Ginsenoside Rg1 mitigates cerebral ischaemia/reperfusion injury in mice by inhibiting autophagy through activation of mTOR signalling.

Reperfusion injury, which is distinct from ischaemic injury, occurs when blood flow is restored in previously ischaemic brain tissue, further compromising neurons and other cells and worsening the injury. There is currently a lack of pharmaceutical agents and therapeutic interventions that specifically mitigate cerebral ischaemia/reperfusion (I/R) injury. Ginsenoside Rg1 (Rg1), a protopanaxatriol-type saponin isolated from Panax ginseng C. A. Meyer, has been found to protect against cerebral I/R injury, but its intricate protective mechanisms remain to be elucidated. Numerous studies have shown that autophagy plays a crucial role in protecting brain tissue during the I/R process and is emerging as a promising therapeutic strategy for effective treatment. In this study, we investigated whether Rg1 protected against I/R damage in vitro and in vivo by regulating autophagy. Both MCAO and OGD/R models were established. SK-N-AS and SH-SY5Y cells were subjected to OGD followed by reperfusion with Rg1 (4-32 µM). MCAO mice were injected with Rg1 (30 mg·kg-1·d-1. i.p.) for 3 days before and on the day of surgery. Rg1 treatment significantly mitigated ischaemia/reperfusion injury both in vitro and in vivo. Furthermore, we demonstrated that the induction of autophagy contributed to I/R injury, which was effectively inhibited by Rg1 in both in vitro and in vivo models of cerebral I/R injury. Rg1 inhibited autophagy through multiple steps, including impeding autophagy initiation, inducing lysosomal dysfunction and inhibiting cathepsin enzyme activities. We revealed that mTOR activation was pivotal in mediating the inhibitory effect of Rg1 on autophagy. Treatment with Torin-1, an autophagy inducer and mTOR-specific inhibitor, significantly reversed the impact of Rg1 on autophagy, decreasing its protective efficacy against I/R injury both in vitro and in vivo. In conclusion, our results suggest that Rg1 may serve as a promising drug candidate against cerebral I/R injury by inhibiting autophagy through activation of mTOR signalling.

YTE-17 inhibits colonic carcinogenesis by resetting antitumor immune response via Wnt5a/JNK mediated metabolic signaling.

The density and composition of lymphocytes infiltrating colon tumors serve as predictive factors for the clinical outcome of colon cancer. Our previous studies highlighted the potent anti-cancer properties of the principal compounds found in Garcinia yunnanensis (YTE-17), attributing these effects to the regulation of multiple signaling pathways. However, knowledge regarding the mechanism and effect of YTE-17 in the prevention of colorectal cancer is limited. In this study, we conducted isobaric tags for relative and absolute quantification (iTRAQ) analysis on intestinal epithelial cells (IECs) exposed YTE-17, both in vitro and invivo, revealing a significant inhibition of the Wnt family member 5a (Wnt5a)/c-Jun N-terminal kinase (JNK) signaling pathway. Subsequently, we elucidated the influence and mechanism of YTE-17 on the tumor microenvironment (TME), specifically focusing on macrophage-mediated T helper 17 (Th17) cell induction in a colitis-associated cancer (CAC) model with Wnt5a deletion. Additionally, we performed the single-cell RNA sequencing (scRNA-seq) on the colonic tissue from the Wnt5a-deleted CAC model to characterize the composition, lineage, and functional status of immune mesenchymal cells during different stages of colorectal cancer (CRC) progression. Remarkably, our findings demonstrate a significant reduction in M2 macrophage polarization and Th17 cell phenotype upon treatment with YTE-17, leading to the restoration of regulatory T (Treg)/Th17 cell balance in azoxymethane (AOM)/dextran sodium sulfate (DSS) model. Furthermore, we also confirmed that YTE-17 effectively inhibited the glycolysis of Th17 cells in both direct and indirect co-culture systems with M2 macrophages. Notably, our study shed light on potential mechanisms linking the non-canonical Wnt5a/JNK signaling pathway and well-established canonical ß-catenin oncogenic pathway in vivo. Specifically, we proposed that Wnt5a/JNK signaling activity in IECs promotes the development of cancer stem cells with ß-catenin activity within the TME, involving macrophages and T cells. In summary, our study undergoes the potential of YTE-17 as a preventive strategy against CRC development by addressing the imbalance with the immune microenvironment, thereby mitigating the risk of malignancies.

