Evaluation of genetic variants in ferroptosis-related genes and house dust mite-induced allergic rhinitis risk.

BACKGROUND: Ferroptosis-related genes disrupt iron homeostasis and enhance lipid peroxidation to initiate respiratory system diseases. However, the association between genetic variants in the ferroptosis-related genes with house dust mite (HDM)-induced allergic rhinitis (AR) susceptibility remains unclear. METHODS: A case-control study, involving 222 cases and 237 healthy controls from a Chinese population, was conducted to evaluate the relationship between single nucleotide polymorphisms (SNPs) in ferroptosis-related genes and HDM-induced AR risk. A gene-based analysis was performed by multi-marker analysis of genomic annotation (MAGMA) to identify candidate associated ferroptosis-related genes. A logistic regression model and joint analysis were used to assess the effect of SNPs on HDM-induced AR susceptibility. RESULTS: Two independent SNPs (rs2305128 in ENPP2 and rs1868088 in EPAS1) were significantly associated with HDM-induced AR risk (OR = 1.82, 95% CI = 1.19-2.79, P = 5.98 × 10-3, PFDR = 4.88 × 10-2; OR = 2.14, 95% CI = 1.23-3.72, P = 6.95 × 10-3, PFDR = 4.87 × 10-2, respectively). Moreover, combined analysis of these two SNPs revealed that an increased risk of HDM-induced AR was positively associated with an increasing number of risk genotypes (Ptrend = 8.48 × 10-5). The stratification analysis showed that the cumulative effect of two SNPs on HDM-induced AR risk was more pronounced among patients presenting more serious symptoms and harboring one or two risk genotypes. CONCLUSIONS: These findings suggest that the genetic variants in ferroptosis-related genes ENPP2 and EPAS1 may increase HDM-induced AR risk and serve as potential predictors of HDM-induced AR susceptibility.

5-epi-ent-Kaurane diterpenoids from the aerial parts of Isodon eriocalyx and their anti-atherosclerotic potential.

The phytochemical investigation of the EtOAc extract from the aerial parts of Isodon eriocalyx afforded seventeen diterpenoids, including eight undescribed compounds. Eriocalyxins H-L have unique structural characteristics featuring a 5-epi-ent-kaurane diterpenoid scaffold with eriocalyxins H-K also possess an unusual 6,11-epoxyspiro-lactone ring while eriocalyxin L, a 1,7:3,20-diepoxy-ent kaurene, features an 1,7-oxygen linkage. The structures of these compounds were elucidated by spectroscopic data interpretation, and the absolute configurations of eriocalyxins H, I, L, and M were confirmed by single-crystal X-ray diffraction. The isolates were screened for their inhibitory activities against VCAM-1 and ICAM-1 at 5 µM. While eriocalyxin O, coetsoidin A and laxiflorin P were found to significantly inhibit both VCAM-1 and ICAM-1, 8 (17),13-ent-labdadien-15 â†’ 16-lactone-19-oic acid displayed evidently inhibitory effect against ICAM-1.

Bartonella Prevalence and Genome Sequences in Rodents in Some Regions of Xinjiang, China.

In this study, we investigated Bartonella infection and its genetic diversity in rodents in Beitun, Xinjiang Uygur Autonomous Region, China. Small mammals were captured using snap traps at four sampling sites in 2018. Spleen and liver tissues were collected and cultured to isolate Bartonella strains. Whole-genome sequencing was performed on the strains identified as Bartonella by gltA gene PCR, and the average nucleotide identity (ANI) of the genomes was calculated by using FastANI v1.33. Phylogenetic trees were constructed for the samples positive for Bartonella spp. by the gltA PCR assay based on 1,290-bp gltA genes, 2,903-bp rpoB genes, and core-genome single nucleotide polymorphisms (SNPs). Among 66 rodents, 11 were positive for Bartonella, with an infection rate of 16.67%. The rodent infection rates in different tissues (χ2 = 2.133; P = 0.242), species (χ2 = 9.631; P = 0.141), and habitats (χ2 = 4.309; P = 0.312) did not show statistical differences. Bartonella spp. isolated from the rodents were phylogenetically divided into six clades (two different Bartonella species were detected in two rodents). By comparing phylogenetic trees based on gltA genes, rpoB genes, and SNPs, we found that the topological structures of several evolutionary trees are different. However, the Bartonella strains isolated in this study were clustered into six clusters in different phylogenetic trees. Broad distributions and high genetic diversity of Bartonella strains were observed among rodents in Beitun, Xinjiang. IMPORTANCE Rodent-borne Bartonella species have been associated with zoonotic diseases. Bartonella species such as Bartonella elizabethae, Bartonella grahamii, and Bartonella tribocorum can cause disease in humans. Humans can be infected by blood-sucking arthropods through the scratches and bites of an infected reservoir host or via contact with infectious rodents. Xinjiang is one of the provinces with the most abundant species of Bartonella in China, but there are few reports about the prevalence of Bartonella in the Beitun area. This research aims to investigate the occurrence and prevalence of Bartonella infection in rodents at these sampling sites and provide a basis for the prevention and control of rodent Bartonella species in Beitun and the surrounding areas of Xinjiang.

