Transcription Factor Pdr3p Promotes Carotenoid Biosynthesis by Activating GAL Promoters in Saccharomyces cerevisiae.

Pleiotropic drug resistance (PDR) family proteins have been extensively studied for their roles in transporting hydrophobic substances, including carotenoids. Overexpression of the PDR family regulator Pdr3p was recently found to boost the biosynthesis of carotenoids, which could not be explained by enhanced product secretion due to the meager extracellular proportions. To provide insights into the possible mechanism, comparative transcriptomics, reverse metabolic engineering, and electrophoretic mobility shift assay (EMSA) were conducted. Transcriptomic data suggested an unexpected correlation between Pdr3p overexpression and the transcriptional levels of GAL promoter-driven genes. This assumption was verified using mCherry and the lycopene synthetic pathway as the reporters. qRT-PCR and EMSA provided further evidence for the activation of GAL promoters by Pdr3p binding to their upstream activation sequences (UASs). This work gives insight into the mechanism of Pdr3p-promoted carotenoid production and highlights the complicated metabolic networking between transcriptional factors and promoters in yeast.

Ileum intussusception secondary to submucosal liposarcoma in adult：A case report.

Intussusception in adults is a rare surgical emergency. Unlike in children, most adult intussusceptions arise from a pathological lead point. Ileal intussusception caused by a submucosal liposarcoma is a particularly rare phenomenon. This report describes the diagnosis and management of adult ileal intussusception secondary to submucosal liposarcoma in adult to provide a reference for future clinical work. A 64-year-old female presented to the emergency department with worsening abdominal pain associated with an 8 h history of intermittent vomiting. Based on physical examination, laboratory investigations, and computed tomography, the most likely diagnosis was ileal intussusception secondary to liposarcoma. Thus, emergency laparotomy was performed. During exploration, an ileal invagination was visualised approximately 30 cm from the ileocecal valve, and a flexible polypoid mass was palpable at the lead point of the intussusception. Subsequently, the patient underwent radical resection of pathological tissues with a primary end-to-end ileal anastomosis. Histopathological examination revealed a well-differentiated submucosal liposarcoma. Postoperatively, the patient recovered uneventfully and was doing well at the 6-month follow-up in the outpatient clinic. Thus, clinicians should consider the origin of submucosal liposarcomas in adult with intussusception. Once ileal intussusception secondary to submucosal liposarcoma is diagnosed, timely radical resection is recommended.

TSLP Induces Epithelial-Mesenchymal Transition in Nasal Epithelial Cells From Allergic Rhinitis Patients Through TGF-ß1/Smad2/3 Signaling.

BACKGROUND: Airway remodeling is demonstrated in Asian patients with allergic rhinitis (AR). The epithelial-mesenchymal transition (EMT) is one of the key mechanisms underlying airway remodeling. Thymic stromal lymphopoietin (TSLP) is an important contributor to airway remodeling. Although increased TSLP is found in AR, little is known about whether TSLP is involved in airway remodeling through induction of the EMT. OBJECTIVE: We investigated the effect of TSLP on the EMT in human nasal epithelial cells (HNECs) from AR patients. METHODS: Human nasal epithelial cells from AR patients were stimulated with TSLP in the absence or presence of the preincubation with a selective inhibitor of transforming growth factor beta 1 (TGF-ß1) receptor (SB431542). The expression of TGF-ß1 in the cells was evaluated by using real-time polymerase chain reaction, Western blotting, and immunocytochemistry. Western blotting and immunocytochemistry were used to assay EMT markers including vimentin, fibroblast-specific protein 1 (FSP1) and E-cadherin, small mothers against decapentaplegic homolog2/3 (Smad2/3), and phosphorylated Smad2/3 in the cells. The levels of extracellular matrix components such as collagens I and III in supernatants were measured by enzyme-linked immunoassay. Morphological changes of the cells were observed under inverted phase-contrast microscope. RESULTS: A concentration-dependent increase of TGF-ß1 mRNA and protein was observed following stimulation with TSLP. Furthermore, TSLP decreased the expression of E-cadherin protein, but upregulated the production of FSP1 and vimentin proteins along with increased levels of collagens I and III, and the morphology of the cells was transformed into fibroblast-like shape. Additionally, a significant increase was found in phosphorylation of Smad2/3 protein. However, these effects were reversed by SB431542 preincubation. CONCLUSION: TSLP-induced HNECs to undergo the EMT process via TGF-ß1-mediated Smad2/3 activation. TSLP is an activator of the EMT in HNECs and might be a potential target for inhibiting EMT and reducing airway remodeling in AR.

