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Abstract As promising energy-storage devices, zinc–air batteries (ZABs) exhibit slow reaction kinetics for oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) occurring at their electrodes. High-performance bifunctional catalysts must thus be synthesized to accelerate the reversible conversion of oxygen and improve the rate and overall performance of ZABs. Herein, we reported the promising prospects of self-supported composite electrodes composed of wood-derived carbon (WDC) and bimetallic cobalt-iron alloys/oxides (CoFe-CoFe2O4@WDC) as efficient electrocatalysts for alkaline ORR/OER. WDC provided a favorable three-phase interface for heterogeneous reactions owing to its layered porous structure and genetic stability, thereby enabling mass diffusion and improving reaction kinetics. The CoFe2O4 spinel surface was reduced to bimetallic CoFe alloy to form abundant heterostructure interfaces that promote electron transfer. Under alkaline conditions, the optimized composite electrode exhibited a remarkable high half-wave potential of 0.85 V and an exceptionally low overpotential of 1.49 V. It also exhibited stable performance over an impressive 2340 cycles in a ZAB. Theoretical calculations also confirmed that the heterointerface addresses the issue of proton scarcity throughout the reaction and actively facilitates the creation of O–O bonds during the reversible transformation of oxygen. This study introduces a new concept for developing bifunctional and efficient electrocatalysts based on charcoal and encourages the sustainable and high-value use of forest biomass resources. Graphical Abstract

Neural tube defects (NTDs) are severe congenital anomalies that result from the failure of early neural tube closure during fetal neurogenesis. They are the most common and severe congenital malformations of the central nervous system. Identifying reliable prenatal diagnostic ultrasound and molecular markers that can predict NTDs is of paramount importance. Early diagnosis of NTDs allows embryonic treatment and prevention strategies, which are crucial for reducing the disability rate associated with these malformations, reducing the burden on individuals and on society. The purpose of this comprehensive review was to summarize the ultrasound biomarkers between 11 and 13 weeks of gestation and the molecular biomarkers used in the diagnosis of NTDs, providing additional insights into early screening for NTDs.

Abstract Background Accurate prediction of tumor molecular alterations is vital for optimizing cancer treatment. Traditional tissue-based approaches encounter limitations due to invasiveness, heterogeneity, and molecular dynamic changes. We aim to develop and validate a deep learning radiomics framework to obtain imaging features that reflect various molecular changes, aiding first-line treatment decisions for cancer patients. Methods We conducted a retrospective study involving 508 NSCLC patients from three institutions, incorporating CT images and clinicopathologic data. Two radiomic scores and a deep network feature were constructed on three data sources in the 3D tumor region. Using these features, we developed and validated the ‘Deep-RadScore,’ a deep learning radiomics model to predict prognostic factors, gene mutations, and immune molecule expression levels. Findings The Deep-RadScore exhibits strong discrimination for tumor molecular features. In the independent test cohort, it achieved impressive AUCs: 0.889 for lymphovascular invasion, 0.903 for pleural invasion, 0.894 for T staging; 0.884 for EGFR and ALK, 0.896 for KRAS and PIK3CA, 0.889 for TP53, 0.895 for ROS1; and 0.893 for PD-1/PD-L1. Fusing features yielded optimal predictive power, surpassing any single imaging feature. Correlation and interpretability analyses confirmed the effectiveness of customized deep network features in capturing additional imaging phenotypes beyond known radiomic features. Interpretation This proof-of-concept framework demonstrates that new biomarkers across imaging features and molecular phenotypes can be provided by fusing radiomic features and deep network features from multiple data sources. This holds the potential to offer valuable insights for radiological phenotyping in characterizing diverse tumor molecular alterations, thereby advancing the pursuit of non-invasive personalized treatment for NSCLC patients.