Profiling Analysis of Circular RNA and mRNA in Human Temporal Lobe Epilepsy with Hippocampal Sclerosis ILAE Type 1.

Hippocampal sclerosis (HS) is the most common surgical pathology associated with temporal lobe epilepsy (TLE). However, the cause of TLE with or without HS remains unknown. Our current study aimed to illustrate the essential molecular mechanism that is potentially involved in the pathogenesis of TLE-HS and to shed light on the transcriptional changes associated with hippocampal sclerosis. Compared to no-HS group, 341 mRNA transcripts and 131 circRNA transcripts were differentially expressed in ILAE type 1 group. The raw sequencing data have been deposited into sequence-read archive (SRA) database under accession number PRJNA699348.Gene Ontology analysis demonstrated that the dysregulated genes were associated with the biological processes of vesicle-mediated transport. Enrichment analysis demonstrated that dysregulated genes were involved mainly in the MAPK signal pathway. Subsequently, A total of 441 known or predicted interactions were formed among DEGs, and the most important module was detected in the PPI network using the MCODE plug-in. There were mainly four functional modules enriched: ER to Golgi transport vesicle membrane, Basal transcription factors, GABA-gated chloride ion channel activity, CENP-A containing nucleosome assembly. A circRNA-mRNA co-expression network was constructed including 5 circRNAs(hsa_circ_0025349, hsa_circ_0002405, hsa_circ_0004805, hsa_circ_0032254, and hsa_circ_0032875) and three mRNAs (FYN, SELENBP1, and GRIPAP1) based on the normalized mRNA signal intensities. This is the first to report the circRNAs and mRNAs expression profile of surgically resected hippocampal tissues from TLE patients of ILAE-1 and no-HS, and these results may provide new insight into the transcriptional changes associated with this pathology.

Pan-cancer analysis of ASB3 and the potential clinical implications for immune microenvironment of glioblastoma multiforme.

Background: Ankyrin repeat and SOCS Box containing 3 (ASB3) is an E3 ubiquitin ligase. It has been reported to regulate the progression of some cancers, but no systematic pan-cancer analysis has been conducted to explore its function in prognosis and immune microenvironment. Method: In this study, mRNA expression data were downloaded from TCGA and GTEx database. Next generation sequencing data from 14 glioblastoma multiforme (GBM) samples by neurosurgical resection were used as validation dataset. Multiple bioinformatics methods (ssGSEA, Kaplan-Meier, Cox regression analysis, GSEA and online tools) were applied to explore ASB3 expression, gene activity, prognosis of patients in various cancers, and its correlation with clinical information, immune microenvironment and pertinent signal pathways in GBM. The biological function of ASB3 in tumor-infiltrating lymphocytes (TILs) was verified using an animal model. Results: We found that ASB3 was aberrant expressed in a variety of tumors, especially in GBM, and significantly correlated with the prognosis of cancer patients. The level of ASB3 was related to the TMB, MSI and immune cell infiltration in some cancer types. ASB3 had a negative association with immune infiltration and TME, including regulatory T cells (Tregs), cancer-associated fibroblasts, immunosuppressors and related signaling pathways in GBM. ASB3 overexpression reduced the proportion of Tregs in TILs. GSEA and PPI analysis also showed negative correlation between ASB3 expression and oncogenetic signaling pathways in GBM. Conclusion: A comprehensive pan-cancer analysis of ASB3 showed its potential function as a biomarker of cancer prognosis and effective prediction of immunotherapy response. This study not only enriches the understanding of the biological function of ASB3 in pan-cancer, especially in GBM immunity, but also provides a new reference for the personalized immunotherapy of GBM.

