Divergent Total Syntheses of Lycorine Alkaloids via a Sequential C-H Functionalization Strategy.

A Pd-catalyzed one-pot sequential C-H functionalization strategy was utilized to prepare four lycorine alkaloids and one pseudo-lycorine alkaloid from the common intermediate 4. By switching the followed oxidative conditions of air, DMSO/H2O/I2, and DMSO/O2, based on the Pd(PPh3)4/K2CO3/toluene catalytic system, three key intermediates 12a, 12b, and 12c with different substitution patterns could be obtained in a well-controlled manner. As a result, four natural products γ-lycorane, hippadine, anhydrolycorinone, and anhydrolycorine as well as a pseudo-lycorine alkaloid Δ(4a,10b)-6-oxodihydrolycorine were successfully synthesized within 10 steps through this divergent route.

Salicylaldehyde as an SET-HAT Bifunctional Photocatalyst for the Intermolecular Transalkylation of Phthalimide.

Salicylaldehyde works as an efficient photocatalyst for the intermolecular transalkylation of phthalimide. The well-designed dimethyl N-hydroxyphthalimide ester proves to be a good alkylation reagent. It inhibits the competing intramolecular alkylation of alkylating reagent, enabling the site-specific synthesis of N-substituted phthalimide.

LncRNA expression signature identified using genome-wide transcriptomic profiling to predict lymph node metastasis in patients with stage T1 and T2 gastric cancer.

BACKGROUND: Lymph node (LN) status is vital to evaluate the curative potential of relatively early gastric cancer (GC; T1-T2) treatment (endoscopic or surgery). Currently, there is a lack of robust and convenient methods to identify LN metastasis before therapeutic decision-making. METHODS: Genome-wide expression profiles of long noncoding RNA (lncRNA) in primary T1 gastric cancer data from The Cancer Genome Atlas (TCGA) was used to identify lncRNA expression signature capable of detecting LN metastasis of GC and establish a 10-lncRNA risk-prediction model based on deep learning. The performance of the lncRNA panel in diagnosing LN metastasis was evaluated both in silico and clinical validation methods. In silico validation was conducted using TCGA and Asian Cancer Research Group (ACRG) datasets. Clinical validation was performed on T1 and T2 patients, and the panel's efficacy was compared with that of traditional tumor markers and computed tomography (CT) scans. RESULTS: Profiling of genome-wide RNA expression identified a panel of lncRNA to predict LN metastasis in T1 stage gastric cancer (AUC = 0.961). A 10-lncRNA risk-prediction model was then constructed, which was validated successfully in T1 and T2 datasets (TCGA, AUC = 0.852; ACRG, AUC = 0.834). Thereafter, the clinical performance of the lncRNA panel was validated in clinical cohorts (T1, AUC = 0.812; T2, AUC = 0.805; T1 + T2, AUC = 0.764). Notably, the panel demonstrated significantly better performance compared with CT and traditional tumor markers. CONCLUSIONS: The novel 10-lncRNA could diagnose LN metastasis robustly in relatively early gastric cancer (T1-T2), with promising clinical potential.

A Predictive Model for Chronic Hydrocephalus After Clipping Aneurysmal Subarachnoid Hemorrhage.

