"Two-Stage Rocket-Propelled" Strategy Boosting Theranostic Efficacy with Mitochondria-Specific Type I-II Photosensitizers.

Imaging-guided photodynamic therapy (PDT) holds great potential for tumor therapy. However, achieving the synergistic enhancement of the reactive oxygen species (ROS) generation efficiency and fluorescence emission of photosensitizers (PSs) remains a challenge, resulting in suboptimal image guidance and theranostic efficacy. The hypoxic tumor microenvironment also hinders the efficacy of PDT. Herein, we propose a "two-stage rocket-propelled" photosensitive system for tumor cell ablation. This system utilizes MitoS, a mitochondria-targeted PS, to ablate tumor cells. Importantly, MitoS can react with HClO to generate a more efficient PS, MitoSO, with a significantly improved fluorescence quantum yield. Both MitoS and MitoSO exhibit less O2-dependent type I ROS generation capability, inducing apoptosis and ferroptosis. In vivo PDT results confirm that this mitochondrial-specific type I-II cascade phototherapeutic strategy is a potent intervention for tumor downstaging. This study not only sheds light on the correlation between the PS structure and the ROS generation pathway but also proposes a novel and effective strategy for tumor downstaging intervention.

Construction and Evaluation of Peptide-Linked Lactobacillus brevis ß-Galactosidase Heterodimers.

BACKGROUND: ß-galactosidases are enzymes that are utilized to hydrolyze lactose into galactose and glucose, and are is widely used in the food industry. OBJECTIVE: We describe the recombinant expression of an unstudied, heterodimeric ß-galactosidase originating from Lactobacillus brevis ATCC 367 in Escherichia coli. Furthermore, six different constructs, in which the two protein subunits were fused with different peptide linkers, were also investigated. METHODS: The heterodimeric subunits of the ß-galactosidase were cloned in expressed in various expression constructs, by using either two vectors for the independent expression of each subunit, or using a single Duet vector for the co-expression of the two subunits. RESULTS: The co-expression in two independent expression vectors only resulted in low ß-galactosidase activities, whereas the co-expression in a single Duet vector of the independent and fused subunits increased the ß-galactosidase activity significantly. The recombinant ß-galactosidase showed comparable hydrolyzing properties towards lactose, N-acetyllactosamine, and pNP-ß-D-galactoside. CONCLUSION: The usability of the recombinant L. brevis ß-galactosidase was further demonstrated by the hydrolysis of human, bovine, and goat milk samples. The herein presented fused ß-galactosidase constructs may be of interest for analytical research as well as in food- and biotechnological applications.

Comparison of the antipathogenic effect toward Staphylococcus aureus of N-linked and free oligosaccharides derived from human, bovine, and goat milk.

N-linked oligosaccharides (N-glycans) derived from milk were recently found to be antipathogenic. This study compares the antimicrobial activity of N-linked glycans and free oligosaccharides from human, bovine, and goat milk against Staphylococcus aureus. Milk N-glycans showed a bactericidal/bacteriostatic effect on the pathogen when compared to free milk oligosaccharides, evidenced by the clear zone from the halo assay, with the order of human milk >goat milk >bovine milk. None of the free milk oligosaccharide samples were bactericidal/bacteriostatic, despite its positive results in growth curve and minimum inhibitory concentration (MIC) assays which are believed to be related to hyperosmosis. Both N-glycans and free milk oligosaccharides can reduce the adhesion of Staphylococcus aureus to Caco-2 cells, however, N-glycans worked significantly more effective than free milk oligosaccharides. Structural analysis of all free oligosaccharide and N-glycan samples showed the obvious interspecies differences, and the structure/function relationship of the respected N-glycans is of interest for future study. The significant bactericidal/bacteriostatic activity possessed by human, bovine, and goat milk N-linked glycans holds great potential as a novel substitute for antibiotics.

A Potential Role for Mitochondrial Produced Reactive Oxygen Species in Salicylic Acid-Mediated Plant Acquired Thermotolerance.

