Multivalent Aptamer-Based Lysosome-Targeting Chimeras (LYTACs) Platform for Mono- or Dual-Targeted Proteins Degradation on Cell Surface.

Selective protein degradation platforms have opened novel avenues in therapeutic development and biological inquiry. Antibody-based lysosome-targeting chimeras (LYTACs) have emerged as a promising technology that extends the scope of targeted protein degradation to extracellular targets. Aptamers offer an advantageous alternative owing to their potential for modification and manipulation toward a multivalent state. In this study, a chemically engineered platform of multivalent aptamer-based LYTACs (AptLYTACs) is established for the targeted degradation of either single or dual protein targets. Leveraging the biotin-streptavidin system as a molecular scaffold, this investigation reveals that trivalently mono-targeted AptLYTACs demonstrate optimum efficiency in degrading membrane proteins. The development of this multivalent AptLYTACs platform provides a principle of concept for mono-/dual-targets degradation, expanding the possibilities of targeted protein degradation.

Recent progress of aptamer‒drug conjugates in cancer therapy.

Aptamers are single-stranded DNA or RNA sequences that can specifically bind with the target protein or molecule via specific secondary structures. Compared to antibody-drug conjugates (ADC), aptamer‒drug conjugate (ApDC) is also an efficient, targeted drug for cancer therapy with a smaller size, higher chemical stability, lower immunogenicity, faster tissue penetration, and facile engineering. Despite all these advantages, several key factors have delayed the clinical translation of ApDC, such as in vivo off-target effects and potential safety issues. In this review, we highlight the most recent progress in the development of ApDC and discuss solutions to the problems noted above.

Formaldehyde Cross-Linking-Assisted Phase Separation for Protein Aptamer Selection.

With the merits of easy synthesis, strong modifiability, and high affinity, aptamers have been broadly applied for protein targeting in bioanalysis, diagnosis, and therapeutics. The selection of protein-targeted aptamers is currently largely dependent on solid-liquid separation by using different types of nano- or micro-beads. However, the use of beads inescapably introduces unwanted nonspecific binding and thus affects selection efficiency. In order to sidestep this obstacle, we herein report an integrated technique to facilitate the discovery and development of protein-targeting aptamers by incorporating formaldehyde cross-linking with phase separation (FCPS). The feasibility and universality of FCPS were confirmed by the successful selection of two aptamers that could target various antibodies. Unlike traditional approaches, the proposed technique avoids the use of beads and enables the rapid generation of aptamers after only one to three rounds of selection. The as-selected aptamers were further used to regulate and control antibody activity, showing potential applications in biomedicine.

Ultrahigh-Performance Supercritical Fluid Chromatography and Detection of Multiple Biogenic Amines in Gentamicin Sulfate: Method Development Using Computer-Assisted Modeling.

In order to solve the problem of difficult separation of various biogenic amines (BAs), which have similar structures or very different polarities, in gentamicin, by conventional liquid chromatography, a new ultrahigh-performance supercritical fluid chromatography (UHPSFC) method was developed. In this method, 10 BAs were derivatized precolumn using dansyl chloride and separated using a UHPSFC system. By computational simulation, complete separation of 10 BAs was successfully achieved. Detection was performed using a photodiode array (PDA) and single-quadrupole mass spectrometry (MS) together with electrospray ionization (ESI). A wide linear range (10-2500 ng/mL) was achieved, with the limits of detection (LODs) between 1.2 and 10.0 ng/mL and the limits of quantification (LOQs) between 5.0 and 25.0 ng/mL. Apart from high sensitivity, this UHPSFC-PDA/ESI-MS detection method also displayed high accuracy, the matrix effect was reduced by an appreciable extent, and the recovery rates of the 10 BAs were between 84.1 and 117.1%. For comparison, high-performance liquid chromatography-tandem mass spectrometry (MS/MS) was also used for the detection of underivatized BAs in gentamicin, showing good linearity and high sensitivity (LODs from 0.05 to 1.00 ng/mL and LOQs from 1.00 to 12.50 ng/mL) for all BAs except for spermine and spermidine. Although single-quadrupole MS is inferior to MS/MS in terms of sensitivity, the UHPSFC method could detect more BAs. It also achieved the quantification limits required for impurity determination, demonstrating a potential strategy to offer a map overview of possible BA presence in fermentation antibiotics.

Molecular Engineering of Aptamer Self-Assemblies Increases in Vivo Stability and Targeted Recognition.

