The GluN2B-Containing NMDA Receptor Alleviates Neuronal Apoptosis in Neonatal Hypoxic-Ischemic Encephalopathy by Activating PI3K-Akt-CREB Signaling Pathwa.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a disease caused by insufficient blood supply in the brain in newborns during the perinatal period. Severe HIE leads to patient death, and patients with mild HIE are at increased risk of cognitive deficits and behavioral abnormalities. The NMDA receptor is an important excitatory receptor in the central nervous system, and in adult hypoxic-ischemic injury both subtypes of the NMDA receptor play important but distinct roles. The GluN2A-containing NMDA receptor (GluN2A-NMDAR) could activate neuronal protective signaling pathway, while the GluN2B-NMDAR subtype is coupled to the apoptosis-inducing signaling pathway and leads to neuronal death. However, the expression level of GluN2B is higher in newborns than in adults, while the expression of GluN2A is lower. Therefore, it is not clear whether the roles of different NMDA receptor subtypes in HIE are consistent with those in adults. We investigated this issue in this study and found that in HIE, GluN2B plays a protective role by mediating the protective pathway through binding with PSD95, which is quite different to that in adults. The results of this study provided new theoretical support for the clinical treatment of neonatal hypoxic ischemia.

[A single-center retrospective study of percutaneous drainage clinical characteristics of grade B and C postoperative pancreatic fistula and determination of the optimal intervention time].

Objective: To classified the fluid location of of grade B and C postoperative pancreatic fistula (POPF) and propose processing flow. Methods: Data from 232 patients who underwent pancreatic surgery from January 2018 to December 2022 at Department of General Surgery & Hepato-billo-pancreatic,Beijing Hospital were collected retrospectively. Forty-six patients who suffered from grade B and C POPF underwent ultrasound-guided drainage. There were 32 males and 14 females, with an age of (60.2±13.7)years (range:18 to 85 years). The imaging data of postoperative CT were collected and the the fluid location was classified. Then analyzed the drainage status when patents were diagnosed as POPF. Machine learning was performed and a random forest model was applied to construct the relationship between intervention time and mortality. The optimal intervention time was calculated. The patients were then divided into early and late intervention groups and clinical data and outcomes were compared using the t test,Mann-Whitney U test, χ2 test or Fisher's exact test between the two groups. Results: Based on the results of the random forest model, the optimal puncture time was within 5.38 days after the diagnosis of POPF. Based on the optimal time, 21 patients were subsumed into early intervention group and 25 patients were subsumed into late intervention group. The location of fluid collection was classified into four types: peripancreatic (32.7%,15/46), extra-pancreatic and epigastric (41.3%,19/46), extra-pancreatic and hypogastic (13.0%,6/46) and diffused (13.0%,6/46). The status of the drainage included normal in 10 patients (21.8%), displaced drain in 18 patients (39.1%) and drain removed or blocked in 18 patients (39.1%). The perioperative mortality rate was 19.0% (4/21) in the early intervention group and 8.0%(2/25) in the late. The late intervention group had significantly higher rates of positive drainage fluid cultures (88.0%(22/25) vs. 42.9%(10/21), χ2=10.584, P=0.001), secondary surgery (24.0%(6/25) vs. 0(0/21), P=0.025), and readmission within 90 days(32.0%(8/25) vs. 4.8%(1/21),χ2=5.381, P=0.020) than the early group, and a significantly longer postoperative hospital stay(M(IQR))(24(20)days vs. 39(53)days,Z=3.023,P=0.003). Conclusions: The location of the POPF fluid collection is classified into four types. Early radiological evaluation can detect abdominal effusion promptly,and early puncture and drainage will be beneficial in improving outcomes in these patents.

[Analysis of natural regression and influencing factors of HSIL in the cervix of childbearing age patients].

