 2-6 dendrites. Sb.1 are gustatory receptors, Sb.2 are olfactory receptors, and the three other sensilla have dual taste and mechanical functions. Sco, Sch, and Str are mechanoreceptors. Sdi are mechanical vibration receptions, given that they are innervated by one dendrite with numerous dendritic branches into the nonporous cuticle. No significant differences among the sexes or species were identified; however, intraspecific variability in the number of Stb.3 and Sdi sensilla was evident. These results will aid future studies of Tomicus beetle behaviors.

Highly Stable Fe/Co-TPY-MIL-88(NH2) Metal-Organic Framework (MOF) in Enzymatic Cascade Reactions for Chemiluminescence-Based Detection of Extracellular Vesicles.

Metal-Organic Frameworks (MOFs) can deliver many advantages when acting as enzyme mimics to assist with signal amplification in molecular detection: they have abundant active catalytic sites per unit volume of the material; their structures and elemental compositions are highly tunable, and their high specific surface area and porous property can assist with target separation and enrichment. In the present work, we have demonstrated that, by adding the pore partition agent, 2,4,6-tris(4-pyridyl)pyridine (TPY) during synthesis of the bimetallic Fe/Co-MIL-88(NH2) MOF to block the open metal sites, a highly porous MOF of Fe/Co-TPY-MIL-88(NH2) can be produced. This material also exhibits high stability in basic solutions and biofluids and possesses high peroxidase-mimicking activity, which can be utilized to produce long-lasting chemiluminescence (CL) from luminol and H2O2. Moreover, acting as the peroxidase-mimic, the Fe/Co-TPY-MIL-88(NH2) MOF can form the enzymatic cascade with glucose oxidase (GOx) for biomarker detection. When applied to detect extracellular vesicles (EVs), the MOF material and GOx are brought to the proximity on the EVs through two surface proteins, which triggers the enzyme cascade to produce high CL from glucose and luminol. EVs within the concentration range of 5 × 105 to 4 × 107 particles/mL can be detected with an LOD of 1 × 105 particles/mL, and the method can be used to analyze EV contents in human serum without sample preparation and EV purification. Overall, our work demonstrates that the high versatility and tunability of the MOF structures could bring in significant benefits to biosensing and enable ultrasensitive detection of biomarkers with judicious material designs.

High-Performance Cannabinoid Sensor Empowered by Plant Hormone Receptors and Antifouling Magnetic Nanorods.

The misuse of cannabinoids and their synthetic variants poses significant threats to public health, necessitating the development of advanced techniques for detection of these compounds in biological or environmental samples. Existing methods face challenges like lengthy sample pretreatment and laborious antifouling steps. Herein, we present a novel sensing platform using magnetic nanorods coated with zwitterionic polymers for the simple, rapid, and sensitive detection of cannabinoids in biofluids. Our technique utilizes the engineered derivatives of the plant hormone receptor Pyrabactin Resistance 1 (PYR1) as drug recognition elements and employs the chemical-induced dimerization (CID) mechanism for signal development. Additionally, the magnetic nanorods facilitate efficient target capture and reduce the assay duration. Moreover, the zwitterionic polymer coating exhibits excellent antifouling capability, preserving excellent sensor performance in complex biofluids. Our sensors detect cannabinoids in undiluted biofluids like serum, saliva, and urine with a low limit of detection (0.002 pM in saliva and few pM in urine and serum) and dynamic ranges spanning up to 9 orders of magnitude. Moreover, the PYR1 derivatives demonstrate high specificity even in the presence of multiple interfering compounds. This work opens new opportunities for sensor development, showcasing the excellent performance of antifouling magnetic nanorods that can be compatible with different recognition units, including receptors and antibodies, for detecting a variety of targets.

Selective Molecular Recognition and Indicator Displacement Sensing of Neurotransmitters in Cellular Environments.

Flexible, water-soluble hosts are capable of selective molecular recognition in cellular environments and can detect neurotransmitters such as choline in cells. Both cationic and anionic water-soluble self-folded deep cavitands can recognize suitable styrylpyridinium dyes in cellular interiors. The dyes selectively accumulate in nucleotide-rich regions of the cell nucleus and cytoplasm. The hosts bind the dyes and promote their relocation to the outer cell membrane: the lipophilic cavitands predominantly reside in membrane environments but are still capable of binding suitable targets in other cellular organelles. Incubating the cells with structurally similar biomarkers such as choline, cholamine, betaine, or butyrylcholine illustrates the selective recognition. Choline and butyrylcholine can be bound by the hosts, but minimal binding is seen with betaine or cholamine. Varying the dye allows control of the optical detection method, and both "turn-on" sensing and "turn-off" sensing are possible.