Functionally modified aptamer conjugates are promising tools for targeted imaging or treatment of various diseases. However, broad applications of aptamer molecules are limited by their in vivo instability. To overcome this challenge, current strategies mostly rely on covalent chemical modification of aptamers, a complicated process that requires case-by-case sequence design, multiple-step synthesis, and purification. Herein, we report a covalent modification-free strategy to enhance the in vivo stability of aptamers. This strategy simply utilizes one-step molecular engineering of aptamers with gold nanoclusters (GNCs) to form GNCs@aptamer self-assemblies. Using Sgc8 as a representative aptamer, the resulting GNCs@Sgc8 assemblies enhance cancer-cell-specific binding and sequential internalization by a receptor-mediated endocytosis pathway. Importantly, the GNCs@aptamer self-assemblies resist nuclease degradation for as long as 48 h, compared to the degradation of aptamer alone at 3 h. In parallel, the tumor-targeted recognition and retention of GNCs@aptamer self-assemblies are dramatically enhanced, indicated by a 9-fold signal increase inside the tumor compared to the aptamer alone. This strategy is to avoid complicated chemical modification of aptamers and can be extended to all aptamers. Our work provides a simple, effective, and universal strategy for enhancing the in vivo stability of any aptamer or its conjugates, thus expanding their imaging and therapeutic applications.

Lipidomics Revealed Aberrant Metabolism of Lipids Including FAHFAs in Renal Tissue in the Progression of Lupus Nephritis in a Murine Model.

Lupus nephritis (LN) is an inflammatory renal disease of patients with systemic lupus erythematosus with lots of immune complexes deposited in kidneys. Accumulated studies have demonstrated the close relationships among dyslipidaemia, inflammation, and autoimmune response, and oxidative stress in the patients. Lipids play numerous important roles in biological process and cellular functions. Herein, shotgun lipidomics was employed to quantitatively analyze cellular lipidomes in the renal tissue of MRL/lpr mice in the progression of LN (including pre-LN and LN state) with/without treated with glucocorticoids (GCs). The levels of cytokines (i.e., TNF-α (Tumor necrosis factor alpha) and IL-6 (Interleukin 6)) in the serum were measured by ELISA (enzyme-linked immunosorbent assay) kits. Renal histopathological changes and C3 deposition in the glomeruli of the mice were also determined. Lipidomics analysis revealed that the ectopic fat deposition and the aberrant metabolism of lipids that were relevant to oxidative stress (e.g., 4-hydroxyalkenal, ceramide, lysophospholipid species, etc.) always existed in the development of LN. Moreover, the anti-inflammatory FAHFA (fatty acid ester of hydroxyl fatty acid) species in the kidney tissue could largely reflect the severity of LN. Thus, they were a potential early biomarker for LN. In addition, the study also revealed that treatment with GCs could prevent the progression of LN, but greatly aggravate the aberrant metabolism of the lipids, particularly when used for a long time.

Sensitive analysis of fatty acid esters of hydroxy fatty acids in biological lipid extracts by shotgun lipidomics after one-step derivatization.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are an important family of endogenous lipids, possessing antidiabetic and anti-inflammatory functions. Therefore, analysis of FAHFAs in biological samples obtained under healthy and disease states can uncover underlying mechanisms of various relevant disorders (e.g., diabetes and autoimmune diseases). Up to now, due to their extremely low abundance, the determination of the changed levels of these species is still a huge challenge, even though great efforts have been made by utilizing liquid chromatography-tandem mass spectrometry with or without derivatization. Herein, we described a novel method for analysis of FAHFAs present in lipid extracts of biological examples after solid-phase extraction and chemical derivatization with one authentic FAHFA specie as an internal standard based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The approach possessed marked sensitivity, high specificity, and broad linear dynamic range of over 3 orders without obvious matrix effects. Moreover, after chemical derivatization, the molecular masses of FAHFAs shift from an overlapped region with ceramide species to a new region without overlaps, removing these contaminating signals from ceramides, and thereby reducing the false results of FAHFAs. Finally, this novel method was successfully applied for determining FAHFAs levels in varieties of representative biological samples, including plasma from lean and overweight/obese individuals of normoglycemia, and tissue samples (such as liver and white adipose tissue from diabetic (db/db) mice). We revealed significant alterations of FAHFAs in samples under patho(physio)logical conditions compared to their respective controls. Taken together, the developed method could greatly contribute to studying altered FAHFA levels under a variety of biological/biomedical conditions, and facilitate the understanding of these lipid species in the patho(physio)logical process.

From Endocytosis to Nonendocytosis: The Emerging Era of Gene Delivery.

Over the past two decades, gene therapy, as a promising way to regulate or replace abnormal gene, has made impressive progress with numerous clinic trials. However, the success of gene therapy was hugely limited by its low translocation into cytoplasm. Therefore, technologies to efficiently protect and deliver therapeutic nucleic acids have been extensively investigated, but most of the delivery strategies involve endosomal entrapment, leading to low delivery efficiency. In this review, we discuss the latest advances in nonendocytosis-dependent strategies for delivering nucleic acids into cells. A highlight is provided on the cellular uptake systems facilitated by the endosome-Golgi-endoplasmic reticulum pathway, pH low insertion peptides, cell-penetrating peptides, scavenger receptor-mediated nonendocytosis, membrane fusion, and the emerged thiol-disulfide exchange. The mechanisms, pros, and cons of these systems are discussed. Finally, current challenges and future perspectives for the translation of nonendocytic gene delivery vectors, especially thiol-mediated cellular uptake, into clinical applications are discussed.

