The emergence of drug-resistant pathogens has stimulated the need for the development of new antimicrobial agents. Epigenetic modulation by suppressing epigenetic inhibitors, such as 5-azacytidine (5-aza), has been shown to activate silent biosynthetic gene clusters within a fungus and causes the production of novel secondary metabolites. This research examined this epigenetic modification strategy in the poorly studied filamentous fungus, Ceratorhiza hydrophila, which may help induce the additional production of bioactive compounds.

ClinicalTrials.gov NCT03548818.

Secreted frizzled-related protein 4 (sFRP4) plays a fundamental role in the regulation of Wnt signalling, which is crucial for cellular proliferation and differentiation. The sFRP4 has garnered significant interest as a therapeutic target for metabolic diseases and cancer due to its mechanism of action. Although existing sFRP4 modulators show limited specificity and notable off-target effects, our study explores the potential of known bioactive compounds as more selective and less toxic alternatives. This study is based on the analysis of expression profiles, which demonstrated that the sFRP4 gene exhibits aberrant expression in multiple cancers, including breast cancer. The protein's primary involvement in cancer signaling pathways was determined through pathway enrichment analysis. The study employed molecular docking analyses and MD simulations to identify breast cancer-fighting small molecules with docking energies of less than -6 kcal/mol, targeting the sFRP4 binding hotspot using 100 natural or synthetic small molecules. Out of 100 screened compounds, Silibinin and Isotretinoin were selected based on docking results and further validated in vitro. In vitro investigations were carried out using the colorimetric MTT assay to assess cell viability and cytotoxicity based on metabolic activity. The potential of Silibinin and isotretinoin to upregulate the tumour suppressor sFRP4 was further examined using ELISA and real-time quantitative PCR. Our study identified potential compounds for high-potential drug candidates against sFRP4, demonstrating their effectiveness in cancer cell death and upregulating sFRP4 expression through improved drug design methods and experimental studies. In conclusion, our in-silico findings could facilitate the discovery of potential therapeutic agents against breast cancer. Silibinin and Isotretinoin impede cancer cell development in vitro; nonetheless, this study demonstrated that they directly upregulate sFRP4 and induce apoptosis in breast cancer cells.

Nitrogen (N) fixation with non-thermal plasmas has been proposed as a sustainable alternative to meet growing N fertilizer demands for agriculture. This technology generates Plasma Activated Water (PAW) with a range of chemical compositions, including different concentrations of nitrate (NO₃⁻) and hydrogen peroxide (H2O2), among other compounds. Potential use of PAW as an effective crop fertilizer necessitates a robust understanding of the underlying biology of the plant, which is not yet available. The lack of a unified standard in PAW production and the varying chemical make-up that results from different devices and protocols hampers comparative studies and adoption of this technology. The objective of this study was to compare the efficacy of two PAW solutions with differing concentrations of H2O2 produced from a Radio Frequency (RF) glow discharge plasma source. The effect of these solutions on plant growth, ROS accumulation, gene expression and heat stress response were compared to N-equivalent controls in the model plant Arabidopsis to assess their potential as an alternative N fertilizer. While PAW solutions lacking detectable H2O2 enhanced seedling growth, those containing approximately 0.3 µM of H2O2 did not. ROS accumulation in root tissues was similar between PAW and chemically equivalent solutions, suggesting H2O2 is the primary ROS present in the PAW at the time of treatment. Gene expression studies showed induction of genes involved in N uptake and assimilation in PAW-treated seedlings. Pre-treatment of seedlings with PAW solutions containing H2O2 improved root growth under heat stress which indicates that this treatment may induce plant stress response pathways. Finally, mature plants showed similar growth when fertilized with PAW lacking H2O2 or NO3- control regimes for over 5 weeks indicating equivalency in chemical composition, plant nutrient uptake and utilization. Overall, these results demonstrate that PAW is an effective alternative to NO3- fertilizers for plant cultivation but the levels of H2O2 need to be carefully controlled.

The Sorbin and SH3 domain containing 1 (SORBS1), a protein linked to insulin signaling CBL interaction, was investigated for its role in pancreatic cancer apoptosis. This study explored polyphyllin H (PPH)'s ability to restore SORBS1-knockdown-mediated repair functions.

