Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin.

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

Identification of new AAV vectors with enhanced blood-brain barrier penetration efficiency via organ-on-a-chip.

To overcome limitations in the generalizability and efficiency of current AAV vectors, in this current study, we constructed an AAV variant library by the insertion of random heptapeptide sequences in the receptor-binding domain of the AAV9 capsid gene. We then applied a recently developed organ-on-a-chip in vitro model of the human blood-brain barrier (BBB) to iteratively enrich for variants that efficiently cross the BBB and transduce astrocyte cells. Through multiple rounds of screening, we obtained two candidate AAV variants, AAV-M6 and AAV-M8, which showed significantly higher BBB penetration efficiency than AAV9 or AAV-PHP.eB. Quantitative PCR (qPCR) assay showed that AAV-M6 could accumulate to a 5 times higher titer, while AAV-M8 reached a 3 times higher titer, than AAV-PHP.eB in the neural chamber of the model. The transduction assay further verified that the AAV-M6 candidate vector was able to infect HA-1800 cells after crossing the BBB, suggesting it could potentially transduce brain parenchymal cells after crossing the hCMEC/D3 layer at higher efficiency than AAV-PHP.eB. Molecular simulations suggested that the human receptor proteins, LY6D and M6PR, could bind the AAV-M6 heptapeptide insertion with high affinity. This study provides two promising candidate AAV vectors and demonstrates the use of this in vitro BBB model for scalable, high-throughput screening of gene therapies. These tools can drive investigations of the mechanisms underlying BBB permeability and the cell-type specificity of virus vectors.

pH-Responsive and Recyclable Hydrogels for Gas Releasing and Scavenging.

Gas-releasing/scavenging hydrogels have wide applications in biomedical and industrial fields. However, the covalently crosslinked nature of these existing materials makes them difficult to degrade or recycle, leading to a waste of raw materials and aggravating environmental pollution. Herein, a new class of pH-responsive and recyclable hydrogels with versatile gas-releasing and scavenging properties is reported, utilizing pH changes to reversibly control disassembly and reassembly of the hydrogel network. The initial hydrogels are constructed via the one-pot radical polymerization and contain dynamic molecular networks based on hydrophobic interactions, which can disassemble when the materials are placed in low pH solutions. The disassembled copolymer chains can reform hydrogels, following supplementation with fresh mineral salts and micelle monomers in neutral solutions. Moreover, the mineral salts used to reform hydrogels can function as gas donors or scavengers, endowing these hydrogels with versatile gas-releasing and consuming properties. Overall, this research provides a facile and environmentally friendly method to recycle hydrogels with gas-releasing and gas-scavenging properties, which have potential applications in diverse fields, including wound healing, wastewater management, and gas therapy for diseases.

Surgical Repair of Giant Asymptomatic Ascending Aortic Aneurysm Accompanied with Chronic Stanford Type A Aortic Dissection: A Case Report.

BACKGROUND: Ascending aortic aneurysm accompanied with stanford type A aortic dissection is a life-threatening condition. The most common presenting symptom is pain. Here, we report a very rare case of giant asymptomatic ascending aortic aneurysm accompanied with chronic stanford type A aortic dissection. CASE PRESENTATION: A 72-year-old woman was founded to have ascending aortic dilation on a routine physical examination. On admission, CTA showed an ascending aortic aneurysm accompanied with stanford type A aortic dissection, the diameter of which was approximately 10 cm. Transthoracic echocardiography showed an ascending aortic aneurysm, aortic sinus and sinus junction dilation, moderate aortic valve regurgitation, left ventricle enlargement, left ventricular wall hypertrophy, and mitral and tricuspid valve mild regurgitation. The patient underwent surgical repair in our department, was discharged, and recovered well. CONCLUSION: This was a very rare case of a giant asymptomatic ascending aortic aneurysm accompanied with chronic stanford type A aortic dissection that was successfully managed by total aortic arch replacement.

Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell.

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules.

[Modeling and Predicting of MODIS Leaf Area Index Time Series Based on a Hybrid SARIMA and BP Neural Network Method].

