T cell receptor dwell times control the kinase activity of Zap70.

Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.

Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA.

Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.

Deubiquitinating enzyme OTUD4 stabilizes RBM47 to induce ATF3 transcription: a novel mechanism underlying the restrained malignant properties of ccRCC cells.

Dysregulation of deubiquitination contributes to various diseases, including cancer, and aberrant expression of deubiquitinating enzymes is involved in carcinoma progression. As a member of the ovarian tumor (OTU) deubiquitinases, OTUD4 is considered a tumor suppressor in many kinds of malignancies. The biological characteristics and mechanisms of OTUD4 in clear cell renal cell carcinoma (ccRCC) remain unclear. The downregulation of OTUD4 in ccRCC was confirmed based on the TCGA database and a validation cohort of 30-paired ccRCC and para-carcinoma samples. Moreover, OTUD4 expression was detected by immunohistochemistry in 50 cases of ccRCC tissues, and patients with lower levels of OTUD4 showed larger tumor size (p = 0.015). TCGA data revealed that patients with high expression of OTUD4 had a longer overall survival rate. In vitro and in vivo studies revealed that downregulation of OTUD4 was essential for tumor cell growth and metastasis in ccRCC, and OTUD4 overexpression inhibited these malignant phenotypes. We further found that OTUD4 sensitized ccRCC cells to Erastin-induced ferroptosis, and ferrostain-1 inhibited OTUD4-induced ferroptotic cell death. Mechanistic studies indicated that OTUD4 functioned as an anti-proliferative and anti-metastasic factor through the regulation of RNA-binding protein 47 (RBM47)-mediated activating transcription factor 3 (ATF3). OTUD4 directly interacted with RBM47 and promoted its stability via deubiquitination events. RBM47 was critical in ccRCC progression by regulating ATF3 mRNA stability, thereby promoting ATF3-mediated ferroptosis. RBM47 interference abolished the suppressive role of OTUD4 overexpression in ccRCC. Our findings provide mechanistic insight into OTUD4 of ccRCC progression and indicate a novel critical pathway OTUD4/RBM47/ATF3 may serve as a potential therapeutic pathway for ccRCC.

Construction of Hydration Layer for Proton Transport by Implanting the Hydrophilic Center Ag0 in Nickel Metal-Organic Frameworks.

The directional arrangement of H2O molecules can effectively regulate the ordered protons transfer to improve transport efficiency, which can be controlled by the interaction between materials and H2O. Herein, a strategy to build a stable hydration layer in metal-organic framework (MOF) platforms, in which hydrophilic centers that can manipulate H2O molecules are implanted into MOF cavities is presented. The rigid grid-Ni-MOF is selected as the supporting material due to the uniformly distributed cavities and rigid structures. The Ag0 possesses potential combination ability with the hydrophilic substances, so it is introduced into the MOF as hydration layer centers. Relying on the strong interaction between Ag0 and H2O, the H2O molecules can rearrange around Ag0 in the cavity, which is intuitively verified by DFT calculation and molecular dynamics simulation. The establishment of a hydration layer in Ag@Ni-MOF regulates the chemical properties of the material and gives the material excellent proton conduction performance, with a proton conductivity of 4.86 × 10-2 S cm-1.

Dual Oxytocin Receptor-G Protein Signaling in the Autoregulation of Activities of Oxytocin Neurons.

Oxytocin (OT), a hypothalamic nonaneuropeptide, can extensively modulate mental and physical activities; however, the regulation of its secretion from hypothalamic OT neurons remains poorly understood. OT neuronal activity is generally modulated by neurochemical environment, synaptic inputs, astrocytic plasticity, and interneuronal interactions. By changing intracellular signals and ion channel activity, these extracellular factors dynamically regulate OT neuronal activity and OT release in a microdomain-specific manner. In this process, OT receptor (OTR) and OTR-coupled G proteins are pivotal, typically observed during lactation. Suckling-elicited somatodendritic release of OT causes sequential activation of Gq and Gs proteins to increase the firing rate gradually and trigger burst firing transiently, and then of Gi/o protein to cause post-burst inhibition as a result of potential bolus somatodendritic release of OT during the burst-like discharges. Under chronic social stress like mother-baby separation and cesarean section, excessive somatodendritic secretion of OT and over-excitation of OT neurons cause post-excitation inhibition of OT neuronal activity and reduction of OT secretion. In this process, dominance of G protein that couples to OTR is switched from Gq to Gi/o type because of inhibition of OTR-Gq signaling following negative feedback of downstream Gq signaling or crosstalk of Gq with Gs and Gi signals. This review summarizes our current understandings of OT/OTR signaling in the autoregulation of OT neuronal activity under physiological and pathological conditions.

