Mitochondrial KATP channel-mediated autophagy contributes to angiotensin II-induced vascular dysfunction in mice.

BACKGROUND AND AIM: The present study aimed to investigate whether the mitochondrial KATP channel contributes to angiotensin II (Ang II)-induced vascular dysfunction, the development of hypertension, and atherosclerosis. METHODS AND RESULTS: ApoE (-/-) mice fed a high-fat diet were chronically infused with Ang II for eight weeks and concomitantly treated with losartan (ARB), apocynin, or 5-hydroxy decanoate (5-HD), or 3-methyladenine (3-MA). Systolic blood pressure was measured, and pathological changes of aortic or liver tissue were observed. Nitric oxide (NO), superoxide dismutase 2 (SOD2) levels and vasorelaxation rate were measured, and protein and mRNA expressions were examined by western blot and RT-PCR. Ang II-induced development of hypertension was suppressed not only by ARB, and apocynin but also by 5-HD or 3-MA. Ang II infusion decreased aortic NO production and relaxation, as well as SOD2 activity in liver, which were improved by all treatments. In addition, Ang II-induced activation of autophagy was suppressed by 5-HD in aortic tissue, furthermore, Ang II increases the atherosclerotic index in plasma and exacerbates the development of atherosclerosis by increases of fat deposition in the aorta and liver. Lipid metabolism-related mRNA expressions (LXR-α, LDLR, SRBI, Acca, and FASN) were changed by Ang II. Similarly, not only ARB, and apocynin, but also 5-HD and 3-MA suppressed Ang II-induced these changes. CONCLUSIONS: Our present findings evidence that mitochondrial KATP channel-mediated autophagy contributes to Ang II-induced vascular dysfunction, development of hypertension, and atherosclerosis.

Prognostic value of right ventricular trabecular complexity in patients with arrhythmogenic cardiomyopathy.

OBJECTIVES: The present study aimed to investigate the incremental prognostic value of the right ventricular fractal dimension (FD), a novel marker of myocardial trabecular complexity by cardiac magnetic resonance (CMR) in patients with arrhythmogenic cardiomyopathy (ACM). METHODS: Consecutive patients with ACM undergoing CMR were followed up for major cardiac events, including sudden cardiac death, aborted cardiac arrest, and appropriate implantable cardioverter defibrillator intervention. Prognosis prediction was compared by Cox regression analysis. We established a multivariable model supplemented with RV FD and evaluated its discrimination by Harrell's C-statistic. We compared the category-free, continuous net reclassification improvement (cNRI) and integrated discrimination index (IDI) before and after the addition of FD. RESULTS: A total of 105 patients were prospectively included from three centers and followed up for a median of 60 (48, 66) months; experienced 36 major cardiac events were recorded. Trabecular FD displayed a strong unadjusted association with major cardiac events (p < 0.05). In the multivariable Cox regression analysis, RV maximal apical FD maintained an independent association with major cardiac events (hazard ratio, 1.31 (1.11-1.55), p < 0.002). The Hosmer-Lemeshow goodness of fit test displayed good fit (X2 = 0.68, p = 0.99). Diagnostic performance was significantly improved after the addition of RV maximal apical FD to the multivariable baseline model, and the continuous net reclassification improvement increased 21% (p = 0.001), and the integrated discrimination index improved 16% (p = 0.045). CONCLUSIONS: In patients with ACM, CMR-assessed myocardial trabecular complexity was independently correlated with adverse cardiovascular events and provided incremental prognostic value. CLINICAL RELEVANCE STATEMENT: The application of FD values for assessing RV myocardial trabeculae may become an accessible and promising parameter in monitoring and early diagnosis of risk factors for adverse cardiovascular events in patients with ACM. KEY POINTS: • Ventricular trabecular morphology, a novel quantitative marker by CMR, has been explored for the first time to determine the severity of ACM. • Patients with higher maximal apical fractal dimension of RV displayed significantly higher cumulative incidence of major cardiac events. • RV maximal apical FD was independently associated with major cardiac events and provided incremental prognostic value in patients with ACM.

Psychological Journey and Coping Styles of Parents of Infants With Biliary Atresia: A Single-Center Qualitative Study.