Study of Neuronal Apoptosis ceRNA Network in Hippocampal Sclerosis of Human Temporal Lobe Epilepsy by RNA-Seq.

Hippocampal sclerosis (HS) is one of the most common pathological type of intractable temporal lobe epilepsy (TLE), often characterized by hippocampal atrophy, neuronal apoptosis, and gliogenesis. However, the molecular mechanisms of neuronal apoptosis in patients with HS are still not fully understood. We therefore conducted a pilot study focusing on the neuronal apoptosis ceRNA network in the sclerotic hippocampus of intractable TLE patients. In this research, RNA sequencing (RNA-seq) was utilized to quantify the expression levels of lncRNAs, miRNAs, and mRNAs in TLE patients with HS (HS-TLE) and without HS (non-HS-TLE), and reverse transcription-quantitative PCR (qRT-PCR). The interactions of differential expression (DE) lncRNAs-miRNAs or DEmiRNAs-mRNAs were integrated by StarBase v3.0, and visualized using Cytoscape. Subsequently, we annotate the functions of lncRNA-associated competitive endogenous RNA (ceRNA) network through analysis of their interactions with mRNAs. RNA-seq analyses showed 381 lncRNAs, 42 miRNAs, and 457 mRNAs were dysregulated expression in HS-TLE compared to non-HS-TLE. According to the ceRNA hypothesis, 5 HS-specific ceRNA network were constructed. Among them, the core ceRNA regulatory network involved in neuronal apoptosis was constituted by 10 DElncRNAs (CDKN2B-AS1, MEG3, UBA6-AS1, etc.), 7 DEmiRNAs (hsa-miR-155-5p, hsa-miR-195-5p, hsa-miR-200c-3p, etc.), and 3 DEmRNAs (SCN2A, DYRK2, and MAPK8), which belonging to apoptotic and epileptic terms. Our findings established the first ceRNA network of lncRNA-mediated neuronal apoptosis in HS-TLE based on transcriptome sequencing, which provide a new perspective on the disease pathogenesis and precise treatments of HS.

Development of a 3 RNA Binding Protein Signature for Predicting Prognosis and Treatment Response for Glioblastoma Multiforme.

Purpose: Glioblastoma multiforme (GBM) is the most widely occurring brain malignancy. It is modulated by a variety of genes, and patients with GBM have a low survival ratio and an unsatisfactory treatment effect. The irregular regulation of RNA binding proteins (RBPs) is implicated in several malignant neoplasms and reported to exhibit an association with the occurrence and development of carcinoma. Thus, it is necessary to build a stable, multi-RBPs signature-originated model for GBM prognosis and treatment response prediction. Methods: Differentially expressed RBPs (DERBPs) were screened out based on the RBPs data of GBM and normal brain tissues from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Program (GTEx) datasets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses on DERBPs were performed, followed by an analysis of the Protein-Protein Interaction network. Survival analysis of the DERBPs was conducted by univariate and multivariate Cox regression. Then, a risk score model was created on the basis of the gene signatures in various survival-associated RBPs, and its prognostic and predictive values were evaluated through Kaplan-Meier analysis and log-rank test. A nomogram on the basis of the hub RBPs signature was applied to estimate GBM patients' survival rates. Moreover, western blot was for the detection of the proteins. Results: BICC1, GNL3L, and KHDRBS2 were considered as prognosis-associated hub RBPs and then were applied in the construction of a prognostic model. Poor survival results appeared in GBM patients with a high-risk score. The area under the time-dependent ROC curve of the prognostic model was 0.723 in TCGA and 0.707 in Chinese Glioma Genome Atlas (CGGA) cohorts, indicating a good prognostic model. What was more, the survival duration of the high-risk group receiving radiotherapy or temozolomide chemotherapy was shorter than that of the low-risk group. The nomogram showed a great discriminating capacity for GBM, and western blot experiments demonstrated that the proteins of these 3 RBPs had different expressions in GBM cells. Conclusion: The identified 3 hub RBPs-derived risk score is effective in the prediction of GBM prognosis and treatment response, and benefits to the treatment of GBM patients.