Proteasomes, multi-subunit proteases, are key actors of cellular protein catabolism and a number of regulatory processes. The detection of subtle proteasome functioning in tumors may contribute to our understanding of the mechanisms of cancer development. The current study aimed to identify the role of low molecular mass protein 2 (LMP2), a proteasome immune subunit, in the development of mouse colon 26 (C26) adenocarcinoma.

Melatonin, a multifunctional signalling molecule in plants, has been increasingly recognized for its role in improving stress tolerance, regulating hormone signalling, and enhancing crop productivity. Exogenous melatonin application represents a promising strategy to enhance crop productivity under global agricultural challenges. This study aimed to investigate the physiological and molecular mechanisms by which melatonin improves yield in Brassica napus. under optimal conditions. Two-week old plants were treated with 10 μM melatonin for 7 days and phenotype was observed. The plants exhibited significant increases in plant height, leaf number, pods per plant, seeds per pod, and 100-seed weight compared to controls. Transcriptomic analysis revealed 2924 differentially expressed genes (DEGs; 1655 upregulated, 1269 downregulated) from 66 258 genes in response to exogenously applied melatonin. Functional enrichment highlighted profound upregulation of photosynthesis-related pathways, including photosystem I/II components (PsbO, PsaH), electron transport genes (PetE, PetH), and F-type ATPase subunits. Melatonin also reconfigured phytohormone signaling, upregulating auxin (AUX1; BnaA10g27610D), ABA (ABF; BnaA06g04750D), cytokinin (CRF1; BnaA06g34500D, A-ARR; BnaC03g48210D, Bna08g14280D, Bna09g36380D, BnaCnng49490D, BnaA06g16900D, and BnaA06g06240D), and gibberellin-associated genes while downregulating ABA repressors (PYR/PYL; BnaA06g40360D, BnaC07g19450, PP2C; BnaA06g23040D, and BnaA01g37370D). Transcription factor profiling showed activation of growth-promoting families (NAC, TCP, bHLH) and suppression of stress-responsive TFs (MYB, AP2/ERF, WRKY). Validation via RT-qPCR confirmed RNA-seq reliability (R² = 0.96). Our study demonstrated that low-dose melatonin enhances B. napus yield by coordinately boost photosynthetic efficiency, optimizing hormone signaling, and activating growth-promoting transcription factors to prioritize reproductive development.

Nonexpressor of pathogenesis-related genes 1 (NPR1) is a master regulator of salicylic acid (SA)- facilitated plant hormone signaling and plays a crucial role in plant defense through the activation of systemic acquired resistance (SAR). Although NPR1-like genes are associated with stress responses in a variety of plant species, no thorough genome-wide investigation of these genes has been undertaken in pearl millet (Pennisetum glaucum). This study discovered seven PgNPR1-like genes on four pearl millet chromosomes (Chr1, Chr2, Chr4, and Chr6), which exhibit close affinity to NPRs from other plants and have common gene structures, conserved motifs, and domains. The promoter regions of PgNPR1-like genes have numerous cis-acting elements connected with biotic and abiotic stresses, natural plant growth, and development. The qPCR results showed that PgNPR1-like genes were differentially expressed in distinct tissues, developmental stages, and under various biotic and abiotic stresses. Some putative NPR1-like genes, such as Pgl_GLEAN_10029279, Pgl_GLEAN_10004488, Pgl_GLEAN_10004489, and Pgl_GLEAN_10015079, showed considerable expression in response to abiotic stimuli such as heat, drought, and salinity. The PgNPR1-like gene Pgl_GLEAN_10029279 was observed to be differently expressed upon treatment of hormones such as SA and MeJA. Pgl_GLEAN_10029279 was also significantly expressed after Magnaporthe grisea infection, which causes blast in pearl millet. In silico expression study of the PgNPR1-like genes after Sclerospora graminicola infection, causing downy-mildew disease, revealed that Pgl_GLEAN_10029279 and Pgl_GLEAN_10004489 were significantly upregulated. In addition, the docking results also showed that Pgl_GLEAN_10029279 and Pgl_GLEAN_10007810 out of all seven PgNPRs have strong interactions with the ligand SA, which proves their potential involvement in SA signaling and hence plant defense. These results offer a firm framework for comprehending the roles and development of PgNPR1-like genes in pearl millet.