The modeling and predicting of vegetation Leaf area index (LAI) is an important component of land surface model and assimilation of remote sensing data. The MODIS LAI product (i.e. MOD15A2) is one of the most widely used LAI data sources. However, the time series of MODIS LAI contains some data of low quality. For example, because of the influence of the cloud, aerosol, etc., the MODIS LAI presents the characteristics of the discontinuous in time and space. In fact, the time series of MODIS LAI include both linear and nonlinear components, which cannot be accurately modeled and predicted by either linear method or nonlinear method alone. In this paper, the original LAI time series data were first smoothed with Savitzky-Golay (SG) filtration and linear interpolation; SARIMA, BP neural network and a hybrid method of SARIMA-BP neural network were then used for modeling and predicting MODIS LAI time series. The SARIMA-BP neural network combined both SARIMA and BP neural network, which could model the linear and the nonlinear component of MODIS LAI time series respectively. That is, the final result of SARIMA-BP neural network was the sum of results of the two methods. Experiments showed that the time series of MODIS LAI that were smoothed with the SG filtration and linear interpolation were more smooth than original time series, with a determination coefficient up to 0.981, closer to 1 than that of SARIMA (0.941) and BP neural network (0.884); the correlation coefficient between SARIMA-BP neural network and the observation is 0.991, higher than that of between SARIMA (0.971) or BP neural network (0.942) SARIMA and the observation. Thus, it can be concluded that, the proposed SARIMA-BP neural network method can better adapt to the LAI time series, and it outperforms the SARIMA and BP neural network methods.

Long-term, efficient inhibition of microRNA function in mice using rAAV vectors.

Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA 'tough decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuDs depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122 TuD but not anti-let-7 TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High-throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuDs induced miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.

A single intravenous rAAV injection as late as P20 achieves efficacious and sustained CNS Gene therapy in Canavan mice.

Canavan's disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤ 1 month) AspA(-/-) mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.

Chemical proteomic profiling of protein dopaminylation in colorectal cancer cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid residue of H3 ( i.e. , glutamine) and dopamine. In our previous studies, we discovered that the dynamics of this post-translational modification (including installation, removal, and replacement) were regulated by a single enzyme, transglutaminase 2 (TGM2), through reversible transamination. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of TGM2 in colon cancer cells. Due to the enzyme promiscuity of TGM2, non-histone proteins have also been identified as targets of dopaminylation on glutamine residues, however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of modification sites of these dopaminylated proteins were identified, attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that to block the installation of protein dopaminylation may become a promising anti-cancer strategy.

pH-Controlled chemoselective rapid azo-coupling reaction (CRACR) enables global profiling of serotonylation proteome in cancer cells.

Serotonylation has been identified as a novel protein post-translational modification (PTM) for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibody for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to label protein serotonylation in a bioorthogonal manner. This chemical biology tool can be utilized alternatively for the antibody-free enrichment of serotonylated proteins, which depends on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 500 serotonylated proteins were identified from HCT 116 cells. Importantly, a number of modification sites of these serotonylated proteins were determine, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Bioorthogonal labeling and enrichment of histone monoaminylation reveal its accumulation and regulatory function in cancer cell chromatin.

Histone monoaminylation ( i . e ., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

[Evaluating the utility of MODIS vegetation index for monitoring agricultural drought].

The exclusive shortwave bands provided by MODIS sensors offer new opportunities for agricultural drought monitoring, since they are very sensitive to vegetation moisture. In the present work, we selected Songnen Plain in Northeast China as study area aiming at monitoring agricultural drought of dry farmland here. Four types of vegetation water indices and vegetation greenness indices were calculated from the 8-day composite MODIS product (MODO9A1) in vegetation growing season between 2001 and 2010, respectively. Multi-scale standardized precipitation index (SPI) derived from precipitation data of weather stations was used as reference data to estimate drought sensitivity of various vegetation indices, and a pixel-to-weather station paired correlation approach was used to calculate the Pearson correlation coefficient between vegetation index and SPIs. The result indicated that vegetation water indices established by near infrared and shortwave infrared bands outperformed vegetation greenness indices based on visible and near infrared bands. Of these indices, NDII7 performs the best with highest correlation coefficients across all SPIs. The authors' results demonstrated the potential of MODIS shortwave spectral bands in monitoring agricultural drought, and this provides new insights to future research.