Noncanonical translation of circRNAs drive antitumor immunity.

Identifying tumor-specific antigenic peptides beyond mutational events is essential for developing effective antitumor immunotherapies. In a recent study, Huang et al. identified an immunogenic cryptic peptide encoded by the circular RNA circFAM53B, through integrating mass spectrometry and ribosome sequencing data from breast cancer samples, and demonstrated its antitumor immunogenicity.

Low-dose radiation from CT examination induces DNA double-strand breaks and detectable changes of DNA methylation in peripheral blood cells.

BACKGROUND: Radiation burden from CT examinations increases rapidly with the increased clinical use frequency. Previous studies have disclosed the association between radiation exposure and increased double-strand breaks (DSBs) and changes in DNA methylation. However, whether the induced DSBs by CT examination recover within 24h and whether a CT examination induces detectable gene-specific methylation changes are still unclear. The aim of the present study was to analyze γ-H2AX in the peripheral blood lymphocyte (PBL) of healthy adults before and after CT examination and to discover the differentially methylated positions (DMPs) along with an analysis of DNA methylation changes caused by CT examination. MATERIALS AND METHODS: Peripheral blood samples of 4 ml were drawn from 20 healthy volunteers at three time points: before CT examination, after CT examination 1h, and after CT examination 24h. γ-H2AX immunofluorescence and Illumina Infinium Human Methylation EPIC BeadChip (850k BeadChip) were used respectively for the test of DSBs and the epigenome-wide DNA methylation analysis. Linear mixed-effect (LME) models were used to evaluate the impacts of doses represented by different parameters and foci on genome-wide DNA methylation. RESULTS: The number of γ-H2AX foci per cell at 1h showed linear dose-responses for the radiation doses represented by CT index volume (CTDIvol), dose length product (DLP), and blood absorbed dose, respectively. Residual γ-H2AX foci was observed after CT examination at 24h (p < .001). DMPs and γ-H2AX foci changes could be found within 1h. One CpG site related to PAX5 was significantly changed by using most of the parameters in LME models and did not recover till 24h. CONCLUSIONS: Residual γ-H2AX foci exist after CT examination at 24h. The DNA methylation changes induced by CT examination may not recover within 24h. The DNA methylation had been changed as early as at 1h. The PAX5-related CpG site may be a potential biomarker of low-dose radiation. CLINICAL RELEVANCE: The biological effects and the cancer risks of CT examination are still unclear. The present study is an effort to document the CT scan-induced events in 24h in vivo. The CT scanning area should be strictly limited, and non-essential repeated operations shouldn't be performed within 24h.

Single-cell characterization of tumor evolution from precursor diseases.

Elucidating the characteristics of precursor diseases is important to the understanding of malignant transformation during tumor progression. In a recent study, Dang et al. characterized the cellular and molecular evolution from precursor conditions to multiple myeloma (MM) by integrating single-cell RNA sequencing (scRNA-seq) and B cell-receptor sequencing (scBCR-seq).

Pulsatilla Decoction and its bioactive component ß-peltatin induce G2/M cell cycle arrest and apoptosis in pancreatic cancer.

BACKGROUND: Pancreatic cancer (PAC), a malignancy that is fatal and commonly diagnosed at a late stage. Despite considerable advancements in cancer treatment, the survival rate of PAC remains largely consistent for the past 60 years. The traditional Chinese medicine formula Pulsatilla Decoction (PD) has been clinically used to treat inflammatory diseases for millennia and recently as a supplementary anti-cancer treatment in China. However, the bioactive ingredients and mechanisms underlying its anti-cancer effect remains unclear. METHODS: The composition and quality control of PD were verified through analysis by high performance liquid chromatography. Cell viability was determined using Cell Counting Kit-8 assay. The cell cycle distribution was analyzed through PI staining and flow cytometry analysis, while apoptotic cells were measured by double staining with Annexin V-FITC and PI. We used immunoblotting to examine protein expressions. The in vivo effects of ß-peltatin and podophyllotoxin were evaluated on a subcutaneously-xenografted BxPC-3 cell nude mice model. RESULTS: The current study demonstrated that PD markedly inhibited PAC cell proliferation and triggered their apoptosis. Four herbal PD formula was then disassembled into 15 combinations of herbal ingredients and a cytotoxicity assay showed that the Pulsatillae chinensis exerted the predominant anti-PAC effect. Further investigation indicated that ß-peltatin was potently cytotoxic with IC50 of ~ 2 nM. ß-peltatin initially arrested PAC cells at G2/M phase, followed by apoptosis induction. Animal study confirmed that ß-peltatin significantly suppressed the growth of subcutaneously-implanted BxPC-3 cell xenografts. Importantly, compared to podophyllotoxin that is the parental isomer of ß-peltatin but clinically obsoleted due to its severe toxicity, ß-peltatin exhibited stronger anti-PAC effect and lower toxicity in mice. CONCLUSIONS: Our results demonstrate that Pulsatillae chinensis and particularly its bioactive ingredient ß-peltatin suppress PAC by triggering cell cycle arrest at G2/M phase and apoptosis.

