Quercetin protects against LPS-induced lung injury in mice via SIRT1-mediated suppression of PKM2 nuclear accumulation.

The role of NOD-like receptor protein 3 (NLRP3)-mediated macrophages pyroptosis in acute lung injury (ALI) is well-established. Quercetin (Que) is a natural bioflavonoid compound with anti-inflammatory and antioxidative properties that reportedly inhibits the NLRP3 inflammasome in sepsis-induced organ dysfunctions such as ALI. However, the mechanism underlying the inhibitory effect of quercetin on NLRP3 activation remains unclear. In this study, we established an endotoxin-induced ALI mouse model with an in vitro LPS challenge. We demonstrated that the administration of quercetin could significantly reduce pulmonary injury and decrease the production of pro-inflammatory cytokines. Moreover, we found that quercetin could inhibit the activation of the NLRP3 inflammasome by suppressing the nuclear accumulation of PKM2 and increasing SIRT1 levels. Importantly, treatment with SRT1720 (a specific SIRT1 activator) could inhibit the nuclear accumulation of PKM2 and the activation of NLRP3. Besides, preventing PKM2 dimerization with ML265 yielded an anti-inflammatory effect, similar to findings observed for SRT1720. In addition, we found that SIRT1 silencing or inhibition by EX527 could increase NLRP3 activation and nuclear accumulation of PKM2 and override quercetin-mediated anti-inflammatory activity. These findings indicated that quercetin could downregulate NLRP3 inflammasome activation by inhibiting the nuclear accumulation of PKM2 and upregulating SIRT1 expression, expanding the treatment landscape for ARDS.

[Constructing an ecological security pattern in Shenzhen, China, by matching the supply and demand of ecosystem services]. / åŸºäºŽç”Ÿæ€ç³»ç»ŸæœåŠ¡ä¾›éœ€åŒ¹é çš„æ·±åœ³å¸‚ç”Ÿæ€å®‰å ¨æ ¼å±€æž„å»º.

Constructing an ecological security pattern by matching the supply and demand of ecosystem services is of great significance for Shenzhen, China. To determine the strategic positioning of sustainable development pioneers and benchmarks for human well-being in a pilot demonstration area of socialism with Chinese characteristics, we selected water yield, carbon sequestration, and recreational services to identifiy the integrated supply and demand status of ecosystem services in Shenzhen using the InVEST model, ecological supply-demand ratio, cell phone signalling data analysis, and circuit theory. An ecological security pattern based on the matching of supply and demand of ecosystem services was constructed. We further examined the impacts of different delimitation boundaries on the area and number of ecological sources. The results showed that the comprehensive ecological supply-demand ratio in Shenzhen generally showed a distribution pattern of abundant in the east and scarce in the west. Among them, water yield services met the demand in terms of quantity and spatial distribution, but carbon sequestration services can not. Recreational services met the demand in terms of quantity, but their spatial distribution was extremely uneven. We identified 25 ecological sources covering a total area of 347.62 km2 and 34 ecological corridors with a total length of 346.06 km. The ecological source area was large in the east and small in the west, while the length of the corridors showed the opposite distribution pattern. Different delineation boundaries had different effects on the area and number of ecological sources. Primary, secondary, and tertiary ecological source areas cover 347.62, 520.84, and 557.58 km2, respectively, showing a gradual increasing trend, the numbers of ecological sources were 25, 35, and 32, showing an increasing then decreasing trend. In Shenzhen, there were important ecological patches that were not included in the ecological control line, and some patches within the ecological control line had poor ecological sustainability. Targeted protection and management policies could be formulated based on such pattern. The results could provide spatial guidance for the delimitation of ecological management and control units in Shenzhen.

Low androgen level impairs erectile function of rat by regulating the Ng/CaN/AKT/eNOS pathway in penile corpus cavernosum.

