Internal validation of the GA118-24B Genetic Analyzer, a stable capillary electrophoresis system for forensic DNA identification.

The GA118-24B Genetic Analyzer (hereafter, "GA118-24B") is an independently developed capillary electrophoresis instrument. In the present research, we designed a series of validation experiments to test its performance at detecting DNA fragments compared to the Applied Biosystems 3500 Genetic Analyzer (hereafter, "3500"). Three commercially available autosomal short tandem repeat multiplex kits were used in this validation. The results showed that GA118-24B had acceptable spectral calibration for three kits. The results of accuracy and concordance studies were also satisfactory. GA118-24B showed excellent precision, with a standard deviation of less than 0.1 bp. Sensitivity and mixture studies indicated that GA118-24B could detect low-template DNA and complex mixtures as well as the results generated by 3500 in parallel experiments. Based on the experimental results, we set specific analytical and stochastic thresholds. Besides, GA118-24B showed superiority than 3500 within certain size ranges in the resolution study. Instead of conventional commercial multiplex kits, GA118-24B performed stably on a self-developed eight-dye multiplex system, which were not performed on 3500 Genetic Analyzer. We compared our validation results with those of previous research and found our results to be convincing. Overall, we conclude that GA118-24B is a stable and reliable genetic analyzer for forensic DNA identification.

Engineering Proteins for Cell Entry.

Proteins are essential for life, as they participate in all vital processes in the body. In the past decade, delivery of active proteins to specific cells and organs has attracted increasing interest. However, most proteins cannot enter the cytoplasm due to the cell membrane acting as a natural barrier. To overcome this challenge, various proteins have been engineered to acquire cell-penetrating capacity by mimicking or modifying natural shuttling proteins. In this review, we provide an overview of the different types of engineered cell-penetrating proteins such as cell-penetrating peptides, supercharged proteins, receptor-binding proteins, and bacterial toxins. We also discuss some strategies for improving endosomal escape such as pore formation, the proton sponge effect, and hijacking intracellular trafficking pathways. Finally, we introduce some novel methods and technologies for designing and detecting engineered cell-penetrating proteins.

Peroxymonosulfate-Activation-Induced Phase Transition of Mn3O4 Nanospheres on Nickel Foam with Enhanced Catalytic Performance.

The transformations of physicochemical properties on manganese oxides during peroxymonosulfate (PMS) activation are vital factors to be concerned. In this work, Mn3O4 nanospheres homogeneously loaded on nickel foam are prepared, and the catalytic performance for PMS activation is evaluated by degrading a target pollutant, Acid Orange 7, in aqueous solution. The factors including catalyst loading, nickel foam substrate, and degradation conditions have been investigated. Additionally, the transformations of crystal structure, surface chemistry, and morphology on the catalyst have been explored. The results show that sufficient catalyst loading and the support of nickel foam play significant roles in the catalytic reactivity. A phase transition from spinel Mn3O4 to layered birnessite, accompanied by a morphological change from nanospheres to laminae, is clarified during the PMS activation. The electrochemical analysis reveals that more favorable electronic transfer and ionic diffusion occur after the phase transition so as to enhance catalytic performance. The generated SO4•- and •OH radicals through redox reactions of Mn are demonstrated to account for the pollutant degradation. This work will provide new understandings of PMS activation by manganese oxides with high catalytic activity and reusability.

Development and validation of novel 8-dye short tandem repeat multiplex system for forensic applications.

DNA profiling of short tandem repeats (STRs) is the primary method for genotyping forensic samples. However, degraded DNA and trace samples are still major problems for commercial 5- or 6-dye STR kits. In order to improve the performance of this method, we developed a novel 8-dye STR multiplex system containing 18 autosomal loci (D3S1358, D1S1656, TPOX, D16S539, vWA, D6S1043, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, TH01, D21S11, D12S391, and PentaD) and the sex-determining locus Amelogenin, with all fragments smaller than 330 bases. Validation was carried out as recommended by the Scientific Working Group on DNA Analysis Methods. The results showed that complete profiles were obtainable when the input DNA was as low as 0.0625 ng. Full profiles were obtained even in the presence of inhibitors such as humic acid (< 300 ng/µl), hematin (< 100 µM), and indigo (0.01%). The 8-dye STR multiplex system also showed good performance in the detection degraded DNA samples. These results indicate that the 8-dye STR multiplex system is suitable for human DNA genotyping, including for difficult forensic materials.

