Comparing the long-term follow-up anal function between robot-assisted and laparoscopic surgery for low rectal cancer: A meta-analysis and systematic review.

BACKGROUND: Robotic-assisted surgery (RAS) is increasingly used for treating low rectal cancer. Its comparative effectiveness against laparoscopic surgery (LAS) in enhancing long-term anal function remains uncertain. METHODS: A meta-analysis was conducted to compare long-term anal function outcomes between patients undergoing RAS and LAS. Meta-regression and sensitivity analyses were performed to assess available evidence. Studies published up to September 2023 in English or Chinese were included. RESULTS: Seven studies were identified. RAS patients exhibited lower low anterior resection syndrome (LARS) scores (standardised mean difference [SMD] = -1.39; 95% confidence interval [CI]: -2.64 to -0.15) and Wexner scores (SMD = -0.74; 95% CI: -1.20 to -0.27) compared with LAS patients. However, RAS did not significantly reduce major LARS risk (odds ratio = 0.85; 95% CI: 0.68-1.04). CONCLUSIONS: RAS slightly improved postoperative anal function compared with LAS. Further studies with large samples are warranted to confirm or update our findings.

Label-Free Direct Identification of MicroRNAs Based on a Narrow Constant-Inner-Diameter Emitter Mass Spectrometry Analysis.

MicroRNAs (miRNAs) are a class of endogenous noncoding small RNAs that play important roles in various biological processes and diseases. Direct determination of miRNAs is a cost-efficient and accurate method for analysis. Herein, we established a novel method for the analysis of miRNAs based on a narrow constant-inner-diameter mass spectrometry emitter. We utilized the gravity-assisted sleeving etching method to prepare a constant-inner-diameter mass spectrometry emitter with a capillary inner diameter of 5.5 µm, coupled it with a high-voltage power supply and a high-resolution mass spectrometer, and used it for miRNA direct detection. The method showed high sensitivity and reproducibility for the analysis of four miRNAs, with a limit of detection of 100 nmol/L (170 amol) for the Hsa-miR-1290 analysis. Compared with commercial ion sources, our method achieved higher sensitivity for miRNA detection. In addition, we analyzed the total miRNAs in the A549 cells. The result indicated that both spiked and endogenous miRNAs could be quantified with high accuracy. As a result, this method offers a promising platform for highly sensitive and accurate miRNA analysis. Furthermore, this approach can be extended to the analysis of other small oligonucleotides and holds the potential for studying clinical samples and facilitating disease diagnosis.

Monolithic 3D nanoelectrospray emitters based on a continuous fluid-assisted etching strategy for glass droplet microfluidic chip-mass spectrometry.

Glass microfluidic chips are suitable for coupling with mass spectrometry (MS) due to their flexible design, optical transparency and resistance to organic reagents. However, due to the high hardness and brittleness of glass, there is a lack of simple and feasible technology to manufacture a monolithic nanospray ionization (nESI) emitter on a glass microchip, which hinders its coupling with mass spectrometry. Here, a continuous fluid-assisted etching strategy is proposed to fabricate monolithic three-dimensional (3D) nESI emitters integrated into glass microchips. A continuous fluid of methanol is adopted to protect the inner wall of the channels and the bonding interface of the glass microfluidic chip from being wet-etched, forming sharp 3D nESI emitters. The fabricated 3D nESI emitter can form a stable electrospray plume, resulting in consistent nESI detection of acetylcholine with an RSD of 4.5% within 10 min. The fabricated 3D emitter is integrated on a glass microfluidic chip designed with a T-junction droplet generator, which can realize efficient analysis of acetylcholine in picoliter-volume droplets by nESI-MS. Stability testing of over 20 000 droplets detected by the established system resulted in an RSD of 9.1% over approximately 180 min. The detection of ten neurochemicals in rat cerebrospinal fluid droplets is achieved. The established glass droplet microfluidic chip-MS system exhibits potential for broad applications such as in vivo neurochemical monitoring and single-cell analysis in the future.

Parameters for selecting biological features in multifunctional bio-inspired design: a convergent evolution approach.