Objective: To investigate the natural regression and related factors of high-grade squamous intraepithelial lesion (HSIL) in the cervix of childbearing age women, and to evaluate the applicability of conservative management for future fertility needs. Methods: This study included 275 patients of reproductive age with fertility needs, who were diagnosed as HSIL by biopsy from April 30, 2015 to April 30, 2022, including 229 cases (83.3%) cervical intraepithelial neoplasia (CIN) Ⅱ and 46 cases (16.7%) CIN Ⅱ-Ⅲ. They were followed-up without immediate surgery in the First Affiliated Hospital of Nanjing Medical University. The median follow-up time was 12 months (range: 3-66 months). The regression, persistence and progression of lesions in patients with HSIL were analyzed during the follow-up period, the influencing factors related to regression and the time of regression were analyzed. Results: (1) Of the 275 HSIL patients, 213 cases (77.5%, 213/275) experienced regression of the lesion during the follow-up period. In 229 CIN Ⅱ patients, 180 cases (78.6%) regressed, 21 cases (9.2%) persisted, and 28 cases (12.2%) progressed. In 46 CIN Ⅱ-Ⅲ patients, 33 cases (71.7%) regressed, 12 cases (26.1%) persisted, and 1 case (2.2%) progressed to invasive squamous cell carcinoma stage Ⅰ a1. There was no significant difference in the regression rate between the two groups (χ2=1.03, P=0.309). (2) The average age at diagnosis, age <25 years old at diagnosis were independent influencing factor of HSIL regression in univariate analysis (all P<0.05). There was no significant difference between HSIL regression and pathological grading, the severity of screening results, human papillomavirus (HPV) genotype, colposcopy image characteristics, number of biopsies during follow-up and pregnancy experience (all P>0.05). (3) The median regression times for patients aged ≥25 years and <25 years at diagnosis were 15 and 12 months, respectively. Kaplan-Meier analysis showed that age ≥25 years at diagnosis significantly increased the median regression time compared to <25 years (χ2=6.02, P=0.014). Conclusions: For HSIL patients of childbearing age, conservative management without immediate surgical intervention is preferred if CINⅡ is fully evaluated through colposcopy examination. Age ≥25 years at diagnosis is a risk factor affecting the prognosis of HSIL patients.

[Relationship between matrix Gla protein and clinical features of ulcerative colitis].

Objective: To analyze the relationship between matrix Gla protein(MGP) and clinical characteristics of patients with ulcerative colitis (UC). Methods: Fifty-one UC patients who were admitted to the gastroenterology department of Peking union medical college hospital from July 1, 2015 to May 31, 2017 were included. Twenty-seven healthy subjects in the same period were included as normal controls. The expression of MGP mRNA in the colonic mucosa was detected by real-time fluorescence quantitative PCR. Clinical data of the patients were collected during a 2-year follow-up. The public data set containing MGP gene expression profile of UC patients was downloaded from the GEO database, and was divided into four groups according to the microscopic Mayo score. The differential expression of MGP in each group was analyzed. Results: All the fifty-one UC patients were followed up. The expression of MGP mRNA in the colonic mucosa of UC patients treated with hormone and immunosuppressive agents or biological agents or surgery was higher than that of patients treated with mesalazine. MGP mRNA expression was positively correlated with C-reactive protein level. It was also higher in the colonic mucosa of UC patients with clostridium difficilis or cytomegalovirus infection than that of patients without opportunistic infection. The difference of MGP mRNA expression between groups in GEO public data set was statistically significant(P<0.01), showing an up-regulation trend with the aggravation of inflammation. The expression level of MGP was moderately correlated with the microscopic Mayo score. The relationship between MGP mRNA expression and lifestyle, lesion range, erythrocyte sedimentation rate, parenteral manifestations and recurrence frequency of UC patients was not obvious. Conclusions: MGP is associated with colonic inflammation and its abnormal expression can help to predict the disease activity of patients with UC.

[Evaluation of the effect of sleep on inflammatory bowel disease patients based on propensity score matching].