To characterize the function of salicylic acid (SA) in acquired thermotolerance, the effects of heat shock (HS) on wild-type and sid2 (for SA induction deficient 2) was investigated. After HS treatment, the survival ratio of sid2 mutant was lower than that of wild-type. However, pretreatment with hydrogen peroxide (H2O2) rescued the sid2 heat sensitivity. HsfA2 is a key component of acquired thermotolerance in Arabidopsis. The expression of HsfA2 induced by SA was highest among those of heat-inducible Hsfs (HsfA2, HsfA7a, HsfA3, HsfB1, and HsfB2) in response to HS. Furthermore, the application of AsA, an H2O2 scavenger, significantly reduced the expression level of HsfA2 induced by SA. Although SA enhanced the survival of sid2 mutant, no significant effect on the hsfA2 mutant was observed, suggesting that HsfA2 is responsible for SA-induced acquired thermotolerance as a downstream factor. Further, real-time PCR analysis revealed that after HS treatment, SA also up-regulated mRNA transcription of HS protein (Hsp) genes through AtHsfA2. Time course experiments showed an increase in the fluorescence intensity of DCF in the mitochondria occurred earlier than in other regions of the protoplasts in response to SA. The cytochrome reductase activity analysis in isolated mitochondria demonstrated that SA-induced mitochondrial ROS possibly originated from complex III in the respiration chain. Collectively, our data suggest that SA functions and acts upstream of AtHsfA2 in acquired thermotolerance, which requires a pathway with H2O2 production involved and is dependent on increased expression of Hsp genes.

Mitochondrial-derived reactive oxygen species play a vital role in the salicylic acid signaling pathway in Arabidopsis thaliana.

Plant mitochondria constitute a major source of ROS and are proposed to act as signaling organelles in the orchestration of defense response. At present, the signals generated and then integrated by mitochondria are still limited. Here, fluorescence techniques were used to monitor the events of mitochondria in vivo, as well as the induction of mitochondrial signaling by a natural defensive signal chemical salicylic acid (SA). An inhibition of respiration was observed in isolated mitochondria subjected to SA. The cytochrome reductase activity analysis in isolated mitochondria demonstrated that SA might act directly on the complex III in the respiration chain by inhibiting the activity. With this alteration, a quick burst of mitochondrial ROS (mtROS) was stimulated. SA-induced mtROS caused mitochondrial morphology transition in leaf tissue or protoplasts expressing mitochondria-GFP (43C5) and depolarization of membrane potential. However, the application of AsA, an H2O2 scavenger, significantly prevented both events, indicating that both of them are attributable to ROS accumulation. In parallel, SA-induced mtROS up-regulated AOX1a transcript abundance and this induction was correlated with the disease resistance, whereas AsA-pretreatment interdicted this effect. It is concluded that mitochondria play an essential role in the signaling pathway of SA-induced ROS generation, which possibly provided new insight into the SA-mediated biological processes, including plant defense response.

Increased expression of TRPC5 in cortical lesions of the focal cortical dysplasia.

Focal cortical dysplasias (FCDs) are frequently associated with the medical refractory epilepsy in both children and adults. Transient receptor potential canonical channel 5 (TRPC5), a receptor-operated cation channel, has been well recognized as a regulator in the central nervous system. Here, we examined the expression and cellular distribution of TRPC5 in the specimens from patients with FCDIa (n = 14), FCDIIa (n = 12), and FCDIIb (n = 12) compared with the age-matched control cortex (CTX). TRPC5 mRNA and protein levels were significantly higher in FCDs compared with CTX. Immunohistochemical data showed that TRPC5 was strongly expressed in the misshapen cells, particularly in neuronal microcolumns, dysmorphic neurons, and balloon cells. Moreover, the double-label immunofluorescence analyses demonstrated that TRPC5 localized on NeuN-positive neurons. In addition, its co-localization with glutamate and gamma-aminobutyric acid (GABA) indicated that TRPC5 was distributed on both glutamatergic and GABAergic neurons. Taken together, these results suggested that increased expression of TRPC5 in FCDs and the cell-specific distribution patterns of TRPC5 in the misshapen neurons in FCDs could potentially contribute to the epileptogenesis of FCDs.