Detection of serum Aspergillus-specific IgM and IgG antibody levels for the diagnosis of chronic pulmonary aspergillosis developed in patients with tuberculosis.

Chronic pulmonary aspergillosis (CPA) is common among individuals with underlying lung diseases. The clinical manifestations of CPA include systemic symptoms (e.g., weight loss, fatigue, fever), chronic productive cough, chest discomfort, and occasional haemoptysis, which are similar to the manifestations of pulmonary tuberculosis (PTB) and are often misdiagnosed as PTB. Considering the striking similarities between CPA and PTB in clinical manifestations and imaging features, more specific microbiological and serological detections are needed for a definitive diagnosis. This study aimed to explore the clinical characteristics of CPA in TB as well as the diagnostic significance of Aspergillus-specific IgG and Aspergillus-specific IgM.A total of 140 patients diagnosed with TB by culture between December 2017 and February 2019 were included. Enrolled patients were categorized into two groups (CPA group and non-CPA group) according to CPA diagnostic criteria. All collected specimens were subjected to Aspergillus-specific IgG and IgM detection testing.The median concentration of Aspergillus-specific IgG in the CPA group (211.04 AU/ml) was significantly higher than that in the non-CPA group (77.88 AU/ml) (Z value - 6.397, P < 0.001). The sensitivity and specificity of Aspergillus-specific IgG for CPA diagnosis were 81.82% and 72.97%, respectively. In the chronic cavitary pulmonary aspergillosis (CCPA) group, the IgG positivity rate (≥ 120 AU/ml) was 96.2%, which was 21.4% in the non-CCPA patients (P < 0.001).The detection of Aspergillus-specific IgG serological changes is feasible and facilitates reliable differentiation between Aspergillus and Mycobacterium tuberculosis infection. However, Aspergillus-specific IgM has limited diagnostic value, with unsatisfactory sensitivity results.

Hyaluronic Acid-Based Nanocarriers for Anticancer Drug Delivery.

Hyaluronic acid (HA), a main component of the extracellular matrix, is widely utilized to deliver anticancer drugs due to its biocompatibility, biodegradability, non-toxicity, non-immunogenicity and numerous modification sites, such as carboxyl and hydroxyl groups. Moreover, HA serves as a natural ligand for tumor-targeted drug delivery systems, as it contains the endocytic HA receptor, CD44, which is overexpressed in many cancer cells. Therefore, HA-based nanocarriers have been developed to improve drug delivery efficiency and distinguish between healthy and cancerous tissues, resulting in reduced residual toxicity and off-target accumulation. This article comprehensively reviews the fabrication of anticancer drug nanocarriers based on HA in the context of prodrugs, organic carrier materials (micelles, liposomes, nanoparticles, microbubbles and hydrogels) and inorganic composite nanocarriers (gold nanoparticles, quantum dots, carbon nanotubes and silicon dioxide). Additionally, the progress achieved in the design and optimization of these nanocarriers and their effects on cancer therapy are discussed. Finally, the review provides a summary of the perspectives, the lessons learned so far and the outlook towards further developments in this field.

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies.

Terpenoids are a diverse group of compounds with isoprene units as basic building blocks. They are widely used in the food, feed, pharmaceutical, and cosmetic industries due to their diverse biological functions such as antioxidant, anticancer, and immune enhancement. With an increase in understanding the biosynthetic pathways of terpenoids and advances in synthetic biology techniques, microbial cell factories have been built for the heterologous production of terpenoids, with the oleaginous yeast Yarrowia lipolytica emerging as an outstanding chassis. In this paper, recent progress in the development of Y. lipolytica cell factories for terpenoid production with a focus on the advances in novel synbio tools and metabolic engineering strategies toward enhanced terpenoid biosynthesis is reviewed.

Arbuscular Mycorrhizal Fungi Compete Asymmetrically for Amino Acids with Native and Invasive Solidago.