Objective: To evaluate the sleep status and the correlation of sleep status with inflammatory bowel disease (IBD). Methods: A case-control study was carried out, including 568 IBD patients including 188 ucerative colitis (UC) patients and 380 Crohn's disease (CD) patients at 36 hospitals from January 1, 2018 to August 31, 2019, and 671 family members and healthy controls from the same cities. The survey consisted of three parts including general demographic data, clinical characteristics and sleep related factors by questionnaires. The items for sleep related factor included sleep duration on work days, sleep duration on weekends, sleep quality, sleep adequacy, snoring, sleep apnea and sleep hand foot movement. R language was used for propensity score and SPSS was used for statistical analysis. Results: The proportion of poor sleep quality before onset in UC and CD was higher than that in healthy control group (CD: 14.5% vs 5.3%, P<0.001; UC: 15.1% vs 5.4%, P<0.001). In CD group, the proportion of snoring before onset (31.3% vs 44.3%, P<0.001) and apnea (0.9% vs 5.5%, P=0.001) was lower than that in heathy control group. The proportion of athetosis in CD was higher than that in healthy control group (35.4% vs 28.9%, P=0.045). Conclusion: Patients with UC and CD have poor sleep quality before the onset of the disease, especially in CD patients.

[Analysis on clinical features of patients with ulcerative colitis associated colorectal cancer].

Objective: To analyze the clinical features of ulcerative colitis associated colorectal cancer (UC-CRC). Methods: A total of 869 inpatients with Ulcerative Colitis (UC) in Peking Union Medical Hospital from January 1998 to January 2018 were continuously enrolled. Clinical data and the outcome of colorectal cancer (CRC) were collected via medical records and telephone follow-up. Chi-square test and logistic regression model were used to analyze the data. Results: There were 16 patients in 869 UC inpatients who were diagnosed with CRC during a period of 7 548 person years and the incidence rate of UC-CRC was 1.84%. Compared to UC inpatients without CRC, a longer course of disease (OR=1.087, 95% CI:1.046-1.129) , a lower usage rate of 5-Aminosalicylic Acid(5-ASA) (OR=0.218, 95% CI:0.052-0.915) and a higher incidence rate of intestinal stenosis (OR=16.533, 95% CI:3.824-71.478) were found in UC inpatients with CRC. Conclusions: A long disease course is a risk factor for UC patients developing CRC, while 5-ASA therapy can reduce the risk of suffering from CRC. For UC patients with intestinal stenosis, CRC should be warned for occurring.

Downregulation of miR-1294 associates with prognosis and tumor progression in epithelial ovarian cancer.

OBJECTIVE: Increasing evidence has revealed that microRNAs (miRNAs) act as key players in the regulation of tumor growth and metastasis in epithelial ovarian cancer (EOC). However, the clinical role and functional effects of miR-1294 in EOC remain unknown. PATIENTS AND METHODS: We examined the expression of miR-1294 in 69 cases of EOC tissues and cell lines by quantitative Real-time polymerase chain reaction (qRT-PCR). The associations of miR-1294 expression with clinicopathologic features and overall survival of EOC patients were analyzed. Biological functional effects of miR-1294 expression on cell growth were analyzed using Cell Counting Kit-8 (CCK8) assays and flow cytometry assays in vitro. RESULTS: In the present study, we identified that miR-1294 expression was lower in 76 specimens of EOC compared to adjacent normal tissues. Lower miR-1294 expression was related to FIGO stage, lymph node metastasis and shorter overall survival rate in EOC patients. Multivariate Cox analysis demonstrated that miR-1294 expression was an independent prognostic indicator of EOC patients. Gain function assays showed that miR-1294 overexpression inhibited cell proliferation and cell cycle progression in EOC. CONCLUSIONS: Our results indicated that miR-1294 acted as a prognostic biomarker and potential target of EOC treatment.

[A 3.0 T MRI study on the alterations of the volume and morphology of fifteen subcortical nucleus in patients with early post-stroke depression].