BACKGROUND: Biliary atresia is a rare and serious neonatal disease that affects the quality of life of both infants and parents. There is currently limited literature on the experiences of parents with infants diagnosed with biliary atresia. PURPOSE: To explore the psychological journey and coping styles of parents of infants with biliary atresia in a single center in Shanghai, China. METHODS: A qualitative study design was used. Face-to-face and semistructured interviews were conducted with 10 parents of infants with biliary atresia. Colaizzi's method of data analysis was utilized, using NVivo 11.0 software. RESULTS: The psychological journey and coping styles of parents could be divided into 4 stages. Different themes were extracted at different stages: before diagnosis, parents experienced complex emotions and actively sought treatment; at the diagnosis stage, negative emotions dominated and parents convinced themselves to accept reality; in the postoperative stage, positive emotions, accepting reality, active response, and the need to learn to take care of their infant gradually appeared; and at the discharge stage, parents accepted the coexistence of positive and negative emotions and the variety of needs that emerged. IMPLICATIONS FOR PRACTICE: The findings of the study may help healthcare professionals identify and focus on the psychological needs of parents of infants with biliary atresia, leading them to implement effective coping strategies to increase the caregiving ability of parents. IMPLICATIONS FOR RESEARCH: Future research should explore the effects of supportive interventions for parents of infants with serious chronic illnesses.

A novel zein-selenium complex nanoparticle with controllable size: Quantitative design, physical properties and cytotoxicity in vitro.

In this study, after proposing a method for the preparation of selenium nanoparticles (Se NPs) with stable properties using zein, the physico-chemical properties of zein-Se NPs were tested. The complex structure of zein-Se NPs was deduced by SEM, and the binding mechanism was determined by FT-IR and XPS. The particle size of zein-Se NPs could be regulated from 11.4 ± 0.1 nm to 138.7 ± 0.9 nm under different preparation parameters, the reason for the change in particle size had been speculated. The pH responsiveness and 30-day storage stability of the zein-Se NPs were discussed. The zein-Se NPs still had strong DPPH radical scavenging activity after heat treatment. The zein-Se NPs were cell-friendly and was able to effectively protect cells from H2O2-induced cell-death. This study performed an extensive determination of the underlying physico-chemical properties of zein-Se NPs, we anticipate this approach will open up new possibilities in using natural material to stabilize Se NPs.

Fractal analysis in cardiovascular magnetic resonance: prognostic value of biventricular trabecular complexity in hypertrophic cardiomyopathy.

Background: Trabecular complexity can be quantified by fractal analysis based on cine images of cardiovascular magnetic resonance (CMR), yielding fractal dimension (FD) index. We aimed to investigate the prognostic value of biventricular FD in patients with hypertrophic cardiomyopathy (HCM). Methods: This retrospective study included 284 (192 men, median age 53 years) patients with HCM who underwent CMR, with median follow-up of 24 months. Biventricular trabeculae complexity was quantified as FD using short-axis cine images. The primary end point included sudden cardiac death (SCD) events. The secondary end point included both SCD events and rehospitalization due to heart failure. Cox regressions were performed. Prediction models were established by adding ventricular FDs to ESC predictors and late gadolinium enhancement (LGE) percentage and the C indices were calculated. Results: Cox regressions revealed that left ventricular (LV) maximal apical FD (HR range 1.114-1.133; all P<0.05) and right ventricular (RV) global FD (HR range 1.135-1.150; all P<0.05) were significant prognostic factors of both end points after adjustment for the European Society of Cardiology (ESC) predictors (age, maximum LV wall thickness, LV atrial size, peak left ventricular outflow tract (LVOT) gradient, family history of SCD, unexplained syncope, non-sustained ventricular tachycardia), and LGE percentage. The prediction model with the addition of biventricular FDs (C-index: 0.864-0.877) had the best performance. Conclusions: LV maximal apical FD and RV global FD were independent predictors of SCD events and rehospitalization due to heart failure in patients with HCM. The addition of biventricular FDs to the conventional prediction model contributed incremental prognosis value in HCM.

Gao-Zi-Yao improves learning and memory function in old spontaneous hypertensive rats.