Strategies to Improve/Eliminate the Limitations in Shotgun Lipidomics.

Direct infusion-based shotgun lipidomics is one of the most powerful and useful tools in comprehensive analysis of lipid species from lipid extracts of various biological samples with high accuracy/precision. However, despite many advantages, the classical shotgun lipidomics suffers some general dogmas of limitations, such as ion suppression, ambiguous identification of isobaric/isomeric lipid species, and ion source-generated artifacts, restraining the applications in analysis of low-abundance lipid species, particularly those less ionizable or isomers that yield almost identical fragmentation patterns. This article reviews the strategies (such as modifier addition, prefractionation, chemical derivatization, charge feature utilization) that have been employed to improve/eliminate these limitations in modern shotgun lipidomics approaches (e.g., high mass resolution mass spectrometry-based and multidimensional mass spectrometry-based shotgun lipidomics). Therefore, with the enhancement of these strategies for shotgun lipidomics, comprehensive analysis of lipid species including isomeric/isobaric species is achieved in a more accurate and effective manner, greatly substantiating the aberrant lipid metabolism, signaling trafficking, and homeostasis under pathological conditions.

Effects of intrathecal isoflurane administration on nociception and Fos expression in the rat spinal cord.

BACKGROUND AND OBJECTIVE: Isoflurane has been used as an inhaled anaesthetic for nearly 30 years. Isoflurane inhalation during anaesthesia also produces an anti-nociceptive effect. Whether this occurs at the spinal or supraspinal level remains unknown. With a novel type of liquid isoflurane, the present study examined the effects of intrathecal isoflurane on the nociceptive response and Fos expression in the rat spinal cord. METHODS: Thirty-six rats were randomly assigned to three groups as follows: group A (n = 6), intrathecal physiological saline 50 µl kg⁻¹; and group B and C (n = 6 each), intrathecal isoflurane at doses of 25 µl kg⁻¹ or 50 µl kg⁻¹, respectively. Noxious thermal (Hargreaves test), mechanical (von Frey test) and chemical (formalin 5%, 50 µl) stimuli were applied to a hind paw after intrathecal isoflurane injection to study its anti-nociceptive effect. In addition, the expression of Fos protein and c-fos mRNA in the spinal dorsal horns was detected by immunohistochemistry and real-time reverse transcriptase PCR, respectively. RESULTS: Compared with the physiological saline control, intrathecal isoflurane significantly suppressed spontaneous paw flinches in rats induced by formalin injection and paw withdrawal induced by thermal and mechanical stimuli in a dose-dependent manner. Immunohistochemistry and real-time reverse transcriptase PCR revealed that isoflurane administration inhibited formalin injection-induced c-fos expression in the spinal cord. CONCLUSIONS: These data suggest that isoflurane can exert anti-nociceptive effects at the spinal level by preventing neuronal activation.

Ethylene perception is involved in female cucumber flower development.

It is well established that ethylene promotes female flower development in cucumber. However, little is known about how the gaseous hormone selectively affects female flowers, and what mechanism it uses. Previously, we found organ-specific DNA damage in the primordial anther of female cucumber flowers. This finding led to a hypothesis that ethylene might promote female flower development via the organ-specific induction of DNA damage in primordial anthers. In this study, we tested this hypothesis first by demonstrating ethylene induction of DNA damage via the ethylene signaling pathway using cucumber protoplasts. Then, using representative component genes of the ethylene signaling pathway as probes, we found that one of the ethylene receptors, CsETR1, was temporally and spatially downregulated in the stamens of stage-6 female cucumber flowers, especially along with the increase of the nodes. Furthermore, by constructing transgenic Arabidopsis plants with organ-specific expression of antisense CsETR1 under the control of an AP3 promoter to downregulate ETR1 expression in the stamens, we generated Arabidopsis 'female flowers', in which the abnormal stamens mimic those of female cucumber flowers. Our data suggest that ethylene perception is involved in the arrest of stamen development in female cucumber flowers through the induction of DNA damage. This opens up a novel perspective and approach to solve the half-century-long puzzle of how gaseous ethylene selectively promotes female flowers in the monoecious cucumber plant.

Stamen development in Arabidopsis is arrested by organ-specific overexpression of a cucumber ethylene synthesis gene CsACO2.