Population genetics of 26 Y-STR loci for the Han ethnic in Hunan province, China.

To study the population data of Y-chromosome STRs (Y-STRs) of Han population resided in Hunan province, we analyzed haplotypes of 26 Y-STRs (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, DYS388, DYS449, DYS460, and YGATAH4) in 310 unrelated male individuals using a commercially available Goldeneye® DNA ID 26Y system. The calculated average gene diversity values ranged from 0.4211 to 0.9590 for DYS438 and DYS385a/b loci, respectively. The discriminatory capacity was 96.77 % with 300 observed haplotypes. Population relationships between Hunan Han and eight other populations available from Y-chromosome haplotype reference database (YHRD) were compared. The results showed that the Han population resided in the Hunan district is significantly different from other populations. Our results also indicated that these 26 Y-STR loci were highly genetically polymorphic in the Hunan Han population and of great value in forensic application.

Engineering Microorganisms for Cancer Immunotherapy.

Cancer immunotherapy presents a promising approach to fight against cancer by utilizing the immune system. Recently, engineered microorganisms have emerged as a potential strategy in cancer immunotherapy. These microorganisms, including bacteria and viruses, can be designed and modified using synthetic biology and genetic engineering techniques to target cancer cells and modulate the immune system. This review delves into various microorganism-based therapies for cancer immunotherapy, encompassing strategies for enhancing efficacy while ensuring safety and ethical considerations. The development of these therapies holds immense potential in offering innovative personalized treatments for cancer.

Graphene oxide quantum dots (GOQDs) induce behavioral disorders via the disturbance of kynurenine pathway in zebrafish larvae.

The emergence of graphene quantum dots (GQDs) expands the use of graphene derivatives in nanomedicine for its direct therapeutic applications in treating neurodegeneration, inflammation, metabolic dysfunction, and among others. Nevertheless, the biosafety assessment of GQDs remains deficient mostly because of the diverse surface characteristics of the nanoparticles. Our prior work demonstrated that GQDs can induce strong thigmotactic effects in zebrafish larvae over a wide range of concentrations, yet the underlying metabolic mechanisms remain largely unknown. In this study, we conducted a further exploration about graphene oxide quantum dots (GOQDs) for its potential neurotoxic effect on the behaviors of zebrafish larvae by combining neurotransmitter-targeted metabolomics with locomotion analysis. After continuous exposure to a concentration gradient of GOQDs (12.5 - 25 - 50 - 100 - 200 µg/mL) for 7 days, the thigmotactic activities of zebrafish larvae were observed across all exposure concentrations relative to the control group, while the basal locomotor activities, including distance moved and average velocity, were significantly changed by low concentrations of GOQDs. Targeted metabolomics was performed using zebrafish larvae at 7 days post-fertilization (dpf) that were exposed to 12.5 and 200 µg/mL, both of which were found to perturb the kynurenine pathway by regulating the levels of kynurenine, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid (QA). Furthermore, the thigmotaxis of larval fish induced by GOQDs during exposure could be counteracted by supplementing Ro-61-8048, an agonist acting on kynurenine 3-monooxygenase (KMO). In conclusion, our study establishes the involvement of the kynurenine pathway in GOQDs-induced thigmotaxis, which is independent of the transcriptional modulation of glutamate receptor families.

A minireview of the medicinal and edible insects from the traditional Chinese medicine (TCM).

Entomoceuticals define a subset of pharmaceuticals derived from insects. The therapeutic effect of insect-derived drugs has been empirically validated by the direct use of various folk medicines originating from three sources in particular: the glandular secretions of insects (e.g., silk, honey, venom), the body parts of the insect or the whole used live or by various processing (e.g., cooked, toasted, ground), and active ingredients extracted from insects or insect-microbe symbiosis. Insects have been widely exploited in traditional Chinese medicine (TCM) relative to other ethnomedicines, especially in the prospect of insect species for medicinal uses. It is noticeable that most of these entomoceuticals are also exploited as health food for improving immune function. In addition, some edible insects are rich in animal protein and have high nutritional value, which are used in the food field, such as insect wine, health supplements and so on. In this review, we focused on 12 insect species that have been widely used in traditional Chinese herbal formulae but have remained less investigated for their biological properties in previous studies. We also combined the entomoceutical knowledge with recent advances in insect omics. This review specifies the underexplored medicinal insects from ethnomedicine and shows their specific medicinal and nutritional roles in traditional medicine.