Objectives: To examine the disparities between COVID-19 studies conducted in high-income countries (HICs) and low-and middle-income countries (LMICs). Methods: We used the International Clinical Trials Registry Platform to identify COVID-19-related studies registered from December 31, 2019 to December 31, 2021. The World Bank definition was used to classify countries as high-, upper-middle-, lower-middle-, and low-income. The last three were considered to be LMICs. We examined the disparities in response speed, classification of medicines and vaccines, and registration and results reporting compliance between COVID-19 studies conducted in HICs and LMICs. Results: We included 12,396 COVID-19 studies, with 6631 (53.5%) from HICs. HIC-registered studies reached a peak of 1039 in April 2020, whereas LMICs had only 440 studies. Of the 6969 interventional trials, those from HICs showed higher registration compliance (2199, 62.3% vs 1979, 57.6%, P

As the newly built tokamak in China, HL-3 will explore high-performance operation scenarios, such as super H-mode. The energy confinement and core parameters in the super H-mode can be much larger than that in the normal H-mode. Based on the pedestal simulation code EPED, the operation space of the super H-mode is obtained in HL-3. Magnetic shear decreases with increasing triangularity; consequently, a super H-mode can be achieved. The threshold of triangularity for accessing a super H-mode in HL-3 is around 0.4. By using BOUT++, a nonlinear simulation study of the pedestal instabilities in the super H-mode equilibrium is executed for the first time. As expected, the low n peeling mode, which can cause much of the energy loss (17%) from the pedestal region, is dominant in the super H-mode. Such a large collapse in the pedestal region would lead to a transition from super H-mode to H-mode. It is crucial to expand the parameter space of the super H-mode or mitigate the edge-localized mode (ELM) size for sustaining the super H-mode operations. The E × B velocity shear is found to play an important role in controlling the ELMs in HL-3. The small E × B velocity shear leads to a large growth rate but results in a small ELM size around the peeling boundary. The ELM size is closely related to both the growth rate of peeling-ballooning mode and the duration time of the linear phase. In contrast, a large E × B velocity shear can stabilize the instabilities near the ballooning boundary. Next, the parameter space of the super H-mode can be enlarged.

Abstract Background Neuromyelitis optica spectrum disorders (NMOSD), a group of autoimmune neurological diseases, involve the optic nerve, spinal cord, and brain. Meningitis is rarely reported as the primary clinical manifestation of both anti-aquaporin-4 (AQP4)/ anti-myelin oligodendrocyte glycoprotein (MOG) antibody-negative NMOSD (NMOSDneg). Case presentation A 30-year-old man initially presented with fever, headache, and neck stiffness. Lumbar puncture revealed mixed cell reaction and decreased glucose levels. As a result, tuberculous meningitis was suspected. After 1 month, the patient developed longitudinally extensive transverse myelitis and area postrema syndrome. This was followed by the presentation of meningitis-like symptoms once again in the third attack, but his condition eventually improved after corticosteroid treatment without relapse for 2 years. However, he was readmitted to our hospital owing to symptoms of diplopia, hiccup, and numbness in the right hand. Brain magnetic resonance imaging (MRI) revealed that the area postrema still contained lesions. Spinal MRI revealed several segmental enhancements at the C4–C5, T1, and T5 levels. Anti-AQP4 and anti-MOG antibodies were persistently absent in the serum and cerebrospinal fluid (CSF). The patient was finally diagnosed with NMOSDneg. Conclusions Meningitis could be a recurrent manifestation of NMOSDneg and requires more careful evaluation.

Abstract Background Entomopathogenic nematodes (EPNs) emerge as compatible alternatives to conventional insecticides in controlling Holotrichia parallela larvae (Coleoptera: Scarabaeidae). However, the immune responses of H. parallela against EPNs infection remain unclear. Results In present research, RNA-Seq was firstly performed. A total of 89,427 and 85,741 unigenes were achieved from the midgut of H. parallela larvae treated with Heterorhabditis beicherriana LF for 24 and 72 h, respectively; 2545 and 3156 unigenes were differentially regulated, respectively. Among those differentially expressed genes (DEGs), 74 were identified potentially related to the immune response. Notably, some immune-related genes, such as peptidoglycan recognition protein SC1 (PGRP-SC1), pro-phenoloxidase activating enzyme-I (PPAE-I) and glutathione s-transferase (GST), were induced at both treatment points. Bioinformatics analysis showed that PGRP-SC1, PPAE-I and GST were all involved in anti-parasitic immune process. Quantitative real-time PCR (qRT-PCR) results showed that the three immune-related genes were expressed in all developmental stages; PGRP-SC1 and PPAE-I had higher expressions in midgut and fat body, respectively, while GST exhibited high expression in both of them. Moreover, in vivo silencing of them resulted in increased susceptibility of H. parallela larvae to H. beicherriana LF. Conclusion These results suggest that H. parallela PGRP-SC1, PPAE-I and GST are involved in the immune responses to resist H. beicherriana LF infection. This study provides the first comprehensive transcriptome resource of H. parallela exposure to nematode challenge that will help to support further comparative studies on host-EPN interactions.

