Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli.

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Continuous programmable mid-infrared thermal emitter and camouflage based on the phase-change material In3SbTe2.

In3SbTe2 (IST), a new non-volatile phase-change material (PCM), promises highly tunable infrared optical properties and offers a distinct path to the significant modulation of its optical scattering fingerprint, suggesting tremendous applications. In this Letter, we demonstrate and optimize a four-layer emitter based on IST, achieving an ultra-wide average emissivity variation of more than 94% in the middle-infrared region (MIR, 3-5 µm). This remarkable emissivity difference can be further continuously modified by changing the structural composition in terms of the amorphous and crystalline states of the IST layers. Based on this continuous programmable emission, the MIR emission characteristics of marble, maple leaf, and blue polyvinyl chloride are successfully imitated together on a desert background, demonstrating the programmable and multi-level MIR optical camouflage capabilities of IST. This work provides a promising platform for continuously modulating emission characteristics and offers a reference for the subsequent application of programmable optical devices.

Evaluating blood supply changes in the osteonecrosis of the femoral head using gadobutrol-based steady-state MR angiography.

OBJECTIVES: Assess the feasibility of using gadobutrol-based steady-state (SS) MR angiography (MRA) to evaluate the blood supply changes of osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: Participants were recruited in this prospective study from December 2021 to May 2022 in a single center. The number of superior retinacular arteries (SRAs), inferior retinacular arteries (IRAs), anterior retinacular arteries (ARAs), and overall retinacular arteries (ORAs), as well as the affected rates of SRA and IRA, were determined and compared between healthy and ONFH hips and between hips across the Association Research Circulation Osseous (ARCO) staging I-IV. RESULTS: Twenty healthy and 64 ONFH hips were evaluated in 54 participants. There were significant differences between ARCO I-IV for the number of ORAs (mean of 3.5, 2.3, 1.7, and 0.8 for ARCO I-IV, respectively; p < .001), SRAs (median of 2.5, 1, 0.5, and 0 for ARCO I-IV, respectively; p < .001), and the affected rate of SRAs (20.00%, 65.22%, 77.78%, 92.31% for ARCO I-IV, respectively, p = 0.002). There were significant differences between ONFH and healthy hips for the number of ORAs (median of 5 vs. 2; p < .001), SRAs (median of 3 vs. 1; p < .001), IRAs (median of 1 vs. 1; p < .001), ARAs (median of 0 vs. 0; p = 0.04), and also the affected rate of SRAs (5.00% vs. 67.20%, p < .001) and IRAs (30% vs. 84.4%, p < .001). CONCLUSION: Gadobutrol-enhanced SS MRA is a feasible method for evaluation of hemodynamics in ONFH. CLINICAL RELEVANCE STATEMENT: Gadobutrol-enhanced magnetic resonance angiography can evaluate blood supply changes of ONFH and therefore helps to aid in the diagnosis and guide treatment of ONFH. KEY POINTS: • Gadobutrol-enhanced magnetic resonance angiography showed changes in the retinacular artery related to the severity of femoral osteonecrosis. • Gadobutrol-enhanced magnetic resonance angiography revealed a reduced blood supply to the ischemic necrotic femoral head compared to the healthy counterparts.

Deep learning-based inverse design optimization of efficient multilayer thermal emitters in the near-infrared broad spectrum.

This study proposes a deep learning architecture for automatic modeling and optimization of multilayer thin film structures to address the need for specific spectral emitters and achieve rapid design of geometric parameters for an ideal spectral response. Multilayer film structures are ideal thermal emitter structures for thermophotovoltaic application systems because they combine the advantages of large area preparation and controllable costs. However, achieving good spectral response performance requires stacking more layers, which makes it more difficult to achieve fine spectral inverse design using forward calculation of the dimensional parameters of each layer of the structure. Deep learning is the main method for solving complex data-driven problems in artificial intelligence and provides an efficient solution for the inverse design of structural parameters for a target waveband. In this study, an eight-layer thin film structure composed of SiO2/Ti and SiO2/W is rapidly reverse engineered using a deep learning method to achieve a structural design with an emissivity better than 0.8 in the near-infrared band. Additionally, an eight-layer thin film structure composed of 3 × 3 cm SiO2/Ti is experimentally measured using magnetron sputtering, and the emissivity in the 1-4 µm band was better than 0.68. This research provides implications for the design and application of micro-nano structures, can be widely used in the fields of thermal imaging and thermal regulation, and will contribute to developing a new paradigm for optical nanophotonic structures with a fast target-oriented inverse design of structural parameters, such as required spectral emissivity, phase, and polarization.