Combining different biological features exhibiting different functions is necessary to generate uncommon and unique multifunctional bio-inspired conceptual designs. Different biological features independently evolve characteristics to solve the same need/necessity. This phenomenon is called convergent evolution. Without parameters, selecting a suitable feature from those that exhibit the same function and have the same geometric relevance becomes quite difficult. This research investigates and identifies the parameters that have the potential to support choosing the suitable biological feature and to support the multifunctional design concept generation. In this paper, parameters are hypothesized by studying the mechanisms of tissue formation responsible for generating structural features in a biological system. These parameters are used in the Expandable Domain Integrated Design (xDID) ideation model to aid designers in choosing and combining suitable biological features for multifunctional concepts. A case study is presented to validate the effectiveness of the parameters in the selection process .

Establishment of a diagnostic model of endometriosis based on disulfidptosis-related genes.

OBJECTIVES: We aimed to establish a diagnostic model of endometriosis (EM) based on disulfidptosis-related genes (DRGs). MATERIALS AND METHODS: The mRNA expression data of EM were downloaded from the gene expression omnibus database and subjected to differential analysis, and co-expression analysis was performed based on 10 disulfidptosis genes to acquire DRGs. The differentially expressed DRGs were subjected to biofunctional analysis. Lasso analysis and support vector machine-recursive feature elimination (SVM-RFE) analysis were employed to extract the intersection of feature genes as biomarkers, and the diagnostic values of biomarkers for EM were evaluated based on receiver operating characteristic curves. The correlations between biomarkers and the immune microenvironment were assessed by Pearson analysis of biomarkers and immune cell infiltration levels. RESULTS: Transforming growth factor ß stimulated protein clone 22 domain family member 4 (TSC22D4), and F-box/SPRY domain-containing protein 1 (FBXO45) worked as the diagnostic classifiers in EM, with an obvious decrease in FBXO45 expression and an evident increase in TSC22D4 expression. The areas under the curves of FBXO45 and TSC22D4 were 0.752 and 0.706, respectively, and the area of FBXO45 combined with TSC22D4 reached 0.865, suggesting that TSC22D4 and FBXO45 had high predictive values. The diagnostic markers were closely correlated with immune cell infiltration. CONCLUSION: The diagnostic markers constructed based on disulfidptosis are good predictors for EM, which have close correlations with EM.

A hierarchical carbon foam-hosted Co2P nanoparticles monolithic electrode for ampere-level and super-durable electrocatalytic hydrogen production.

In this work, we develop a hierarchical carbon foam-based monolithic electrode (Co2P@HCF) from Co2+-adsorbed polyvinyl alcohol (PVA) sponge via the successive carbonization and phosphorization. Owing to the 3D hierarchical porous structure, excellent electrolyte wettability, good mechanical strength, and intimate embedding of highly dispersed Co2P nanoparticles, the Co2P@HCF electrode delivers a high current density of 1.0 A cm-2 for the hydrogen evolution reaction (HER) at ultralow overpotentials of 189.6 and 218.6 mV in 0.5 M H2SO4 and 1.0 M KOH solutions, respectively, with remarkable durability for 100 h.

Genome-wide identification of PYL/PYR-PP2C (A)-SnRK2 genes in Eutrema and their co-expression analysis in response to ABA and abiotic stresses.

ABA signaling core components PYR/PYL, group A PP2C and SnRK2 play important roles in various environmental stress responses of plants. This study identified 14 PYR/PYL, 9 PP2C (A), and 10 SnRK2 genes from halophytic Eutrema. Phylogenetic analysis showed 4 EsPYR/PYL, 4 EsPP2C (A) and 3 EsSnRK2 subfamilies characterized, which was supported by their gene structures and protein motifs. Large-scale segmental duplication event was demonstrated to be a major contributor to expansion of the EsPYL-PP2C (A)-SnRK2 gene families. Synteny relationship analysis revealed more orthologous PYL-PP2C (A)-SnRK2 gene pairs located in collinear blocks between Eutrema and Brassica than that between Eutrema and Arabidopsis. RNA-seq and qRT-PCR revealed EsABI1, EsABI2 and EsHAL2 showed a significantly up-regulated expression in leaves and roots in response to ABA, NaCl or cold stress. Three markedly co-expression modules of ABA/R-brown, NaCl/L-lightsteelblue1 and Cold/R-lightgreen were uncovered to contain EsPYL-PP2C (A)-SnRK2 genes by WGCNA analysis. GO and KEGG analysis indicated that the genes of ABA/R-brown module containing EsHAB1, EsHAI2 and EsSnRK2.6 were enriched in proteasome pathway. Further, EsHAI2-OE transgenic Arabidopsis lines showed significantly enhanced seeds germination and seedlings growth. This work provides a new insight for elucidating potential molecular functions of PYL-PP2C (A)-SnRK2 responding to ABA and abiotic stresses.