Arbuscular mycorrhizal fungi (AMF) and soil amino acids both affect plant performance. However, little is known about how AMF compete for amino acids with native and invasive congeners. We conducted a factorial experiment (inoculation, native and invasive species, and amino acids) to examine the competition for amino acids between soil microbes and both native and invasive congeners. The competition for amino acids between AMF and invasive Solidago canadensis was weaker than that observed between AMF and native S. decurrens. This asymmetric competition increased the growth advantage of S. canadensis over S. decurrens. The efficacy (biomass production per unit of nitrogen supply) of amino acids compared to ammonium was smaller in S. canadensis than in S. decurrens when both species were grown without inoculation, but the opposite was the case when both species were grown with AMF. AMF and all microbes differentially altered four phenotypic traits (plant height, leaf chlorophyll content, leaf number, and root biomass allocation) and the pathways determining the effects of amino acids on growth advantages. These findings suggest that AMF could enhance plant invasiveness through asymmetric competition for amino acids and that amino acid-driven invasiveness might be differentially regulated by different microbial guilds.

Interleukin-17A up-regulates thymic stromal lymphopoietin production by nasal fibroblasts from patients with allergic rhinitis.

PURPOSE: Emerging evidence has shown that interleukin (IL)-17A is implicated in the pathogenesis of allergic rhinitis (AR). Thymic stromal lymphopoietin (TSLP) orchestrates the immune response toward a Th2 phenotype. Although increased TSLP is found in AR, the contribution of IL-17A in TSLP production by nasal fibroblasts is not well understood. We aimed to investigate the effect and mechanism of IL-17A on TSLP production by human nasal fibroblasts (HNFs) from AR patients. METHODS: HNFs from AR patients were cultured and stimulated with IL-17A in the absence or presence of a Janus kinase (JAK) 2 or JAK1/3 inhibitor. Western blotting was used to assay phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and nuclear factor-kappa B (NF-κB) p65 in HNFs. The TSLP expression in the cells and culture supernatants was evaluated by real-time polymerase chain reaction and enzyme-linked immunoassay. RESULTS: Stimulation with IL-17A induced STAT3 phosphorylation, which was inhibited by the pretreatment with JAK2 inhibitor AZD1480 or JAK1/3 inhibitor tofacitinib. IL-17A promoted the nuclear translocation of NF-κBp65 protein, leading to increased TSLP production, while the pre-incubation with AZD1480 prior to IL-17A attenuated these effects. However, the pre-incubation with tofacitinib before IL-17A stimulation had no impact on the expression of NF-κBp65 and TSLP. CONCLUSIONS: IL-17A up-regulated TSLP production by HNFs through JAK2/NF-κB pathway. Although IL-17A induced STAT3 activation through JAK1/2/3, IL-17A-mediated TSLP expression was not dependent on STAT3 signaling. These observations would provide mechanistic insight into therapeutic strategies to improve the immune and inflammation associated with Th17A in the management of AR.

Rational design of the carbonyl reductase EbSDR8 for efficient biosynthesis of enantiopure (R)-3-chloro-1-phenyl-1-propanol.

(R)-3-Chloro-1-phenyl-1-propanol ((R)-CPPO) is an important chiral intermediate for antidepressants. For its efficient biosynthesis, the carbonyl reductase EbSDR8 was engineered to asymmetrically reduce the unnatural substrate 3-chloro-1-phenyl-1-propanone (3-CPP) at high concentrations. Molecular docking and molecular dynamics simulations of the resulting mutants suggested enlarged substrate binding pocket and more reasonable interactions between the enzyme and the substrate or cofactor as the reasons for the enhanced catalytic activity and thus the remarkably improved conversion of high-concentration 3-CPP. Using the best mutant EbSDR8G94A/L153I/Y188A/Y202M as the whole-cell biocatalyst, reduction of 3-CPP (1.0 M) was conducted using 100% isopropanol as both the solvent and co-substrate for NADH regeneration, delivering (R)-CPPO with ˃ 99% eep and 95.5% conversion. This result suggests EbSDR8G94A/L153I/Y188A/Y202M as a potential biocatalyst for green production of (R)-CPPO at the industrial scale. KEY POINTS: • Rational design of EbSDR8 by modulating steric hindrance and molecular interactions; • Non-aqueous biocatalysis using isopropanol as both the solvent and co-substrate; • Whole-cell catalyzed production of 161 g/L enantiopure (R)-CPPO from 1.0 M of 3-CPP. Graphical Abstract.

