Phase-separated ParB enforces diverse DNA compaction modes and stabilizes the parS-centered partition complex.

The tripartite ParABS system mediates chromosome segregation in the majority of bacterial species. Typically, DNA-bound ParB proteins around the parS sites condense the chromosomal DNA into a higher-order multimeric nucleoprotein complex for the ParA-driven partition. Despite extensive studies, the molecular mechanism underlying the dynamic assembly of the partition complex remains unclear. Herein, we demonstrate that Bacillus subtilis ParB (Spo0J), through the multimerization of its N-terminal domain, forms phase-separated condensates along a single DNA molecule, leading to the concurrent organization of DNA into a compact structure. Specifically, in addition to the co-condensation of ParB dimers with DNA, the engagement of well-established ParB condensates with DNA allows for the compression of adjacent DNA and the looping of distant DNA. Notably, the presence of CTP promotes the formation of condensates by a low amount of ParB at parS sites, triggering two-step DNA condensation. Remarkably, parS-centered ParB-DNA co-condensate constitutes a robust nucleoprotein architecture capable of withstanding disruptive forces of tens of piconewton. Overall, our findings unveil diverse modes of DNA compaction enabled by phase-separated ParB and offer new insights into the dynamic assembly and maintenance of the bacterial partition complex.

The salt sensitivity of Drd4-null mice is associated with the upregulations of sodium transporters in kidneys.

To explore the mechanism of the hypertension in dopamine receptor-4 (Drd4) null mice, we determined the salt sensitivity and renal sodium transport proteins in Drd4-/- and Drd4+/+ mice with varied salt diets. On normal NaCl diet (NS), mean arterial pressures (MAP, telemetry) were higher in Drd4-/- than Drd4+/+; Low NaCl diet (LS) tended to decrease MAP in both strains; high NaCl diet (HS) elevated MAP with sodium excretion decreased and pressure-natriuresis curve shifted to right in Drd4-/- relative to Drd4+/+ mice. Drd4-/- mice exhibited increased renal sodium-hydrogen exchanger 3 (NHE3), sodium-potassium-2-chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC), and outer medullary α-epithelial sodium channel (αENaC) on NS, decreased NKCC2, NCC, αENaC, and αNa+-K+-ATPase on LS, and increased αENaC on HS. NKCC2, NCC, αENaC, and αNa+-K+-ATPase in plasma membrane were greater in Drd4-/- than in Drd4+/+ mice with HS. D4R was expressed in proximal and distal convoluted tubules, thick ascending limbs, and outer medullary collecting ducts and colocalized with NKCC2 and NCC. The phosphorylation of NKCC2 was enhanced but ubiquitination was reduced in the KO mice. There were no differences between the mouse strains in serum aldosterone concentrations and urinary dopamine excretions despite their changes with diets. The mRNA expressions of renal NHE3, NKCC2, NCC, and αENaC on NS were not altered in Drd4-/- mice. Thus, increased protein expressions of NHE3, NKCC2, NCC and αENaC are associated with hypertension in Drd4-/- mice; increased plasma membrane protein expression of NKCC2, NCC, αENaC, and αNa+-K+-ATPase may mediate the salt sensitivity of Drd4-/- mice.

Candesartan upregulates angiotensin-converting enzyme 2 in kidneys of male animals by decreased ubiquitination.

Candesartan is a common angiotensin-II receptor-1 blocker used for patients with cardiovascular and renal diseases. Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of blood pressure (BP), and also a major receptor for coronaviruses. To determine whether and how candesartan upregulates ACE2, we examined BP and ACE2 in multi-organs from male and female C57BL/6J mice treated with candesartan (1 mg/kg, i.p.) for 7 days. Relative to the vehicle, candesartan lowered BP more in males than females; ACE2 protein abundances were increased in kidneys, not lungs, hearts, aorta, liver, spleen, brain, or serum, only from males. Ace2-mRNA was similar in kidneys. Candesartan also decreased BP in normal, hypertensive, and nephrotic male rats. The renal ACE2 was increased by the drug in normal and nephrotic male rats but not spontaneously hypertensive ones. In male mouse kidneys, ACE2 was distributed at sodium-hydrogen-exchanger-3 positive proximal-convoluted-tubules; ACE2-ubiquitination was decreased by candesartan, accompanied with increased ubiquitin-specific-protease-48 (USP48). In candesartan-treated mouse renal proximal-convoluted-tubule cells, ACE2 abundances and activities were increased while ACE2-ubiquitination and colocalization with lysosomal and proteosomal markers were decreased. The silence of USP48 by siRNA caused a reduction of ACE2 in the cells. Thus, the sex-differential ACE2 upregulation by candesartan in kidney from males may be due to the decreased ACE2-ubiquitination, associated with USP48, and consequent degradation in lysosomes and proteosomes. This is a novel mechanism and may shed light on candesartan-like-drug choice in men and women prone to coronavirus infections.