Berberine (BBR) is an isoquinoline alkaloid with a variety of biological activities, including anti-microbial and anti-tumoral activities. However, the cellular targets of BBR and the roles of BBR in the radiosensitivity of breast cancer cells are not well defined. In this study, we investigated the effects of BBR on the radiosensitivity of BT549 triple-negative breast cancer cells. Through RNA-seq results, we found that BBR significantly down-regulated the expression of DNA ligases. The results of western blot and comet assay confirmed that BBR attenuated DNA ligase III (LIGIII) expression and caused DNA damage in a dose-dependent manner. The results of electron microscopy showed that BBR decreased mitochondrial copies and induced mitochondrial autophagy. Clonal formation analysis showed that BBR sensitised breast cancer cells to irradiation. The genetic complementation and interference assays showed that the effect of BBR on the radiosensitivity of BT549 breast cancer cells is dependent on the expression of LIGIII. These results contribute to the understanding of the effects of BBR on cellular DNA repair and the clinical application of BBR in breast cancer therapy.

In this study, we evaluated the antibacterial activities of plant essential oils (EOs) from the Lamiaceae family against Agrobacterium tumefaciens to find new eco-friendly antimicrobials. Thymus vulgaris L. (thyme white) EO demonstrated the most potent fumigant antibacterial activity among these. Key compounds identified in thyme white EO, including thymol, carvacrol, S-(-)-α-pinene, R-(+)-α-pinene, and (-)-limonene, also exhibited strong fumigant antibacterial activity against A. tumefaciens. The inhibition zone diameters observed for thymol, carvacrol, R-(+)-α-pinene, S-(-)-α-pinene, thyme white EO, and S-(-)-limonene were 2.85, 2.74, 1.64, 1.55, 1.15, and 0.86 cm, respectively, at a concentration of 5 mg per paper disc. Only thyme white EO, thymol, and carvacrol exhibited contact antibacterial activity against A. tumefaciens. The minimum inhibition concentrations (MIC) of thyme white EO, thymol, carvacrol, and streptomycin were 800 μg/mL, 350 μg/mL, 350 μg/mL, and 3.125 μg/mL, respectively. Using confocal laser scanning microscopy, we observed intracellular reactive oxygen species (ROS) production and membrane damage in A. tumefaciens treated with thyme white EO, thymol, and carvacrol. Additionally, we analyzed the differential gene expression level in A. tumefaciens treated with thyme white EO, thymol, and carvacrol, comparing them to untreated cells to elucidate the mode of action. In particular, the expression of manganese transport-related genes (sitABCD and mntH) was significantly downregulated, which impaired the function of sod. As a result, the system responsible for detoxifying ROS was disrupted. This led to excessive accumulation of ROS, causing damage to the cell membrane.

As the weed Echinochloa phyllopogon has rapidly developed multi- and cross-resistance to several herbicides, we aimed to determine the mechanism underlying penoxsulam resistance in weeds. There was no target mutation in the tested population, and P450 enzyme activity was significantly higher in the penoxsulam-treated resistant population, confirming that non-target-site resistance was dominant. The antioxidant enzyme activity of the resistant population was higher than that of the sensitive population following the application of the penoxsulam and cleared H2O2 faster. The antioxidant enzyme system of the sensitive population was inhibited in the early stage of the stress and the malondialdehyde content was increased, leading to severe cellular damage. Transcriptome sequencing indicated upregulated expression of metabolic genes related to resistance including CYP709B2, CYP71C4, CYP72A15, and AKR4C10 in resistant versus sensitive populations. Overexpression of CYP72A15 in Arabidopsis thaliana induced significant resistance to penoxsulam, bensulfuron-methyl, and quinclorac. Transgenic Oryza sativa once again demonstrated the important role of CYP72A15 in E. phyllopogon herbicide resistance. The current study highlights the role of CYP450 family enzymes in the herbicide resistance of weeds.