Cucumber (Cucumis sativus L.) has served as a model to understand hormone regulation in unisexual flower development since the 1950s and the role of ethylene in promoting female flower development has been well documented. Recent studies cloned the F-locus in gynoecious lines as an additional copy of the ACC synthase (ACS) gene, which further confirmed the role of ethylene in the promotion of female cucumber flowers. However, no direct evidence was generated to demonstrate that increases in endogenous ethylene production could induce female flowers by arresting stamen development. To clarify the relationship between ethylene production and stamen development, we overexpressed the ethylene synthesis cucumber gene CsACO2 to generate transgenic Arabidopsis, driven by the organ-specific promoter P ( AP3 ). We found that organ-specific overexpression of CsACO2 significantly affected stamen but not carpel development, similar to that in the female flowers of cucumber. Our results suggested that increases in ethylene production directly disturb stamen development. Additionally, our study revealed that among all floral organs, stamens respond most sensitively to exogenous ethylene.

The dynamic pollen tube cytoskeleton: live cell studies using actin-binding and microtubule-binding reporter proteins.

Pollen tubes elongate within the pistil to transport sperm cells to the embryo sac for fertilization. Growth occurs exclusively at the tube apex, rendering pollen tube elongation a most dramatic polar cell growth process. A hallmark pollen tube feature is its cytoskeleton, which comprises elaborately organized and dynamic actin microfilaments and microtubules. Pollen tube growth is dependent on the actin cytoskeleton; its organization and regulation have been examined extensively by various approaches, including fluorescent protein labeled actin-binding proteins in live cell studies. Using the previously described GFP-NtADF1 and GFP-LlADF1, and a new actin reporter protein NtPLIM2b-GFP, we re-affirm that the predominant actin structures in elongating tobacco and lily pollen tubes are long, streaming actin cables along the pollen tube shank, and a subapical structure comprising shorter actin cables. The subapical collection of actin microfilaments undergoes dynamic changes, giving rise to the appearance of structures that range from basket- or funnel-shaped, mesh-like to a subtle ring. NtPLIM2b-GFP is used in combination with a guanine nucleotide exchange factor for the Rho GTPases, AtROP-GEF1, to illustrate the use of these actin reporter proteins to explore the linkage between the polar cell growth process and its actin cytoskeleton. Contrary to the actin cytoskeleton, microtubules appear not to play a direct role in supporting the polar cell growth process in angiosperm pollen tubes. Using a microtubule reporter protein based on the microtubule end-binding protein from Arabidopsis AtEB1, GFP-AtEB1, we show that the extensive microtubule network in elongating pollen tubes displays varying degrees of dynamics. These reporter proteins provide versatile tools to explore the functional connection between major structural and signaling components of the polar pollen tube growth process.

[Effects of MSC on cell cycle, phenotype and cytokine secretion of T cells activated by PHA].

AIM: To study the effect of human bone marrow derived mesenchymal stem cell (MSC) on T cell cycle and activation, and to investigate the inhibitory effect of MSC on T cell proliferation and the underlying mechanism. METHODS: Human bone marrow derived MSC were isolated by gradient centrifugation. then in vitro MSC were cultured, expanded,and were used in test after third passage. FCM analysis and ELISA were used to investigate the effects of MSC on the early activation marker expression of T cells, cell cycle and cytokine secretion. RESULTS: T cells stimulated by PHA in the presence of MSC were arrested at G0/G1 phase. The expression of the early activation marker CD25 and CD69 of T cells was inhibited in the presence of MSC both in CD4(+) and CD8(+) T cell subpopulation. MSC caused a sharp decrease of cytokine secretion in IL-2 and IFN-gamma. CONCLUSION: Human bone marrow derived MSC can suppress the activation and proliferation of T cells by altering T cell cycle.

Attachment and growth of human bone marrow derived mesenchymal stem cells on regenerated antheraea pernyi silk fibroin films.

Silk fibroin of the silkworm Bombyx mori has been studied extensively, while the research on Antheraea pernyi silk fibroin (A. pernyi SF) in biomaterials is only at an early stage. In this study, the attachment, morphology, growth and phenotype of human bone marrow derived mesenchymal stem cells (hBMSCs) cultured on the regenerated A. pernyi SF films were studied in vitro. The results indicated that the attachment of hBMSCs on the regenerated A. pernyi SF films was almost the same as that on the collagen films. MTT and cell counting analyses demonstrated that the growth of hBMSCs on the regenerated A. pernyi SF films was better than that on controls. Moreover, electron scanning microscopy and fluorescence-activated cell sorting assays showed that the regenerated A. pernyi SF supported hBMSCs growth and functional maintenance compared with the controls. These data suggest that the regenerated A. pernyi SF, like Bombyx mori silk fibroin (B. mori SF) and collagen, can support hBMSCs attachment, growth and phenotypic maintenance, and has better biocompatibilities for hBMSCs in vitro culture.