Chromosome-scale genomics, metabolomics, and transcriptomics provide insight into the synthesis and regulation of phenols in Vitis adenoclada grapes.

Vitis adenoclada is a wild grape unique to China. It exhibits well resistance to heat, humidity, fungal disease, drought, and soil infertility. Here, we report the high-quality, chromosome-level genome assembly of GH6 (V. adenoclada). The 498.27 Mb genome contained 221.78 Mb of transposable elements, 28,660 protein-coding genes, and 481.44 Mb of sequences associated with 19 chromosomes. GH6 shares a common ancestor with PN40024 (Vitis vinifera) from approximately 4.26-9.01 million years ago, whose divergence occurred later than Vitis rotundifolia and Vitis riparia. Widely-targeted metabolome and transcriptome analysis revealed that the profiles and metabolism of phenolic compounds in V. adenoclada varieties significantly were differed from other grape varieties. Specifically, V. adenoclada varieties were rich in phenolic acids and flavonols, whereas the flavan-3-ol and anthocyanin content was lower compared with other varieties that have V. vinifera consanguinity in this study. In addition, ferulic acid and stilbenes content were associated with higher expressions of COMT and STSs in V. adenoclada varieties. Furthermore, MYB2, MYB73-1, and MYB73-2 were presumably responsible for the high expression level of COMT in V. adenoclada berries. MYB12 (MYBF1) was positively correlated with PAL, CHS, FLS and UFGT.Meanwhile, MYB4 and MYBC2-L1 may inhibit the synthesis of flavan-3-ols and anthocyanins in two V. adenoclada varieties (YN2 and GH6). The publication of the V. adenoclada grape genome provides a molecular foundation for further revealing its flavor and quality characteristics, is also important for identifying favorable genes of the East Asian species for future breeding.

Discovery of STRO-002, a Novel Homogeneous ADC Targeting Folate Receptor Alpha, for the Treatment of Ovarian and Endometrial Cancers.

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non-small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).

Design and optimization of mid-infrared hot electron detector based on Al/GaAs fishnet nanostructure for CO2 sensing.

Hot electron detectors (HEDs) based on plasmon resonance can circumvent a semiconductor's bandgap limitation and have high sensitivity, suitable for infrared gas detectors. Unfortunately, there are few literature reports on research in the mid-infrared (MIR) region. Herein, we design and optimize a HED based on Al/GaAs fishnet nanostructure for MIR CO2 sensing, and its optical-electrical properties are numerically studied. Surface plasmons not only achieve strong absorptance at CO2 emission wavelength but also greatly improve the photoelectric responsivity over a plane structure detector (∼42times). By changing the thickness of the GaAs layer, the detection wavelength can also be actively adjusted, achieving a larger range of multi-gas detection. The effect of external voltage is also considered. This work highlights a potential engineering application value and offers a path toward more compact and efficient MIR gas detectors.

Widely Targeted Metabolomics Profiling Reveals the Effect of Powdery Mildew on Wine Grape Varieties with Different Levels of Tolerance to the Disease.