GA participates in FR light-induced internode elongation of cucumber by regulating the expression of genes/proteins related to aquaporins, expansins, cell wall biosynthesis, hormone metabolism, and signal transduction. This study investigated the effects of the interaction between far-red (FR) light and gibberellin (GA) on the internode elongation of cucumber (Cucumis sativus L. 'Zhongnong No. 26') seedlings through combined physiological, biochemical, transcriptomic, and proteomic analyses. The results revealed that FR light and GA significantly promoted internode elongation in cucumber seedlings, whereas a GA biosynthesis inhibitor (PAC) inhibited the promoting effect of FR light. Hormone content determination revealed that FR light and GA decreased the contents of abscisic acid (ABA), indole-3-acetic acid (IAA), cytokinin (CTK), and jasmonate (JA) in cucumber seedling internodes. Bioinformatics analysis revealed that the expression patterns of the Co-DEGs and Co-DEPs were consistent in the FR (WL combined with FR light) and WLG (WL, in which plants were sprayed GA) groups, as well as in the FRP (FR, in which plants were sprayed PAC) and WL (full-spectrum LED white light) groups, suggesting that the mechanisms of FR and GA were similar in these Co-DEGs and Co-DEPs. Further analysis of these Co-DEGs and Co-DEPs revealed that they were involved mainly in cell wall biosynthesis and modification, lignin synthesis, hormone metabolism, and signal transduction pathways. In conclusion, this study revealed the important role of GA in FR light-induced internode elongation in cucumber seedlings, and this promoting effect was achieved mainly through the regulation of aquaporins, expansins, hormone metabolism, and signal transduction-related genes/proteins. This study provides new insights into the molecular mechanism of FR light-induced internode elongation in cucumber seedlings.

Microglial activation-induced neuroinflammation and impaired neuronal mitophagy are recognized as pivotal pathogeneses in Parkinson's disease (PD). However, the role of microglial mitophagy in microglial activation during PD development remains unclear, and therapeutic interventions targeting this interaction are lacking. Rhapontigenin (Rhap), a stilbenoid enriched in Vitis vinifera, exhibits dual anti-neuroinflammatory and mitophagy-enhancing properties, but its therapeutic potential and mechanisms in PD are unexplored. This study aimed to investigate the therapeutic efficacy of Rhap on neurodegeneration in a PD model and explore its underlying mechanism. Here, we showed that Rhap administration significantly ameliorated motor deficits, dopaminergic neuron loss, and neuroinflammation in MPTP-induced PD mice. Mechanistically, Rhap suppressed neuroinflammation by inhibiting the cGAS-STING-NF-κB signaling axis in both PD model mice and MPP⁺-induced BV2 microglia. Crucially, its anti-inflammatory effects depend on the PINK1-mediated enhancement of microglial mitophagy to control cytosolic mtDNA leakage. Specifically, Rhap bound to PINK1 strengthened the PINK1-DRP1 interaction, promoted mitochondrial fission in damaged organelles, and enhanced mitophagy clearance. This mitophagy activation prevents cytosolic leakage of mitochondrial DNA (mtDNA), thereby attenuating mtDNA-cGAS-STING-NF-κB-derived neuroinflammation and subsequent neurodegeneration in PD. PINK1 deficiency in BV2 microglia abolished Rhap's ability to suppress mtDNA-cGAS-STING-NF-κB activation and enhance mitophagy. Overall, our study reveals a previously unrecognized mechanism by which Rhap ameliorates PD-associated neurodegeneration through dual modulation of PINK1/DRP1-dependent microglial mitophagy and the mtDNA-cGAS-STING-NF-κB neuroinflammatory axis, suggesting a potential therapeutic strategy for PD and related neurodegenerative disorders.

Low density lipoprotein receptor-related protein 2 (LRP2) is a 600 kilodalton multi-ligand endocytic membrane receptor expressed in several cell types during fetal development, including neuroepithelial cells, and in select absorptive epithelial cells in the adult. In epithelial cancers, LRP2 expression is associated with a differentiated tumor cell state and better prognosis. In previous work, we found that while LRP2 is not expressed in benign naevi, it is frequently acquired in melanoma. However, the molecular drivers of LRP2 expression in melanoma and characteristics of LRP2-expressing melanoma have yet to be described. Here, we show that LRP2 expression is related to a transitory melanoma cell state defined by co-expression of melanocyte lineage and neural crest transcriptional programs. Further, we reveal that melanoma LRP2 expression is increased in T cell-inflamed tumors and is directly upregulated through interferon-gamma signaling. Correlation of melanoma LRP2 expression with clinicopathological variables demonstrates that LRP2 expression is associated with low Breslow thickness and low clinical stage in primary melanomas. Taken together, the present study describes the characteristics of LRP2-expressing melanoma and reveals interferon-gamma signaling as a novel strong positive regulator of LRP2 expression in melanoma.