Association of dietary inflammatory index and the SARS-CoV-2 infection incidence, severity and mortality of COVID-19: a systematic review and dose-response meta-analysis.

BACKGROUND: Several studies have reported the association between dietary inflammatory index (DII) and the SARS-CoV-2 infection risk, severity or mortality of COVID-19, however, the outcomes remain controversial. OBJECTIVE: We sought to examine whether a dose-response association of DII and SARS-CoV-2 infection exists. DESIGN: A dose-response meta-analysis was performed to investigate the association of DII and SARS-CoV-2 infection. We conducted a systematic search of PubMed, Embase and Web of Science up to March 15th, 2023. The odds ratios (OR) of DII and COVID-19 risk and severity were computed. RESULTS: Totally, 5 studies were included (1 from UK and 4 from Iran), consisting of 197,929 participants with 12,081 COVID-19 cases. Although there was heterogeneity among studies, the results indicated that higher DII was independently related to higher SARS-CoV-2 infection incidence (OR = 1.57, 95% CI: 1.14, 2.17) and COVID-19 severity (OR = 1.11, 95% CI: 1.07, 1.15) but not COVID-19 mortality (risk ratio = 1.13, 95% CI: 1.00, 1.27). The incidence of SARS-CoV-2 infection increased by 31% for each 1-point increase in the E-DII (OR = 1.31, 95% CI: 1.20, 1.43). CONCLUSIONS: This meta-analysis suggests that an elevated DII score is associated with increased SARS-CoV-2 infectious risk and severity of COVID-19. There were not enough studies on COVID-19 mortality. Further large prospective studies in different countries are warranted to validate our results.

Cement-augmented locked plate fixation proximal humerus fractures in elderly patient: a systematic review and meta-analysis.

BACKGROUND: This systemic review and meta-analysis aimed to evaluate the clinical outcomes of proximal humeral fracture in elderly patient fixation using locked plate with or without cement augmentation. METHODS: The databases of PubMed, Embase, and Cochrane Library were searched in August 2023 for literature comparing the clinical outcomes of patients with PHFs treated with locked plate alone and locked plate augmented with cement. Data describing study design; level of evidence; inclusion criteria; demographic information; final follow-up; revision rate; implant failure rate; avascular necrosis rate; total complication rate; constant score; and disability of arm, shoulder, and hand (DASH) score were collected. RESULTS: Eight studies (one randomized-controlled trial and seven observational studies), involving 664 patients, were identified. Compared with locked plates alone, using cement-augmented locked plates reduced the implant failure rate (odds ratio (OR) = 0.19; 95% confidence interval (CI) 0.10-0.39; P < 0.0001) and total complication rate (OR = 0.45; 95% CI 0.29-0.69; P = 0.0002) and improved DASH scores (mean difference (MD) = 2.99; 95% CI 1.00-4.98; P = 0.003). However, there was no significant difference in clinical outcomes, including revision rate, avascular necrosis rate, and constant score. CONCLUSION: In this review and meta-analysis, fixation of the PHFs in elderly patients using locked plates with or without cement augmentation has no significant difference in revision rate, but the implant failure and total complication rates may be lesser on using the cement-augmented locked plate for fixation than on using a locked plate alone. Good results are expected for most patients treated with this technique. TRIAL REGISTRATION: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)21 guidelines were followed to conduct this systematic review and meta-analysis and was registered as a protocol in PROSPERO (CRD42022318798).

Graphiumisides A-D, Monoterpene Glycosides from an Animal-Derived Endophytic Fungus Graphium sp. GD-11.