Chronic hydrocephalus after clipping aneurysmal subarachnoid hemorrhage (aSAH) often results in poor outcomes. This study was to establish and validate model to predict chronic hydrocephalus after aSAH by least absolute shrinkage and selection operator logistic regression. The model was constructed from a retrospectively analyzed. Two hundred forty-eight patients of aSAH were analyzed retrospectively in our hospital from January 2019 to December 2021, and the patients were divided into chronic hydrocephalus (CH) group (n=55) and non-CH group (n=193) according to whether occurred CH within 3 months. In summary, 16 candidate risk factors related to chronic hydrocephalus after aSAH were analyzed. Univariate analysis was performed to judging the risk factors for CH. The least absolute shrinkage and selection operator regression was used to filter risk factors. Subsequently, the nomogram was designed by the above variables. And area under the curve and calibration chart were used to detect the discrimination and goodness of fit of the nomogram, respectively. Finally, decision curve analysis was constructed to assess the practicability of the risk of chronic hydrocephalus by calculating the net benefits. Univariate analysis showed that age (60 y or older), aneurysm location, modified Fisher grade, Hunt-Hess grade, and the method for cerebrospinal fluid drainage, intracranial infections, and decompressive craniectomy were significantly related to CH ( P <0.05). Whereas 5 variables [age (60 y or older), posterior aneurysm, modified Fisher grade, Hunt-Hess grade, decompression craniectomy] from 16 candidate factors were filtered by LASSO logistic regression for further research. Area under the curve of this model was 0.892 (95% confidence interval: 0.799-0.981), indicating a good discrimination ability. Meanwhile, the result of calibration indicated a good fitting between the prediction probability and the actual probability. Finally, decision curve analysis showed a good clinical efficacy. In summary, this model could conveniently predict the occurrence of chronic hydrocephalus after aSAH. Meanwhile, it could help physicians to develop personalized treatment and close follow-up for these patients.

Depth-driven responses of microbial residual carbon to nitrogen addition approaches in a tropical forest: Canopy addition versus understory addition.

Canopies play an important role in nitrogen (N) redistribution in forest ecosystems, and ignoring the canopy's role might bias estimates of the ecological consequences of anthropogenic atmospheric N deposition. We investigated the effects of the approach of N addition (Canopy addition vs. Understory addition) and level of N addition (25 kg N ha-1yr-1 vs. 50 kg N ha-1yr-1) on microbial residual carbon (MRC) accumulation in topsoil and subsoil. We found that the response of MRC to both approach and level of N addition varied greatly with soil depth in a tropical forest over eight years of continuous N addition. Specifically, N addition enhanced the accumulation of fungal and total MRC and their contribution to soil organic C (SOC) pools in the topsoil, whereas it decreased the contribution of fungal and total MRC to SOC in the subsoil. The contrasting effects of N addition on MRC contribution at varying soil depths were associated with the distinct response of microbial residues production. Understory N addition showed overall greater effects on MRC accumulation than canopy N addition did. Our results suggest that the canopy plays an important role in buffering the impacts of anthropogenic atmospheric N deposition on soil C cycling in tropical forests. The depth-dependent response of microbial residues to N addition also highlights the urgent need for further studies of different response mechanisms at different soil depths.

Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis.

OBJECTIVES: Dysregulated chondrocyte metabolism is closely associated with the pathogenesis of osteoarthritis (OA). Suppressing chondrocyte catabolism to restore cartilage homeostasis has been extensively explored, whereas far less effort has been invested toward enhancing chondrocyte anabolism. This study aimed to repurpose clinically approved drugs as potential stimulators of chondrocyte anabolism in treating OA. METHODS: Screening of a Food and Drug Administration-approved drug library; Assays for examining the chondroprotective effects of digoxin in vitro; Assays for defining the therapeutic effects of digoxin using a surgically-induced OA model; A propensity-score matched cohort study using The Health Improvement Network to examine the relationship between digoxin use and the risk of joint OA-associated replacement among patients with atrial fibrillation; identification and characterisation of the binding of digoxin to low-density lipoprotein receptor-related protein 4 (LRP4); various assays, including use of CRISPR-Cas9 genome editing to delete LRP4 in human chondrocytes, for examining the dependence on LRP4 of digoxin regulation of chondrocytes. RESULTS: Serial screenings led to the identification of ouabain and digoxin as stimulators of chondrocyte differentiation and anabolism. Ouabain and digoxin protected against OA and relieved OA-associated pain. The cohort study of 56 794 patients revealed that digoxin use was associated with reduced risk of OA-associated joint replacement. LRP4 was isolated as a novel target of digoxin, and deletion of LRP4 abolished digoxin's regulations of chondrocytes. CONCLUSIONS: These findings not only provide new insights into the understanding of digoxin's chondroprotective action and underlying mechanisms, but also present new evidence for repurposing digoxin for OA.

Characteristics of Nosocomial Infections in Children Screened for SARS-CoV-2 Infection in China.