The human microbiome and cancer: a diagnostic and therapeutic perspective.

Recent evidence has shown that the human microbiome is associated with various diseases, including cancer. The salivary microbiome, fecal microbiome, and circulating microbial DNA in blood plasma have all been used experimentally as diagnostic biomarkers for many types of cancer. The microbiomes present within local tissue, other regions, and tumors themselves have been shown to promote and restrict the development and progression of cancer, most often by affecting cancer cells or the host immune system. These microbes have also been shown to impact the efficacy of various cancer therapies, including radiation, chemotherapy, and immunotherapy. Here, we review the research advances focused on how microbes impact these different facets and why they are important to the clinical care of cancer. It is only by better understanding the roles these microbes play in the diagnosis, development, progression, and treatment of cancer, that we will be able to catch and treat cancer early.

Dynamic and Wearable Electro-responsive Hydrogel with Robust Mechanical Properties for Drug Release.

Electro-responsive dynamic hydrogels, which possess robust mechanical properties and precise spatiotemporal resolution, have a wide range of applications in biomedicine and energy science. However, it is still challenging to design and prepare electro-responsive hydrogels (ERHs) which have all of these properties. Here, we report one such class of ERHs with these features, based on the direct current voltage (DCV)-induced rearrangement of sodium dodecyl sulfate (SDS) micelles, where the rearrangement can tune the hydrogel networks that are originally maintained by the SDS micelle-assisted hydrophobic interactions. An enlarged mesh size is demonstrated for these ERHs after DCV treatment. Given the unique structure and properties of these ERHs, hydrophobic cargo (thiostrepton) has been incorporated into the hydrogels and is released upon DCV loading. Additionally, these hydrogels are highly stretchable (>6000%) and tough (507 J/m2), showing robust mechanical properties. Moreover, these hydrogels have a high spatiotemporal resolution. As the cross-links within our ERHs are enabled by the non-covalent (i.e., hydrophobic) interactions, these hydrogels are self-healing and malleable. Considering the robust mechanical properties, precise spatiotemporal resolution, dynamic nature (e.g., injectable and self-healing), and on-demand drug delivery ability, this class of ERHs will be of great interest in the fields of wearable bioelectronics and smart drug delivery systems.

Structural basis for the neurotropic AAV9 and the engineered AAVPHP.eB recognition with cellular receptors.

Clade F adeno-associated virus (AAV) 9 has been utilized as therapeutic gene delivery vector, and it is capable of crossing blood brain barrier (BBB). Recently, an AAV9-based engineering serotype AAVPHP.eB with enhanced BBB crossing ability further expanded clade F AAVs' usages in the murine central nervous system (CNS) gene delivery. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of the AAVPHP.eB and its parental serotype AAV9 in native form or in complex with their essential receptor AAV receptor (AAVR). These structures reveal the molecular details of their AAVR recognition, where the polycystic kidney disease repeat domain 2 (PKD2) of AAVR interacts with AAV9 and AAVPHP.eB virions at the 3-fold protrusions and the raised capsid regions between the 2- and 5-fold axes, termed the 2/5-fold wall. The interacting patterns of AAVR to AAV9 and AAVPHP.eB are similar to what was observed in AAV1/AAV2-AAVR complexes. Moreover, we found that the AAVPHP.eB variable region VIII (VR-VIII) may independently facilitate the new receptor recognition responsible for enhanced CNS transduction. Our study provides insights into the recognition principles of multiple receptors for engineered AAVPHP.eB and parental serotype AAV9, and further reveal the potential molecular basis underlying their different tropisms.

A GA-insensitive dwarf mutant of Brassica napus L. correlated with mutation in pyrimidine box in the promoter of GID1.