Sensitive electrochemical measurement of nitric oxide released from living cells based on dealloyed PtBi alloy nanoparticles.

Nitric oxide (NO), as a vital signaling molecule related to different physiological and pathological processes in living systems, is closely associated with cancer and cardiovascular disease. However, the detection of NO in real-time remains a difficulty. Here, PtBi alloy nanoparticles (NPs) were synthesized, dealloyed, and then fabricated to NP-based electrodes for the electrochemical detection of NO. Transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and nitrogen physical adsorption/desorption show that dealloyed PtBi alloy nanoparticles (dPtBi NPs) have a porous nanostructure. Electrochemical impedance spectroscopy and cyclic voltammetry results exhibit that the dPtBi NP electrode possesses unique electrocatalytic features such as low charge transfer resistance and large electrochemically active surface area, which lead to its excellent NO electrochemical sensing performance. Owing to the higher density of catalytical active sites formed PtBi bimetallic interface, the dPtBi NP electrode displays superior electrocatalytic activity toward the oxidation of NO with a peak potential at 0.74 V vs. SCE. The dPtBi NP electrode shows a wide dynamic range (0.09-31.5 µM) and a low detection limit of 1 nM (3σ/k) as well as high sensitivity (130 and 36.5 µA µM-1 cm-2). Moreover, the developed dPtBi NP-based electrochemical sensor also exhibited good reproducibility (RSD 5.7%) and repeatability (RSD 3.4%). The electrochemical sensor was successfully used for the sensitive detection of NO produced by live cells. This study indicates a highly effective approach for regulating the composition and nanostructures of metal alloy nanomaterials, which might provide new technical insights for developing high-performance NO-sensitive systems, and have important implications in enabling real-time detection of NO produced by live cells.

Biomimetic Nanocomposites Camouflaged with Hybrid Cell Membranes for Accurate Therapy of Early-Stage Glioma.

Glioma features high fatality rate and short survival time of patients due to its fast growth speed and high invasiveness, hence timely treatment of early-stage glioma is extremely important. However, the blood brain barrier (BBB) severely prevents therapeutic agents from entering the brain; meanwhile, the non-targeted distribution of agents always causes side effects to vulnerable cerebral tissues. Therefore, delivery systems that possess both BBB penetrability and precise glioma targeting ability are keenly desired. We herein proposed a hybrid cell membrane (HM) camouflage strategy to construct therapeutic nanocomposites, in which HM consisting of brain metastatic breast cancer cell membrane and glioma cell membrane was prepared with a simple membrane fusion pathway. By coating HM onto drug-loaded nanoparticles, the as-obtained biomimetic therapeutic agent (termed HMGINPs) inherited satisfying BBB penetrability and homologous glioma targeting ability simultaneously from the two source cells. HMGINPs exhibited good biocompatibility and superior therapeutic efficacy towards early-stage glioma.

A metabolic perspective of selection for fruit quality related to apple domestication and improvement.

BACKGROUND: Apple is an economically important fruit crop. Changes in metabolism accompanying human-guided evolution can be revealed using a multiomics approach. We perform genome-wide metabolic analysis of apple fruits collected from 292 wild and cultivated accessions representing various consumption types. RESULTS: We find decreased amounts of certain metabolites, including tannins, organic acids, phenolic acids, and flavonoids as the wild accessions transition to cultivated apples, while lysolipids increase in the "Golden Delicious" to "Ralls Janet" pedigree, suggesting better storage. We identify a total of 222,877 significant single-nucleotide polymorphisms that are associated with 2205 apple metabolites. Investigation of a region from 2.84 to 5.01 Mb on chromosome 16 containing co-mapping regions for tannins, organic acids, phenolic acids, and flavonoids indicates the importance of these metabolites for fruit quality and nutrition during breeding. The tannin and acidity-related genes Myb9-like and PH4 are mapped closely to fruit weight locus fw1 from 3.41 to 3.76 Mb on chromosome 15, a region under selection during domestication. Lysophosphatidylethanolamine (LPE) 18:1, which is suppressed by fatty acid desaturase-2 (FAD2), is positively correlated to fruit firmness. We find the fruit weight is negatively correlated with salicylic acid and abscisic acid levels. Further functional assays demonstrate regulation of these hormone levels by NAC-like activated by Apetala3/Pistillata (NAP) and ATP binding cassette G25 (ABCG25), respectively. CONCLUSIONS: This study provides a metabolic perspective for selection on fruit quality during domestication and improvement, which is a valuable resource for investigating mechanisms controlling apple metabolite content and quality.