Colocalization of Protein and microRNA Markers Reveals Unique Extracellular Vesicle Sub-Populations for Early Cancer Detection.

Extracellular vesicles (EVs) play important roles in cell-cell communication but they are highly heterogeneous, and each vesicle has dimensions smaller than 200 nm thus encapsulates very limited amounts of cargos. We report the technique of NanOstirBar (NOB)-EnabLed Single Particle Analysis (NOBEL-SPA) that utilizes NOBs, which are superparamagnetic nanorods easily handled by a magnet or a rotating magnetic field, to act as isolated "islands" for EV immobilization and cargo confinement. NOBEL-SPA permits rapid inspection of single EV with high confidence by confocal fluorescence microscopy, and can assess the colocalization of selected protein/microRNA (miRNA) pairs in the EVs produced by various cell lines or present in clinical sera samples. Specific EV sub-populations marked by the colocalization of unique protein and miRNA combinations have been revealed by the present work, which can differentiate the EVs by their cells or origin, as well as to detect early-stage breast cancer (BC). We believe NOBEL-SPA can be expanded to analyze the co-localization of other types of cargo molecules, and will be a powerful tool to study EV cargo loading and functions under different physiological conditions, and help discover distinct EV subgroups valuable in clinical examination and therapeutics development.

Adaptive COVID-19 Mitigation Strategies: Tradeoffs between Trigger Thresholds, Response Timing, and Effectiveness.

Background. To support proactive decision making during the COVID-19 pandemic, mathematical models have been leveraged to identify surveillance indicator thresholds at which strengthening nonpharmaceutical interventions (NPIs) is necessary to protect health care capacity. Understanding tradeoffs between different adaptive COVID-19 response components is important when designing strategies that balance public preference and public health goals. Methods. We considered 3 components of an adaptive COVID-19 response: 1) the threshold at which to implement the NPI, 2) the time needed to implement the NPI, and 3) the effectiveness of the NPI. Using a compartmental model of SARS-CoV-2 transmission calibrated to Minnesota state data, we evaluated different adaptive policies in terms of the peak number of hospitalizations and the time spent with the NPI in force. Scenarios were compared with a reference strategy, in which an NPI with an 80% contact reduction was triggered when new weekly hospitalizations surpassed 8 per 100,000 population, with a 7-day implementation period. Assumptions were varied in sensitivity analysis. Results. All adaptive response scenarios substantially reduced peak hospitalizations relative to no response. Among adaptive response scenarios, slower NPI implementation resulted in somewhat higher peak hospitalization and a longer time spent under the NPIs than the reference scenario. A stronger NPI response resulted in slightly less time with the NPIs in place and smaller hospitalization peak. A higher trigger threshold resulted in greater peak hospitalizations with little reduction in the length of time under the NPIs. Conclusions. An adaptive NPI response can substantially reduce infection circulation and prevent health care capacity from being exceeded. However, population preferences as well as the feasibility and timeliness of compliance with reenacting NPIs should inform response design. Highlights: This study uses a mathematical model to compare different adaptive nonpharmaceutical intervention (NPI) strategies for COVID-19 management across 3 dimensions: threshold when the NPI should be implemented, time it takes to implement the NPI, and the effectiveness of the NPI.All adaptive NPI response scenarios considered substantially reduced peak hospitalizations compared with no response.Slower NPI implementation results in a somewhat higher peak hospitalization and longer time spent with the NPI in place but may make an adaptive strategy more feasible by allowing the population sufficient time to prepare for changing restrictions.A stronger, more effective NPI response results in a modest reduction in the time spent under the NPIs and slightly lower peak hospitalizations.A higher threshold for triggering the NPI delays the time at which the NPI starts but results in a higher peak hospitalization and does not substantially reduce the time the NPI remains in force.

Exchange of hosts: can agaonid fig wasps reproduce successfully in the figs of non-host Ficus?

In the obligate mutualism between figs (Ficus) and their specific pollinators (Chalcidoidea, Agaonidae), each species of fig wasp typically reproduces in figs of a single host species. Host specificity is maintained largely because pollinators are attracted to tree-specific volatiles released from their host figs, but whether the wasps can reproduce if they enter figs of non-host species is unclear. We investigated the reproductive success of Ceratosolen emarginatus (associated with Ficus auriculata) and Ceratosolen sp. (associated with F. hainanensis) in atypical hosts by experimentally introducing foundresses into host and non-host figs. F. auriculata figs entered by Ceratosolen sp. were more likely to abort than if entered by C. emarginatus, but abortion of F. hainanensis figs was not affected by pollinator species. Single C. emarginatus foundresses produced more but smaller offspring in F. hainanensis than in their normal host. Conversely Ceratosolen sp. produced fewer but larger offspring in F. auriculata than in their normal host, probably as a result of having longer to develop. Mean style length differences, relative to the lengths of the wasps' ovipositors, may have dictated the number of offspring produced, with oviposition made easier by the shorter styles in F. hainanensis figs. Our results imply that, in addition to morphological constraints and tree-specific volatiles, reduced reproductive success in atypical hosts can be another factor maintaining host specificity, but for other species only behavioural changes are required for host switching to occur.