BACKGROUND This study summarizes the characteristics of children screened for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and reports the case of 1 child who was diagnosed with SARS-CoV-2 infection in Guangzhou Women and Children's Medical Center and the cases of his family members. MATERIAL AND METHODS The medical records of 159 children who were admitted to our hospital from January 23 to March 20, 2020, were retrospectively analyzed. Samples from pharyngeal or/and anal swabs were subjected to reverse-transcription polymerase chain reaction (RT-PCR) testing for SARS-CoV-2 within 12 h of patient admission; a second RT-PCR test was done 24 h after the first test. RESULTS Of the 159 patients, 151 patients had epidemiological histories, 14 patients had cluster onset, and 8 patients had no epidemiological history but had symptoms similar to coronavirus disease 2019 (COVID-19). The most common symptom was fever (n=125), followed by respiratory and gastrointestinal symptoms. A 7-year-old boy in a cluster family from Wuhan was confirmed with asymptomatic SARS-CoV-2 infection with ground-glass opacity shadows on his lung computed tomography scan, and his swab RT-PCR test had not turned negative until day 19 of his hospitalization. In patients who did not test positive for SARS-CoV-2, influenza, respiratory syncytial virus, and adenovirus were observed. A total of 158 patients recovered, were discharged, and experienced no abnormalities during follow-up. CONCLUSIONS For SARS-CoV-2 nosocomial infections, taking a "standard prevention & contact isolation & droplet isolation & air isolation" strategy can prevent infection effectively. Children with clustered disease need close monitoring.

An Extra-Large-Pore Pure Silica Zeolite with 16×8×8-Membered Ring Pore Channels Synthesized using an Aromatic Organic Directing Agent.

Extra-large-pore zeolites for processing large molecules have long been sought after by both the academia and industry. However, the synthesis of these materials, particularly extra-large-pore pure silica zeolites, remains a big challenge. Herein we report the synthesis of a new extra-large-pore silica zeolite, designated NUD-6, by using an easily synthesized aromatic organic cation as structure-directing agent. NUD-6 possesses an intersecting 16×8×8-membered ring pore channel system constructed by four-connected (Q4 ) and unusual three-connected (Q3 ) silicon species. The organic cations in NUD-6 can be removed in nitric acid to yield a porous material with high surface area and pore volume. The synthesis of NUD-6 presents a feasible means to prepare extra-large pore silica zeolites by using assembled aromatic organic cations as structure-directing agents.

The role of sema4D in vasculogenic mimicry formation in non-small cell lung cancer and the underlying mechanisms.

Vasculogenic mimicry (VM) is a special vascular pattern in malignant tumors, which is composed of highly aggressive tumor cells. This tumor cell-mediated blood supply pattern is closely associated with a poor prognosis in cancer patients. The interaction of axon guidance factor Sema4D and its high affinity receptor plexinB1 could activate small GTPase RhoA and its downstream ROCKs; this process has an active role in the migration of endothelial cells and tumor angiogenesis. Here, we have begun to uncover the role of this pathway in VM formation in non-small cell lung cancer (NSCLC). First, we confirmed this special form of vasculature in NSCLC tissues and found the existence of VM channels in tumor tissues was correlated with Sema4D expression. Further, we found that inhibition of Sema4D in the human NSCLC cells H1299 and HCC827 reduces VM formation both in vitro and in vivo. Moreover, we demonstrated that downregulating the expression of plexinB1 by siRNA expressing vectors and inhibiting the RhoA/ROCK signaling pathway using fasudil can reduce VM formation of H1299 and HCC827 cells. Finally, we found that suppression of Sema4D leads to less stress fibers and depleted the motility of H1299 and HCC827 cells. Collectively, our study implicates Sema4D plays an important role in the process of VM formation in NSCLC through activating the RhoA/ROCK pathway and regulating tumor cell plasticity and migration. Modulation of the Sema4D/plexinB1 and downstream RhoA/ROCK pathway may prevent the tumor blood supply through the VM pattern, which may eventually halt growth and metastasis of NSCLC.

Catecholamines contribute to the neovascularization of lung cancer via tumor-associated macrophages.