AIMS: Gao-Zi-Yao has long been a unique way for treating various diseases. The present study is to explore the effect of Gao-Zi-Yao on learning and memory function in old spontaneous hypertensive rats (SHR) and its possible mechanism. METHOD: Male old SHR were received different doses of Gao-Zi-Yao for 4 weeks. Systolic blood pressure (SBP) and heart rate were monitored. Serum levels of nitric oxide (NO), interleukin (IL)-1ß, IL-2, and tumor necrotic factor (TNF)-α were measured. Morris water maze was performed to test the learning and memory function of the rats. Number of neurons in hippocampus was counted by Nissl staining. Western blot was applied to detect the expressions of learning and memory function related proteins, N-methyl-d-aspartate receptor 2B (NMDAR 2B), glutamate receptor 1 (GluR1), phosphorylated-calmodulin-dependent protein kinase II (p-CaMK II), and phosphorylated-cAMP responsive element-binding protein (p-CREB) in rat hippocampus. RESULTS: Data showed that Gao-Zi-Yao reduced SBP in old SHR, elevated NO level, and suppressed levels of IL-1ß, IL-2, TNF-α. The results of Morris water maze experiment showed that Gao-Zi-Yao dose-dependently improved learning and memory function. Number of neurons in the hippocampal dentate gyrus (DG) region of the old SHR was increased by Gao-Zi-Yao treatment. In addition, Gao-Zi-Yao elevated the protein expressions of NMDAR 2B, GluR1, p-CaMK II, and p-CREB in hippocampus. CONCLUSION: Gao-Zi-Yao decreases SBP and improves the learning and memory function of the old SHR by regulation of oxidative stress, inflammatory factors and neuron number in hippocampal DG area and the expression of learning and memory function related proteins.

Traditional Chinese Medicine Formulation Huangqi Jianzhong Tang Improves Cardiac Function after Myocardial Infarction in Rats.

Huangqi Jianzhong Tang (HQJZT) is a traditional Chinese herbal formula consisting of seven different herbs: Radix Astragali, Radix Paeoniae Alba, Ramulus Cinnamomi, Fructus Jujubae, Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, Rhizoma Zingiberis Recens, and Saccharum Granorum. The present study aims to evaluate the possible effects of HQJZT on cardiac function in acute myocardial infarction (AMI) and related mechanism. AMI model was established by ligation of the left anterior descending coronary artery followed by one-week HQJZT treatment. Survival rate was calculated. Rat heart function was assessed by heart performance analysis system. 5-Triphenyltetrazolium chloride (TTC) staining was used to observe myocardial infarct size. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and western blot were applied to evaluate tissue apoptotic level. Treatment with high dose of HQJZT improved cardiac function, reduced infarct size, number of apoptotic cells and expression of apoptotic proteins, Bax (a proapoptotic protein), and increased expression of antiapoptotic protein, Bcl2. However, enalapril (an angiotensin-converting enzyme inhibitor) treatment did not show marked improvement of these parameters. Our present data suggest that HQJZT has potential therapeutic effects to improve cardiac function by regulation of apoptotic signaling pathway.

Cholecystokinin 1 Receptor - A Unique G Protein-Coupled Receptor Activated by Singlet Oxygen (GPCR-ABSO).

Plasma membrane-delimited generation of singlet oxygen by photodynamic action with photosensitizer sulfonated aluminum phthalocyanine (SALPC) activates cholecystokinin 1 receptor (CCK1R) in pancreatic acini. Whether CCK1R retains such photooxidative singlet oxygen activation properties in other environments is not known. Genetically encoded protein photosensitizers KillerRed or mini singlet oxygen generator (miniSOG) were expressed in pancreatic acinar tumor cell line AR4-2J, CCK1R, KillerRed or miniSOG were expressed in HEK293 or CHO-K1 cells. Cold light irradiation (87 mW⋅cm-2) was applied to photosensitizer-expressing cells to examine photodynamic activation of CCK1R by Fura-2 fluorescent calcium imaging. When CCK1R was transduced into HEK293 cells which lack endogenous CCK1R, photodynamic action with SALPC was found to activate CCK1R in CCK1R-HEK293 cells. When KillerRed or miniSOG were transduced into AR4-2J which expresses endogenous CCK1R, KillerRed or miniSOG photodynamic action at the plasma membrane also activated CCK1R. When fused KillerRed-CCK1R was transduced into CHO-K1 cells, light irradiation activated the fused CCK1R leading to calcium oscillations. Therefore KillerRed either expressed independently, or fused with CCK1R can both activate CCK1R photodynamically. It is concluded that photodynamic singlet oxygen activation is an intrinsic property of CCK1R, independent of photosensitizer used, or CCK1R-expressing cell types. Photodynamic singlet oxygen CCK1R activation after transduction of genetically encoded photosensitizer in situ may provide a convenient way to verify intrinsic physiological functions of CCK1R in multiple CCK1R-expressing cells and tissues, or to actuate CCK1R function in CCK1R-expressing and non-expressing cell types after transduction with fused KillerRed-CCK1R.