Objective: To investigate the alterations of the volumes and 3D shapes of fifteen subcortical nucleus in patients with post-stroke depression (PSD) and to explore the pathogenesis regularity and mechanism of early PSD. Methods: From 2015 to 2017, a total of 28 patients with PSD and 18 stroke patients without depression (PSND), 13 patients with depression (De) and 11 cases of healthy volunteers (NC) were enrolled to perform 3.0 T high resolution MRI.Computer automatic segmentation and vertex analysis were used to segment and measure the volume of bilateral nucleus accumbens, caudate nucleus, putamen, globus pallidus, thalamus, hippocampus, mygdale and brainstem. Results: The volume of bilateral nucleus accumbens and bilateral thalamus, left pallidum were different among groups with statistical difference (P<0.05). The nucleus volume of the PSD group was (415±128) mm(3) (L-Nac)/(303±90) mm(3) (R-Nac), (7 590±867) mm(3) (L-Th)/(7 459±905) mm(3) (R-Th), (1 675±328) mm(3) (L-Pa), which was smaller than that of PSND group (433±100) mm(3) /(307±88) mm(3), (7 999±961) mm(3) /(7 753± 955) mm(3), (1 790±286) mm(3) and other groups.The nuclei with significantly statistical differences between inter-group were found in following: between PSD group and NC group, right accumbens and bilateral thalamus (P<0.01); between PSD group and De group, right accumbens and right thalamus (P<0.001), left accumbens, left pallidum and left thalamus (P<0.01); between PSND group and NC group, right accumbens (P<0.05); between PSND group and De group, right accumbens (P<0.001), left accumbens and right thalamus (P<0.05). Significant differences in morphology changes of nuclei (P<0.05) by F test mainly located on the top and tail of right accumbens, the anterior and middle body of right caudate nucleus, the most part of bilateral thalamus, the ventromedial body of bilateral hippocampus, the anterior and body of left caudate nucleus, especially in left thalamus. Conclusion: PSD has abnormal volume and morphological structure of subcortical nuclei, which supports the role of subcortical structures changes in the pathophysiology and pathogenesis of early PSD.

Independent prognostic Factor of low-expressed LncRNA ZNF667-AS1 for cervical cancer and inhibitory function on the proliferation of cervical cancer.

OBJECTIVE: To investigate the expression of long non-coding RNA ZNF667-AS1 in cervical cancer and its effect on the proliferation of cervical cancer cell line, SiHa cells. MATERIALS AND METHODS: The expression level of ZNF667-AS1 from two microarray datasets (GSE63514 and GSE6791) and TCGA (The Cancer Genome Atlas) were selected to analyze the difference between cervical cancer tissues and normal cervical tissues with bioinformatics methods. Then, the prognosis of ZNF667-AS1 was calculated in TCGA. The expression of LncRNA ZNF667-AS1 in 30 normal cervical tissues and 60 cervical cancer tissue samples was explored using qRT-PCR. In addition, analysis of the clinical data found that the expression of LncRNA ZNF667-AS1 was correlated with the total survival, tumor size and International Federation of Gynecology and Obstetrics (FIGO) stage. At last, the proliferative ability was detected by CCK8 and colon formation assay. RESULTS: Search the relevant microarray datasets using the keywords "cervical cancer" and "GPL570" from the Gene Expression Omnibus (GEO) database. Afterwards, two microarray datasets (GSE63514 and GSE6791) were selected to analyze the differentially expressed genes in cervical cancer tissues and normal cervical tissues using bioinformatics methods. The results showed that the expression of LncRNA ZNF667-AS1 in cervical cancer was significantly lower than that in normal cervical tissues. 30 normal cervical tissues and 60 cervical cancer tissue samples were selected to extract total RNA for qRT-PCR experiment, and found that the expression of LncRNA ZNF667-AS1 in cervical cancer tissues was lower than that in normal cervical tissues, which was consistent with that of TCGA. Analysis of the clinical data found that the expression of LncRNA ZNF667-AS1 was correlated with the total survival, tumor size and FIGO stage. Compared with the negative control group, the proliferation ability and cell cloning ability of cells with over-expressed LncRNA ZNF667-AS1 were significantly decreased (p<0.001), indicating that overexpression of ZNF667-AS1 inhibited the proliferation of SiHa cells. CONCLUSIONS: Expression of LncRNA ZNF667-AS1 was significantly lower in cervical cancer tissues, and its expression was negatively correlated with the overall survival, tumor size and FIGO stage. ZNF667-AS1 inhibited the proliferation of cervical cancer cells and was expected to be the biomarker and potential therapeutic target for predicting cervical cancer and determining its prognosis.

[Clinical and anatomic study of preserving parathyroid specific adipose attachments in total thyroidectomy].