Development and validation of a nomogram for specific survival in osteosarcoma patients less than 60 years old: a population-based study.

PURPOSE: The present study aimed to develop a nomogram to predict the overall survival of patients with osteosarcoma, especially those less than 60 years old. METHODS: 903 osteosarcoma patients less than 60 years old were collected from the Surveillance, Epidemiology, and End Results (SEER) database.Univariate and multivariate analyses identified the independent prognostic factors of osteosarcoma. Nomogram was used to predict 3- and 5-year overall survival (OS) of osteosarcoma.The accuracy of the model was determined using the concordance index (C­index), calibration curves, the area under the receiver operating characteristic curves (ROC),as well as decision curve analysis (DCA). RESULTS: Osteosarcoma patients less than 60 years old were randomly assigned into a training cohort (n=635) or validation cohort (n=268). Age, tumor site, tumor grade, tumor size, and tumor stage were identified as independent prognostic factors via univariate and multivariate Cox analyses (all p<0.05) and then included in the prognostic nomogram. The concordance indices(C-index) for OS prediction in the training cohort was 0.788 (95% CI 0.751-0.852) and in the external validation cohort was 0.779 (95% CI 0.712-0.846). Calibration plots and the area under the ROC revealed excellent consistency between actual survival and nomogram prediction. Finally, DCA demonstrated that the prognostic nomogram was clinically meaningful. CONCLUSION: A nomogram could accurately predict the OS of osteosarcoma patients less than 60 years old and contribute to making better clinical treatment decisions for the treating doctors.

New dammarane-type triterpenoid saponins from Gynostemma pentaphyllum and their Sirt1 agonist activity.

Gynostemma pentaphyllum (Thunb.) Makino (Cucurbitaceae family) is a perennial creeping plant with a common Chinese name of "south ginseng". To date, more than 250 individual saponins with dammarane-type skeleton have been isolated from G. pentaphyllum. The purpose of this study was the isolation and structural characterization of novel, minor gypenosides from G. pentaphyllum and evaluation of their Sirt1 agonist activity. Individual saponins from G. pentaphyllum were isolated and purified by a variety of chromatography techniques, and their structures were elucidated by means of various spectroscopic analysis and comparision with the reported data. Sirt1 enzyme activity detection kit was used to preliminarily evaluate the Sirt1 agonist activity of thirty three individual saponins purified from G. pentaphyllum. Fourteen new triterpenoid saponins named gypenoside CII-CXV (1-14) along with twenty six known compounds (15-40) were isolated from G. pentaphyllum. Thirty three of all the isolates were screened for Sirt1 agonist activity, and the results showed that three dammarane-type saponins (2, 18, 37) and one cucurbitane-type saponin 33 exhibited satisfactory Sirt1 agonist activity. These findings suggested that G. pentaphyllum was worthy of further investigation to find small molecule Sirt1 agonist and facilitate their utilization as "south ginseng".

Two-year outcome of Trabeculo-Canalectomy for Chinese Glaucoma Patients.

To evaluate the efficacy of trabeculo-canalectomy in treating glaucoma patients, a retrospective investigation of 53 glaucoma patients (53 eyes) who underwent trabeculo-canalectomy was conducted at the First Affiliated Hospital of Nanjing Medical University, China, from April 2017 to January 2019. Intraocular pressure (IOP), visual acuity, surgical success rates, medications, and complications were monitored at post-operative 1 day, 1 week, 1, 3, 6, 12 and 24 months. Surgical success criteria were defined as 6 mm Hg≤IOP≤21 mmHg with or without additional medications. Our results showed that average IOP was statistically significant between pre-operative visit and each follow-up visit (all P <0.05). The total success rate of trabeculo-canalectomy at 1, 3, 6, 12 and 24 months was 92.5%, 86.8%, 94.3%, 92.5% and 90.6% respectively. After 3 months post-operatively, all patients had no obvious filtering blebs. The main early complications included postoperative hyphema (7.5%), elevated IOP (5.7%) and anterior chamber exudation (3.8%), which were all cured after conservative treatment. No blebitis, shallow anterior chamber, choroidal detachment and endophthalmitis were observed. Logistic regression analysis showed that patients with secondary glaucoma were more likely to undergo surgical failure 24 months post-operatively (P= 0.008). Thus, we conclude that trabeculo-canalectomy is effective and safe for the treatment of glaucoma.