DOPAMINE D4 RECEPTOR DOWN-REGULATES RENAL SODIUM CHLORIDE COTRANSPORTER VIA UBIQUITINATION-ASSOCIATED LYSOSOME DEGRADATION.

BACKGROUND: The thiazide-sensitive sodium chloride cotransporter (NCC) is the major apical sodium transporter located in the mammalian renal distal convoluted tubule (DCT). The amount of sodium reabsorbed in the DCT through NCC plays an important role in the regulation of extracellular fluid volume and blood pressure. Dopamine and its receptors constitute a renal antihypertensive system in mammals. The disruption of Drd4 in mice causes kidney-related hypertension. However, the pathogenesis of D4R-deficiency associated hypertension is not well documented. METHOD: We assessed the effects of D4R on NCC protein abundances and activities of DCT in mice with renal or global Drd4-deficiencies and expressing human D4.7 variant and in cultured mouse DCT cells, and explored the molecular mechanism. RESULTS: NCC inhibitor hydrochlorothiazide enhanced the natriuresis in Drd4-/- mice. Renal NCC protein was greater while ubiquitination of NCC was less in Drd4-/- than Drd4+/+ mice. Silencing of D4R in cultured mouse DCT cells increased NCC protein but decreased NCC ubiquitination. D4R agonist had opposite effects that were blocked by the antagonist. In mouse kidneys and DCT cells D4R and NCC colocalized and co-immunoprecipitated. Moreover, D4R-agonist promoted the binding between the two proteins demonstrated by fluorescence resonance energy transfer. D4R agonism internalized NCC, decreased NCC in the plasma membrane, increased NCC in lysosomes and reduced NCC-dependent-intracellular-sodium transport. The lysosomal inhibitor chloroquine prevented the D4R-induced NCC-reduction. A shortened NCC half-life was suggested by its decay under cycloheximide-chase. Ubiquitin-specific-protease 48 (USP48, a deubiquitinating enzyme) was increased in the kidneys and cells with Drd4-deficiency while D4R stimulation decreased it in vitro and reduction of USP48 with siRNA decreased NCC expression. The mice carrying human D4.7 variant or with renal supcapsular-Drd4-siRNA-delivery developed hypertension with increased NCC. CONCLUSION: Our data demonstrates that D4R downregulates NCC by promoting USP48-associated deubiquitination and subsequent internalization, lysosome relocation and degradation.

Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G-Quadruplex.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

M2: Mixed Models With Preferences, Popularities and Transitions for Next-Basket Recommendation.

Next-basket recommendation considers the problem of recommending a set of items into the next basket that users will purchase as a whole. In this paper, we develop a novel mixed model with preferences, popularities and transitions (M2) for the next-basket recommendation. This method models three important factors in next-basket generation process: 1) users' general preferences, 2) items' global popularities and 3) transition patterns among items. Unlike existing recurrent neural network-based approaches, M2 does not use the complicated networks to model the transitions among items, or generate embeddings for users. Instead, it has a simple encoder-decoder based approach (ed-Trans) to better model the transition patterns among items. We compared M2 with different combinations of the factors with 5 state-of-the-art next-basket recommendation methods on 4 public benchmark datasets in recommending the first, second and third next basket. Our experimental results demonstrate that M2 significantly outperforms the state-of-the-art methods on all the datasets in all the tasks, with an improvement of up to 22.1%. In addition, our ablation study demonstrates that the ed-Trans is more effective than recurrent neural networks in terms of the recommendation performance. We also have a thorough discussion on various experimental protocols and evaluation metrics for next-basket recommendation evaluation.

GYY4137, a H2S donor, ameliorates kidney injuries in diabetic mice by modifying renal ROS-associated enzymes.