Single-Cell Metabolomics-Based Strategy for Studying the Mechanisms of Drug Action.

Studying the mechanisms of drug antitumor activity at the single-cell level can provide information about the responses of cell subpopulations to drug therapy, which is essential for the accurate treatment of cancer. Due to the small size of single cells and the low contents of metabolites, metabolomics-based approaches to studying the mechanisms of drug action at the single-cell level are lacking. Herein, we develop a label-free platform for studying the mechanisms of drug action based on single-cell metabolomics (sMDA-scM) by integrating intact living-cell electro-launching ionization mass spectrometry (ILCEI-MS) with metabolomics analysis. Using this platform, we reveal that non-small-cell lung cancer (NSCLC) cells treated by gefitinib can be clustered into two cell subpopulations with different metabolic responses. The glutathione metabolic pathway of the subpopulation containing 14.4% of the cells is not significantly affected by gefitinib, exhibiting certain resistance characteristics. The presence of these cells masked the judgment of whether cysteine and methionine metabolic pathway was remarkably influenced in the analysis of overall average results, revealing the heterogeneity of the response of single NSCLC cells to gefitinib treatment. The findings provide a basis for evaluating the early therapeutic effects of clinical medicines and insights for overcoming drug resistance in NSCLC subpopulations.

The complete mitochondrial genome of Cuspidaria undata (Bivalvia, Anomalodesmata, Cuspidariidae).

The mitochondrial genome of Cuspidaria undata (Verrill, 1884) was sequenced in full using Illumina HiSeq 2500. The circular mitochondrial DNA (mtDNA) was 16,266 bp in size, encoded 37 genes, and contained 13 protein-coding genes (PCGs), 2 rRNAs and 22 tRNAs. The gene order of the 13 PCGs in this species exhibited extensive rearrangement and differences in comparison to other Cuspidariidae, indicating that gene order is not conserved within this family. Phylogenetic analysis based on 13 PCGs and 2 rRNAs recovered a monophyletic Cuspidariidae.

Diagnostic value of combined pelvic floor ultrasound parameters for pelvic floor dysfunction and risk factors.

OBJECTIVE: To investigate the diagnostic value of pelvic floor ultrasound parameters in combination for pelvic floor dysfunction (PFD), and to explore the risk factors. METHODS: Forty PFD patients treated from April2019to December 2020(case group) and another 40 healthy women (control group) were enrolled. Their clinical data were collected, and both groups received three-dimensional (3D) ultrasound of the pelvic floor. The diagnostic value of pelvic floor ultrasound parameters for PFD was assessed by receiver operating characteristic (ROC) curves. The risk factors of PFD were evaluated by multivariate logistic regression analysis. RESULTS: The area under the ROC curve (AUC), sensitivity, and specificity of the parameters in combination for predicting PFD were 0.851 [95% confidence interval (CI): 0.743-0.959], 0.901, and 0.812, respectively, indicating acceptable accuracy. Results of logistic regression analysis revealed that spontaneous delivery, lateral episiotomy/laceration, and large bladder neck rotation angle, posterior urethrovesical angle (PUA), bladder neck tilt angle, bladder neck distance (BND), levator hiatus area (LHA) (at anal contraction), R-LHA and V-LHA were risk factors for PFD (p < 0.05), while physical exercise was a protective factor (p < 0.05). ROC curve analysis revealed that the AUC, sensitivity, and specificity of the forest map model were 0.822 (95% CI: 0.759-0.885), 0.942, and 0.601, respectively, indicating acceptable accuracy of the model. Internal data validation of the model demonstrated consistence of the predicted occurrence of PFD with the actual one. CONCLUSIONS: Spontaneous delivery, lateral episiotomy/laceration, and large bladder neck rotation angle, PUA, bladder neck tilt angle, BND, LHA (at anal contraction), R-LHA and V-LHA were risk factors for PFD.