Permanent Photodynamic Cholecystokinin 1 Receptor Activation: Dimer-to-Monomer Conversion.

The G protein-coupled cholecystokinin 1 receptor (CCK1R) is activated permanently by type II photodynamic action (i.e., by singlet oxygen) in the freshly isolated rat pancreatic acini, in contrast to reversible activation by CCK. But how CCK1R is photodynamically activated is not known. Therefore, in the present work, we subjected membrane proteins extracted from isolated rat pancreatic acini to photodynamic action with photosensitiser sulphonated aluminium phthalocyanine (SALPC), and used reducing gel electrophoresis and Western blot to detect possible changes in CCK1R oligomerization status. Photodynamic action (SALPC 1 µM, light 36.7 mW cm- 2 × 10 min) was found to convert dimeric CCK1R nearly quantitatively to monomers. Such conversion was dependent on both irradiance (8.51-36.7 mW cm- 2) and irradiation time (1-20 min). Minimum effective irradiance was found to be 11.1 mW cm- 2 (× 10 min, with SALPC 1 µM), and brief photodynamic action (SALPC 1 µM, 36.7 mW cm- 2 × 1 min) was effective. Whilst CCK stimulation of purified membrane proteins alone had no effect on CCK1R dimer/monomer balance, sub-threshold photodynamic action (SALPC 100 nM, 36.7 mW cm- 2 × 10 min) plus CCK revealed a bell-shaped CCK dose response curve for CCK1R monomerization, which was remarkably similar to the dose response curve for CCK-stimulated amylase secretion in isolated rat pancreatic acini. These two lines of evidence together suggest that during photodynamic CCK1R activation, CCK1R is permanently monomerized, thus providing a unique approach for permanent G protein-coupled receptor (GPCR) activation which has not been achieved before.

Heme oxygenase-1 promotes tumor progression and metastasis of colorectal carcinoma cells by inhibiting antitumor immunity.

Heme oxygenase-1 (HO-1) is upregulated in colorectal carcinoma (CRC) cells. However, the role of HO-1 in the metastatic potential of CRC remains to be elucidated. In this study, we investigated the potential of HO-1 to control the antitumor immunity of CRC. Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the immune surveillance system. Hemin-induced HO-1 expression suppressed the expression of ICAM-1 in human CRC cells. HO-1 regulated ICAM-1 expression via tristetraprolin, an mRNA-binding protein, at the posttranscriptional level in CRC cells. The upregulated HO-1 expression in CRC cells markedly decreased the adhesion of peripheral blood mononuclear lymphocytes (PBMLs) to CRC cells and PBML-mediated cytotoxicity against CRC cells. Production of CXCL10, an effector T cell-recruiting chemokine, was significantly reduced by the increased HO-1 expression. The expression of the CXCL10 receptor, CXCR3, decreased significantly in PBMLs that adhered to CRC cells. HO-1 expression correlated negatively, although nonsignificantly, with ICAM-1 and CXCL10 expression in xenograft tumors. Taken together, our data suggest that HO-1 expression is functionally linked to the mediation of tumor progression and metastasis of CRC cells by inhibiting antitumor immunity.

PF2405, standardized fraction of Scutellaria baicalensis, ameliorates colitis in vitro and in vivo.