BACKGROUND: The mechanism by which low androgen status inhibit erectile function has not yet been clearly elucidated. Neurogranin (Ng) is a Ca2+ -sensitive calmodulin binding protein that is expressed in endothelial cells and regulates eNOS function. OBJECTIVES: To investigate whether low androgen status inhibit erectile function by regulating the Ng/CaN/AKT/eNOS pathway in the penile cavernous tissue of rats. MATERIALS AND METHODS: Thirty-six 8-week-old male Sprague-Dawley rats were randomly divided into six groups as follows (n = 6): 4-week control group (4w-control), 4-week castration group (4w-cast), 4-week castration+testosterone replacement group (4w-cast+T), 8-week control group (8w-control), 8-week castration group (8w-cast), and 8-week castration+testosterone replacement group (8w-cast+T). Four weeks and eight weeks after surgery, the ratio of the maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) was examined. The level of NO and the expression of Ng, calcineurin (CaN), AKT, p-AKT(S473), eNOS, and p-eNOS(Ser1177) in the penile cavernous tissue of each group were determined. RESULTS: Ng and CaN were mainly expressed in the membrane and cytoplasm of endothelial cells and smooth muscle cells in the penile cavernous tissue of rats. The ICPmax/MAP and the concentration of NO in the cast group were significantly lower than those in the control group and cast+T replacement group (p < 0.01). The expression of Ng and the ratios of p-AKT/AKT and p-eNOS/eNOS in the penile cavernous tissue of rats in the cast group were significantly lower than those in the control group and cast+T replacement group (p < 0.01). The expression of CaN in the penile cavernous tissue of rats in the cast group was significantly increased compared with that in the control group and the cast+T replacement group (p < 0.01). CONCLUSION: Inhibiting the expression of Ng and subsequently upregulating the expression of CaN in the rat penile cavernous tissue was one of the upstream mechanisms of low androgen status inhibiting erectile function by inhibiting the AKT/eNOS signaling pathway.

Effectiveness and Safety of Tourniquet Application Strategies in Total Knee Arthroplasty: a Meta-analysis.

BACKGROUND: We conducted the present meta-analysis to assess the effectiveness and safety of long-duration tourniquet and short-duration tourniquet for patients with total knee arthroplasty (TKA). METHODS: Published randomized controlled trials (RCTs) were identified from the following electronic databases: PubMed, Embase, Web of Science and Cochrane Library from inception to January 1, 2021. Studies comparing long-duration tourniquet and short-duration tourniquet in total knee arthroplasty were included. Either a random-effects model or a fixed-effects model was used for meta-analysis depending on the heterogeneity. Statistical analysis was assessed using RevMan 5.3 software. RESULTS: A total of 8 RCTs with a total of 484 patients were eligible and ultimately included in the meta-analysis. Compared with patients in the long-duration tourniquet, those in the short-duration tourniquet were significantly correlated with postoperative drainage volume (WMD = -71.85, 95% CI - 76.52 to - 67.17, P = 0.31), but an increased risk of total blood loss (WMD = 68.13, 95% CI 5.15-131.12, P = 0.03) and intraoperative blood loss (WMD = 137.13, 95% CI 69.06-205.20, P < 0.00001). Two groups showed no significant difference in operation time, visual analogue scale score of knee in early, transfusion rate and incidence of deep venous thrombosis. CONCLUSIONS: Our meta-analysis suggested that the short-duration tourniquet in TKA significantly decreased the postoperative drainage volume, increased the total blood loss and intraoperative blood loss. However, it did not marked decrease the occurrence of transfusion rate. Although the incidence of deep venous thrombosis was reduced in some study, it was not statistically significant. Additional high quality RCTs are needed to identify the hypothesis. Therefore, we recommend prolonged use of tourniquets during TKA surgery.

[Network Meta-analysis of Chinese patent medicines of Ziziphi Spinosae Semen in treatment of primary insomnia].