Hydrogen-rich water alleviates chilling injury-induced lignification of kiwifruit by inhibiting peroxidase activity and improving antioxidant system.

BACKGROUND: Kiwifruit is prone to chilling stress and displays chilling injury (CI) such as lignification; however, the underlying physicochemical mechanism remains largely unknown. Here, the changes in levels of quality attributes, lignin biosynthesis, antioxidant system and sugars were compared in kiwifruit between control and hydrogen-rich water (HRW) treatments during cold storage for 90 days at 0 °C. RESULTS: The results reveal that HRW is an effective measure for CI alleviation, as indicated by the decrease in lignification level with repressed peroxidase activity but enhanced polyphenol oxidase activity. The amelioration of membrane peroxidation was suggested by the repressed levels of H2 O2 and malondialdehyde. They were accompanied by the improvement of antioxidant system, which is supported by the enhancement of sugars including fructose and glucose. CONCLUSION: In conclusion, HRW can enhance chilling tolerance, as suggested by the alleviation of lignification through inhibiting peroxidase activity and elevating the antioxidant system to attenuate membrane peroxidation. © 2022 Society of Chemical Industry.

Attomole-Level Multiplexed Detection of Neurochemicals in Picoliter Droplets by On-Chip Nanoelectrospray Ionization Coupled to Mass Spectrometry.

While droplet microfluidics is becoming an effective tool for biomedical research, sensitive detection of droplet content is still challenging, especially for multiplexed analytes compartmentalized within ultrasmall droplets down to picoliter volumes. To enable such measurements, we demonstrate a silicon-based integrated microfluidic platform for multiplexed analysis of neurochemicals in picoliter droplets via nanoelectrospray ionization (nESI)-mass spectrometry (MS). An integrated silicon microfluidic chip comprising downscaled 7 µm-radius channels, a compact T-junction for droplet generation, and an integrated nESI emitter tip is used for segmentation of analytes into picoliter compartments and their efficient delivery for subsequent MS detection. The developed system demonstrates effective detection of multiple neurochemicals encapsulated within oil-isolated plugs down to low picoliter volumes. Quantitative measurements for each neurochemical demonstrate limits of detection at the attomole level. Such results are promising for applications involving label-free and small-volume detection for monitoring a range of brain chemicals.

Molecular Cloning and Expression Analysis of the Cryptochrome Gene CiPlant-CRY1 in Antarctic Ice Alga Chlamydomonas sp. ICE-L.

Cryptochrome (CRY) is a kind of flavin-binding protein that can sense blue light and near-ultraviolet light, and participates in the light response of organisms and the regulation of the circadian clock. The complete open reading frame (ORF) of CiPlant-CRY1 (GenBank ID OM389130.1), encoding one kind of CRY, was cloned from the Antarctic ice alga Chlamydomonas sp. ICE-L. The quantitative real-time PCR study showed that the expression level of the CiPlant-CRY1 gene was the highest at 5 °C and salinity of 32‱. CiPlant-CRY1 was positively regulated by blue or yellow light, suggesting that it is involved in the establishment of photomorphology. The CiPlant-CRY1 gene can respond to polar day and polar night, indicating its expression is regulated by circadian rhythm. The expression level of CiPlant-CRY1 was most affected by UVB irradiation, which may be related to the adaptation of ice algae to a strong ultraviolet radiation environment. Moreover, the recombinant protein of CiPlant-CRY1 was expressed by prokaryotic expression. This study may be important for exploring the light-induced rhythm regulation of Antarctic ice algae in the polar marine environment.

Abscisic acid alleviates chilling injury in cold-stored peach fruit by regulating ethylene and hydrogen peroxide metabolism.