Influence of diabetes mellitus on energy metabolism in patients with alcoholic liver cirrhosis.

OBJECTIVE: The objective was to explore the characteristics of energy metabolism in patients with alcoholic liver cirrhosis (ALC) and diabetes mellitus (DM). METHODS: Thirty-four male patients with ALC and DM, 30 male patients with ALC without DM and 10 male healthy controls (HC) were enrolled in this study. Resting energy expenditure (REE), respiratory quotient (RQ) were measured by indirect calorimetry. Data were analyzed using the Student's t-test, Mann-Whitney U-test and χ2 tests between two groups. Logistic regression analysis was used to analyze the risk factors for hypermetabolism. RESULTS: Measured REE was significantly higher in patients with ALC and DM (1740 ± 338 kcal/d) than in patients with ALC (1400 ± 304 kcal/d, P < 0.01). Fasting blood glucose was an independent factor predicting hypermetabolism in all of the patients with ALC (P = 0.005). RQ was lower in patients with ALC and DM (0.80 ± 0.06) than in patients with ALC (0.83 ± 0.05, P = 0.027) and the HC (0.86 ± 0.03, P = 0.001). In the ALC and DM group, measured REE as percentage of predicted REE by Harris-Benedict formula was higher in patients with HbA1c ≥ 7.5% than in those with HbA1c < 7.5% (126.36 ± 15.19% vs. 109.48 ± 23.89%, P = 0.040). CONCLUSION: REE was increased and RQ was significantly decreased in patients with ALC and DM. These changes were associated with poor glucose control. HbA1c less than 7.5% may reduce the risk of hypermetabolism.

Efficacy of path-oriented psychological self-help interventions to improve mental health of empty-nest older adults in the Community of China.

BACKGROUND: China has the world's largest aging population and the number of empty-nest older adults is on the rise. In comparison to the aging population in general, empty-nest older adults have a lower level of subjective well-being and poorer mental health status due to a lack of emotional support from their children. The aim of this study is to conduct an empirical study to evaluate the efficacy of the 'Path-oriented Psychological Self-help Intervention' (P-oPSI) led by nurses on the mental health of empty-nest older adults in the community, to provide a scientific foundation for improving their quality of life. METHODS: A Quasi-Experimental controlled intervention study was conducted from 2015 to 2017. A total of 76 empty-nest older adults from 2 districts were recruited using a convenience sampling and assigned to 2 groups based on their residential communities in the city of Chifeng in the Inner Mongolia Autonomous Region, China. The wait list control group participated in a mental health lecture to gain knowledge and learn techniques of mental health promotion. The intervention group additionally received 1 month of training in a nurse-led 'P-oPSI' for a month. Both groups were followed-up for 3 months. Mental health status, coping styles, and psychological self-help ability of the participating empty nest older adults were assessed at the baseline, 1 month, and 3-months follow up, respectively. Two-way analysis of variance and a simple effect test were used to analyse the differences of the two groups. RESULTS: The P-oPSI yielded a greater benefit for the mental health status, coping styles, and psychological self-help ability of the participants in the intervention group. Combined with a simple effect test, the scores of the mental health status, positive coping style, and psychological self-help ability of those in the intervention group significantly increased at 1 month after the baseline (F mental health status = 7.59, F positive coping style = 7.24, F psychological self-help ability = 7.07); and the sustainable effect of this program lasted for 3 months after the intervention (F mental health status = 13.24, F positive coping style = 10.42, F psychological self-help ability = 10.45), which reached statistical significance (P < 0.01). CONCLUSIONS: The P-oPSI program significantly improved the level of mental health of empty-nest older adults in China. This intervention provides a new approach of self-management to improve mental health of older adults in community settings. TRIAL REGISTRATION: chictr.org.cn: ChiCTR1900025552. Retrospectively registered 1 September 2019.

Interleukin-17A potentiates interleukin-13-induced eotaxin-3 production by human nasal epithelial cells from patients with allergic rhinitis.