Four new monoterpene rhamnosides, graphiumisides A-D (1-4), along with four known steroid compounds (5-8) were isolated from the fermentation extract of animal-derived endophytic fungus, Graphium sp. GD-11. The chemical structures of all compounds were elucidated using 1D and 2D NMR, HRESIMS spectroscopic analyses, and other spectroscopic methods. Compounds 1-4 exhibit a distinctive structure connected by one p-menthane type monoterpene and one L-rhamnose. This is the first report of monoterpene glycosides from Graphium sp. All compounds (1-8) were tested for cytotoxic activities against four cancer cell lines (HepG2, SMMC7721, SW480, and A549), and only compound 1 showed weak anti-tumor activity against SMMC7721 cells.

Uniformly Distributed Mixed Matrix Membranes via a Solution Processable Strategy for Propylene/Propane Separation.

Aggregation of filler particles during the formation of mixed matrix membranes is difficult to avoid when filler loadings exceed a 10-15 wt %. Such agglomeration usually leads to poor membrane performance. In this work, using a ZIF-67 metal-organic framework (MOF) as filler along with surface modification of Ag4 tz4 to improve processability and selective olefin adsorption, we demonstrate that highly loaded with a very low agglomeration degree membranes can be synthesized displaying unmatched separation selectivity (39) for C3 H6 /C3 H8 mixtures and high permeability rates (99 Barrer), far surpassing previous reports in the literature. Through molecular dynamics simulation, the enhanced compatibility between ZIF-67 and polymer matrix with adding Ag4 tz4 was proven and the tendency in gas permeability and C3 H6 selectivity in the mixed matrix membranes (MMMs) were well explained. More importantly, the membrane showed a wide range of pressure and temperature resistance, together with remarkable long-term stability (>900 h). The modification method might help solve interface issues in MMMs and can be extended to the fabrication of other fillers to achieve high performance MMMs for gas separation.

Highly Stable MOF-Type Lead Halide Luminescent Ferroelectrics.

Lead halide molecular ferroelectrics represent an important class of luminescent ferroelectrics, distinguished by their high chemical and structural tunability, excellent processability and distinctive luminescent characteristics. However, their inherent instability, prone to decomposition upon exposure to moisture and light, hinders their broader ferroelectric applications. Herein, for the first time, we present a series of isoreticular metal-organic framework (MOF)-type lead halide luminescent ferroelectrics, demonstrating exceptional robustness under ambient conditions for at least 15 months and even when subjected to aqueous boiling conditions. Unlike conventional metal-oxo secondary building units (SBUs) in MOFs adopting highly centrosymmetric structure with limited structural distortion, our lead halide-based MOFs occupy structurally deformable [Pb2X]+ (X=Cl-/Br-/I-) SBUs that facilitate a c-axis-biased displacement of Pb2+ centers and substantially contribute to thermoinducible structural transformation. Importantly, this class of MOF-type lead halide ferroelectrics undergo ferroelectric-to-paraelectric phase transitions with remarkably high Curie temperature of up to 505 K, superior to most of molecular ferroelectrics. Moreover, the covalent bonding between phosphorescent organic component and the light-harvesting inorganic component achieves efficient spin-orbit coupling and intersystem crossing, resulting in long-lived afterglow emission. The compelling combination of high stability, ferroelectricity and afterglow emission exhibited by lead halide MOFs opens up many potential opportunities in energy-conversion applications.

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase.

The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT-nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.

Structural basis of main proteases of HCoV-229E bound to inhibitor PF-07304814 and PF-07321332.

PF-07321332 and PF-07304814, inhibitors against SARS-CoV-2 developed by Pfizer, exhibit broad-spectrum inhibitory activity against the main protease (Mpro) from various coronaviruses. Structures of PF-07321332 or PF-07304814 in complex with Mpros of various coronaviruses reveal their inhibitory mechanisms against different Mpros. However, the structural information on the lower pathogenic coronavirus Mpro with PF-07321332 or PF-07304814 is currently scarce, which hinders our comprehensive understanding of the inhibitory mechanisms of these two inhibitors. Meanwhile, given that some immunocompromised individuals are still affected by low pathogenic coronaviruses, we determined the structures of lower pathogenic coronavirus HCoV-229E Mpro with PF-07321332 and PF-07304814, respectively, and analyzed and defined in detail the structural basis for the inhibition of HCoV-229E Mpro by both inhibitors. Further, we compared the crystal structures of multiple coronavirus Mpro complexes with PF-07321332 or PF-07304814 to illustrate the differences in the interaction of Mpros, and found that the inhibition mechanism of lower pathogenic coronavirus Mpro was more similar to that of moderately pathogenic coronaviruses. Our structural studies provide new insights into drug development for low pathogenic coronavirus Mpro, and provide theoretical basis for further optimization of both inhibitors to contain potential future coronaviruses.