Colorectal cancer (CRC) is a common malignancy often characterized by metastasis and poor prognosis. This study attempts to ascertain the anticancer impacts of theaflavin (TF) on CRC cells and examine the fundamental molecular mechanisms, focusing on the function of DDIT4 in CRC progression. This study utilized RNA sequencing for gene expression profiling, differential expression analysis, and Venn diagram analysis for overlapping genes. Protein interactions were explored, while cell viability was evaluated using colony formation assays and Cell Counting Kit-8 (CCK-8). Flow cytometry was employed for apoptosis analysis, and Transwell assays measured cell migration and invasion. ATP synthesis, lactate production, and glucose uptake were analyzed to evaluate metabolic changes, with protein and RNA expression identified by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). This study reveals that TF effectively inhibits CRC cell invasion, migration, and proliferation in a dose- and time-dependent manner. TF induces apoptosis by suppressing the antiapoptotic protein Bcl-2 and enhancing proapoptotic proteins (Cleaved Caspase-3, Bax, and Caspase-9). Through bioinformatics analysis, DDIT4 was identified as a key target gene. Additionally, correlation analysis highlighted a positive relationship between DDIT4 and the glycolysis/gluconeogenesis pathway. TF downregulates DDIT4 expression, which suppresses CRC cell proliferation and glycolysis. Moreover, DDIT4 overexpression partially reverses the suppressive impacts of TF on glycolysis and cell viability. These observations imply that TF suppresses CRC progression by targeting DDIT4 and regulating glycolytic activity, highlighting its promise as a medicinal substance for the treatment of CRC.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that, for the western blots shown in Fig. 4C, the two left lanes in the 'Fibrillarin' gel slice appeared to be strikingly similar to the mirrored two right lanes in the 'Actin' gel slice, albeit the orientations of the blots were horizontally reversed, such that data which were intended to have shown the results of differently performed experiments appeared to have been derived from the same original source. Moreover, the control β-actin blots featured in Fig. 5A of the above paper were strikingly similar to control blots used in a different context that appeared in a subsequently published paper featuring some of the same authors in American Journal of Cancer Research. The authors were contacted by the Editorial Office to offer an explanation for the apparent anomaly in the presentation of the data in this paper, although up to this time, no response from them has been forthcoming. Owing to the fact that the Editorial Office has been made aware of potential issues surrounding the scientific integrity of this paper, we are issuing an Expression of Concern to notify readers of this potential problem while the Editorial Office continues to investigate this matter further. [International Journal of Molecular Medicine 29: 65‑72, 2012; DOI: 10.3892/ijmm.2011.806].

Leukemia is a malignant clonal disease originating from hematopoietic stem cells, whose complex pathogenesis is associated with multiple factors. Epigenetic regulation has been found to play an important role in the occurrence and development of leukemia, and has become a major focus of research. Fucoidan (FPS), a natural sulfated polysaccharide primarily extracted from marine brown algae, is rich in L‑fucose and sulfate groups. It has a variety of biological activities, including antioxidant, antiviral, immunomodulatory and antitumor activities. Notably, FPS exhibits antileukemic potential by epigenetically inhibiting the protein expression of DNA methyltransferases, regulating methylation levels at the promoter regions of specific genes such as peroxiredoxin 2, influencing the activity of histone‑modifying enzymes, and controlling the expression of non‑coding RNAs (ncRNAs), including microRNAs and long ncRNAs. These effects collectively suppress the proliferative and differentiation of leukemic cells. The present review examines the epigenetic regulatory mechanisms by which FPS may inhibit leukemia, including DNA methylation, histone modification and ncRNA‑associated mechanisms. In addition, it also discusses the potential advantages and challenges of FPS in the treatment of leukemia, as well as future research directions for FPS in leukemia therapy, aiming to provide a stronger theoretical basis for its clinical application.