PURPOSE: Elevated catecholamines in the tumor microenvironment often correlate with tumor development. However, the mechanisms by which catecholamines modulate lung cancer growth are still poorly understood. This study is aimed at examining the functions and mechanisms of catecholamine-induced macrophage polarization in angiogenesis and tumor development. EXPERIMENTAL DESIGN: We established in vitro and in vivo models to investigate the relationship between catecholamines and macrophages in lung cancer. Flow cytometry, cytokine detection, tube formation assay, immunofluorescence, and western blot analysis were performed, and animal models were also used to explore the underlying mechanism of catecholamine-induced macrophage polarization and host immunological response. RESULTS: Catecholamines were shown to be secreted into tumor under the control of the sympathetic nerve system to maintain the pro-tumoral microenvironment. In vivo, the chemical depletion of the natural catecholamine stock with 6OHDA could reduce the release of catecholamines within tumor tissues, restrain the function of alternatively activated M2 macrophage, attenuate tumor neovascularization, and inhibit tumor growth. In vitro, catecholamine treatment triggered the M2 polarization of macrophages, enhanced the expression of VEGF, promoted tumor angiogenesis, and these catecholamine-stimulated effects could be reversed by the adrenergic receptor antagonist propranolol. In addition to regulating tumor-associated macrophages (TAM) recruitment, decreasing catecholamine levels could also shift the immunosuppressive microenvironment by decreasing myeloid-derived suppressor cells' (MDSCs) recruitment and facilitating dendritic cells' (DCs) activation, potentially resulting in a positive antitumor immune response. CONCLUSION: Our study demonstrates the potential of adrenergic stress and catecholamine-driven adrenergic signaling of TAMs to regulate the immune status of a tumor microenvironment and provides promising targets for anticancer therapies.

Posterior Ischemic Optic Neuropathy Following Radiofrequency Thermocoagulation for the Treatment of Trigeminal Neuralgia.

Radiofrequency thermocoagulation is one of the effective methods to treat trigeminal neuralgia. However, the complication of posterior ischemic optic neuropathy after treatment has not been reported, at present. In the present study, preoperative examination and intraoperative operation were successful, but visual acuity decreased after the operation. The cause was considered to be vasospasm induced by atherosclerosis, blood viscosity, and mental stress, which caused local ischemia, and subsequently, ischemic optic neuropathy.

[Critical quality evaluation and application value of network Meta-analyses in traditional Chinese medicine].

To introduce the application status of network Meta-analysis( NMA) in the field of traditional Chinese medicine,and to discuss the application value of NMA in the field of traditional Chinese medicine,this study comprehensively reviewed the systematic reviews with application of NMA in the field of traditional Chinese medicine. CNKI,Wan Fang,Sino Med,VIP,Embase,PubMed and Cochrane Library and the reference list of previous studies were searched. The AMSTAR scale was used to evaluate the quality of literature methodology,and PRISMA-NMA checklist was used to measure the degree of report specification. Overall,122 articles were included,including 80 in Chinese and 42 in English. The included studies centered on cancer,bone and joint disease,cardiovascular disease,respiratory disease,mental disease and digestive disease. Additionally,the intervention can be categorized into three groups,traditional Chinese medicine injection,oral Chinese medicine or prescription,and traditional physical therapy including acupuncture.Nearly one-third of the researches' intervention program is aimed at comparing the effect of Chinese and Western combined therapy and monotherapy. The overall methodology quality grade is medium and the report quality is average,with methodology reporting and result reporting especially need to be improved. The subgroup analysis shows that the methodology quality of the English literatures is evidently higher than Chinese literatures,and the quality of the literatures published after 2015 is higher than those published in or before 2015.This study indicates that the NMA can compare multiple treatments simultaneously,which accords with characteristics of the clinical practice in traditional Chinese medicine that is complex and individual. NMA in the field of traditional Chinese medicine is still in the process of development. With higher level of quality control and reporting as well as the improvement of the statistical methodology and the accumulation of original researches,NMA application in the field of traditional Chinese medicine will be promising.

Unlocking the potential antioxidant and anti-inflammatory activities of Rhododendron molle G. Don.