Instabilities in multiplies of ion cyclotron frequency range are identified and termed as core ion cyclotron emission (ICE) in recent HL-2A neutral beam injection heated experiments. Characteristics of the core ICE are presented, including frequency dependence and harmonics features. The detected frequencies are found to agree well with the multiplies of the deuterium cyclotron frequency around the magnetic axis. Additionally, the core ICE exhibits a predominantly compressional property. Observations of distinct spectrum features and individual excitation of each harmonic have demonstrated that the core ICE harmonics are independent multiple modes. Notably, the variation of plasma current is a necessary condition for exciting the 4th harmonic ICE individually. The results suggest that the drive mechanism of core ICE varies between the different frequency ranges.

Abstract Objective Previous studies showed that flight muscles degenerate after migration in some aphid species; however, the underlying molecular mechanism remains virtually unknown. In this study, using the wheat aphid, Sitobion avenae, we aim to investigate aphid flight muscle degeneration and the underlying molecular mechanism. Results Sitobion avenae started to differentiate winged or wingless morphs at the second instar, the winged aphids were fully determined at the third instar, and their wings were fully developed at the fourth instar. After migration, the aphid flight muscles degenerated via programmed cell death, which is evidenced by a Terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling assay. Then, we identified a list of differentially expressed genes before and after tethered flights using differential-display reverse transcription-PCR. One of the differentially expressed genes, ubiquitin-ribosomal S27a, was confirmed using qPCR. Ubiquitin-ribosomal S27a is drastically up regulated following the aphids’ migration and before the flight muscle degeneration. Our data suggested that aphid flight muscles degenerate after migration. During flight muscle degeneration, endogenous proteins may be degraded to reallocate energy for reproduction.

Anthocyanins belong to a group of flavonoids, which are the most important flower pigments. Clarifying the potential anthocyanins biosynthesis molecular mechanisms could facilitate artificial manipulation of flower pigmentation in plants. In this paper, we screened a differentially expressed gene, MhTCP4, from the transcriptome data of Malus halliana petals at different development stages and explored its role in anthocyanins biosynthesis. The transcriptome data and qRT-PCR analysis showed that the expression level of MhTCP4 gradually decreased from the flower color fades. Tissue specific expression analysis showed MhTCP4 was expressed in the petal, leaf, and fruit of M. halliana, and was highly expressed in the scarlet petal. Overexpression of MhTCP4 promoted anthocyanins accumulation and increased pigments in infected parts of M. ‘Snowdrift’ and M. ‘Fuji’ fruit peels. In contrast, when endogenous MhTCP4 was silenced, the anthocyanins accumulation was inhibited and pigments decreased in the infected peels. The qRT-PCR analysis revealed that overexpression or silence of MhTCP4 caused expression changes of a series of structural genes included in anthocyanins biosynthesis pathway. The yeast two-hybrid assays indicated that MhTCP4 did not interact with MhMYB10. Furthermore, the yeast one-hybrid assays indicated that MhTCP4 did not directly bind to the promoter of MhMYB10, but that of the anthocyanins biosynthesis genes, MhCHI and MhF3′H. Dual luciferase assays further confirmed that MhTCP4 can strongly activate the promoters of MhCHI and MhF3′H in tobacco. Overall, the results suggest that MhTCP4 positively regulates anthocyanins biosynthesis by directly activated MhCHI and MhF3′H in M. halliana flowers.