Standardized extraction procedures for herb are as important as their authentication to maintain their quality and ensure their safe use. We had prepared a standardized and purified Scutellaria baicalensis Georgi extract, PF2405, which was enriched with three major components, baicalein, oroxylin A and wogonin. In the present study, we investigated the potential anti-inflammatory effects of PF2405 in vitro and in two different experimental animal models of inflammatory bowel disease. Effect of PF2405 studied in tumor necrosis factor (TNF)-α-induced HT-29 cells in vitro. In vivo experimental colitis models were induced by administration of trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS). PF2405 (50 µg/ml) decreased TNF-α-induced cyclooxygenase (COX)-2 expressions through inhibition of phosphorylation of c-Jun N-terminal kinases and p38 mitogen-activated protein kinase in HT-29 cells. Combination of baicalein (20 µg/ml), oroxylin A (8 µg/ml), and wogonin (2 µg/ml) markedly inhibits TNF-α-induced COX-2 expression when compared with individual components. PF2405 (25 mg/kg b.w.) treatment significantly reduced histopathological severity; suppressed expression of COX-2, TNF-α, and interleukin-1ß in TNBS-induced mice. Moreover, PF2405 (25 mg/kg b.w.) has both potent preventive and therapeutic activities in DSS-induced colitis. Collectively, PF2405 shows prominent anti-inflammatory effect that can be used as a new therapeutic approach for intestinal inflammatory disorders.

An essential role of NAD(P)H oxidase 2 in UVA-induced calcium oscillations in mast cells.

Solar UVA radiation (320-400 nm) is known to have immunomodulatory effects, but the detailed mechanisms involved are not fully elucidated. UVA irradiation has been shown to induce calcium oscillations in rat peritoneal mast cells due to NAD(P)H oxidase (NOX) activation, but the specific NOX isoforms have not been identified. In the present work effects of UVA irradiation were investigated in isolated rat peritoneal mast cells, in cultured rat mast cell line RBL-2H3, and in mouse bone marrow-derived mast cells (BMMC). It was found that UVA irradiation by alternate 340/380 nm (3.2-5.6 µW cm(-2)) or by LED (380 nm, 80 µW cm(-2)) induced calcium oscillations in isolated rat peritoneal mast cells, in RBL-2H3, and in BMMC. Such UVA-induced calcium oscillations resembled closely those induced by surface IgE receptor (FcεRI) activation. It was found that RBL-2H3 expressed high levels of gp91(phox) (NOX2), p22(phox), p67(phox), p47(phox), p40(phox), Rac1, Rac2, moderate levels of DUOX2, but did not express NOX1, NOX3, NOX4, or DUOX1. The specific cellular localizations of gp91(phox) (NOX2), p22(phox), p47(phox), p67(phox), p40(phox) and Rac1/2 were confirmed by immunocytochemistry. UVA-induced reactive oxygen species (ROS) production in RBL-2H3 was completely suppressed by the NOX inhibitor diphenyleneiodonium chloride (DPI) or by the antioxidant N-acetyl-l-cysteine (NAC). siRNA suppression of gp91(phox) (NOX2), p22(phox) and p47(phox) expression inhibited markedly UVA-induced calcium oscillations, ROS and IL-6/LTC4 production in RBL-2H3. Taken together these data indicate that NOX2 plays an essential role in UVA irradiation-induced calcium oscillations, ROS and mediator production in mast cells.

Hirsutenone reduces deterioration of tight junction proteins through EGFR/Akt and ERK1/2 pathway both converging to HO-1 induction.