Rhododendron molle G. Don is an important traditional Chinese medicinal plant, which has been applied to treat some inflammatory diseases. In the present study, ethanol extracts of R. molle flower (RFE) and leaf (RLE) were used for phytochemical, antioxidant and anti-inflammatory analysis. The antioxidant activity was investigated using the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (OH-)-scavenging activity, super oxide anion radical (O2.-)-scavenging activity and iron reducing power (FRAP). Production of nitric oxide (NO) was an indicator to evaluate the anti-inflammatory activity. The results showed that compared with RFE, RLE was more active against DPPH (56.66%), FRAP (51.29%) and hydroxyl radical (OH-) (69.66%) at 100µg/mL. In the same time, RLE and RFE had significant anti-inflammatory activity which could reduce nitrite production from 8.76µM to 5.08µM and 6.01µM, respectively. In addition, GC-MS analysis showed that 43 compounds were identified in R. molle. Among them, 11 compounds had antioxidant and 5 compounds had anti-inflammatory effect. Results showed that ethanol extracts of R. molle have significant antioxidant and anti-inflammatory activity. These results would be helpful for further investigation on the anti-inflammatory mechanism of R. molle.

Alterations in leaf nitrogen metabolism indicated the structural changes of subtropical forest by canopy addition of nitrogen.

Globally, nitrogen deposition increment has caused forest structural changes due to imbalanced plant nitrogen metabolism and subsequent carbon assimilation. Here, a 2 consecutive-year experiment was conducted to reveal the effects of canopy addition of nitrogen (CAN) on nitrogen absorption, assimilation, and allocation in leaves of three subtropical forest woody species (Castanea henryi, Ardisia quinquegona, and Blastus cochinchinensis). We hypothesized that CAN altered leaf nitrogen absorption, assimilation and partitioning of different plants in different ways in subtropical forest. It shows that CAN increased maximum photosynthetic rate (Amax), photosynthetic nitrogen use efficiency (PNUE), and metabolic protein content of the two understory species A. quinquegona and B. cochinchinensis. By contrary, for the overstory species, C. henryi, Amax, PNUE, and metabolic protein content were significantly reduced in response to CAN. We found that changes in leaf nitrogen metabolism were mainly due to the differences in enzyme (e.g. Ribulose-1,5-bisphosphate carboxylase, nitrate reductase, nitrite reductase and glutamine synthetase) activities under CAN treatment. Our results indicated that C. henryi may be more susceptible to CAN treatment, and both A. quinquegona and B. cochinchinensis could better adapt to CAN treatment but in different ways. Our findings may partially explain the ongoing degradation of subtropical forest into a community dominated by small trees and shrubs in recent decades. It is possible that persistent high levels of atmospheric nitrogen deposition will lead to the steady replacement of dominant woody species in this subtropical forest.

The effects of sclerostin antibody plus parathyroid hormone (1-34) on bone formation in ovariectomized rats.

Previous studies have demonstrated the effects of sclerostin antibody (Scl-Ab) and parathyroid hormone (1-34, PTH) on healing in osteoporosis; however, reports about the combined effects of Scl-Ab plus PTH on osteoporosis are limited. This study was designed to investigate the impact of combined treatment with Scl-Ab and PTH on osteoporosis healing in ovariectomized (OVX) rats. After bilateral ovariectomy, 12 weeks were allowed to pass for the establishment of standard conditions for osteoporosis in animal models. The rats then randomly received a vehicle (control), Scl-Ab (25 mg/kg body weight, twice weekly), PTH (60 µg/kg, three times per week) or PTH plus Scl-Ab until death at 12 weeks. The blood and distal femurs of the rats were harvested for evaluation. The results of treatment for osteoporosis were evaluated by serum analysis, histology, microcomputed tomography (micro-CT) and biomechanical tests. Results from this study indicated that PTH + Scl-Ab had stronger effects on the prevention and treatment of osteoporosis than either of the monotherapies in OVX rats. The PTH + Scl-Ab produced the strongest effects on bone volume fraction (BV/TV), bone trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp), bone mineral density (BMD) and strength of distal femurs and increased the levels of procollagen type I N­terminal propeptide (PINP) and osteocalcin. In contrast, monotherapy with PTH or Scl-Ab showed no differences between treated groups in the assessment of the metaphysis of contralateral femurs by histology, serum, biomechanical tests and micro-CT. These results seem to indicate that Scl-Ab plus PTH has an additive effect on osteoporosis in OVX rats.