Protein-based delivery systems for RNA delivery.

RNA-based therapeutics have emerged as promising approaches to modulate gene expression and generate therapeutic proteins or antigens capable of inducing immune responses to treat a variety of diseases, such as infectious diseases, cancers, immunologic disorders, and genetic disorders. However, the efficient delivery of RNA molecules into cells poses significant challenges due to their large molecular weight, negative charge, and susceptibility to degradation by RNase enzymes. To overcome these obstacles, viral and non-viral vectors have been developed, including lipid nanoparticles, viral vectors, proteins, dendritic macromolecules, among others. Among these carriers, protein-based delivery systems have garnered considerable attention due to their potential to address specific issues associated with nanoparticle-based systems, such as liver accumulation and immunogenicity. This review provides an overview of currently marketed RNA drugs, underscores the significance of RNA delivery vector development, delineates the essential characteristics of an ideal RNA delivery vector, and introduces existing protein carriers for RNA delivery. By offering valuable insights, this review aims to serve as a reference for the future development of protein-based delivery vectors for RNA therapeutics.

Graphene quantum dots (GQDs) induce thigmotactic effect in zebrafish larvae via modulating key genes and metabolites related to synaptic plasticity.

Graphene quantum dots (GQDs) recently gain much attention for its medicinal values in treating diseases such as neurodegeneration and inflammations. However, owing to the high permeability of GQDs across the blood-brain barrier, whether its retention in the central nervous system (CNS) perturbs neurobehaviors remains less reported. In the study, the locomotion of zebrafish larvae (Danio rerio) was fully evaluated when administrated by two GQDs in a concentration gradient, respectively as reduced-GQDs (R-GQDs): 150, 300, 600, 1200, and 2400 g/L, and graphene oxide QDs (GOQDs): 60, 120, 240, 480, and 960 g/L. After exposure, the larvae were kept for locomotion analysis within one week's depuration. Substantial data showed that the basal locomotor activity of zebrafish larvae was not significantly changed by both two GQDs at low concentrations while weakened greatly with the increase of concentrations, and the total ATP levels of zebrafish larvae were also found to decrease significantly when exposed to the highest concentrations of GQDs. Next, the thigmotactic effect was observed to be remarkably induced in larvae by both two GQDs at any concentrations during exposure, and remained strong in larvae treated by high concentrations of R-GQDs after 7 days' depuration. To be noted, we found that GQDs affected the synaptic plasticity via downregulating the mRNA levels of NMDA and AMPA receptor family members as well as the total glutamine levels in zebrafish larvae. Together, our study presented robust data underlying the locomotor abnormalities aroused by GQDs in zebrafish larvae and indicated the potential adverse effects of GQDs on synaptic plasticity.

Synthesis of CdSe and CdSe/ZnS Quantum Dots with Tunable Crystal Structure and Photoluminescent Properties.

Mastery over the structure of nanocrystals is a powerful tool for the control of their fluorescence properties and to broaden the range of their applications. In this work, the crystalline structure of CdSe can be tuned by the precursor concentration and the dosage of tributyl phosphine, which is verified by XRD, photoluminescence and UV-vis spectra, TEM observations, and time-correlated single photon counting (TCSPC) technology. Using a TBP-assisted thermal-cycling technique coupled with the single precursor method, core-shell QDs with different shell thicknesses were then prepared. The addition of TBP improves the isotropic growth of the shell, resulting in a high QY value, up to 91.4%, and a single-channel decay characteristic of CdSe/ZnS quantum dots. This work not only provides a facile synthesis route to precisely control the core-shell structures and fluorescence properties of CdSe nanocrystals but also builds a link between ligand chemistry and crystal growth theory.

In vivo toxicity assessment of four types of graphene quantum dots (GQDs) using mRNA sequencing.