High-Performance Bifunctional Porous Iron-Rich Phosphide/Nickel Nitride Heterostructures for Alkaline Seawater Splitting.

Seawater is the most abundant natural water resource in the world, which is an inexhaustible and low-cost feedstock for hydrogen production by alkaline water electrolysis. It is appearling to develop robust and stable electrocatalysts for alkaline seawater electrolysis. However, the development of seawater electrolysis is seriously impeded by anodic chloride corrosion and chlorine evolution reaction, and few non-noble electrocatalysts show prominent catalytic performance and excellent durability. Here, a heterogeneous electrocatalyst constructed by in situ growing highly dispersed iron-rich bimetallic phosphide nanoparticles on metallic Ni3 N (Fe2-2 x Co2 x P/Ni3 N), which exhibits outstanding bifunctional catalytic activities for alkaline seawater splitting, is reported. The optimal (Fe0.74 Co0.26 )2 P/Ni3 N and Fe2 P/Ni3 N electrocatalysts demand only 113 and 212 mV to afford 100 mA cm-2 for hydrogen and oxygen evolution reactions (HER and OER) in 1 m KOH, respectively, thus substantially expediting overall water/seawater electrolysis at 100 mA cm-2 with 1.592/1.645 V. Particularly, Fe2 P/Ni3 N displays an unprecedented overpotential of 302 mV at 500 mA cm-2 , which represents the best alkaline seawater oxygen evolution activity among the ever-reported non-noble electrocatalysts; and thus substantially expedites overall water/seawater splitting at 500 mA cm-2 with 1.701/1.768 V, surpassing most of the reported non-noble lectrocatalysts. This work provides a new approach for developing high-performance electrocatalysts for seawater splitting.

Highly sensitive and selective detection of nitrite using a fiber optofluidic laser.

Nitrite ion (NO2-) is a common contaminant that can significantly threaten human health and the environment. In this study, we demonstrate a chemical sensing platform to monitor the nitrite concentration using a fiber optofluidic laser (FOFL). An optical fiber, integrated into a microchannel, is used both as an optical micro-cavity and the sensing element. Rhodamine 6 G (Rh6G) in an aqueous micellar solution is used as the laser gain medium. The light intensity change of the lasing spectra is employed as an indicator for the NO2- ion concentration sensing. The lasing properties under different NO2- ion concentrations are experimentally and theoretically investigated to examine the sensing performance of the FOFL. The results show that the limit detection of the FOFL sensor is 0.54 µM, which is 2-order-of-magnitude lower than fluorescence measurement. The sensing mechanism of Rh6G for NO2- detection is studied by using density functional theory (DFT). The calculation results indicate that nitrite influences the electronic distribution of Rh6G based on the heavy atom effect, which leads to the fluorescence quenching of Rh6G in the excited state. In addition, the detection system exhibits favorable selectivity for NO2- ions.

Excitatory Effects of Astrocytic Hydrogen Sulfide on the Electrical Activity of Oxytocin Neurons in the Supraoptic Nucleus.