Powdery mildew is an economic threat for viticulture because it not only affects grape yield, but also causes a series of impacts on the qualities of fruit and wine, especially the flavors and various metabolites. Different grape varieties may have different levels of powdery mildew resistance/tolerance and their components of their metabolome are also various. In this study, two wine grape varieties, Guipu No.6 (GP6) and Marselan (Mar) with different levels of powdery mildew tolerance, were used to compare the quality differences in metabolism level by using the widely targeted metabolomics method. The results show that GP6 has a better powdery mildew leaf tolerance than Mar. A total of 774 metabolites were detected by using a UPLC-QQQ-MS-based metabolomics approach, and 57 differential metabolites were identified as key metabolites that were accumulated after infection with powdery mildew in GP6 and Mar, including phenolic acids, flavonoids, terpenoids, stilbenes, lipids, nucleotides and derivatives, lignans and coumarins, and quinones. This finding indicates that the defense mechanisms of grape fruit are mainly associated with phenylpropane-flavonoid metabolism. Specifically, stilbenes had greater variations after powdery mildew infection in GP6; while in Mar, the variations of flavonoids, especially kaempferol-3-O-glucuronide and luteolin-7-O-glucuronide, were more remarkable. The above results demonstrate that stilbenes may play a more important role than flavonoids in resisting powdery mildew infection in GP6's fruits, and the drastic variations of these phenolic compounds in different wine grapes after powdery mildew infection might also lead to quality difference in the flavors. This study can provide new insights into the understanding of the cause of powdery mildew tolerance in different grape varieties and the effects on the quality of wine grapes infected with the disease exerted by metabolism level.

Multi-Modal Vehicle Trajectory Prediction by Collaborative Learning of Lane Orientation, Vehicle Interaction, and Intention.

Accurate trajectory prediction is an essential task in automated driving, which is achieved by sensing and analyzing the behavior of surrounding vehicles. Although plenty of research works have been invested in this field, it is still a challenging subject due to the environment's complexity and the driving intention uncertainty. In this paper, we propose a joint learning architecture to incorporate the lane orientation, vehicle interaction, and driving intention in vehicle trajectory forecasting. This work employs a coordinate transform to encode the vehicle trajectory with lane orientation information, which is further incorporated into various interaction models to explore the mutual trajectory relations. Extracted features are applied in a dual-level stochastic choice learning to distinguish the trajectory modality at both the intention and motion levels. By collaborative learning of lane orientation, interaction, and intention, our approach can be applied to both highway and urban scenes. Experiments on the NGSIM, HighD, and Argoverse datasets demonstrate that the proposed method achieves a significant improvement in prediction accuracy compared with the baseline.

Indexing Ultrafast Shape-Based Descriptors in MongoDB to Identify TLR4 Pathway Agonists.

A method is presented for an ultrafast shape-based search workflow for the screening of large compound collections, i.e., those of vendors. The three-dimensional shape of a molecule dictates its biological activity by enabling the molecule to fit into binding pockets of proteins. Quite often, distinctly different chemical compounds that have similar shapes can bind in a similar way. OpenEye pioneered an algorithm for comparing shapes of molecules by overlaying them in a computer and measuring differences between a query molecule and a target molecule. Overlaying shapes is a computationally intensive process and represents a bottleneck in searching for similar molecules. More recent publications describe alternative methods of overlaying molecules, which are accomplished by comparing shape-based descriptors. These methods were implemented in the Open Drug Discovery Toolkit (ODDT) package. We utilized a combination of open-source software packages like ODDT and RDkit to implement a workflow for ultrafast conformer generation and matching that does not require storing precomputed conformers on the file system or in memory. Moreover, the generated descriptors could be optionally stored in MongoDB for performing searches in the future. To speed up the search, we created a set of indexes from the transformed shape-based descriptors. We are in the process of calculating descriptors for multiple vendors, including Enamine's "REAL" collection of 1.2 billion compounds. Currently, the shape similarity search on more than 70 million compounds takes less than 8 s! We exemplified our methodology with the screen of compounds that can act as putative TLR4 agonists. The search was based on a literature-known small-molecule TLR4 agonist series. In due course, we identified compounds with novel structural motifs that were active in mouse and human TLR4 reporter cell lines.

Development of an E. coli strain for cell-free ADC manufacturing.

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody-drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.

How Facial Attractiveness Affects Time Perception: Increased Arousal Results in Temporal Dilation of Attractive Faces.