Relationship between development of the condylar cortex and the changes in condyle morphology: a cone-beam computed tomography (CBCT) observational study.

Background: A correct understanding of the mandibular condyle morphology may help clinicians judge the normal range of morphological variations of asymptomatic patients or the pathological conditions correctly. Hence, the aim of the present study was to evaluate the status of condyle cortication and condyle morphology, and to investigate the relationship between the development of the condylar cortex and the changes of condyle morphology. Methods: The present study was an observational study. A total of 1,010 temporomandibular joint (TMJ) cone-beam computed tomography (CBCT) images were collected retrospectively. The mandibular condyle morphology was observed in axial (concave, convex, plane and others for anterior and posterior facets), coronal (plane, convex, angled and round for superior facet) and sagittal (round and plane for superior facet) views, and the condylar cortication was grouped into three types (undeveloped, developing and developed). Analytical statistics were performed to detect a relationship between the cortication status and morphology of the condyles. Results: For males and females, the mean age was 15.11±2.71 and 14.25±2.60 years (for condylar bone without cortication), 19.45±3.92 and 18.65±3.45 years (with developing cortical bone), 23.63±3.36 and 23.86±3.73 years (with developed cortical bone), respectively. The condyle morphology with a plane form in the anterior aspect, a convex form in the posterior aspect, a convex form in the coronal view and a round form in the sagittal view was the most often recorded condyle morphologies (13.2%). After the cortical bone of condyle completely forms, the plane form was significantly increased in the superior surface in both sagittal and coronal views. Conclusions: The condylar shape gradually changes with growth and development of the condyle bone cortex. The more mature the bone cortex is, the higher the probability that the condyle will have an uneven shape, which may mean that the condyle morphology may change due to remodeling during growth and development.

Garcinol and its analogues: Synthesis, cytotoxic activity and mechanistic investigation.

Garcinol is a polyisoprenylated benzophenone isolated from Garcinia. It has been reported to have a variety of intriguing biological effects, including anticancer, anti-inflammatory, and antioxidant capabilities. The purpose of this research is to thoroughly evaluate garcinol and a series of its analogues in terms of synthesis, structural diversity, biosynthesis, and potential for preventing carcinoma cell proliferation. Garcinopicrobenzophenone and eugeniaphenone, which contain a unique cyclobutyl unit at C-5, were initially synthesized using the procedures utilized in the synthesis of garcinol. All the natural analogs of garcinol were produced at completion of the synthesis, and their structures and absolute configurations were clarified. Based on the synthesis, a possible biogenetic synthesis pathway towards cambogin, 13,14-didehydroxyisogarcinol via O-cyclization, and garcinopicrobenzophenone or eugeniaphenone via C-cyclization was proposed. The cytotoxicity of polyisoprenylated benzophenones produced in our group was tested, and the structure-activity relationship was summarized. The mechanism by which garcinol, cambogin, and 21' induce apoptosis was studied. Cambogin and 21' were shown to have a greater capacity to cause apoptosis in pancreatic cancer BXPC3 cells, and the suppression of BXPC3 cells by 21' might be attributed to the target of STAT3 signaling. Garcinol could cause pyroptosis and apoptosis in pancreatic cancer cells at the same time, which was the first time that garcinol was identified as a possible chemotherapeutic agent that could significantly promote pyroptosis in cancer cells.