To systematically evaluate the efficacy of oral Chinese patent medicines of Ziziphi Spinosae Semen in the treatment of primary insomnia by using network Meta-analysis. Nine databases(CNKI, Wanfang, VIP, CBM, PubMed, EMbase, Web of Science, Medline and Cochrane Library) were systematically and comprehensively undertaken to identify the literatures published from the establishment of each database to August, 2020. Randomized controlled trials(RCTs) on oral Chinese patent medicine of Ziziphi Spinosae Semen in the treatment of primary insomnia, either alone or in combination with conventional sedative hypnotics, were screened out according to inclusion criteria and exclusion criteria. Literature screening, data extraction and the evaluation of the risk of bias for the included studies were conducted independently by 2 researchers. Traditional Meta-analysis and Bayesian network Meta-analysis were then conducted with use of Stata 15.0 and R software. Finally, a total of 42 RCTs were included, involving 9 kinds of oral Chinese patent medicines and 4 196 patients. The results of Meta-analysis showed that(1) in terms of improving Pittsburgh sleep quality index scale score, the efficacy of the combination of drugs was significantly superior to that of sedative-hypnotics or most of Chinese patent medicines used alone, and Bailemian Capsules combined with sedative-hypnotics had the best effect; both the efficacy of Shenqi Wuweizi Tablets and Compound Zaoren Anshen Capsules alone were significantly superior to that of conventional sedative-hypnotics treatment, and Shenqi Wuweizi Tablets had the best effect.(2)In terms of safety, single use or combined use of Chinese patent medicine had a certain improvement as compared with conventional sedative-hypnotics treatment. Due to the large clinical heterogeneity, the studies could not be combined quantitatively, and no serious adverse reactions occurred in all patients enrolled in the study. The results showed that Chinese patent medicine of Ziziphi Spinosae Semen combined with conventional sedative-hypnotics could significantly improve the short-term sleep quality of patients with primary insomnia. In the comparison among single use of drugs, Shenqi Wuweizi Tablets had the largest possibility for best effect. However, the lack of evidences for international promotion, the influence of different types of sedative-hypnotics treatment and intervention time on the curative effect, and the patients' different acceptance of combined treatment should be considered before clinical application. Limited by the number and quality of the included studies, the above conclusions need to be further verified by more large sample-size and high-quality studies.

Cardiovascular complications of SARS-CoV-2 infection (COVID-19): a systematic review and meta-analysis.

Coronavirus Disease 2019 (COVID-19) originated in Wuhan, China in December 2019 and rapidly spread worldwide. Herein, we conducted a systematic review and meta-analysis to find the association between COVID-19 and cardiovascular complications. We conducted a systematic literature search of the PubMed and Embase databases from 01 December 2019 to 30 November 2020. We then statistically analyzed the incidence of cardiovascular complications in COVID-19 patients. We included 3044 confirmed COVID-19 cases from 12 studies. The most common cardiovascular complications in COVID-19 patients were myocardial injury (21.2%, 95% CI 12.3-30.0%) and arrhythmia (15.3%, 95% CI 8.4-22.3%), followed by heart failure (14.4%, 95% CI 5.7-23.1%) and acute coronary syndrome (1.0%, 95% CI 0.5-1.5%). The pooled incidence of heart failure, arrhythmia and myocardial injury in non-survivors were 47.8% (95% CI 41.4-54.2%), 40.3% (95% CI 1.6-78.9%) and 61.7% (95% CI 46.8-76.6%), respectively. Also, the data separately showed significantly higher incidence of heart failure and cardiac injury in non-survivors (relative risks = 5.13, 95% CI 2.46-10.7, Z = 4.36, P = 0.017) and (relative risks = 6.91, 95% CI 3.19-14.95, Z = 4.91, P = 0.009). Myocardial injury and arrhythmia were the most common complications in COVID-19 patients. Myocardial injury and heart failure were more common in patients who died, regardless of a history of cardiovascular disease. The incidence of heart failure and myocardial injury were higher in non-survivors compared to the survivors. Accordingly, in addition to basic support, cardiac reactions of patients with confirmed COVID-19 with or without underlying cardiovascular diseases should be closely monitored.

Weighted gene co-expression network analysis identified six hub genes associated with rupture of intracranial aneurysms.

Intracranial aneurysms (IAs) are characterized by localized dilation or ballooning of a cerebral artery. When IAs rupture, blood leaks into the space around the brain to create a subarachnoid hemorrhage. The latter is associated with a higher risk of disability and mortality. The aims of this study were to gain greater insight into the pathogenesis of ruptured IAs, and to clarify whether identified hub genes represent potential biological markers for assessing the likelihood of IA progression and rupture. Briefly, the GSE36791 and GSE73378 datasets from the National Center of Biotechnology Information Gene Expression Omnibus database were reanalyzed and subjected to a weighted gene co-expression network analysis to test the association between gene sets and clinical features. The clinical significance of these genes as potential biomarkers was also examined, with their expression validated by quantitative real-time PCR. A total of 14 co-expression modules and 238 hub genes were identified. In particular, three modules (labeled turquoise, blue, and brown) were found to highly correlate with IA rupture events. Additionally, six potential biomarkers were identified (BASP1, CEBPB, ECHDC2, GZMK, KLHL3, and SLC2A3), which are strongly associated with the progression and rupture of IAs. Taken together, these findings provide novel insights into potential molecular mechanisms responsible for IAs and they highlight the potential for these particular genes to serve as biomarkers for monitoring IA rupture.