Roles of Arabidopsis bax inhibitor-1 in delaying methyl jasmonate-induced leaf senescence.

Previous studies have reported that methyl jasmonate (MeJA) can promote plant senescence. Arabidopsis thaliana BI1 (AtBI1) participates in leaf senescence and JA signal pathway. Our recent report has suggested that AtBI1 plays a crucial role in MeJA-induced leaf senescence. Concomitantly, cytosolic calcium ([Ca²âº]cyt) and MPK6, a mitogen-activated protein kinase (MAPK), participate in the process of MeJA-induced leaf senescence. And AtBI1 might play its roles in delaying MeJA-induced leaf senescence by suppressing the [Ca²âº]cyt-dependent activation of MPK6. Our study contributes to the understanding of the function and mechanism of AtBI1 in plant senescence. Though some of signaling molecules have been elucidated in this type of plant senescence, the mechanism of AtBI-1 functions in reducing the [Ca²âº]cyt during MeJA-induced leaf senescence needs further improvement, and the source and location of Ca²âº are still not clear enough. By using the Arabidopsis and MeJA as the research model, the subsequent researches have been performed to investigate the upstream regulation and downstream function of Ca²âº in this type of plant senescence.

Over-expression of Arabidopsis Bax inhibitor-1 delays methyl jasmonate-induced leaf senescence by suppressing the activation of MAP kinase 6.

Methyl jasmonate (MeJA) is an important signalling molecule that has been reported to be able to promote plant senescence. The cell death suppressor Bax inhibitor-1 (BI1) has been found to suppress stress factor-mediated cell death in yeast and Arabidopsis. However, the effect and the genetic mechanism of Arabidopsis thaliana BI1 (AtBI1) on leaf senescence remain unclear. It was found here that the AtBI1 mutant, atbi1-2 (a gene knock-out), showed accelerated progression of MeJA-induced leaf senescence, while the AtBI1 complementation lines displayed similar symptoms as the WT during the senescence process. In addition, over-expression of the AtBI1 gene delayed the onset of MeJA-induced leaf senescence. Further analyses showed that during the process of MeJA-induced senescence, the activity of MPK6, a mitogen-activated protein kinase (MAPK), increased in WT plants, whereas it was significantly suppressed in AtBI1-overexpressing plants. Under the MeJA treatment, cytosolic calcium ([Ca(2+)](cyt)) functioned upstream of MPK6 activation and the elevation of [Ca(2+)](cyt) was reduced in AtBI1-overexpressing leaves. These results suggested a role of AtBI1 over-expression in delaying MeJA-induced leaf senescence by suppressing the [Ca(2+)](cyt)-dependent activation of MPK6, thus providing a new insight into the function and mechanism of AtBI1 in plant senescence.

MAP Kinase 6-mediated activation of vacuolar processing enzyme modulates heat shock-induced programmed cell death in Arabidopsis.

• Vacuolar processing enzyme (VPE), a cysteine protease, has been intensively studied in plant hypersensitive cell death, but the role and molecular mechanism of VPEs in response to abiotic stresses remain unclear. This work investigated the involvement of VPEs in Arabidopsis response to heat stress. • Under heat shock (HS), Arabidopsis VPE activity and the transcript level of γVPE were both upregulated, and γVPE deficiency suppressed vacuolar disruption and delayed caspase-3-like activation in HS-induced programmed cell death (PCD). Moreover, the change of VPE activity generally paralleled the alteration of caspase-1-like activity under HS treatment, indicating that HS-induced VPE activity might exhibit the caspase-1-like activity. • Further studies showed that MAP Kinase 6 (MPK6) activity was increased after HS treatment, and experiments with inhibitors and mutants suggested that MPK6 was responsible for the γVPE activation after HS treatment. In response to HS stress, reactive oxygen species (ROS) production, increase of cytoplasmic calcium concentration ([Ca(2+) ](cyt)) and the upregulation of calmodulin 3 (CaM3) transcript level occurred upstream of MPK6 activation. • Our results suggested that activation of Arabidopsis γVPE was mediated by MPK6 and played an important role in HS-induced Arabidopsis PCD, providing new insight into the mechanistic study of plant VPEs.