Prolonged effect associated with inflammatory response observed after exposure to low dose of tritium ß-rays.

Background: The value of relative biological effectiveness of tritium increases at low dose domain, which results in the suspicion of weighting factor of 1 for tritium after low dose exposure. Thus, present study was carried out to analyze the differences in the cellular responses at early and late period between low dose of tritium ß-rays and γ-rays radiation.Methods: MCF-10A cells were exposed to low dose of tritium ß-rays or γ-rays, then cellular behaviors, such as DNA double strand breaks (DSBs), apoptosis, reactive oxygen species (ROS) level and inflammatory relevant gene expression were analyzed at early and late period post-irradiation.Results: At early period the elimination of DSB foci produced by HTO is longer than γ-rays. High ROS level and a continual change of cell cycle distribution are observed in HTO radiation group. Based on the results of RNA sequencing, Ingenuity Pathway Analysis (IPA) indicates TNFR1 signaling and production of nitric oxide and ROS are activated as an acute response at 24 h post radiation. Moreover, it also shows a disturbance in cholesterol biosynthesis. The results of 30 days point that there is a lasting active inflammatory response, accompanying with a persistent high expression of relevant cytokines, such as TNF and IL1R.Conclusion: Compared to an acute response induced by γ-rays, a persistent inflammatory response exists in HTO-irradiated cells when cultured for 30 days, which might be related to accumulation of tritium in the form of organically bound tritium (OBT) in cellular DNA or lipids.

The potential roles of circular RNAs in osteonecrosis of the femoral head (Review).

Circular RNAs (circRNAs) are categorized as non­coding RNAs that, unlike widely known canonical linear RNAs, form a covalently closed continuous loop without 5' or 3' polarities, which enables them to resist digestion by RNA exonucleases. Although the functions of circRNAs remain largely unknown, accumulated evidence has demonstrated that circRNAs can act as microRNA sponges, which allows them to regulate numerous biological processes and disease mechanisms, including apoptosis, angiogenesis, invasion, metastasis and stem cell differentiation. Although research into circRNAs is in its infancy, studies have identified critical roles for circRNAs in the initiation and progression of disease. The present study delineated the characteristics and functions of circRNAs, and focused on the potential relationship between circRNAs and osteonecrosis of the femoral head (ONFH). CircRNAs represent a novel avenue for studying the mechanisms underlying ONFH as well as possible treatments.

The application of nasoseptal "rescue" flap technique in endoscopic transsphenoidal pituitary adenoma resection.

To explore the reliability and superiority of nasoseptal "rescue" flap technique in neuroendoscopic transnasal pituitary adenoma resection. Retrospective clinical analysis of 113 cases of endoscopic transsphenoid pituitary adenoma resection with the application of nasoseptal "rescue" flap technology. The reliability and the superiority of the technique were evaluated according to the duration of nasal cavity and sphenoid sinus stage, the incidence of postoperative anosmia, and cerebrospinal rhinorrhea. The duration of nasal and sphenoid sinus stage was 15-30 min, averaging 24 min. There were 27 cases of intro-operative cerebrospinal fluid leakage, including 24 cases of low-flow cerebrospinal fluid leak and 3 cases of high-flow cerebrospinal fluid leak. Twenty-three cases were converted from nasoseptal "rescue" flap to nasal septum flap. There were 17 cases of postoperative olfactory decline or disappearance, 1 case of epistaxis and 1 case of cerebrospinal rhinorrhea. The application of nasoseptal "rescue" flap technique can proceed sellar floor reconstruction when the diaphragma sellae rupture occurs during the operation. There is no obvious increase of the duration of sphenoid sinus and nasal stage and the rate of postoperative olfactory loss. This technique can be used as a conventional technique for endoscopic transsphenoid pituitary adenoma resection.

Diagnostic Performance of PET/CT Using 18F-FACBC in Prostate Cancer: A Meta-Analysis.