Toll-like receptor-4 pathway as a possible molecular mechanism for brain injuries after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is known as an acute catastrophic neurological disease that continues to be a serious and significant health problem worldwide. The mechanisms contributing to brain injury after SAH remain unclear despite decades of study focusing on early brain injury (EBI) and delayed brain injury (DBI). Neuroinflammation is a well-recognized consequence of SAH and may be responsible for EBI, cerebral vasospasm, and DBI. Toll-like receptors (TLRs) play a crucial role in the inflammatory response by recognizing damage-associated molecular patterns derived from the SAH. TLR4 is the most studied Toll-like receptor and is widely expressed in the central nervous system (CNS). It can be activated by the extravasated blood components in myeloid differentiation primary response-88/Toll/interleukin-1 receptor-domain-containing adapter-inducing interferon-ß (MyD88/TRIF)-dependent pathway after SAH. Transcription factors, such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) and interferon regulatory factor (IRF), that regulate the expression of proinflammatory cytokine genes are initiated by the activation of TLR4, which cause the brain damage after SAH. TLR4 may therefore be a useful therapeutic target for overcoming EBI and DBI in post-SAH neuroinflammation, thereby improving SAH outcome. In the present review, we summarized recent findings from basic and clinical studies of SAH, with a primary focus on the biological characteristics and functions of TLR4 and discussed the mechanisms associated with TLR4 signaling pathway in EBI and DBI following SAH.

[Impacts of Shenzhen basic ecological control line on landscape ecological risk]. / 深圳市基本生态控制线对景观生态风险的影响.

The Shenzhen Basic Ecological Control Line (SZBECL) has been in operation for more than ten years since its implementation in 2005, which has a significant protective effect on the ecological space. Landscape ecological risk refers to the possible adverse consequences caused by the natural or human-induced interactions between landscape pattern and ecological processes. The scien-tific management of ecological risk is an important prerequisite for the sustainable development of human beings. Therefore, it is of great significance to explore the changes of landscape ecological risk inside and outside the SZBECL. Combining two indicators (ecological sensitivity and landscape vulnerability), we comprehensively evaluated the landscape ecological risk of Shenzhen in 2005 and 2015. The difference-in-difference method was used to explore the impacts of the SZBECL on the landscape ecological risk inside the line. Results showed that the landscape ecological risk in Shen-zhen presented a spatial structure of "high west and low east", which decreased by 13.5% during 2005 to 2015. Inside and outside the SZBECL, the declines of the landscape ecological risk were 0.7% and 14.4%, respectively. For the five subzones, declines were more significant outside the SZBECL, except for the eastern coastal zone. After controlling the effects of other variables, at the municipal level, there was 1.8% higher landscape ecological risk inside the line compared with the region outside the line. Furthermore, at the subzone level, there was 1.6%, 1.6%, 1.4%, and 1.9% higher landscape ecological risk inside the line in the central urban zone, the western coastal zone, the middle zone, and the eastern zone, respectively. There was no significant difference between the inside and outside of the line in the eastern coastal zone. With the implementation of SZBECL, habitat quality and landscape vulnerability inside the line were lowered, but NDVI and population density did not change.

Downregulation of long noncoding RNA H19 rescues hippocampal neurons from apoptosis and oxidative stress by inhibiting IGF2 methylation in mice with streptozotocin-induced diabetes mellitus.