GQDs show great potential in drug carriers, bioimaging, biosensors, theranostics, and are recently reported as promising therapeutic agents to treat amyloid-related diseases such as Parkinson's disease and inflammations such as colitis. However, current toxicity data about GQDs based on in vivo toxicity assessments remain scarce. In the study, we examined the mRNA expression changes of zebrafish embryos exposed to four types of GQDs, including raw graphene quantum dots (R-GQDs), graphene oxide quantum dots (GOQDs), carboxyl GQDs (C-GQDs), and aminated GQDs (A-GQDs). Firstly, we treated embryos with the four GQDs at three concentrations (50, 100, and 200 µg/mL), and found that only A-GQDs caused embryonic developmental arrest at 100 and 200 µg/mL with significantly decreased survival rates and heartbeat rates, as well as the elevated malformation rates. Next, we analyzed the mRNA sequencing data acquired from zebrafish embryos exposed to the four GQDs for 7 days at 100 µg/mL, and found that all GQDs can act on potassium (K+) and calcium (Ca2+) channels, and spliceosomes with varying degrees of regulatory effects. Compared to other GQDs, A-GQDs can strongly perturb the anticoagulant protein C (PC) pathway via activating most genes associated with complement and coagulation system, cell adhesion molecules (CAMs), and MAPK. In conclusion, this study provided substantial transcriptomic data underlying the common signaling pathways induced by various types of GQDs and pointed out the specific toxicity of A-GQDs on hemostatic system.

Targeted disruption of mitochondria potently reverses multidrug resistance in cancer therapy.

BACKGROUND AND PURPOSE: Multidrug resistance (MDR) is a major obstacle to the successful treatment of cancer. Ample evidence shows that ATP-binding cassette (ABC) transporters and high-energy states in cells are linked to cancer drug resistance. Our previous work reported an engineered therapeutic protein named PAK, which selectively inhibited tumour progression by targeting mitochondria. EXPERIMENTAL APPROACH: We studied the effects of PAK on reversing drug resistance in MDR phenotypic cells and xenograft mouse models. Effects of PAK on the process of mitochondrial energy production, ABC transporter expression, and drugs enrichment were investigated in cancer cells. RNA-seq and co-immunoprecipitation were employed to analyse the mechanism of PAK on the redistribution of ABC transporters. KEY RESULTS: PAK promoted the enrichment of drugs in MDR cancer cells, thus enhancing the sensitivity of cancer cells to chemotherapy. Furthermore, PAK was colocalized in the mitochondria and initiated mitochondrial injury by selectively inhibiting the mitochondrial complex V. Also, ABCB1 and ABCC1 were redistributed from the plasma membrane to the cytoplasm through the disruption of lipid rafts, which was attributed to the low energy state and decreased cholesterol levels. CONCLUSIONS AND IMPLICATIONS: Our results revealed a previously unrecognized pattern of reversal of drug resistance and have suggested mitochondria as a clinically relevant target for the treatment of MDR malignant tumours.

Molecular basis of neurophysiological and antioxidant roles of Szechuan pepper.

The balance between anti- and pro-oxidant activities is of great important to maintain the biochemical and physiological homeostasis in the human body. Developing new therapeutic strategies to reduce health risks caused by free radicals has always been research focus over the past decades. Szechuan pepper, a characteristic pungent-flavored spice in Sichuanese cuisine, recently attracts the attention of researchers for its widespread therapeutic effects on acute and chronic diseases. The plant produces the innocuous 'tingling and numbing' sensations across the oral cavity by stirring specific neuron types, which are mechanically distinct from those excited by capsaicin. Furthermore, the extracts or the compounds of Szechuan pepper are biochemically proven to possess strong antioxidant activities that could scavenge free radicals and inhibit overactive peroxidase system in pathological models. Herein, the review emphasizes the molecular basis underlying the neurophysiological and antioxidant activities of the plant by a comprehensive analysis of various signaling pathways in disease models treated by Szechuan pepper. Further, we performed a broadening analysis to unearth potential signaling pathways associated with the antioxidant roles of the plant.

Na3SbSe4-xS x as Sodium Superionic Conductors.