Oxidative stress-induced disruption of epithelial tight junctions (TJ) plays a critical role in the pathogenesis of intestinal disorders, including inflammatory bowel disease (IBD). The current study investigated the protective effect of hirsutenone against disruption of the intestinal barrier in vitro and in a mouse model of colitis. Caco-2 cells were stimulated with tert-butyl hydroperoxide (t-BH). Hirsutenone prevented the t-BH-induced increase in permeability by inhibiting the reduction in zonula occludens-1 (ZO-1) expression, and rapidly stimulated tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Hirsutenone-mediated protection against the loss of ZO-1 depends on the activation of both ERK1/2 and Akt signaling pathways. Interestingly, hirsutenone-mediated activation of Akt, but not ERK1/2, signaling was EGFR-dependent. Hirsutenone increased heme oxygenase-1 (HO-1) expression through both EGFR/Akt- and ERK1/2-dependent pathways, contributing to the protective effects against TJ dysfunction. Colitis was induced in mice by intrarectal administration of 2,4,6,-trinitrobenzene sulfonic acid (TNBS). Hirsutenone administration improved the clinical parameters and tissue histological appearance, increased HO-1 expression, attenuated reduction of ZO-1 and occludin mRNA, and promoted BrdU incorporation in the colonic epithelium of TNBS-treated mice. Taken together, our results demonstrate that hirsutenone reverse disordered intestinal permeability by activating EGFR/Akt and ERK1/2 pathways, which are involved in the regulation of HO-1 expression. These findings highlight the potential of hirsutenone for clinical applications in the treatment of IBD.

PF2401-SF, standardized fraction of Salvia miltiorrhiza shows anti-inflammatory activity in macrophages and acute arthritis in vivo.

Standardization of processing methods for herbs is as important as authentication to maintain their quality and ensure their safe use. We had previously prepared a standardized and purified Salvia miltiorrhiza Bunge extract, PF2401-SF, and showed that it protects against liver injury in vivo, at a greater potency than an ethanol extract. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). In this study, we investigated potential anti-inflammatory effects of PF2401-SF in vitro and in vivo. We demonstrated that PF2401-SF shows anti-inflammatory potency on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. A mechanistic study indicated that PF2401-SF induced heme oxygenase (HO)-1 expression through extracellular signal-regulated kinases (ERK1/2) phosphorylation. Moreover, we also evaluated that PF2401-SF significantly reduced inflammation on carrageenan- or dextran-induced acute arthritis in rats. Our results suggest that PF2401-SF may be a potential candidate for the treatment of various inflammatory diseases.

PF2401-SF, standardized fraction of Salvia miltiorrhiza, induces apoptosis of activated hepatic stellate cells in vitro and in vivo.

During the course of our attempts to develop a potential herbal medicine, we had previously prepared PF2401-SF, a standardized fraction of S. miltiorrhiza, and reported its hepatoprotective activity in vitro as well as in vivo. Since apoptosis of activated hepatic stellate cells (HSCs) is a well-accepted anti-fibrotic strategy, in this study, we investigated the direct effect of PF2401-SF on t-HSC/Cl-6 cells in vitro and on CCl4-induced liver injury in vivo. We evaluated the activation and cleavage of hallmarkers of apoptosis, namely, caspase 3, 8, 9 and PARP. Upregulation of the pro-apoptotic Bax protein and downregulation of the anti-apoptotic Bcl2 protein were also analyzed. Furthermore, in the PF2401-SF treated rats, apoptosis induction of activated HSCs was demonstrated by reduced distribution of α-SMA-positive cells and the presence of high number of TUNEL-positive cells in vivo. Our data suggest that PF2401-SF can mediate HSCs apoptosis induction, and may be a potential herbal medicine for the treatment of liver fibrosis.

Apoptotic effect of propyl gallate in activated rat hepatic stellate cells.

Hepatic stellate cells (HSCs) play a central role in liver fibrosis. Inhibition of HSC growth and induction of apoptosis have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. Propyl gallate (PG) is an antioxidant widely used in processed foods, cosmetics and medicinal preparations. However, the anti-fibrotic effect of PG in liver injury is unclear. In this study, we investigated whether PG could induce apoptosis in activated HSCs. Treatment of activated HSCs with PG inhibited cell viability in a dose- and time-dependent manner. PG induced apoptosis as demonstrated by morphological changes, poly(ADP-ribose) polymerase (PARP) cleavage, caspase-3 cleavage, increased Bad expression, and decreased Bcl-2 protein expression. Through stimulation of the activation of c-Jun NH2-terminal protein kinase (JNK) and p38 mitogen-activated protein kinases (MAPK) by PG treatment, we demonstrated that JNK and p38 MPAK are not involved in PG-induced apoptosis using their specific inhibitors. Taken together, these findings indicate that PG induces apoptosis in activated HSCs. The potential anti-fibrotic effect of PG warrants further evaluation.