Ethylene and nitric oxide interact to regulate the magnesium deficiency-induced root hair development in Arabidopsis.

Nitric oxide (NO) and ethylene respond to biotic and abiotic stresses through either similar or independent processes. This study examines the mechanism underlying the effects of NO and ethylene on promoting root hair development in Arabidopsis under magnesium (Mg) deficiency. The interaction between NO and ethylene in the regulation of Mg deficiency-induced root hair development was investigated using NO- and ethylene-related mutants and pharmacological methods. Mg deficiency triggered a burst of NO and ethylene, accompanied by a stimulated development of root hairs. Interestingly, ethylene facilitated NO generation by activation of both nitrate reductase and nitric oxide synthase-like (NOS-L) in the roots of Mg-deficient plants. In turn, NO enhanced ethylene synthesis through stimulating the activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase (ACS). These two processes constituted an NO-ethylene feedback loop. Blocking either of these two processes inhibited the stimulation of root hair development under Mg deficiency. In conclusion, we suggest that Mg deficiency increases the production of NO and ethylene in roots, each influencing the accumulation and role of the other, and thus these two signals interactively regulate Mg deficiency-induced root hair morphogenesis.

Optical Peregrine rogue waves of self-induced transparency in a resonant erbium-doped fiber.

The resonant interaction of an optical field with two-level doping ions in a cryogenic optical fiber is investigated within the framework of nonlinear Schrödinger and Maxwell-Bloch equations. We present explicit fundamental rational rogue wave solutions in the context of self-induced transparency for the coupled optical and matter waves. It is exhibited that the optical wave component always features a typical Peregrine-like structure, while the matter waves involve more complicated yet spatiotemporally balanced amplitude distribution. The existence and stability of these rogue waves is then confirmed by numerical simulations, and they are shown to be excited amid the onset of modulation instability. These solutions can also be extended, using the same analytical framework, to include higher-order dispersive and nonlinear effects, highlighting their universality.

Complementary optical rogue waves in parametric three-wave mixing.

We investigate the resonant interaction of two optical pulses of the same group velocity with a pump pulse of different velocity in a weakly dispersive quadratic medium and report on the complementary rogue wave dynamics which are unique to such a parametric three-wave mixing. Analytic rogue wave solutions up to the second order are explicitly presented and their robustness is confirmed by numerical simulations, in spite of the onset of modulation instability activated by quantum noise.

Bis(difluoromethyl)trimethylsilicate Anion: A Key Intermediate in Nucleophilic Difluoromethylation of Enolizable Ketones with Me3 SiCF2 H.

A pentacoordinate bis(difluoromethyl)silicate anion, [Me3 Si(CF2 H)2 ](-) , is observed for the first time by the activation of Me3 SiCF2 H with a nucleophilic alkali-metal salt and 18-crown-6. Further study on its reactivity by tuning the countercation effect led to the discovery and development of an efficient, catalytic nucleophilic difluoromethylation of enolizable ketones with Me3 SiCF2 H by using a combination of CsF and 18-crown-6 as the initiation system. Mechanistic investigations demonstrate that [(18-crown-6)Cs](+) [Me3 Si(CF2 H)2 ](-) is a key intermediate in this catalytic reaction.

Rapid and specific detection of porcine parvovirus using real-time PCR and high resolution melting (HRM) analysis.

BACKGROUND: Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world. RESULTS: We developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the detection of PPV. Primers targeting the VP gene were highly specific, as evidenced by the negative amplification of closely related viruses, such as porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal. Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods. The assay has the accuracy of 100% in reference to labeled real time PCR, when it was tested on 45 clinical samples. CONCLUSIONS: The present study demonstrated that the established assay integrating real-time PCR and HRM is relatively cost-effective and more stable, which provides an alternative tool for rapid, simple, specific and sensitive detection of PPV.