Background: Diagnostic performance of PET/CT using 18F-fluciclovine (18F-FACBC) in patients with prostate cancer (PCa) has been evaluated in only a few studies. There is no consensus on the diagnostic value of 18F-FACBC PET/CT in PCa recurrence or metastasis (except for bone metastasis), the primary diagnosis of the lesion. Hence, a meta-analysis was conducted to evaluate the performance of 18F-FACBC PET/CT. Methods: The literature published from June 2015 to June 2019 on using 18F-FACBC PET/CT for the diagnosis of PCa was retrieved from PubMed and EMBASE. Pooled sensitivity (Sen), specificity (Spe), positive and negative likelihood ratios (LR+ and LR-), area under the curve (AUC), and diagnostic odds ratio (DOR) of 18F-FACBC PET/CT in patients with PCa were calculated. An SROC map was made, and a meta-regression analysis was carried out. A Fagan plot and likelihood ratio dot plot were drawn. Sensitivity and funnel plot analysis were made. Meta-disc, Review Manager 5.3, and STATA 13 were used for the meta-analysis. Results: A total of nine articles met the strict criteria for diagnostic meta-analysis, which included 363 patients and 345 lesions. Pooled Sen, Spe, LR+, LR-, DOR were 0.88, 0.73, 3.3, 0.17, and 20, respectively. Lesions detected on the PET/CT image included primary lesions and metastases. For the lesion, the doctors considered the abnormal part as a lesion on the PET/CT image by their own experience and expertise, including primary lesions and metastases. For the patient, patients who participated in the trial can be diagnosed as PCa through 18F-FACBC. Conclusion: This study comprehensively evaluated the diagnostic value of 18F-FACBC PET/CT on PCa. Our analysis suggests that 18F-FACBC PET/CT is a valuable agent in diagnosing PCa. More studies are needed for further validation.

An open-walled ionization chamber appropriate to tritium monitoring for glovebox.

An open-walled ionization chamber is developed to monitor the tritium concentration in gloveboxes in tritium processing systems. Two open walls are used to replace the sealed wall in common ionization chambers, through which the tritium gas can diffuse into the chamber without the aid of pumps and pipelines. Some basic properties of the chamber are examined to evaluate its performance. Results turn out that an open-walled chamber of 1 l in volume shows a considerably flat plateau over 700 V for a range of tritium concentration. The chamber also gives a good linear response to gamma fields over 4 decades under a pressure condition of 1 atm. The pressure dependence characteristics show that the ionization current is only sensitive at low pressures. The pressure influence becomes weaker as the pressure increases mainly due to the decrease in the mean free path of beta particles produced by tritium decay. The minimum detection limit of the chamber is 3.7x10(5) Bq/m(3).

Inhibition of voltage-gated L-type calcium channels by labedipinedilol-A involves protein kinase C in rat cerebrovascular smooth muscle cells.

Labedipinedilol-A, a novel calcium channel blocker with alpha/beta-adrenoceptor blockade properties, inhibits L-type calcium channels (LTCCs) in rat cerebrovascular smooth muscle cells (CSMCs). We used conventional whole cell patch-clamp electrophysiology to investigate Ba(2+) currents (I(Ba)) through LTCCs in rat CSMCs enzymatically dissociated from rat cerebral arteries. Labedipinedilol-A (1, 10 microM) reversibly inhibited I(Ba) in a voltage-dependent manner without modifying the I(Ba) current-voltage relationship. The I(Ba) was also abolished by the LTCC blocker nifedipine (1 microM), but enhanced by the LTCC activator Bay K8644 (100 nM). Labedipinedilol-A shifted the steady-state inactivation curve of I(Ba) to more negative potentials. Additionally, labedipinedilol-A had greater inhibitory activity on I(Ba) holding at -40 mV than at -80 mV. This might contribute to labedipinedilol-A's more selective effect on vascular muscles compared to cardiac muscles. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and norepinephrine-enhanced I(Ba) were also inhibited by labedipinedilol-A. Pretreatment with the PKC inhibitor chelerythrine (5 microM) attenuated labedipinedilol-A-mediated I(Ba) inhibition. However, the Rho kinase inhibitor Y-27632 (30 microM) had little effect on labedipinedilol-A inhibition of I(Ba). Labedipinedilol-A inhibition of voltage-dependent LTCCs may be, at least in part, due to its modulation of the PKC pathway.