Peach (Prunus persica (L.) Batsch) is susceptible to chilling injury under improper low-temperature storage (2°C-5°C). Previous research has shown that abscisic acid (ABA) alleviates chilling injury in fruits and vegetables, but the potential mechanism is still unclear. To explore its effectiveness and potential mechanism in alleviating chilling injury during cold storage, exogenous ABA was applied to peach fruit by immersion in 100 µmol L-1 solutions for 10 min. In our experiment, ABA alleviated chilling injury by reducing hydrogen peroxide (H2O2) content and ethylene production. In addition, ABA inhibited the expression of the ethylene synthesis-related genes PpACO1 and PpEIN2. At the same time, ABA activated the antioxidant enzymatic pathway and the ascorbate-glutathione (AsA-GSH) cycle, the transcript abundance encoding genes related to antioxidant enzyme activities also changed correspondingly. The results suggested that ABA alleviated chilling injury by scavenging excessive H2O2 by promoting antioxidant enzymes and the AsA-GSH pathway.

Intact living-cell electrolaunching ionization mass spectrometry for single-cell metabolomics.

While single-cell mass spectrometry can reveal cellular heterogeneity and the molecular mechanisms of intracellular biochemical reactions, its application is limited by the insufficient detection sensitivity resulting from matrix interference and sample dilution. Herein, we propose an intact living-cell electrolaunching ionization mass spectrometry (ILCEI-MS) method. A capillary emitter with a narrow-bore, constant-inner-diameter ensures that the entire living cell enters the MS ion-transfer tube. Inlet ionization improves sample utilization, and no solvent is required, preventing sample dilution and matrix interference. Based on these features, the detection sensitivity is greatly improved, and the average signal-to-noise (S/N) ratio is about 20 : 1 of single-cell peaks in the TIC of ILCEI-MS. A high detection throughput of 51 cells per min was achieved by ILCEI-MS for the single-cell metabolic profiling of multiple cell lines, and 368 cellular metabolites were identified. Further, more than 4000 primary single cells digested from the fresh multi-organ tissues of mice were detected by ILCEI-MS, demonstrating its applicability and reliability.

Complement-Opsonized NIR-IIb Emissive Immunotracers for Dynamically Monitoring Neutrophils in Inflammation-Related Diseases.

Real-time monitoring of neutrophil dynamics is crucial for timely diagnosis and effective treatment of inflammation-related diseases, which requires a reliable tracer for in vivo tracking of neutrophils. However, immunotracers for neutrophils are extremely limited because of the difficulty in labeling the cells. Inspired by the natural biological function of the complement system, a strategy of enhancing the complement C3 opsonization of lanthanide-doped nanoparticles (LnNPs) by modulating their surface chemistry, thus developing a near infrared-IIb emissive nanotracer for neutrophils, is reported herein. Four kinds of surface-modified LnNPs are fabricated, among which phospholipids DOPG-modified LnNPs (LnNPs@PG) with weak antifouling ability and hydroxyl groups adsorb more complement C3 proteins and form covalent linkages with C3b active fragments under inflammation conditions, inducing enhanced complement C3 opsonization. Therefore, LnNPs@PG with enhanced complement C3 opsonization are capable of efficiently labeling inflammation-stimulated neutrophils in vivo through complement-receptors-mediated phagocytosis and achieve dynamic monitoring neutrophils during cutaneous wound healing and cerebral ischemia/reperfusion.

Adaptative Mechanisms of Halophytic Eutrema salsugineum Encountering Saline Environment.

Salt cress (Eutrema salsugineum), an Arabidopsis-related halophyte, can naturally adapt to various harsh climates and soil conditions; thus, it is considered a desirable model plant for deciphering mechanisms of salt and other abiotic stresses. Accumulating evidence has revealed that compared with Arabidopsis, salt cress possesses stomata that close more tightly and more succulent leaves during extreme salt stress, a noticeably higher level of proline, inositols, sugars, and organic acids, as well as stress-associated transcripts in unstressed plants, and they are induced rapidly under stress. In this review, we systematically summarize the research on the morphology, physiology, genome, gene expression and regulation, and protein and metabolite profile of salt cress under salt stress. We emphasize the latest advances in research on the genome adaptive evolution encountering saline environments, and epigenetic regulation, and discuss the mechanisms underlying salt tolerance in salt cress. Finally, we discuss the existing questions and opportunities for future research in halophytic Eutrema. Together, the review fosters a better understanding of the mechanism of plant salt tolerance and provides a reference for the research and utilization of Eutrema as a model extremophile in the future. Furthermore, the prospects for salt cress applied to explore the mechanism of salt tolerance provide a theoretical basis to develop new strategies for agricultural biotechnology.