Time perception plays a fundamental role in people's daily life activities, and it is modulated by changes in environmental contexts. Recent studies have observed that attractive faces generally result in temporal dilation and have proposed increased arousal to account for such dilation. However, there is no direct empirical result to evidence such an account. The aim of the current study, therefore, was to clarify the relationship between arousal and the temporal dilation effect of facial attractiveness by introducing a rating of arousal to test the effect of arousal on temporal dilation (Experiment 1) and by regulating arousal via automatic expression suppression to explore the association between arousal and temporal dilation (Experiment 2). As a result, Experiment 1 found that increased arousal mediated the temporal dilation effect of attractive faces; Experiment 2 showed that the downregulation of arousal attenuated the temporal dilation of attractive faces. These results highlighted the role of increased arousal, which is a dominating mechanism of the temporal dilation effect of attractive faces.

Altered Brain Functional Connectivity Density in Fast-Ball Sports Athletes With Early Stage of Motor Training.

The human brain shows neuroplastic adaptations caused by motor skill training. Of note, there is little known about the plastic architecture of the whole-brain network in resting state. The purpose of the present study was to detect how motor training affected the density distribution of whole-brain resting-state functional connectivity (FC). Resting-state functional magnetic resonance imaging data was assessed based on a comparison of fast-ball student athletes (SA) and non-athlete healthy controls (NC). The voxel-wise data-driven graph theory approach, global functional connectivity density (gFCD) mapping, was applied. Results showed that the SA group exhibited significantly decreased gFCD in brain regions centered at the left triangular part of the inferior frontal gyrus (IFG), extending to the opercular part of the left IFG and middle frontal gyrus compared to the NC group. In addition, findings suggested the idea of an increased neural efficiency of athletes' brain regions associated with attentional-motor modulation and executive control. Furthermore, behavioral results showed that in the SA group, faster executive control reaction time relates to smaller gFCD values in the left IFG. These findings suggested that the motor training would decrease the numbers of FC in IFG to accelerate the executive control with high attentional demands and enable SA to rapidly focus the attention to detect the intriguing target.

Comparison of transcriptional expression patterns of phenols and carotenoids in 'Kyoho' grapes under a two-crop-a-year cultivation system.

To fully utilize the characteristic climatic conditions in the southern region of China, a two-crop-a-year cultivation system technique for 'Kyoho' grape was developed during the past decade. After summer harvest in June, appropriate pruning and chemical treatments promote flowering and fruiting, which enables a second harvest in late December. Due to climatic differences between the two crop growing seasons, grape phenol and carotenoid metabolism differ greatly. The reported study analyzed the transcriptome of the carotenoid and phenylpropanoid/flavonoid pathways in grapes at four different stages during the two growing seasons. Compared with those in summer grapes, expression levels of the majority of genes involved in the carotenoid metabolic pathway in winter grapes were generally upregulated. This result was associated with lower rainfall and much more abundant sunlight during the second growing season. On the other hand, summer cropping strongly triggered the expression of upstream genes in the phenylpropanoid/flavonoid pathway at E-L 33 and E-L 35. Transcript levels of flavonoid 3',5'-hydroxylase (F3'5'H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3-hydroxylase (F3H) and glutathione S-transferase (GST) were upregulated in winter grapes at the mature stage. Together, these results might indicate that more flavonoids would be synthesized in ripe winter grapes during the mature stage of the second crop under much drier conditions, longer sunlight hours and lower temperature. These data provide a theoretical foundation for the secondary metabolism of berries grown under two-crop-a-year cultivation systems.

Exploration of the carmaphycins as payloads in antibody drug conjugate anticancer agents.