BACKGROUND: Interleukin (IL)-17A is involved in the pathogenesis of allergic rhinitis (AR). Increased expression of IL-17A is correlated with disease severity and nasal eosinophilia. However, the molecular mechanisms by which IL-17A contributes to T-helper 2 cytokine IL-13-driven pathology in AR remain unclear. We sought to obtain mechanistic insight into how IL-17A and IL-13 regulate the epithelial production of eotaxin-3 representing eosinophilic inflammation in AR. METHODS: Human nasal epithelial cells (HNECs) from AR patients were cultured and stimulated with IL-17A, IL-13, or IL-17A and IL-13. Phosphorylated signal transducer activator of transcription 6 (p-STAT6) and suppressor of cytokine signaling 1 (SOCS1) in HNECs were assayed using Western blotting. Immunocytochemistry was used to determine p-STAT6-positive expression in the cells. Eotaxin-3 expression in the cells and culture supernatants was evaluated using real-time polymerase chain reaction and enzyme-linked immunosorbent assays. RESULTS: Stimulation with IL-13 alone induced STAT6 phosphorylation and promoted p-STAT6 nuclear translocation, leading to eotaxin-3 production by HNECs. These effects were further enhanced by cotreatment with IL-13 and IL-17A, whereas IL-17A alone had no impact on STAT6 or eotaxin-3 expression. Incubation with IL-17A or IL-13 increased the level of SOCS1 protein in the cells, whereas the addition of IL-17A attenuated IL-13-induced SOCS1 expression. CONCLUSION: IL-17A potentiated IL-13-driven STAT6 activation through the downregulation of SOCS1 expression, leading to enhancement of eotaxin-3 production by HNECs. These factors contributed to eosinophilic inflammation in AR.

Histamine H4 receptor regulates Th2-cytokine profile through thymic stromal lymphopoietin in allergic rhinitis.

PURPOSE: Epithelial thymic stromal lymphopoietin (TSLP) promotes Th2 inflammatory responses through induction of OX40 ligand (OX40L) on dendritic cells in allergic rhinitis (AR). Emerging evidence supports the important role of histamine H4 receptor (H4R) in allergic inflammation. This study aimed to investigate the effects of H4R in Th2-cytokine profile mediated by TSLP in AR. METHODS: Human nasal epithelial cells (HNECs) from AR patients were stimulated with histamine in the presence or absence of H4R agonist (4-methylhistamine, 4-MH) and antagonist (NJ7777120, JNJ) or H1R agonist (2-pyridylethylamine). TSLP protein was measured by Western blotting and ELISA. To further elucidate the role of H4R in the in vivo situation of experimental AR, rats were sensitized and treated with JNJ or 4-MH. TSLP and OX40 ligand (OX40L) in the nasal mucosa were assayed by Western blotting. Th2 cytokines including interleukin-4, 5 and 13 in nasal lavage fluids were detected by ELISA. RESULTS: Histamine alone did not induce TSLP production by HNECs. The pre-incubation with 4-MH prior to histamine promoted TSLP expression, which was inhibited by the stimulation with JNJ prior to histamine and 4-MH. The pre-incubation with 2-pyridylethylamine before histamine stimulation had no impact on TSLP production. In AR rats, the levels of TSLP and OX40L protein were increased as well as Th2 cytokines, which was further up-regulated by 4-MH treatment, while JNJ treatment attenuated these effects. CONCLUSIONS: H4R activation induced TSLP production by HNECs, which up-regulated OX40L expression in the nasal mucosa of sensitized rats. These factors promoted Th2-cytokine profile in AR.

Simple strategy for sensitive detection of dopamine using CdTe QDs modified glassy carbon electrode.

BACKGROUND: Cellular and brain metabolism of dopamine can be correlated with a number of neurodegenerative disorders, our study was to explore a simple and efficient method to detect dopamine in real samples. METHODS: A new quantum dots (CdTe QDs) could be prepared using the hydrothermal method, the electrochemical biosensor was established by dropping CdTe QDs on the surface of glassy carbon electrode (GCE). RESULTS: The CdTe QDs/GCE exhibited the excellent electrochemical catalytic activity toward dopamine (DA) with good stability and high sensitivity in presence of interfering substances. The detection limit of DA was calculated by differential pulse voltammetry (DPV) as low as 0.3 µmol L-1 with a linear dynamic range of 1 µmol L-1 to 400 µmol L-1 . CONCLUSION: In this paper, the proposed electrochemical biosensor could be effectively used for the direct and rapid detection of DA in human serum and urine samples.