The diabetes mellitus (DM)-induced reduction of neurogenesis in the hippocampus is consequently accompanied by cognitive decline. The present study set out to define the critical role played by long noncoding RNA H19 (lncRNA H19) in the apoptosis of hippocampal neurons, as well as oxidative stress (OS) in streptozotocin (STZ)-induced DM mice through regulation of insulin-like growth factor 2 (IGF2) methylation. The expression of lncRNA H19 in the hippocampal neurons and surviving neurons were detected. Hippocampal neurons were cultured and transfected with oe-H19, sh-H19, oe-IGF2, or sh-IGF2, followed by detection of the expressions of IGF2 and apoptosis-related genes. Determination of the lipid peroxide and glutathione levels was conducted, while antioxidant enzyme activity was identified. The IGF2 methylation, the binding of lncRNA H19 to DNA methyltransferase, and the binding of lncRNA H19 to IGF2 promoter region were detected. DM mice exhibited high expressions of H19, as well as a decreased hippocampal neurons survival rate. Higher lncRNA H19 expression was found in DM. Upregulated lncRNA H19 significantly increased the expression of Bax and caspase-3 but decreased that of Bcl-2, thus promoting the apoptosis of hippocampal neuron. Besides, upregulation of lncRNA H19 induced OS. LncRNA H19 was observed to bind specifically to the IGF2 gene promoter region and promote IGF2 methylation by enriching DNA methyltransferase, thereby silencing IGF2 expression. Taken together, downregulated lncRNA H19 reduces IGF2 methylation and enhances its expression, thereby suppressing hippocampal neuron apoptosis and OS in STZ-induced (DM) mice.

Expression profile analysis of differentially expressed genes in ruptured intracranial aneurysms: In search of biomarkers.

Intracranial aneurysms (IAs) result from the bulging of arterial walls secondary to several factors such as flow, vessel morphology, and genetics. Subarachnoid hemorrhage occurs when such walls rupture, leading to high disability and mortality. Despite numerous investigations pertaining to the relationship between geometric characteristics and IA rupture, only a few have obtained consistent results. This study aimed to further identify the potential genes associated with the pathogenesis of IAs, which may provide novel molecular biomarkers. We downloaded and reanalyzed six datasets, which were divided into four groups. IA walls and blood samples were screened for differentially expressed genes (DEGs); then, functional and pathway enrichment analyses were conducted. In total, 158 common DEGs were identified from Groups 1-3 and 396 genes (187 upregulated and 209 downregulated genes) were differentially expressed in Group 4. The functional analysis revealed that the DEGs were mainly associated with the major histocompatibility complex class II protein complex and antigen processing and presentation. Finally, we identified nine key genes, both in aneurysm tissue samples and blood samples, of which three were mostly associated with the progression and rupture of IAs. Bioinformatics was used to analyze the datasets of the ruptured IAs and identify potential biomarkers, which may provide information for the early detection and treatment of IAs.

Progress in Understanding the Relationship Between Circular RNAs and Neurological Disorders.

Circular RNAs (circRNAs), a type of endogenous noncoding RNAs distinct from linear forms, are produced by backsplicing events within genes. circRNAs are structurally stable, highly conserved molecules found widely in organisms, and display tissue-type and developmental-stage specific expression patterns, which reveal their significant regulatory functions in gene expression. Based on accumulating evidence, some circRNAs are now believed to be a class of competitive endogenous RNAs that regulate gene expression. For example, circRNAs may prevent microRNAs from inhibiting target RNAs acting as microRNA sponges, or interact with RNA binding proteins and thereby efficiently and post-transcriptionally regulate expression of the parental and other genes. In addition, an increasing number of studies have shown that circRNAs play important roles in the development and progression of neurological disorders. In this review, we provide a comprehensive overview on the biogenesis, characteristics, and functions of circRNAs. We also discuss the critical role of circRNAs in neurological disorders.

Long non-coding RNA H19 induces hippocampal neuronal apoptosis via Wnt signaling in a streptozotocin-induced rat model of diabetes mellitus.

Defects in hippocampal synaptic plasticity and disorders of memory and learning are the central nervous system complications of diabetes mellitus (DM). Here, we used a streptozotocin-induced rat DM model to investigate the effects of long non-coding RNA H19 (lncRNA H19) on learning and memory and apoptosis of hippocampal neurons, and the involvement of the Wnt signaling. Our data demonstrate that lncRNA H19 is highly expressed in rats with DM. Over-expression of lncRNA H19 increased positioning navigation latency in DM rats and decreased duration of space exploration. lncRNA H19 over-expression also increased hippocampal neuronal apoptosis and expression of Wnt3, ß-catenin, TCF-1, Bax, caspase-8 and caspase-3. By contrast, expression of GSK-3ß and Bcl-2 was suppressed in DM rats over-expressing lncRNA H19. These results suggest that lncRNA H19 induces hippocampal neuronal apoptosis via Wnt signaling, and that inhibition of lncRNA H19 may serve as a promising novel target for the treatment of cognitive decline in patients with DM.