Na based all-solid-state batteries are a promising technology for large-scale energy storage applications owing to good safety properties and low cost. High performance solid electrolyte materials with high room temperature ionic conductivity, good electrochemical stability and facile synthesis are highly desired for the commercialization of this technology. In this work, we report the synthesis and characterization of a novel fast Na-ion conductor, cubic Na3SbSe4, with an excellent ionic conductivity of 0.85 mS cm-1 at room temperature, and a group of S doped variants. Na3SbSe4 exhibits good compatibility with metallic Na and good stability in a wide voltage range. The application of this compound as solid electrolyte is demonstrated in all-solid-state Na-ion cells cycled at room temperature.

Graphene oxide assisted template-free synthesis of nanoscale splode-like NiCo2O4 hollow microsphere with superior lithium storage properties.

A facile template-free Ostwald ripening method is developed for the preparation of the reduced graphene oxide supported splode-like NiCo2O4 hollow microsphere (SNHM/rGO). The graphene oxide used in the reaction mixture is found to play a crucial role in the formation of the SNHM/rGO. It promotes the formation of the NiCo-glycerol microspheres suitable for the Ostwald ripening to form the reduced graphene oxide supported hollow NiCo-glycerol microspheres, which is important for the subsequent calcination to form the SNHM/rGO. The obtained SNHM/rGO shows a great promise as the anode for lithium-ion batteries and can deliver a stable reversible capacity of 1048.1 mA h g-1 at the current density of 100 mA g-1. The performance of the SNHM/rGO is much higher than that of most NiCo2O4-based materials reported previously, strongly suggesting that the SNHM/rGO could be used as the anode for practical applications. This is well supported by the higher performance of the LiCoO2//SNHM-rGO full cell. The excellent electrochemical performance can be attributed to the specific structure of the SNHM/rGO, which comprises the splode-like hollow NiCo2O4 microspheres with the reduced graphene oxide integrated.

[Protective effects of ketamine on allergen-induced airway inflammatory injure and high airway reactivity in asthma: experiment with rats].

OBJECTIVE: To observe the effects of ketamine on bronchial hyperresponsiveness and airway inflammation in equal asthma. METHODS: 56 Brown-Norway rats were randomly assigned to seven groups: negative control group (Group A), asthma model group (Group B) and inhalation groups with nebulized ketamine at different concentrations (Group C, D, E) and intraperitoneal injection groups with ketamine at different doses (Group F, G). The rats were sensitized by injection of ovalbumin (OVA) together with aluminum hydroxide and Bordetella pertussis as adjuvants, then challenged by repeated intermittent (thrice weekly) exposure to aerosolized OVA for two weeks. Before challenge, the sensitized rats were exposed to an aerosol of phosphate buffered saline (PBS) or ketamine at the concentrations of 12.5 mg/ml, 25 mg/ml and 50 mg/ml respectively in Groups B, C, D and E. The sensitized rats were intraperitoneally injected with ketamine at the doses of 50 microg/kg or 100 microg/kg respectively in Group F and G. The sensitized rats in Group A received phosphate buffered solution (PBS) by inhalation. The airway reactivity to acetylcholine (ACH) was assessed in vivo 24 hr after the last OVA challenge, then the lungs were removed for measurement of the mRNA and protein expression of iNOS and production of NO and lung sections for histopathologic examination. RESULTS: (1) In the OVA-sensitized and challenged rats, the dose-response curve of the expiratory resistance (Re) shifted to the upper-left +/- ward compared with that of PBS control rats. In addition, the provocation doses required to increase the Re by 100%, 200% and 400% for OVA-sensitized and challenged rats in Group B were significantly lower than those of the PBS control rats (14.65 +/- 1.19 vs 32.28 +/- 1.43, 15.17 +/- 1.19 vs 38.91 +/- 1.39, and 16.28 +/- 1.18 vs 56.53 +/- 1.38, all P < 0.01). The OVA-sensitized rats treated with ketamine before OVA challenge demonstrated a significant decrease in AHR by a rightward shift of the dose-response curves to ACH and significant higher provocation doses compared with that of the OVA control rats (P < 0.05). (2) Marked inflammatory changes in the airways of Group B were present, while obviously lessen inflammatory cell infiltration in peribronchial and perialveolar tissues and improved lung edema were observed in the groups treated with ketamine. (3) Quantitation by densitometry showed that the relative density of iNOS mRNA bands normalized to beta-actin was significantly higher in the OVA control than the PBS control (1.0 +/- 0.07 vs 0.48 +/- 0.07, P < 0.01). Treatment with ketamine significantly decreased the expression of iNOS mRNA in Group C (0.65 +/- 0.07), Group D (0.58 +/- 0.09), Group E (0.56 +/- 1.00), and Group F (0.66 +/- 0.06) when compared with Group B (all P < 0.05). (4) The relative iNOS protein levels (ratios of iNOS/beta-actin) determined by densitometry analysis showed a 4-fold increase in Group A compared with those in the negative group (0.54 +/- 0.08 vs 0.13 +/- 0.08, P < 0.05). When compared with those of the OVA control, the levels of relative iNOS protein expression showed a significant decrease in the lungs from the rats treated with ketamine inhalation at the doses of 12.5 mg/ml (0.20 +/- 0.03) and 25 mg/ml (0.18 +/- 0.03) and with ketamine and intraperitoneally the at dose of 50 microg/kg (0.21 +/- 0.04) (P < 0.05). (5) NO production in pulmonary tissues was significantly higher in the OVA-treated rats compared to the PBS controls (0.39 +/- 0.04 micromol/g protein vs 0.13 +/- 0.01 micromol/g protein, P < 0.01), but this OVA-triggered NO production was significantly decreased by treatment with 12.5 and 25 mg/ml inhaled ketamine (0.19 +/- 0.03 micromol/g and 0.17 +/- 0.03 micromol/g, both P < 0.05) and 50 microg/kg i.p.-injected ketamine (0.16 +/- 0.04 micromol/g, P < 0.05) when compared with the OVA-treated rats. CONCLUSION: Both inhalation and systemic administration of ketamine attenuate inflammatory the lung injury and airway hyperreactivity of the OVA-induced asthma model. The protective effects of ketamine is achieved by inhibiting OVA-provoked over-expression of mRNA and protein of iNOS and reducing the production of NO in pulmonary tissues.