As an economically valuable red seaweed, Neopyropia yezoensis (Rhodophyta) is cultivated in intertidal areas, and its growth and development are greatly influenced by environmental factors such as temperature. Although much effort has been devoted to delineating the influence, the underlying cellular and molecular mechanisms remain elusive. In this study, the gametophyte blades and protoplasts were cultured at different temperatures (13 °C, 17 °C, 21 °C, 25 °C). Only blades cultured at 13 °C maintained a normal growth state (the relative growth rate of thalli was positive, and the content of phycobiliprotein and pigments changed little); the survival and division rates of protoplasts were high at 13 °C, but greatly decreased with the increase in temperature, suggesting that 13 °C is suitable for the growth of N. yezoensis. In our efforts to delineate the underlying mechanism, a partial coding sequence (CDS) of Cyclin B and the complete CDS of cyclin-dependent-kinase B (CDKB) in N. yezoensis were cloned. Since Cyclin B controls G2/M phase transition by activating CDK and regulates the progression of cell division, we then analyzed how Cyclin B expression in the gametophyte blades might change with temperatures by qPCR and Western blotting. The results showed that the expression of Cyclin B first increased and then decreased after transfer from 13 °C to higher temperatures, and the downregulation of Cyclin B was more obvious with the increase in temperature. The phosphorylation of extracellular signal-regulated kinase (ERK) decreased with the increase in temperature, suggesting inactivation of ERK at higher temperatures; inhibition of ERK by FR180204 significantly decreased the survival and division rates of protoplasts cultured at 13 °C. These results suggest that downregulation of Cyclin B and inactivation of ERK might be involved in negatively regulating the survival and division of protoplasts and the growth of gametophyte blades of N. yezoensis at high temperatures.

The flower color of many horticultural plants fades from red to white during the development stages, affecting ornamental value. We selected Malus halliana, a popular ornamental species, and analyzed the mechanisms of flower color fading using RNA sequencing. Forty-seven genes related to anthocyanin biosynthesis and two genes related to anthocyanin transport were identified; the expression of most of these genes declined dramatically with flower color fading, consistent with the change in the anthocyanin content. A number of transcription factors that might participate in anthocyanin biosynthesis were selected and analyzed. A phylogenetic tree was used to identify the key transcription factor. Using this approach, we identified MhMYB10 as directly regulating anthocyanin biosynthesis. MhMYB10 expression was strongly downregulated during flower development and was significantly positively related to the expression of anthocyanin biosynthetic genes and anthocyanin content in diverse varieties of Malus. To analyze the methylation level during flower development, the MhMYB10 promoter sequence was divided into 12 regions. The methylation levels of the R2 and R8 increased significantly as flower color faded and were inversely related to MhMYB10 expression and anthocyanin content. Therefore, we deduce that the increasing methylation activities of these two regions repressed MhMYB10 expression.

Abstract White grubs are largely unsolved problems in vegetable and tuber production in East Africa. Novel Rwandan as well as international entomopathogenic nematodes (EPNs) were screened in nine laboratory bioassays and two small-scale field trials in 2014 and 2015. Soil-based laboratory bioassays revealed that all EPNs were able to infect Anomala graueri larvae (Coleoptera: Scarabeidae), although a relatively high number of infective juveniles were needed. Rwandan EPNs were as infectious as their corresponding international strains. At 100 infective juveniles per larva, the Heterorhabditis bacteriophora strains (Rwanda14-N-C4a and international H06) as well as the international Steinernema carpocapsae All caused 18 to 22% grub mortality within 7 days compared to the control. At 1000 infective juveniles, both H. bacteriophora strains as well as the Rwandan S. carpocapsae RW14-G-R3a-2 killed 34 up to 58%. The Rwandan Steinernema RW14-M-C2a-3 least performed in the bioassays (2 to 6%). In two Irish potato fields, the into-soil-applied 1 × 109 infective juvenile Steinernema RW14-M-C2b-1 per hectare reduced 29 ± 33% and 96 ± 3% of grubs within 30 and 60 days, respectively. About 1 up to 2.5 × 109 infective juvenile Steinernema longicaudum X7 per hectare reduced 77 up to 85% of grubs until day 30, respectively, and 82 up to 95% until day 60. Avermectin + Chlorpyrifos tuber coatings reduced 39% of grubs, and Fipronil + Chlorpyrifos reduced 27%, whereas handpicking did not help much. In conclusion, at least Steinernema RW14-M-C2b-1 and S. longicaudum X7 are promising for managing white grubs in tuber production, this is at a rate of at least 1.5 × 109 EPNs per hectare, but H. bacteriophora RW14-N-C4a needs further field research. Findings will support the biocontrol product development in Rwanda, including registration if any would be needed for native macrobial biocontrol agents.