Objective: To discuss the anatomical characteristic and important role of specific adipose attachments in preserving parathyroid function in total thyroidectomy. Methods: Parathyroid glands of 91 cases underwent total thyroidectomy in Department of General Surgery, Beijing Hospital from January to November 2015 were observed prospectively. There were 19 male and 72 female patients, aging from 25 to 74 years with an average age of (52±12) years. Preoperative ultrasound examination, analysis of intraoperative high definition images, and postoperative pathological examination were used to detect the origins of the parathyroid micro vascular structures and the characteristics of parathyroid specific adipose attachments. Parathyroid specific adipose attachments preserving technique was used in all procedures. The results of parathyroid hormone (PTH), serum calcium and phosphorus were compared pre- and 1-month post-operatively. Paired t test was statistically used to analyze data. Results: Parathyroid micro vascular vessels were closely attached to parathyroid, and they were mainly originated from inside and (or) outside thyroid. Twenty patients received intraoperative high definition images analysis, 62 parathyroids were detected, in which 48 parathyroids (77.4%) had its specific adipose attachments. Lymphatic tissues could be distinguished from adipose tissue using carbon nanoparticles lymphatic tracer technique, which resulted in better preservation of parathyroid specific adipose attachments. There was no significant difference in PTH ((39±17) ng/L vs. (30±16) ng/L), serum calcium ((2.23±0.10) mmol/L vs. (2.20±0.14) mmol/L) and phosphorus ((1.27±0.20) mmol/L vs.(1.26±0.25) mmol/L) pre- and 1-month post-operatively (P>0.05). Conclusions: Preservation of parathyroid specific adipose attachments in total thyroidectomy could result in better protection of parathyroid micro vascular structure and function. It could be benefit to preservation of parathyroid in situ and its function recovery, thus may reduce the incidence of permanent postoperative hypoparathyroidism.

Modulation of photonic nanojets generated by microspheres decorated with concentric rings.

A novel design of decorating microsphere surface with concentric rings to modulate the photonic nanojet (PNJ) is investigated. By introducing the concentric ring structures into the illumination side of the microspheres, a reduction of the full width at half maximum (FWHM) intensity of the PNJ by 29.1%, compared to that without the decoration, can be achieved numerically. Key design parameters, such as ring number and depth, are analyzed. Engineered microsphere with four uniformly distributed rings etched at a depth of 1.2 µm and width of 0.25 µm can generate PNJ at a FWHM of 0.485 λ (λ = 400nm). Experiments were carried out by direct observation of the PNJ with an optical microscope under 405 nm laser illumination. As a result, shrinking of PNJ beam size of 28.0% compared to the case without the rings has been achieved experimentally. Sharp FWHM of this design can be beneficial to micro/nanoscale fabrication, optical super-resolution imaging, and sensing.

Exogenous NO depletes Cd-induced toxicity by eliminating oxidative damage, re-establishing ATPase activity, and maintaining stress-related hormone equilibrium in white clover plants.

Various nitric oxide (NO) regulators [including the NO donor sodium nitroprusside (SNP), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), the NO-synthase inhibitor N (G)-nitro-L-Arg-methyl ester (L-NAME), and the SNP analogues sodium nitrite/nitrate and sodium ferrocyanide] were investigated to elucidate the role of NO in white clover (Trifolium repens L.) plants after long-term (5 days) exposure to cadmium (Cd). A dose of 100 µM Cd stress significantly restrained plant growth and decreased the concentrations of chlorophyll and NO in vivo, whereas it disrupted the balance of stress-related hormones and enhanced the accumulation of Cd, thereby inducing reactive oxygen species (ROS) burst. However, the inhibition of plant growth was relieved by 50 µM SNP through its stimulation of ROS-scavenging compounds (ascorbic acid, ascorbate peroxidase, catalase, glutathione reductase, non-protein thiol, superoxide dismutase, and total glutathione), regulation of H(+)-ATPase activity of proton pumps, and increasing jasmonic acid and proline but decreasing ethylene in plant tissues. Even so, the alleviating effect of SNP on plant growth was counteracted by cPTIO and L-NAME and was not observed with SNP analogues, suggesting that the protective roles of SNP are related to the induction of NO. These results suggest that NO may improve the Cd tolerance of white clover plants by eliminating oxidative damage, re-establishing ATPase activity, and maintaining hormone equilibrium. Improving our understanding of the role of NO in white clover plants is key to expanding the plantations to various regions and the recovery of pasture species in the future.

p53 directly regulates αA- and ßA3/A1-crystallin genes to modulate lens differentiation.