Diabetic nephropathy (DN) is a common microvascular complication of both type 1 and type 2 diabetes mellitus and often advances to end-stage renal disease. Oxidative stress plays an important role in the pathogenesis and progress of DN. Hydrogen sulfide (H2S) is considered as a promising candidate for the management of DN. But the antioxidant effects of H2S in DN have not been fully studied. In mouse model induced by high-fat diet and streptozotocin, GYY4137, a H2S donor, ameliorated albuminuria at weeks 6 & 8 and decreased serum creatinine at week 8, but not hyperglycemia. Renal nitrotyrosine and urinary 8-isoprostane were reduced along with the suppressed levels of renal laminin and kidney-injury-molecule 1. Renal NADPH oxidase (NOX) 2 was lower but heme oxygenase (HO) 2, paraoxonase (PON) 1, PON2 were higher in DN+GYY than DN group. NOX1, NOX4, HO1, superoxide dismutases 1-3 were similar between groups. Except for a rise at HO2, all the affected enzymes were unchanged in mRNA levels. The affected reactive-oxygen-species (ROS) enzymes were mainly located in the renal sodium-hydrogen-exchanger positive proximal tubules with similar distribution but changed immunofluorence in GYY4137 treated DN mice. Kidney morphological alterations in DN mice under light and electrical-microscopes were also improved by GYY4137. Thus, exogenous H2S administration may improve the renal oxidative damage in DN by reducing ROS production and enhancing ROS cleavage in kidney via the affected enzymes. This study may shed a light on therapeutic applications in diabetic nephropathy with H2S donors in the future.

Renal CD81 interacts with sodium potassium 2 chloride cotransporter and sodium chloride cotransporter in rats with lipopolysaccharide-induced preeclampsia.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

A low-salt diet with candesartan administration is associated with acute kidney injury in nephritis by increasing nitric oxide.

A low-salt diet may activate the renin-angiotensin-aldosterone system (RAAS) and is often applied simultaneously with RAAS inhibitors, especially for treatment of proteinuric nephritis. To explore the effect of a low-salt diet combined with angiotensin receptor blockers (ARB) on kidney function, the proteinuric nephritis model was induced by single intravenous injection of doxorubicin, and then the SD rats were administrated with candesartan intraperitoneal injection and fed with different salt diets. Rats with low-salt plus candesartan, not either alone, experienced acute kidney injury (AKI) at day 7 and could not self-restore when extending the experiment time from 7 days to 21 days, unless switching low-salt to normal-salt. Among three nitric oxide synthetases (NOS), endothelial NOS (eNOS) was obviously elevated and PI3K-Akt-eNOS signal pathway was activated. NG-Nitro-L-Arginine Methyl Ester (L-NAME), an eNOS inhibitor, reversed the decreased blood pressure and recovered the kidney dysfunction induced by low-salt with candesartan. The increased TUNEL-positive cells, Bax/Bcl-2 and cleaved-caspase3 protein abundance was ameliorated by L-NAME in vivo. In vitro, sodium nitroprusside, a nitric oxide donor, can also increase Bax/Bcl-2 and cleaved-caspase3 protein level in HK-2 cell. Thus, low-salt diet combined with candesartan in nephritis rats led to AKI, and the mechanism involved the increase of eNOS/NO, which linked to the decrease of blood pressure and the increase of apoptosis. This study provides practical guidance for salt intake in cases of RAS inhibitor usage clinically.

Assessment of Urinary Exosomal NHE3 as a Biomarker of Acute Kidney Injury.

The diagnosis of acute kidney injury (AKI) traditionally depends on the serum creatinine (Scr) and urine output, which lack sufficient sensitivity and specificity. Using urinary exosomes as a biomarker has unique advantages. To assess whether urinary exosomal Na+/H+ exchanger isoform 3 (NHE3) protein could serve as a biomarker of AKI, we constructed four AKI rat models: cisplatin (7.5 mg/kg) injected intraperitoneally (IP), furosemide (20 mg/kg, IP) with a low-NaCl (0.03%) diet, a low-NaCl (0.03%) diet with candesartan (1 mg/kg, IP) and bilateral ischemia and reperfusion (I/R) injury for 40 min. Additionally, we assessed six sepsis-associated AKI patients and six healthy volunteers. Urinary exosomes were extracted by ultracentrifugation, and the NHE3 protein abundance was tested by immunoblotting for all the AKI rats and human subjects. The isolated cup-shaped particles with an average diameter of 70 nm and enrichment in CD63 were identified as exosomes. NHE3 abundance was six times higher in exosomes than in the whole urine. In cisplatin-induced AKI rats, urinary exosomal NHE3 was increased on day 2, one day earlier than the increases in Scr and blood urea nitrogen (BUN). In additional rats, urinary exosomal NHE3 decreased along with the decline in Scr after EPO pretreatment. In volume-depletion AKI induced by furosemide injection with a low-NaCl diet, the urinary exosomal NHE3 expression was higher than that in the control. Under a low-NaCl diet with candesartan-related AKI, the urinary exosomal NHE3 was elevated on day 5, earlier than Scr. In I/R-injury AKI, the urinary exosomal NHE3 was also raised compared with that in the control. In humans, the urinary exosomal NHE3 level was also elevated in sepsis-associated AKI patients in comparison with that in the healthy volunteers. The urinary exosomal NHE3 was increased in multiple AKI; it may be used as a diagnostic biomarker of AKI.