Antibody-drug conjugates (ADCs) represent a new dimension of anticancer chemotherapeutics, with warheads to date generally involving either antitubulin or DNA-directed agents to achieve low-to sub-nanomolar potency. However, other potent cytotoxins working by different pharmacological mechanisms are under investigation, such as α,ß-epoxyketone based proteasome inhibitors. These proteasome active agents are an emerging class of anticancer drug that possesses ultra-potent cytotoxicity to some cancer cell lines. The carmaphycins are representatives of this latter class that we isolated and characterized from a marine cyanobacterium, and these as well as several synthetic analogues exhibit this level of potency. In the current work, we investigated the use of these highly potent cytotoxic compounds as warheads in the design of novel ADCs. We designed and synthesized a library of carmaphycin B analogues that contain amine handles, enabling their attachment to an antibody linker. The basicity of these incorporated amine handles was shown to strongly affect their cytotoxic properties. Linear amines resulted in the greatest reduction in cytotoxicity whereas less basic aromatic amines retained potent activity as demonstrated by a 4-sulfonylaniline derivative. These investigations resulted in identifying the P2 residue in the carmaphycins as the most suitable site for linker attachment point, and hence, we synthesized a highly potent analogue of carmaphycin B that contained a 4-sulfonylaniline handle as an attachment point for the linker antibody.

Evolutionary, interaction and expression analysis of floral meristem identity genes in inflorescence induction of the second crop in two-crop-a-year grape culture system.

The fruitfulness of grapevines (Vitis viniferaL.) is determined to a large extent by the differentiation of uncommitted meristems, especially in the second-crop production of some varieties, where the intermediate of inflorescence and tendril accounts for a significant proportion in two-crop-a-year grape culture system. The differentiation of uncommitted lateral meristem was reported to be regulated by a network, whose backbone was composed of several floral meristem identity genes. In the present study, the phylogenetics of grape floral meristem identity genes with their orthologues in other species, and their conserved domain and interaction networks were analysed. In addition, the effects of chlormequat chloride and pinching treatments on the expression profiles of floral meristem identity genes and content of gibberellic acid (GAs) and zeatin riboside (ZR), as well as the ratio of ZR/GAs in buds that were used to produce the second crop, and the ratio of inflorescence induction of the second crop were studied in 'Summer Black'. The present results showed that floral meristem identity genes of grape and their orthologues in one or more among Malus domastic, Citrus sinensis, heobroma cacaoT, Nicotiana tabacum, Solanum lycopersicum and Glycine hirsutum, probably originated from a common ancestor. Interaction networks of six grape-floral meristem identity genes indicated that the inflorescence induction and floral development were regulated by one more complex network, and expression profiles of genes that involved in this network could be affected by each other. Expression profiles of eight floral meristem identity genes were affected by chlormequat chloride and pinching treatments, and higher expression levels of FT, TFL1A and TFL1B, as well as lower expression levels of LFY from 3 days before full bloom to 11 days after full bloom were thought to play important roles in promoting the formation of inflorescence primordial of the second crop, and higher expression levels of CAL A, SOC1 and TFL1A at 18 days after full bloom (DAF) could promote the development of inflorescence primordial. In addition, lower ratio of ZR/GAs at 3 days before full bloom and 4 days after full bloom could promote the formation of uncommitted lateral meristems in chlormequat chloride and pinching-treated plants, and higher ratio at 11 days after full bloom was the main reason for the formation of more inflorescences after chlormequat chloride treatment.

Toward a Ferrous Iron-Cleavable Linker for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are antigen-targeted therapeutics that employ antibodies to deliver potent, cytotoxic effectors to cells with potentially high specificity. While promising clinical results have been achieved, significant pitfalls remain including internalization of ADCs in nontargeted cells expressing target antigen, which can limit therapeutic windows. Novel ADC linkers that are cleaved selectively in cancer cells but not in normal cells could minimize collateral damage caused by ADC uptake in nontargeted tissues. Here, we describe a prototypical ADC linker based on an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX) that by itself has demonstrated tumor-selective activity in preclinical cancer models. We prepared TRX-linked ADCs by site-selective conjugation to two sites in trastuzumab and compared their activity in Her2 positive and negative cells to ADC controls based on established linker chemistry. Our results confirm that the TRX moiety efficiently releases its payload following ADC uptake, affording picomolar potencies in antigen-positive cells. We also identified a destabilizing interaction between these initial TRX linkers and nearby antibody residues and suggest an approach to improve upon these prototypical designs.

RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates.

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis.

Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species. Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7-66.4% and 56.5-60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death.

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent.