Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.The recent development of small molecule antagonists of prosurvival Bcl-2 family proteins looks promising for the future treatment of cancer. These novel compounds, also known as BH3 3 mimetics, bind to prosurvival Bcl-2 proteins and neutralize them. Gossypol is a natural polyphenol compound isolated from cottonseeds. Various levels of anticancer activity of gossypol have been observed against many different cancer cell lines both in vitro and in vivo (1-3). Evidence is accumulating that gossypol and its derivatives act as BH3 mimetics, killing multiple tumor cell lines, at least in part by activating the Bcl-2-regulated apoptotic pathway (4 -7). Gossypol suppresses both telomerase activity and NF-B activity in human leukemia cells (8, 9). However, the precise mechanisms responsible for the inhibition of cell growth and the stimulation of cell death induced by gossypol are poorly understood. We demonstrate that gossypol potently induces caspase-dependent apoptosis in the absence of Bak and Bax (Bcl-2-associated X protein) by converting Bcl-2 from an inhibitor to an activator of apoptosis (4).Autophagy has only recently become an important area in cancer research and is emerging as a key regulator of death pathways (10 -13). Autophagy is a genetically programmed, evolutionarily conserved process that degrades long-lived cellular proteins and organelles. Autophagy is induced by the formation of the initial membrane nucleation that requires a kinase complex including Beclin 1, a BH3-only protein, and hVps34 to generate the phospholipid PI3P. WIPI-1 (WD repeat domain phosphoinositide-interacting protein 1) binds to PI3P and was found to localize to isolation membranes (14...

Lectin receptor-like kinases (LecRLKs), a plant-specific receptor-like kinase (RLK) sub-family, have been recently found to play crucial roles in plant development and responses to abiotic and biotic stresses. In this review, we first describe the classification and structures of Lectin RLKs. Then we focus on the analysis of functions of LecRLKs in various biological processes and discuss the status of LecRLKs from the ligands they recognize, substrate they target, signaling pathways they are involved in, to the overall regulation of growth-defense tradeoffs. LecRLKs and the signaling components they interact with constitute recognition and protection systems at the plant cell surface contributing to the detection of environmental changes monitoring plant fitness.

Seed protein, oil content and yield are highly correlated agronomically important traits that essentially account for the economic value of soybean. The underlying molecular mechanisms and selection of these correlated seed traits during soybean domestication are, however, less known. Here, we demonstrate that a CCT gene, POWR1, underlies a large-effect protein/oil QTL. A causative TE insertion truncates its CCT domain and substantially increases seed oil content, weight, and yield while decreasing protein content. POWR1 pleiotropically controls these traits likely through regulating seed nutrient transport and lipid metabolism genes. POWR1 is also a domestication gene. We hypothesize that the TE insertion allele is exclusively fixed in cultivated soybean due to selection for larger seeds during domestication, which significantly contributes to shaping soybean with increased yield/seed weight/oil but reduced protein content. This study provides insights into soybean domestication and is significant in improving seed quality and yield in soybean and other crop species.

DNA-based activatable theranostic nanoprobes are still unmet for in vivo applications. Here, by utilizing the "induced-fit effect", a smart split aptamer-based activatable theranostic probe (SATP) was first designed as "nanodoctor" for cancer-activated in vivo imaging and in situ drug release. The SATP assembled with quenched fluorescence and stable drug loading in its free state. Once binding to target proteins on cell surface, the SATP disassembled due to recognition-triggered reassembly of split aptamers with activated signals and freed drugs. As proof of concept, split Sgc8c against CEM cancer was used for theranostic studies. Benefiting from the design without blocking aptamer sequence, the SATP maintained an excellent recognition ability similar to intact Sgc8c. An "incubate-and-detect" assay showed that the SATP could significantly lower background and improve signal-to-background ratio (∼4.8 times of "always on" probes), thus affording high sensitivity for CEM cell analysis with 46 cells detected. Also, its high selectivity to target cells was demonstrated in analyzing mixed cell samples and serum samples. Then, using doxorubicin as a model, highly specific drug delivery and cell killing was realized with minimized toxicity to nontarget cells. Moreover, in vivo and ex vivo investigations also revealed that the SATP was specifically activated by CEM tumors inside mice. Especially, contrast-enhanced imaging was achieved in as short as 5 min, thus, laying a foundation for rapid diagnosis and timely therapy. As a biocompatible and target-activatable strategy, the SATP may be widely applied in cancer theranostics.