Acupuncture for Lateral Epicondylitis: A Systematic Review.

Objective. This systematic review aimed to assess the effectiveness and safety of acupuncture for lateral epicondylitis (LE). Methods. Seven databases and the WHO International Clinical Trials Registry Platform Search Portal were searched to identify relevant studies. The data were extracted and assessed by two independent authors, and Review Manager Software (V.5.3) was used for data synthesis with effect estimate presented as standard mean difference (SMD) and mean difference (MD) with a 95% confidence interval. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) was used to assess the level of evidence. Results. Four RCTs with 309 participants were included with poor methodological quality. Participants who received acupuncture and acupuncture plus moxibustion with material insulation were likely to have an improvement in elbow functional status and/or myodynamia. The overall quality rated by GRADE was from very low to low. Two studies reported that the needle pain would be the main reason for the dropout. Conclusion. For the small number of included studies with poor methodological quality, no firm conclusion can be drawn regarding the effect of acupuncture of elbow functional status and myodynamia for LE. This trial is registered with CRD42015016199.

[Effects of moxibustion on erectile function and NO-cGMP pathway in diabetic rats with erectile dysfunction].

OBJECTIVE: To explore the effect and the mechanism of moxibustion for treatment of diabetic erectile dysfunction (ED). METHODS: Diabetes mellitus (DM) rat model was induced by streptozotozin (STZ) and then penis erectile experiment of apomorphine (APO) was used to select diabetic ED rats model, which were divided into 2 groups: a model group and a moxibustion group, with another normal control group set up. The moxibustion group were treated with moxibustion at "Shenshu" (BL 23) and "Sanyinjiao" (SP 6) with small moxa cone about the size of a wheat grain. The effects on penis erectile, blood sugar and total NOS, cNOS, iNOS, and cGMP were investigated. RESULTS: Moxibustion had a certain improving action on blood sugar, improved significantly erectile function, and more significantly increased NOS, cNOS, iNOS activities and cGMP contents in the penis. CONCLUSION: Moxibustion has a certain action on the erectile function in the diabetic ED rats, which is related with improvement of blood sugar and promoting NO-cGMP pathway.