Holotrichia parallela is one of the agriculturally important scarab beetle pests in China. In this study, HparOBP14 was cloned, which is the most abundantly expressed among the OBP genes in the legs of female H. parallela adults. Sequence comparison and phylogenetic analysis showed that HparOBP14 has a Plus-C structure motif. The expression profile analysis revealed that HparOBP14 expression was the highest in the female antennae and then in the legs. The fluorescence competitive binding experiment of the recombinant HparOBP14 protein showed that HparOBP14 had an affinity with 6-methyl-5-heptene-2-one (plant volatile), 3-methylindole, p-cymene, methanol, formaldehyde, α-pinene, and geraniol (organic fertilizer volatile). Knockdown HparOBP14 expression decreased significantly the EAG response of the injected female adults to p-cymene, methanol, formaldehyde, α-pinene, and geraniol. Similarly, the injected female adults were significantly less attracted to geraniol and methanol. Therefore, HparOBP14 might bind organic matter volatiles during oviposition. These results are not only helpful to analyze the olfactory recognition mechanism of female adult H. parallela when choosing suitable oviposition sites, but also to provide target genes for green prevention and control of H. parallela in the future.

Introduction: Vitamin D deficiency and inflammation are involved with bronchopulmonary dysplasia (BPD) in preterm neonates; however, the clinical evidence still remains scarce. We hypothesized that vitamin D and inflammatory cytokines may be risk factors for BPD in infants.Methods: Preterm infants born between 28 and 31 weeks' gestation were recruited between January 2016 and 2017. Blood samples were all collected at corresponding time points. Vitamin D was measured using an automatic biochemical analyzer, and inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) were measured using ELISA.Results: The baseline characteristics for preterm infants without BPD (non-BPD control, n = 20) or with BPD (n = 19) were similar. In the blood samples collected 24-h post birth, vitamin D was significantly reduced in the BPD neonates (non-BPD vs. BPD, 28.96 ± 3.404 vs. 17.99 ± 2.233 nmol/l, p = 0.0134). Inflammatory cytokines TNF-α, IL-1β, and IL-6 were comparable in both groups. The anti-inflammatory cytokine IL-10, however, was significantly decreased in 24-h blood samples from BPD preterm infants (non-BPD vs. BPD, 44.61 ± 10.48 vs. 11.64 ± 2.351 pg/ml, p = 0.0054). In the BPD infants with mild or moderate disease, vitamin D deficiency was quite similar. IL-10 deficiency, however, was more aggravated in the BPD infants with moderate disease. No changes in Vitamin D or cytokines (TNF-α, IL-1β, IL-6, and IL-10) were observed for blood samples collected 2 or 4 weeks after birth.Conclusion: In our pilot study, Vitamin D and IL-10 levels at 24-h of life were risk factors for the development of BPD in very preterm infants.

Data dissemination in vehicular ad hoc networks has been an interesting research topic since rising applications can be run and served based on the success and efficiency of data dissemination in vehicular ad hoc networks. However, the volatile vehicle density, the highly dynamic topologies, and the packet lossy nature of the vehicular wireless communications pose real challenges for vehicular ad hoc networks to achieve high-speed content downloading and efficient resource utilization. In this work, we design a collision-free distributed relay selection method, by jointly considering the geographical locations, channel conditions, moving velocities, and packets receiving statuses of vehicles, to combat the mobility and lossy channel property of vehicular ad hoc networks. Moreover, we adopt the instantly decodable network coding for the selected relay vehicle to retransmit packets which were lost in the original transmission, resulting significant improvements in both network throughput and transmission delay. Simulation results show that the proposed strategy effectively reduces the delay of data dissemination in highway scenarios and improves the downloading rate, as well as the resource utilization of wireless channels.