A dwarf mutant from Brassica napus, namely NDF-1, which was derived from a high doubled haploid (DH) line '3529'(Brassica napus L.) of which seeds were jointly treated with chemical inducers and fast neutron bombardment, was revealed that dwarfism is under the control of a major gene(designated as ndf1) with a mainly additive effect and non-significant dominance effect. The germination and hypocotyls elongation response of dwarf mutants after exogenous GA and uniconazol application showed NDF-1 was a gibberellin insensitive dwarf. We cloned the Brassica napus GID1 gene, named BnGID1, and found it was the ortholog of AtGID1a. The sequence blasting of the BnGID1 genes from NDF-1 and wild type showed there was no mutant in the gene. But the quantitative RT-PCR analysis of GID1 EST pointed out the mutation was caused by the low-level expression of BnGID1 gene. After sequenced the BnGID1 gene's upstream, we found three bases mutated in the pyrimidine box (P-box) of the BnGID1 promoter, which is linkage with the dwarf mutant.

VHL inactivation induces HEF1 and Aurora kinase A.

The ciliary hypothesis for cystic renal diseases postulates that most of these conditions result from abnormalities in the primary cilium, a microtubule-based structure that acts as a sensor for extracellular cues. Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes to renal cysts and clear cell renal cell carcinoma. VHL plays a critical role in the formation of primary cilia in kidney epithelium, but the underlying mechanisms are poorly understood. Here, we demonstrate that VHL inactivation induces HEF1/Cas-L/NEDD9 and Aurora kinase A via the stabilization of hypoxia-inducible factors 1 and 2. Aurora kinase A is a mitotic kinase commonly upregulated in cancer that causes regression of the primary cilium by promoting histone deacetylase-dependent tubulin depolymerization of the ciliary axoneme. HEF1/Cas-L/NEDD9 is a component of focal adhesions that has a prominent role in inducing metastasis and that colocalizes with Aurora kinase A at the centrosome, thereby enhancing the harmful effect of Aurora kinase A on the cilium. Suppression of this pathway improved the formation of primary cilia and reduced cell motility in VHL-defective renal cancer cells. Our results highlight the gatekeeper role of VHL in the kidney epithelium.

Identification and structural elucidation of a new cetrorelix methylene dimer impurity in cetrorelix acetate by using LC-MS/MS.

Cetrorelix, a potent third generation of luteinizing hormone releasing hormone (LHRH) antagonist, is a synthetic decapeptide used for treatment of infertility, prostatic hypertrophy and sexual hormone-dependent tumors. The approved drug of cetrorelix (Cetrotide, Asta Medica AG, Frankfurt, Germany.), was used for prevention of premature ovulation in patients undergoing a controlled ovarian stimulation (COS), followed by oocyte pick-up and assisted reproductive techniques, and has been shown safe and effective in controlled ovarian stimulation. Nevertheless, the study of aggregation products of cetrorelix was rarely reported. A simple liquid chromatography mass spectrometry (LC-MS/MS) method was developed for separation, identification and characterization of a new cetrorelix methylene dimer impurity in cetrorelix. The chromatographic separation was achieved on an XSelect Peptide CSH ™C18 column (150 × 4.6 mm, 3.5 µm particle size) using gradient elution with a mobile phase of ammonium formate buffer (pH 3.0, 20 mM), acetonitrile at a flow rate 1.0 mL min-1, and an ultraviolet detection wavelength of 226 nm. The new cetrorelix methylene dimer impurity was characterized by LC-MS/MS and it characteristic fragment ions were summarized. A simple, fast and accurate method was established for the determination of the molecular weight and structure of the new cetrorelix methylene dimer impurity. In this study, the results showed that the cetrorelix was highly unstable in formaldehyde conditions. In addition, it is proposed that the impact of formaldehyde in the environment on the quality of cetrorelix acetate for Injection should be evaluated during the production process.

Overexpression of a weed (Solanum americanum) proteinase inhibitor in transgenic tobacco results in increased glandular trichome density and enhanced resistance to Helicoverpa armigera and Spodoptera litura.

In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.