Saikosaponin A enhances Docetaxel efficacy by selectively inducing death of dormant prostate cancer cells through excessive autophagy.

Quiescent cancer cells (QCCs), also known as dormant cancer cells, resist and survive chemo- and radiotherapy, resulting in treatment failure and later cancer recurrence when QCCs resume cell cycle progression. However, drugs selectively targeting QCCs are lacking. Saikosaponin A (SSA) derived from Bupleurum DC., is highly potent in eradicating multidrug-resistant prostate QCCs compared with proliferative prostate cancer cells. By further exacerbating the already increased autophagy through inactivation of Akt-mTOR signaling, SSA triggered cell death in QCCs. Contrarily, inhibition of autophagy or activation of Akt signaling pathway prevented SSA-induced cell death. The multicycle of Docetaxel treatments increased the proportion of QCCs, whereas administering SSA at intervals of Docetaxel treatments aggravated cell death in vitro and led to tumor growth arrest and cell death in vivo. In conclusion, SSA is posed as a novel QCCs-eradicating agent by aggravating autophagy in QCCs. In combination with the current therapy, SSA has potential to improve treatment effectiveness and to prevent cancer recurrence.

The natural compound from Garcinia bracteata mainly induces GSDME-mediated pyroptosis in esophageal cancer cells.

BACKGROUND: Pyroptosis, an inflammatory form of programmed cell death (PCD), is reported to play important roles in the treatment of tumors. In our previous studies, we found that neobractatin (NBT), a caged prenylxanthone isolated from edible fruits of Garcinia bracteata C. Y. Wu ex Y. H. Li, showed anticancer effects against different cancer cells. However, the effect of NBT on pyroptosis is not well understood. PURPOSE: This study aims to investigate whether and how GSDME-mediated pyroptosis contributes to NBT-induced antitumor effects in esophageal cancer (EC) cells. METHODS: Cell viability assay and colony formation assay were used to determine the anticancer effects of NBT in esophageal cancer cells. Lactate dehydrogenase (LDH) release assay and microscopy imaging were used to detect the main characteristic of pyroptosis. CRISPR-Cas9 knockout and siRNA knockdown were performed to verify the roles of GSDME and caspase-3 in NBT-induced pyroptosis. Flow cytometry was used to measure the reactive oxygen species (ROS) level and cell apoptosis. The changes of related protein level were detected by Western blot. Furthermore, animal experiments were used to verify the in vivo effect of NBT. RESULTS: The results showed that NBT reduced the viability of EC cells mainly through GSDME-mediated pyroptosis. Morphologically, NBT induced cell swelling and formed large bubbles emerging from plasma membrane in wild type EC cells. Furthermore, NBT induced the cleavage of GSDME by activating caspase-3 in EC cells. On the other hand, caspase-3 activated by NBT also induced apoptosis especially at high dosage. Knocking down GSDME switched NBT-induced cell death from mainly pyroptosis to apoptosis in vivo and in vitro. Mechanistic studies indicated that NBT led to accumulation of ROS, which then regulated the phosphorylation of both JNK and MEK/ERK. In the absence of ROS or caspase-3, NBT-induced pyroptosis and apoptosis were completely reversed. Moreover, NBT showed a significant antitumor effect in both the KYSE150 and GSDME knockout KYSE150-/- xenograft models by inducing pyroptosis and apoptosis, respectively. CONCLUSION: Our results indicated that natural compound NBT could induce GSDME-mediated pyroptosis and apoptosis in esophageal cancer cells, making it a potential therapeutic drug in clinical treatment.

Polyphenolic-protein-polysaccharide conjugates from Spica of Prunella vulgaris: Chemical profile and anti-herpes simplex virus activities.

Previous studies showed that the water extract (PVW) from Spica of Prunella vulgaris Linn. (Labiatae) exerts anti-herpes simplex virus (HSV) activity. Evaluation the antiviral activity of the graded ethanol precipitations indicated that 30% ethanol precipitate (PVE30) was the active principle of water extract (PVW). Further activity-oriented separation of PVE30 through salting-out method revealed that the anti-HSV activity of P. vulgaris glycoconjugates (PVG) was more potent than PVE30 and PVW, 2-fold and 4-fold, respectively. UPLC-QTOF-MS/MS, FT-IR and NMR techniques identified PVG as a type of polyphenolic-protein-polysaccharides (PPPs) with an average molecular weight of 41.69 kDa. PVG was composed of dibenzylbutyrolactone lignan units, and rich in galacturonic acid, xylose, rhamnose, rhamnose, arabinose, glucose monosaccharide units, glutamic acid and aspartic acid. Further in vitro antiviral testing confirmed that PVG substantially and stably inhibited acyclovir (ACV) resistant HSV strains; its inhibitory action was even better than the positive control ACV. Overall, our findings support PVG as a potential drug resource for anti-HSV therapy.