Loxostege sticticalis Linnaeus is an economically important agricultural pest, and the larvae cause great damage to crops, especially in Northern China. However, effective and environmentally friendly chemical methods for controlling this pest have not been discovered to date. In the present study, we performed HiSeq2500 sequencing of transcriptomes of the male and female adult antennae, adult legs and third instar larvae, and we identified 54 candidate odorant receptors (ORs), including 1 odorant receptor coreceptor (Orco) and 5 pheromone receptors (PRs), 18 ionotropic receptors (IRs), 13 gustatory receptors (GRs), 34 odorant binding proteins (OBPs), including 1 general odorant binding protein (GOBP1) and 3 pheromone binding proteins (PBPs), 10 chemosensory proteins (CSPs) and 2 sensory neuron membrane proteins (SNMPs). The results of RNA-Seq and RT-qPCR analyses showed the expression levels of most genes in the antennae were higher than that in the legs and larvae. Furthermore, PR4, OR1-4, 7-11, 13-15, 23, 29-32, 34, 41, 43, 47/IR7d.2/GR5b, 45, 7/PBP2-3, GOBP1, OBP3, 8 showed female antennae-biased expression, while PR1/OBP2, 7/IR75d/CSP2 showed male antennae-biased expression. However, IR1, 7d.3, 68a/OBP11, 20-22, 28/CSP9 had larvae enriched expression, and OBP15, 17, 25, 29/CSP5 were mainly expressed in the legs. The results shown above indicated that these genes might play a key role in foraging, seeking mates and host recognition in the L. sticticalis. Our findings will provide the basic knowledge for further studies on the molecular mechanisms of the olfactory system of L. sticticalis and potential novel targets for pest control strategies.

As the first crucial barrier in the midgut of insects, the peritrophic membrane (PM) plays an important role in preventing external invasion. PM proteins, as the major components of the PM, determine the structure and function of this membrane. A new PM protein, named LstiCBP, from the PM of Loxostege sticticalis larvae was identified using cDNA library screening. The full cDNA of LstiCBP is 2606 bp in length and contains a 2403 bp ORF that encodes an 808-amino acid preprotein with a 15-amino acid as signal peptide. The deduced protein sequence of the cDNA contains 8 cysteine-rich chitin-binding domains (CBDs). Recombinant LstiCBP was successfully expressed in BL21 cells using recombinant plasmid DNA and showed high chitin-binding activity. LstiCBP expression was detected in the midgut at both the transcriptional and translational levels; however, the biochemical and physiological functions of LstiCBP in L. sticticalis require further investigation.

The Pt particles within diameters of 1⁻3 nm known as Pt nanoclusters (NCs) are widely considered to be satisfactory oxygen reduction reaction (ORR) catalysts due to higher electrocatalytic performance and cost effectiveness. However, the utilization of such smaller Pt NCs is always limited by the synthesis strategies, stability and methanol tolerance of Pt. Herein, unprotected Pt NCs (~2.2 nm) dispersed on carbon nanotubes (CNTs) were prepared via a modified top-down approach using liquid Li as a solvent to break down the bulk Pt. Compared with the commercial Pt/C, the resultant Pt NCs/CNTs catalyst (Pt loading: 10 wt.%) exhibited more desirable ORR catalytic performance in 0.1 M HClO4. The specific activity (SA) and mass activity (MA) at 0.9 V for ORR over Pt NCs/CNTs were 2.5 and 3.2 times higher than those over the commercial Pt/C (Pt loading: 20 wt.%). Meanwhile, the Pt NCs/CNTs catalyst demonstrated more satisfactory stability and methanol tolerance. Compared with the obvious loss (~69%) of commercial Pt/C, only a slight current decrease (~10%) was observed for Pt NCs/CNTs after the chronoamperometric measurement for 2 × 104 s. Hence, the as-prepared Pt NCs/CNTs material displays great potential as a practical ORR catalyst.