INTRODUCTION: In the regulation of oxytocin (OT) neuronal activity, hydrogen sulfide (H2S), a gaseous neurotransmitter, likely exerts an excitatory role. This role is associated with increased expression of astrocytic cystathionine-ß-synthase (CBS), the key enzyme for H2S synthesis. However, it remains unclear whether H2S is mainly produced in astrocytes and contributes to the autoregulation of OT neurons. METHODS: In hypothalamic slices of male rats, OT and H2S-associated drug effects were observed on the firing activity and spontaneous excitatory postsynaptic currents (sEPSCs) of putative OT neurons in the supraoptic nucleus (SON) in whole-cell patch-clamp recording. Expression of glial fibrillary acidic protein (GFAP) in the SON was analyzed in Western blots. In addition, changes in the length of rat pups' hypothalamic astrocytic processes were observed in primary cultures. RESULTS: In brain slices, OT significantly increased the firing rate of OT neurons, which was simulated by CBS allosteric agonist S-adenosyl-L-methionine (SAM) and H2S slow-releasing donor GYY4137 but blocked by CBS inhibitor aminooxyacetic acid (AOAA). L-α-aminoadipic acid (a gliotoxin) blocked SAM-evoked excitation. OT and SAM also increased the frequency and amplitude of sEPSCs; the effect of OT was blocked by AOAA. Both OT and GYY4137 reduced GFAP expression in the SON. Morphologically, OT or GYY4137 time-dependently reduced the length of astrocytic processes in primary cultures. CONCLUSIONS: These findings indicate that the auto-excitatory effect of OT on OT neurons is mediated by H2S from astrocytes at least partially and astrocytic H2S can elicit retraction of astrocytic processes that subsequently increase OT neuronal excitability.

Parapedobacter tibetensis sp. nov., isolated from shore soil of saline lake in Tibet of China and its genome mining of secondary metabolite.

Five aerobic, Gram-stain-negative, non-motile, non-spore-forming, short rod bacteria strains, designated as C3-1-R+6T, C3-2-M9, B3-2-R-7, B3-2-R-21 and C3-2-M2, were isolated from shore soil of LungmuCo Lake in Tibet of China. The 16S rRNA gene sequence comparisons confirmed their affiliation to the genus Parapedobacter of the family Sphingobacteriaceae, and showed that they were most closely related to Parapedobacter lycopersici KACC 18788T with 94.26 % similarities. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between them and the validly published Parapedobacter species were all below the thresholds for delineating species, supporting that they were novel species of genus Parapedobacter. The ANI, AAI and dDDH values between strains C3-1-R+6T and Parapedobacter lycopersici KACC 18788T were 72, 75, and 18% respectively. Meanwhile, the ANI/AAI and dDDH values between these five isolates were higher than the threshold values, showing that they belonged to the same species of Parapedobacter. According to genome comparison, the novel isolates have some special biosynthetic gene clusters of secondary metabolites including bacteriton, aryl-polyene, lantipeptide and t1pks, which were absent from their most related phylogenetic neighbours P. lycopersici KACC 18788T and P. pyrenivorans CGMCC 1.12195T. The main polar lipids contained phosphatidylethanolamine, one unidentified phospholipid, one unidentified aminolipid, one unidentified glycolipid and five unidentified lipids. The predominant respiratory quinone was MK-7. The major cellular fatty acids were iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C17 : 0 3-OH. The genome size of strain C3-1-R+6T was 5 984 948 bp, and its genomic DNA G+C content was 46.21 mol%. To sum up, the five strains were identified as a novel species of the genus Parapedobacter, for which the name Parapedobacter tibetensis sp. nov. was proposed. The type strain was C3-1-R+6T (=CGMCC 1.19194T=KCTC 92150T).

Large dynamic range dual-mode pH sensors via dye-doped ionic liquid fiber optofluidic lasers.

We report a fiber optofluidic laser (FOFL) using an RhB-doped ionic liquid (BmimPF6) as the gain medium and explore its application for large dynamic range highly sensitive pH sensing. Due to the high Q-factor of the FOFL and the unique merits of BmimPF6, lasing emission presents a threshold of only 0.61 µJ mm-1. Particularly, lasing emission behaviors are strongly dependent on the pH value of the gain medium, i.e., in the pH range 4.28-6.37, the lasing central wavelength blue-shifts monotonically with a sensitivity as high as 5.02 nm per pH unit, which we attribute to the conversion of the cationic form of RhB to the zwitterionic form caused by the deprotonation of the COOH group. Under alkaline conditions (pH 7.20-11.17), the lasing emission intensity exhibits a significant decrease and the corresponding lasing central wavelength is also blue-shifted due to the solvent effect. The sensitivity based on the wavelength shift is 3.03 nm per pH unit, which is 4-fold higher than that of fluorescence-based sensing, while the sensitivity based on the variation of the lasing emission intensity is almost three orders of magnitude higher than that of fluorescence-based sensing. Our work presents a novel dual sensing paradigm in response to different pH conditions, which can greatly improve the reliability and discrimination of pH sensing.