Nujiangexanthone A Inhibits Cervical Cancer Cell Proliferation by Promoting Mitophagy.

Nujiangexanthone A (NJXA), a bioactive component isolated from the leaves of Garcinia nujiangensis, has been reported to exhibit anti-inflammatory, antioxidant, and antitumor effects. Our previous work has shown that NJXA induced G0/1 arrest and apoptosis, thus suppressing cervical cancer cell growth. The present study provides new evidence that NJXA can induce cell death in HeLa cells by promoting mitophagy. We first identified that NJXA triggered GFP-LC3 and YFP-Parkin puncta accumulation, which are biomarkers of mitophagy. Moreover, NJXA degraded the mitochondrial membrane proteins Tom20 and Tim23 and mitochondrial fusion proteins MFN1 and MFN2, downregulated Parkin, and stabilized PINK1. Additionally, we revealed that NJXA induced lysosome degradation and colocalization of mitochondria and autophagosomes, which was attenuated by knocking down ATG7, the key regulator of mitophagy. Furthermore, since mitophagy is induced under starvation conditions, we detected the cytotoxic effect of NJXA in nutrient-deprived HeLa cells and observed better cytotoxicity. Taken together, our work contributes to the further clarification of the mechanism by which NJXA inhibits cervical cancer cell proliferation and provides evidence that NJXA has the potential to develop anticancer drugs.

Stereodivergent Strategy in Structural Determination: Asymmetric Total Synthesis of Garcinol, Cambogin, and Related Analogues.

The asymmetric total synthesis of five biologically significant polycyclic polyprenylated acylphloroglucinols (PPAPs), including garcinol and cambogin, was achieved through a highly diastereoselective and stereodivergent strategy. Along the way, an efficient cascade Dieckmann cyclization was employed to construct the bicyclo[3.3.1]nonane core in one step. The synthesis provided a general approach toward the chiral endo-type B PPAPs and their C-30 diastereomers in a single sequence, which resolved the challenges of the absolute configuration determination/structural revision of PPAPs bearing exocyclic stereocenters.

Nujiangexanthone A Inhibits Hepatocellular Carcinoma Metastasis via Down Regulation of Cofilin 1.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with poor prognosis. High expression level of cofilin 1 (CFL1) has been found in many types of cancers. However, the role of CFL1 in HCC hasn't been known clearly. Here, we found that CFL1 was up regulated in human HCC and significantly associated with both overall survival and disease-free survival in HCC patients. Nujiangexanthone A (NJXA), the caged xanthones, isolated from gamboge plants decreased the expression of CFL1, which also inhibited the migration, invasion and metastasis of HCC cells in vitro and in vivo. Down regulation of CFL1 inhibited aggressiveness of HCC cells, which mimicked the effect of NJXA. Mechanism study indicated that, knockdown of CFL1 or treatment with NJXA increased the level of F-actin and disturbed the balance between F-actin and G-actin. In conclusion, our findings reveal the role of CFL1 in HCC metastasis through the CFL1/F-actin axis, and suggest that CFL1 may be a potential prognostic marker and a new therapeutic target. NJXA can effectively inhibit the metastasis of HCC cells by down regulating the expression of CFL1, which indicates the potential of NJXA for preventing metastasis in HCC.

Safrana l Prevents Prostate Cancer Recurrence by Blocking the Re-activation of Quiescent Cancer Cells via Downregulation of S-Phase Kinase-Associated Protein 2.