The peritrophic membrane (PM) plays an important role in protecting insects. The PM proteins are important to determinate the formation and function of the PM. A new PM protein, named Lsti99, was identified from the PM of Loxostege sticticalis larvae by cDNA library screening. The full cDNA of Lsti99 is 1392 bp in length, contains an open reading frame (ORF) of 1245 bp that encodes a preprotein of 415 amino acid residues with a 17-amino acid signal peptide. The sequence of Lsti99 showed no homology to other known PM proteins. The recombinant Lsti99 was successfully expressed in insect cells (Sf9) using recombinant baculoviruses and was used to isolate the antibodies to Lsti99 from the polyclonal antiserum. Lsti99 was expressed mainly in the PM, but weaker bands could be detected in the head and integument as well. The Lsti99 protein could be separated from the PM complex by chitinase in vitro, but M2R did not show effect in vitro confirming the chitin-binding activity of Lsti99. The biochemical and physiological functions of Lsti99 in L. sticticalis require further investigation.

Chemosensory proteins (CSPs) play important roles in chemical communication by insects, as they recognize and transport environmental chemical signals to receptors within sensilla. In this study, we identified HoblCSP1 and HoblCSP2 from a cDNA library of Holotrichia oblita antennae, successfully expressed them in E. coli and purified them by Ni ion affinity chromatography. We then measured the ligand-binding specificities of HoblCSP1 and HoblCSP2 to 50 selected ligands in a competitive binding assay. These results demonstrated that HoblCSP1 and HoblCSP2 have similar ligand-binding spectra. Both proteins displayed the highest affinity for β-ionone, α-ionone and cinnamaldehyde, indicating that they prefer binding to odorants other than sex pheromones. Additionally, immuno-localization revealed that HoblCSP1 is highly concentrated in sensilla basiconica, while HoblCSP2 is specifically localized to sensilla placodea. In conclusion, HoblCSP1 and HoblCSP2 are responsible for binding to general odorants with slightly different specificities due to their different in vivo environments.

It was previously thought that the odorant binding proteins (OBPs) in the sensillum lymph might serve as carriers, which could carry lipophilic odorant molecules to olfactory receptors. In this study, two novel OBP genes of the scarab beetle Holotrichia oblita were screened using an antennal cDNA library. The full cDNA of HoblOBP3 and HoblOBP4 was cloned using reverse transcription PCR and rapid amplification of the cDNA ends. Homology modeling of both OBPs was performed using SWISS-MODEL on-line tools. Next, the two OBPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. The ligand-binding properties of HoblOBP3 and HoblOBP4 in 42 ligands respectively were measured using the fluorescence probe N-phenyl-naphthylamine (1-NPN). The results obtained from competitive binding assays demonstrated that HoblOBP4 showed a broader range of binding affinities to the test compounds, while HoblOBP3 displays more specific binding affinity. Furthermore, other OBPs and CSPs were expressed in Escherichia coli and purified using Ni ion affinity chromatography. Binding curves were measured for binary mixtures of OBPs and CSPs using 1-NPN, and the Scatchard plots exhibited "J"-like nonlinear correlation trends in some samples. In addition, competitive binding assays of the HoblOBP1 and HoblOBP2 mixtures and of the HoblOBP2 and HoblOBP4 mixtures with representative compounds unexpectedly demonstrated good affinity, which revealed extreme differences that were only obtained using the individual proteins. In the immunocytochemical analysis, colocalization of HoblOBP1 and HoblOBP2, and of HoblOBP2 and HoblOBP4, was detected in the sensilla basiconica and sensilla placodea, respectively. All of these results suggested that HoblOBP1 and HoblOBP2, as well as HoblOBP2 and HoblOBP4, may serve as heterodimers in the sensillum lymph.

Odorant binding proteins play a crucial role in transporting semiochemicals across the sensillum lymph to olfactory receptors within the insect antennal sensilla. In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends. Recombinant LstiGOBP2 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR assays indicated that LstiGOBP2 mRNA is expressed mainly in adult antennae, with expression levels differing with developmental age. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants. Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.