Development of a sandwich ELISA for the specific quantitation of hemagglutinin (HA)-tagged proteins during their inducible expression in Escherichia coli.

Heavy single-chain antibodies (VHH or nanobodies) are popular in the medical and analytical fields due to its small size, high solubility, stability, and other advantageous features. However, the usage of VHHs is limited by the low yield of its production and purification. In order to determine the optimal purification strategy for VHH to improve the yield, a method to monitor purification at the intermediate steps is needed. In this study, a simple, sensitive, low-cost sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitate VHHs throughout the purification steps. Under optimized conditions, the assay has a sensitivity of 0.149 OD·mL/ng and a limit of detection (LOD) of 0.029 ng/mL. The average recoveries of the assay against the spiked samples were 101.9-106.0% and 100.7-108.0%. The method was applied to a variety of real samples for the detection of different VHHs in bacterial cell media. High amount of VHHs (up to 41.3 mg/mL), which are comparable to the average yield of VHH in standard production protocols, were detected in the media. This study raises attention to the problem of protein losses in cell culture supernatants and provides a method for the continuous detection of the protein abundance to optimize the expression and purification protocols especially for nanobodies.

Glyphosate decreases survival, increases fecundity, and alters the microbiome of the natural predator Harmonia axyridis (ladybird beetle).

Glyphosate is a widely-used herbicide that shows toxicity to non-target organisms. The predatory natural enemy Harmonia axyridis may ingest glyphosate present in pollen and aphid prey. The present study characterized the responses of adult H. axyridis to environmentally relevant concentrations of glyphosate (5, 10, and 20 mg/L) for one or five days. There were no obvious effects on adult H. axyridis survival rates or fecundity in response to 5 or 10 mg/L glyphosate. However, exposure to 20 mg/L glyphosate significantly reduced the survival rate and increased fecundity. Analysis of the adult H. axyridis microbiota with 16S rRNA sequencing demonstrated changes in the relative and/or total abundance of specific taxa, including Serratia, Enterobacter, Staphylococcus, and Hafnia-Obesumbacterium. These changes in symbiotic bacterial abundance may have led to changes in survival rates or fecundity of this beetle. This is the first report of herbicide-induced stimulation of fecundity in a non-target predatory natural enemy, reflecting potentially unexpected risks of glyphosate exposure in adult H. axyridis. Although glyphosate resistant crops have been widely planted, the results of this study indicate a need to strengthen glyphosate management to prevent over-use, which could cause glyphosate toxicity and threaten environmental and human health.

Assessment of the risk of imidaclothiz to the dominant aphid parasitoid Binodoxys communis (Hymenoptera: Braconidae).

The neonicotinoid of imidaclothiz insecticide with low resistance and high efficiency, has great potential for application in pest control in specifically cotton field. In this systematically evaluate the effects of sublethal doses of imidaclothiz (LC10: 11.48 mg/L; LC30: 28.03 mg/L) on the biology, transcriptome, and microbiome of Binodoxys communis, the predominant primary parasitic natural enemy of aphids. The findings indicated that imidaclothiz has significant deleterious effects on the survival rate, parasitic rate, and survival time of B. communis. Additionally, there was a marked reduction in the survival rate and survival time of the F1 generation, that is, the negative effect of imidaclothiz on B. communis was continuous and trans-generational. Transcriptome analysis revealed that imidaclothiz treatment elicited alterations in the expression of genes associated with energy and detoxification metabolism. In addition, 16S rRNA analysis revealed a significant increase in the relative abundance of Rhodococcus and Pantoea, which are associated with detoxification metabolism, due to imidaclothiz exposure. These findings provide evidence that B. communis may regulate gene expression in conjunction with symbiotic bacteria to enhance adaptation to imidaclothiz. Finally, this study precise evaluation of imidaclothiz's potential risk to B. communis and provides crucial theoretical support for increasing the assessment of imidaclothiz in integrated pest management.