Ischemic or toxic injury: A challenging diagnosis and treatment of drug-induced stenosis of the sigmoid colon.

A 48-year-old woman was admitted with 15-mo history of abdominal pain, diarrhea and hematochezia, and 5-mo history of defecation difficulty. She had been successively admitted to nine hospitals, with an initial diagnosis of inflammatory bowel disease with stenotic sigmoid colon. Findings from computed tomography virtual colonoscopy, radiography with meglumine diatrizoate, endoscopic balloon dilatation, metallic stent implantation and later overall colonoscopy, coupled with the newfound knowledge of compound Qingdai pill-taking, led to a subsequent diagnosis of ischemic or toxic bowel disease with sigmoid colon stenosis. The patient was successfully treated by laparoscopic sigmoid colectomy, and postoperative pathological examination revealed ischemic or toxic injury of the sigmoid colon, providing a final diagnosis of drug-induced sigmoid colon stenosis. This case highlights that adequate awareness of drug-induced colon stenosis has a decisive role in avoiding misdiagnosis and mistreatment. The diagnostic and therapeutic experiences learnt from this case suggest that endoscopic balloon expansion and colonic metallic stent implantation as bridge treatments were demonstrated as crucial for the differential diagnosis of benign colonic stenosis. Skillful surgical technique and appropriate perioperative management helped to ensure the safety of our patient in subsequent surgery after long-term use of glucocorticoids.

Therapeutic experience of 289 elderly patients with biliary diseases.

AIM: To present clinical characteristics, diagnosis and treatment strategies in elderly patients with biliary diseases. METHODS: A total of 289 elderly patients with biliary diseases were enrolled in this study. The clinical data relating to these patients were collected in our hospital from June 2013 to May 2016. Patient age, disease type, coexisting diseases, laboratory examinations, surgical methods, postoperative complications and therapeutic outcomes were analyzed. RESULTS: The average age of the 289 patients with biliary diseases was 73.9 ± 8.5 years (range, 60-102 years). One hundred and thirty-one patients (45.3%) had one of 10 different biliary diseases, such as gallbladder stones, common bile duct stones, and cholangiocarcinoma. The remaining patients (54.7%) had two types of biliary diseases. One hundred and seventy-nine patients underwent 9 different surgical treatments, including pancreaticoduodenectomy, radical resection of hilar cholangiocarcinoma and laparoscopic cholecystectomy. Ten postoperative complications occurred with an incidence of 39.3% (68/173), and hypopotassemia showed the highest incidence (33.8%, 23/68). One hundred and sixteen patients underwent non-surgical treatments, including anti-infection, symptomatic and supportive treatments. The cure rate was 97.1% (168/173) in the surgical group and 87.1% (101/116) in the non-surgical group. The difference between these two groups was statistically significant (χ2 = 17.227, P < 0.05). CONCLUSION: Active treatment of coexisting diseases, management of indications and surgical opportunities, appropriate selection of surgical procedures, improvements in perioperative therapy, and timely management of postoperative complications are key factors in enhancing therapeutic efficacy in elderly patients with biliary diseases.

Dissecting Solidago canadensis-soil feedback in its real invasion.

The importance of plant-soil feedback (PSF) has long been recognized, but the current knowledge on PSF patterns and the related mechanisms mainly stems from laboratory experiments. We aimed at addressing PSF effects on community performance and their determinants using an invasive forb Solidago canadensis. To do so, we surveyed 81 pairs of invaded versus uninvaded plots, collected soil samples from these pairwise plots, and performed an experiment with microcosm plant communities. The magnitudes of conditioning soil abiotic properties and soil biotic properties by S. canadensis were similar, but the direction was opposite; altered abiotic and biotic properties influenced the production of subsequent S. canadensis communities and its abundance similarly. These processes shaped neutral S. canadensis-soil feedback effects at the community level. Additionally, the relative dominance of S. canadensis increased with its ability of competitive suppression in the absence and presence of S. canadensis-soil feedbacks, and S. canadensis-induced decreases in native plant species did not alter soil properties directly. These findings provide a basis for understanding PSF effects and the related mechanisms in the field conditions and also highlight the importance of considering PSFs holistically.