It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the αA and ßA3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.

C-Jun terminal kinases play an important role in regulating embryonic survival and eye development in vertebrates.

The c-Jun N-terminal kinases (JNKs) constitute one of the three major types of mitogen-activated protein kinases. Previous studies showed that JNK mediates multiple signaling transduction pathways implicated in cell proliferation, differentiation, inflammation, stress response and apoptosis in mammals. In the present study, we use goldfish as a model system and demonstrate that JNK kinases are necessary to promote embryonic survival and regulate eye development in vertebrates. During goldfish development, JNK1 and JNK2 are expressed at every stage from cleavage to hatching larvae. JNK3 is turned on at the gastrulation stage and then expressed at similar level to that of JNK2. JNK1 activity remains slightly fluctuated during different developmental stages. Inhibition of JNK activity caused massive apoptosis of blastula cells and significant death of goldfish embryos, which are associated with altered expression of the anti-apoptotic regulator, Mcl-1 and the proapoptotic regulator, Bak. These results provide novel information regarding the mechanisms by which JNKs promote embryonic survival. In addition, the embryos that survived inhibition of JNK activity displayed severe phenotype in the eye with clear microphthalmia and lens coloboma. To confirm that the observed phenotype is derived from JNK activity deficiency, we expressed JNK dominant negative mutant (DNM-JNK) in goldfish. Expression of DNM-JNK also caused similar phenotypes with altered expression of pax-6, Sox-2 and ß-crystallin. Together, our results demonstrate that JNKs play important roles in promoting survival of vertebrate embryos and regulating development of vertebrate eye.

Protein serine/threonine phosphotase-1 is essential in governing normal development of vertebrate eye.

Protein serine/threonine phosphatase-1 (PP-1) is one of the key enzymes responsible for dephosphorylation in vertebrates. Protein dephosphorylation via PP-1 is implicated in many different biological processes including gene expression, cell cycle control, transformation, neuronal transmission, apoptosis, autophage and senescence. However, whether PP-1 directly controls animal development remains to be investigated. Here, we present direct evidence to show that PP-1 plays an essential role in regulating eye development of vertebrates. Using goldfish as a model system, we have shown the following novel results. First, inhibition of PP-1 activity leads to death of a majority of the treated embryos, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of each catalytic subunit of PP-1 with morpholino oligomers leads to partial (PP-lα knockdown) or complete (PP-lß or PP-lγ knockdown) death of the injected embryos. The survived embryos from PP-1α knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each subunit of PP-1 also causes death of majority of the injected embryos and leads to abnormal development of goldfish eye. Mechanistically, Pax-6 is one of the major downstream targets mediating the effects of PP-1 function since the eye phenotype in Pax-6 knockdown fish is similar to that derived from overexpression of PP-1. Together, our results for the first time provide direct evidence that protein phosphatase-1 plays a key role in governing normal eye formation during goldfish development.

The tumor suppressor p53 regulates c-Maf and Prox-1 to control lens differentiation.

The tumor suppressor p53 plays a key role in regulating apoptosis and cell cycle progression. In addition, p53 is implicated in control of cell differentiation in muscle, the circulatory system, ocular lens and various carcinoma tissues. However, the mechanisms by which p53 controls cell differentiation are not fully understood. Here we present evidence that p53 directly regulates c-Maf and Prox1, two important transcription factors controlling differentiation in the ocular lens. First, human and murine c-Maf and Prox1 gene promoters contain authentic p53 DNA binding sites. Second, p53 directly binds to the p53 binding sites found in the promoter regions. Third, exogenous p53 induces dose-dependent expression of the luciferase report gene driven by both c-Maf and Prox1 promoters, and p53 binds to both promoters in the ChIP assays. Fourth, in the in vitro differentiation model, knockdown of p53 significantly inhibits lens differentiation which is associated with downregulated expression of c-Maf and Prox1. Finally, in p53 knockout mice, the expression of c-Maf and Prox1 are significantly altered. Together, our results reveal that p53 regulates lens differentiation through modulation of two important transcription factors, c-Maf and Prox1, and through them p53 thus controls expression of various differentiation-related downstream crystallin genes.