Nitrogen (N) is essential for plant growth, but excessive fertilizer use decreases nitrogen use efficiency (NUE) and raises environmental concerns. This study investigated the effect of exogenous abscisic acid (ABA; 50 μM) application on rapeseed (Brassica napus L.) plants under hydroponic conditions with high (7.5 mM NO3-) and low (0.25 mM NO3-) nitrate. N deficiency significantly reduced growth, chlorophyll, gas exchange, and stomatal size (length and width). Exogenous ABA application under low N conditions increased the N content (23.1%) and the activities of nitrate reductase (NR; 15.7%), glutamine synthetase (GS; 30.3%), and GS/glutamate synthase (GOGAT; 19.3%). It also enhanced activities of antioxidant enzymes: superoxide dismutase (SOD; 18.3%), peroxidase (POD; 30.2%), ascorbate peroxidase (APX; 39%), catalase (CAT; 16.3%), and endogenous hormones: ABA (35.3%), salicylic acid (SA; 20.5%), indole acetic acid (IAA; 49.8%), and jasmonic acid (JA; 90.3%) compared to untreated low N conditions. Histochemical staining (NBT and DAB) confirmed that ABA alleviated oxidative stress under N deficiency. Transcriptomic analysis identified differentially expressed genes related to photosynthesis, antioxidant defense, N metabolism, and ABA signaling (PYL/PYR/RCAR receptors, PP2C phosphatases, SnRK2 kinases). Transcription factors from bZIP, MYB, and AP2/ERF families were significantly regulated. These results highlight the mechanisms of ABA-mediated N stress mitigation and provide a basis for improving NUE in rapeseed through genetic approaches such as overexpression CRISPR/Cas9, supporting sustainable agriculture.

Epitalon, a naturally occurring tetrapeptide, is known for its anti-aging effects on mammalian cells. This happens through the induction of telomerase enzyme activity, resulting in the extension of telomere length. A strong link exists between telomere length and aging-related diseases. Therefore, telomeres are considered to be one of the biomarkers of aging, and increasing or maintaining telomere length may contribute to healthy aging and longevity. Epitalon has been the subject of several anti-aging studies however, quantitative data on the biomolecular pathway leading to telomere length increase, hTERT mRNA expression, telomerase enzyme activity, and ALT activation have not been extensively studied in different cell types. In this article, the breast cancer cell lines 21NT, BT474, and normal epithelial and fibroblast cells were treated with epitalon then DNA, RNA, and proteins were extracted. qPCR and Immunofluorescence analysis demonstrated dose-dependent telomere length extension in normal cells through hTERT and telomerase upregulation. In cancer cells, significant telomere length extension also occurred through ALT (Alternative Lengthening of Telomeres) activation. Only a minor increase in ALT activity was observed in Normal cells, thereby showing that it was specific to cancer cells. Our data suggests that epitalon can extend telomere length in normal healthy mammalian cells through the upregulation of hTERT mRNA expression and telomerase enzyme activity.

Bone metastasis most commonly occurs in castration-resistant prostate cancer (CRPC). The TRPV6 calcium channel is absent in healthy prostate tissue, but its expression increases considerably during cancer progression. We hypothesized that cancer cells induce TRPV6 expression de novo to directly benefit from tightly regulated calcium intake via TRPV6 while providing cancer cells with a selective advantage for metastasis in the calcium-abundant niche, such as bone. Using a cohort of prostate cancer tissue biopsies from patients with a clinical history of at least 10 years after biopsy, we report that TRPV6 expression directly correlates with CRPC tumor aggressiveness and increased risk of metastasis development. The TRPV6 channel is involved in the acquisition of both mesenchymal and invasive phenotypes through increased phosphorylation of CaMK2 followed by the translocation of the transcription factor NF-κB to the nucleus and the expression of EMT markers, MMPs, and transcription factors such as Twist, Snail, and Slug. Moreover, TRPV6 expression was accompanied by increased formation of CXCR4/TRPV6 complexes. In vivo, mice bearing trpv6+/+ tumors presented increased metastasis, notably bone metastasis, whereas trpv6-/- mice developed no metastasis. Targeting TRPV6 with a monoclonal antibody resulted in a significant reduction in the metastatic burden and an increase in overall survival. When AMD3100, a selective inhibitor of the CXCR4 receptor, was combined with AMD3100, a synergistic effect on the suppression of metastasis development was achieved. Thus, the suppression of CRPC metastasis to bone can be achieved via simultaneous targeting of TRPV6/CXCR4, demonstrating that combined therapy is a proof-of-concept approach in vivo.