The re-proliferation of quiescent cancer cells is considered to be the primary contributor to prostate cancer (Pca) recurrence and progression. In this study, we investigated the inhibitory effect of safranal, a monoterpene aldehyde isolated from Crocus sativus (saffron), on the re-proliferation of quiescent Pca cells in vitro and in vivo. The results showed that safranal efficiently blocked the re-activation of quiescent Pca cells by downregulating the G0/G1 cell cycle regulatory proteins CDK2, CDK4, CDK6, and phospho-Rb at Ser807/811 and elevating the levels of cyclin-dependent kinase inhibitors, p21 and p27. Further investigation on the underlying mechanisms revealed that safranal suppressed the mRNA and protein expression levels of Skp2, possibly through the deregulation of the transcriptional activity of two major transcriptional factors, E2F1 and NF-κB subunits. Moreover, safranal inhibited AKT phosphorylation at Ser473 and deregulated both canonical and non-canonical NF-κB signaling pathways. Safranal suppressed the tumor growth of quiescent Pca cell xenografts in vivo. Furthermore, safranal-treated tumor tissues exhibited a reduction in Skp2, E2F1, NF-κB p65, p-IκBα (Ser32), c-MYC, p-Rb (Ser807), CDK4, CDK6, and CDK2 and an elevation of p27 and p21 protein levels. Therefore, our findings demonstrate that safranal suppresses cell cycle re-entry of quiescent Pca cells in vitro and in vivo plausibly by repressing the transcriptional activity of two major transcriptional activators of Skp2, namely, E2F1 and NF-κB, through the downregulation of AKT phosphorylation and NF-κB signaling pathways, respectively.

Nujiangexathone A, a Novel Compound Derived from Garcinia nujiangensis, Induces Caspase-Dependent Apoptosis in Cervical Cancer through the ROS/JNK Pathway.

Nujiangexathone A (NJXA), a novel compound derived from Garcinia nujiangensis, has been demonstrated to inhibit the proliferation of several human cancer cell lines. This study is the first to demonstrate the apoptosis inductive activities of NJXA and the possible underlying mechanisms. Our results demonstrated that NJXA inhibited colony formation by HeLa and SiHa cells in a dose-dependent manner. An Annexin V-FITC/PI staining assay showed that NJXA strongly triggered apoptosis in a dose-dependent manner. Western blotting analyses showed that NJXA induced the caspase-dependent apoptosis of HeLa and SiHa cells by triggering a series of events, including changes in the levels of Bcl-2 family proteins, cytochrome c release, caspase-3 activation, and chromosome fragmentation. Furthermore, we demonstrated that NJXA induced cell apoptosis by activating the reactive oxygen species (ROS)-mediated JNK signaling pathway. Consistent with this finding, a ROS scavenger, N-acetyl-l-cysteine (NAC, 10 mM), hindered NJXA-induced apoptosis and attenuated the sensitivity of HeLa and SiHa cells to NJXA. In vivo results further confirmed that the tumor inhibitory effect of NJXA was partially through the induction of apoptosis. Taken together, our results demonstrated that NJXA induced the apoptosis of HeLa and SiHa cells through the ROS/JNK signaling pathway, indicating that NJXA could be important candidate for the clinical treatment of cervical cancer.

Nujiangexathone A, a novel compound from Garcinia nujiangensis, suppresses cervical cancer growth by targeting hnRNPK.

Cervical cancer is among the most frequently diagnosed cancers in females worldwide. Nujiangexathone A (NJXA), a novel compound from Garcinia nujiangensis, has been shown to have anti-cancer potential. In this study, the anti-tumor effects and the underlying mechanisms of NJXA action were investigated. Our results suggested that NJXA induced G0/G1 cell cycle arrest in HeLa and SiHa cells by down-regulating cyclins B1, E1, and A and cyclin-dependent kinases 2, 4 and 6, while selectively restoring p27. Using two-dimensional gel electrophoresis, we showed that NJXA reduced the expression of heterogeneous nuclear ribonucleoprotein K (hnRNPK) by accelerating ubiquitin-proteasome-dependent hnRNPK degradation, which then induced cell cycle arrest through the c-Myc-cyclin/Cdk-Rb-E2F1 pathway. The loss-of-function study showed NJXA induced cell cycle arrest was mediated by down regulation of hnRNPK protein. In vivo results further confirmed the tumor inhibitory effect of NJXA via the down-regulation of hnRNPK, and NJXA induced no apparent toxicity. Our study suggests that NJXA may be a novel anti-cancer drug candidate, especially for treating cancers with abnormally high hnRNPK expression.