IEX-1 targets mitochondrial F1Fo-ATPase inhibitor for degradation.

IEX-1 (Immediate Early response gene X-1) is a stress-inducible gene. It suppresses production of reactive oxygen species (ROS) and protects cells from apoptosis induced by a wide range of stimuli, but the underlying mechanism is not known. This study reveals that IEX-1 targets the mitochondrial F1Fo-ATPase Inhibitor (IF1) for degradation, resulting in acceleration of ATP hydrolysis, concomitant with reduction in ROS production. A prominent role for IF1 degradation in the function of IEX-1 was corroborated by siRNA-mediated gene silencing of IF1 that recapitulated the effects of IEX-1 on ATP hydrolysis and ROS production. Moreover, progressive C-terminal truncation studies demonstrated that IEX-1 interacted with the C terminus of IF1 and the interaction might render IF1 prone to degradation by an as yet unidentified mitochondrial protease. In support of a physiological importance of IEX-1 in the modulation of IF1 expression, gene-targeted deletion of IEX-1 stabilized IF1 and reduced mitochondrial F1Fo-ATPase activity in vivo. The altered activity of the F1Fo enzyme may account for a metabolic switch from oxidative phosphorylation toward glycolysis in IEX-1 deficient cells. Thus, IEX-1 deficient cells were more susceptible to glucose deprivation than wild type counterparts and displayed increased glucose uptake and lactate production in hypoxic conditions. The cells were also relatively refractory to oligomycin-mediated inhibition of ATP production. The studies offer novel insights into the primary role of IEX-1 in regulating a balance between energy provision and ROS production.

Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis.

In response to changes in the external environment cells must initiate a coordinated program of gene expression for them to adapt. IEX-1 (immediate early response gene X-1) is precisely regulated by multiple transcription factors among which p53, NF-kappaB/rel, Sp1 and c-Myc play central roles, to ensure rapid and transient expression of IEX-1 in cells under a variety of stress conditions. Overexpression of IEX-1 renders some cells sensitive to apoptosis and accelerates cell cycle progression, but reduces proliferation of other cells, whereas disruption of IEX-1 expression is associated with decreases in both apoptosis and cell cycle progression. In sharp contrast to in vitro studies, in vivo constitutive expression of IEX-1 prevents activated T cells but not B cells from apoptosis, as shown using IEX-1-transgenic mice that target IEX-1 expression specifically to lymphocytes driven by the Emu enhancer. The animals developed a lupus-like disease and subsequently a high incidence of T cell lymphomas when they aged, due to insufficient apoptosis of T cells. These varied effects of IEX-1 on cell death and cell cycle progression in a cell-context dependent fashion implicate that IEX-1 is involved in more than one signaling pathway, understanding of which will certainly improve our knowledge with respect to cancer biology, cell death and cell cycle regulation.

Mechanism of anesthetic action: oxygen pathway perturbation hypothesis.

Although more than 150 years have passed since the discovery of general anesthetics, precisely how they work remains a mystery. We propose a novel unitary mechanism of general anesthesia verifiable by experiments. In the proposed mechanism, general anesthetics perturb oxygen pathways in both membranes and oxygen-utilizing proteins, such that the availability of oxygen to its sites of utilization is reduced, which in turn triggers cascading cellular responses through oxygen-sensing mechanisms, resulting in general anesthesia. Despite the general assumption that cell membranes are readily permeable to oxygen, existing publications indicate that these membranes are plausible oxygen-transport barriers. The present hypothesis provides a unified framework for explaining phenomena associated with general anesthesia and experimental results on the actions of general anesthetics. If verified by experiments, the proposed mechanism also has other significant medical and biological implications.