The establishment of the symbiosis between legumes and nitrogen-fixing rhizobia is finely regulated at the transcriptional, posttranscriptional and posttranslational levels. Argonaute5 (AGO5), a protein involved in RNA silencing, can bind both viral RNAs and microRNAs to control plant-microbe interactions and plant physiology. For instance, AGO5 regulates the systemic resistance of Arabidopsis against Potato Virus X as well as the pigmentation of soybean (Glycine max) seeds. Here, we show that AGO5 is also playing a central role in legume nodulation based on its preferential expression in common bean (Phaseolus vulgaris) and soybean roots and nodules. We also report that the expression of AGO5 is induced after 1 h of inoculation with rhizobia. Down-regulation of AGO5 gene in P. vulgaris and G. max causes diminished root hair curling, reduces nodule formation and interferes with the induction of three critical symbiotic genes: Nuclear Factor Y-B (NF-YB), Nodule Inception (NIN) and Flotillin2 (FLOT2). Our findings provide evidence that the common bean and soybean AGO5 genes play an essential role in the establishment of the symbiosis with rhizobia.

The soybean gene GmFWL1 (FW2-2-like1) belongs to a plant-specific family that includes the tomato FW2-2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2-2-like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain-associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.

Intracellular pH (pHi) is an important parameter associated with cellular behaviors and pathological conditions. Sensing pHi and monitoring its changes are essential but challenging due to the lack of high-sensitive probes. Herein, a ratiometric fluorescent probe with ultra pH-sensitivity is developed based on hairpin-contained i-motif strand (I-strand, labeled with Rhodamine Green and BHQ at two termini) and complementary strand (C-strand, labeled with Rhodamine Red at its 5'-end). At neutral pH, both I-strand and C-strand hybridize into a rigid duplex (I-C), which holds the Rhodamine Red and the BHQ in close proximity. As a result, the fluorescence emission (F) of the Rhodamine Red is strongly suppressed, while the Rhodamine Green (F) is in a "signal on" state. However, the slightly acidic pH enforced the I-strand to form an intramolecular i-motif and initiated the dehybridization of I-C duplex, leading to Rhodamine Red in a "signal on" state and a decreased fluorescence of Rhodamine Green. The ratio (F/F) can be used as a signal for pH sensing. Due to the rational internal hairpin design of I-C duplex probe, almost 70-fold change in the ratio was observed in the physiological pH range (6.50-7.40). This probe possesses efficient stability, fast response, and reversible pH measurement capabilities. Furthermore, intracellular application of the ratiometric probe was demonstrated on the example of SMMC-7721 cells. With different recognition elements in engineering of i-motif based platforms, the design might hold great potential to become a versatile strategy for intracellular pH sensing.

Activatable aptamers have emerged as promising molecular tools for cancer theranostics, but reported monovalent activatable aptamer probes remain problematic due to their unsatisfactory affinity and poor stability. To address this problem, we designed a novel theranostic strategy of DNA nanotriangle-scaffolded multivalent split activatable aptamer probe (NTri-SAAP), which combines advantages of programmable self-assembly, multivalent effect and target-activatable architecture.Methods: NTri-SAAP was assembled by conjugating multiple split activatable aptamer probes (SAAPs) on a planar DNA nanotriangle scaffold (NTri). Leukemia CCRF-CEM cell line was used as the model to investigate its detection, imaging and therapeutic effect both in vitro and in vivo. Binding affinity and stability were evaluated using flow cytometry and nuclease resistance assays.Results: In the free state, NTri-SAAP was stable with quenched signals and loaded doxorubicin, while upon binding to target cells, it underwent a conformation change with fluorescence activation and drug release after internalization. Compared to monovalent SAAP, NTri-SAAP displayed greatly-improved target binding affinity, ultralow nonspecific background and robust stability in harsh conditions, thus affording contrast-enhanced tumor imaging within an extended time window of 8 h. Additionally, NTri-SAAP increased doxorubicin loading capacity by ~5 times, which further realized a high anti-tumor efficacy in vivo with 81.95% inhibition but no obvious body weight loss.Conclusion: These results strongly suggest that the biocompatible NTri-SAAP strategy would provide a promising platform for precise and high-quality theranostics.