Maintenance of specificity in sympatric host-specific fig/wasp pollination mutualisms.

Background: Fig/wasp pollination mutualisms are extreme examples of species-specific plant-insect symbioses, but incomplete specificity occurs, with potentially important evolutionary consequences. Why pollinators enter alternative hosts, and the fates of pollinators and the figs they enter, are unknown. Methods: We studied the pollinating fig wasp, Ceratosolen emarginatus, which concurrently interacts with its typical host Ficus auriculata and the locally sympatric alternative host F. hainanensis, recording frequencies of the wasp in figs of the alternative hosts. We measured ovipositor lengths of pollinators and style lengths in female and male figs in the two host species. Volatile organic compounds (VOCs) emitted by receptive figs of each species were identified using GC-MS. We tested the attraction of wasps to floral scents in choice experiments, and detected electrophysiologically active compounds by GC-EAD. We introduced C. emarginatus foundresses into figs of both species to reveal the consequences of entry into the alternative host. Results: C. emarginatus entered a low proportion of figs of the alternative host, and produced offspring in a small proportion of them. Despite differences in the VOC profiles of the two fig species, they included shared semiochemicals. Although C. emarginatus females prefer receptive figs of F. auriculata, they are also attracted to those of F. hainanensis. C. emarginatus that entered male figs of F. hainanensis produced offspring, as their ovipositors were long enough to reach the bottom of the style; however, broods were larger and offspring smaller than in the typical host. Female figs of F. hainanensis failed to produce seeds when visited by C. emarginatus. These findings advance our current understanding of how these species-specific mutualisms usually remain stable and the conditions that allow their diversification.

Functional Morphology of the Antennae and Sensilla of Coeloides qinlingensis Dang et Yang (Hymenoptera: Braconidae).

Coeloides qinlingensis Dang et Yang, 1989 (Hymenoptera: Braconidae) is a biocontrol agent of several scolytid pine pests in Southwest China. We examined the fine morphology of the antennae of adult C. qinlingensis, as well as the type, shape, and distribution of antennal sensilla, via scanning electron microscopy. The antennae of female and male C. qinlingensis are filiform and comprise a scape, pedicel, and 31-36 flagellomeres. We detected sexual dimorphism in antennal flagellar length but not in the length of other subsegments. A total of nine morphological types of antennal sensilla varying in cuticular pore structure are present in both sexes, including nonporous types (sensilla trichodea, sensilla chaetica (2 subtypes), and sensilla coeloconica); apical pore types (sensilla basiconica and sensilla auricillica); and multiporous types (dome-shaped sensilla and sensilla placodea (2 subtypes)). Dome-shaped sensilla and sensilla auricillica are reported for the first time for C. qinlingensis, and their shape differs from that of sensilla in other parasitic wasps. The functional morphology of the sensilla of C. qinlingensis was compared with that of the sensilla of other parasitic wasps, including those that parasitize concealed insects. This information provides a foundation for further research on the chemical communication and behavior of C. qinlingensis.

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds.

A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense-response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense-response applications.

Calibration-free analysis of surface proteins on single extracellular vesicles enabled by DNA nanostructure.

Extracellular vesicles (EVs) are essential intercellular communicators that are of increasing interest as diagnostic biomarkers. Exploring their biological functions and clinical values, however, remains challenging due to their small sizes and high heterogeneity. Herein, we report an ultrasensitive method that employs target-initiated construction of DNA nanostructure to detect single EVs with an input as low as 100 vesicles/µL. Taking advantage of both DNA nanostructure labeling and EV membrane staining, the method can also permit calibration-free analysis of the protein profiles among different EV samples, leading to clear EV differentiation by their cell of origin. Moreover, this method allows co-localization of dual protein markers on the same EV, and the increased number of EVs carrying dual tumor proteins present in human serum could differentiate cancer patients at the early developmental stage from healthy controls. Our results demonstrate the great potential of this single-EV visualization method in non-invasive detection of the EV-based protein biomarkers for cancer diagnosis and treatment monitoring.