Systematic review of randomized controlled trials of traditional Chinese medicine treatment of non-acute bronchial asthma complicated by gastroesophageal reflux.

OBJECTIVE: To assess the effectiveness and safety of Traditional Chinese Medicine (TCM) treatment of non-acute bronchial asthma complicated by gastroesophageal reflux. METHODS: We searched databases from MEDLINE, Cochrane Library, CNKI, VIP, CBM, Wanfang Data, and TCM Database Systems. All randomized, controlled trials (RTCs) of TCM treatment of non-acute asthma complicated by gastroesophageal reflux were included. Data were independently collected by two reviewers. The standards for assessing quality described in the Cochrane Handbook for Systematic Reviews of Interventions were used to evaluate articles. Meta-analyses were conducted using Rev- Man 5.0.17 software. Heterogeneity was assessed, and a corresponding effects model was used to merge and analyze results. Indexes used to evaluate curative effects were: clinical efficacy, symptom scores, pulmonary function values, and adverse incidents. Effectiveness was indicated using risk ratio (RR) or mean difference (MD), and 95% confidence intervals (CIs) were calculated. RESULTS: Six RCTs were included, involving 304 patients with non-acute asthma complicated by gastroesophageal reflux. The treatment groups received Chinese drugs alone or TCM combined with standard Western medical treatment, and the control groups received standard Western medical treatment alone. Standard Western medical treatment included anti-inflammatory drugs and bronchodilators for asthma, and drugs to promote gastric peristalsis and inhibit gastric acid production for gastroesophageal reflux. Methodological quality was low in all six RCTs. Two RCTs showed that clinical efficacy was higher in the treatment group than in the control group (RR: 1.43, 95%CI: 1.10 to 1.87 vs RR: 1.51, 95% CI: 1.09 to 2.08). One RCT showed that the asthma score was lowered more effectively in the treatment group than in the control group (MD:-1.10, 95% CI:-2.04 to-0.16). Two RCTs showed that the gastroesophageal reflux score was reduced more effectively in the treatment group than in the control group (RR:-3.70, 95% CI:-4.30 to 3.10 vs RR:-5.30, 95% CI:-6.32 to -4.28). One RCT showed that some pulmonary function values were improved more effectively in the treatment group than in the control group (P < 0.05). No differences were seen in the various indexes between groups in the other RCTs. No adverse reactions, dropout rates, or follow-up rates were reported in any of the RCTs. CONCLUSIONS: The clinical symptoms of non-acute asthma complicated by gastroesophageal reflux can be improved by some Chinese drugs. Curative effects can be increased by combining the use of TCM with Western medicine. Because of the small quantity and low quality of research reported to date, it is necessary to conduct further RCTs to confirm these results. The results of this systematic review indicate that the quality of future clinical trials should be improved by including larger patient numbers, correctly randomizing patients into study groups, using blinding methods to measure and assess outcomes, and using accepted indexes to evaluate curative effects.

Study on the drug selection law for treatment of chronic gastritis with spleen deficiency and stomach dryness by complex system entropy cluster.

OBJECTIVE: To study on Prof. GAO Zhong-ying's drug selection law for treatment of chronic gastritis with data mining technique. METHODS: The 407 medical records of chronic gastritis treated by Prof. GAO Zhong-ying were collected and the study on these drugs in the recipes was carried out with data mining method. Among them, the recipe composed of one drug was studied with frequency statistical method, correlativity between drug pairs with improved mutual information, correlativity among multi-drugs with complex system entropy cluster technique. RESULTS: In treatment of chronic gastritis by Prof. GAO Zhong-ying there were 30 drugs with a higher use frequency of over 38 times, 94 commonly-used drug pairs with correlation coefficient of over 0.05, 11 commonly-used drug core combinations. CONCLUSION: The results attained with data mining technique for studying experience of famous and old TCM physicians conform to the clinical practice and the method is of an important significance for summarization of famous and old TCM physicians' experiences.