HAM: Hybrid Associations Models for Sequential Recommendation.

Sequential recommendation aims to identify and recommend the next few items for a user that the user is most likely to purchase/review, given the user's purchase/rating trajectories. It becomes an effective tool to help users select favorite items from a variety of options. In this manuscript, we developed hybrid associations models (HAM) to generate sequential recommendations. using three factors: 1) users' long-term preferences, 2) sequential, high-order and low-order association patterns in the users' most recent purchases/ratings, and 3) synergies among those items. HAM uses simplistic pooling to represent a set of items in the associations, and element-wise product to represent item synergies of arbitrary orders. We compared HAM models with the most recent, state-of-the-art methods on six public benchmark datasets in three different experimental settings. Our experimental results demonstrate that HAM models significantly outperform the state of the art in all the experimental settings. with an improvement as much as 46.6%. In addition, our run-time performance comparison in testing demonstrates that HAM models are much more efficient than the state-of-the-art methods. and are able to achieve significant speedup as much as 139.7 folds.

Improving information retrieval from electronic health records using dynamic and multi-collaborative filtering.

Due to the rapid growth of information available about individual patients, most physicians suffer from information overload and inefficiencies when they review patient information in health information technology systems. In this paper, we present a novel hybrid dynamic and multi-collaborative filtering method to improve information retrieval from electronic health records. This method recommends relevant information from electronic health records to physicians during patient visits. It models information search dynamics using a Markov model. It also leverages the key idea of collaborative filtering, originating from Recommender Systems, for prioritizing information based on various similarities among physicians, patients and information items. We tested this new method using electronic health record data from the Indiana Network for Patient Care, a large, inter-organizational clinical data repository maintained by the Indiana Health Information Exchange. Our experimental results demonstrated that, for top-5 recommendations, our method was able to correctly predict the information in which physicians were interested in 46.7% of all test cases. For top-1 recommendations, the corresponding figure was 24.7%. In addition, the new method was 22.3% better than the conventional Markov model for top-1 recommendations.

Glucose-induced decrease of cystathionine ß-synthase mediates renal injuries.

Exogenous hydrogen sulfide (H2 S) protects kidneys from diabetic injuries in animal models. In order to explore the role of endogenous H2 S in diabetic nephropathy, we determined the renal H2S producing enzymes in vivo and in vitro. In diabetic mice, H2 S levels in blood and kidney were decreased while cystathionine ß-synthase (CBS), mainly located in mouse renal proximal convoluted tubules (PCT), was reduced selectively. In cultured mouse PCT cells treated with high glucose, CBS protein and activity was reduced while ubiquitinated CBS was increased, which was abolished by a proteasome inhibitor MG132 at 1 hour; high glucose drove CBS colocalized with proteasome 26S subunit ATPase6, indicating an involvement of ubiquitination proteasome degradation. At 48 hours, high glucose also selectively decreased CBS protein, concentration-dependently, but increased the ubiquitination of CBS; silence of CBS by siRNA increased nitrotyrosine, a marker for protein oxidative injury. Nitrotyrosine was also increased by high glucose treatments. The increases of nitrotyrosine either by cbs-siRNA or by glucose were restored by GYY4137, indicating that the H2 S donor may protect kidney from oxidative injury induced by CBS deficiency. In diabetic kidneys, ubiquitinated CBS and nitrotyrosine were increased but restored by GYY4137. The treatment also ameliorated albuminuria and renal morphologic changes in diabetic mice. Our findings suggest that high glucose induces reduction of renal CBS protein and activity in vivo and in vitro that is critical to the pathogenesis of diabetic kidney disease.

Hybrid collaborative filtering methods for recommending search terms to clinicians.

With increasing and extensive use of electronic health records (EHR), clinicians are often challenged in retrieving relevant patient information efficiently and effectively to arrive at a diagnosis. While using the search function built into an EHR can be more useful than browsing in a voluminous patient record, it is cumbersome and repetitive to search for the same or similar information on similar patients. To address this challenge, there is a critical need to build effective recommender systems that can recommend search terms to clinicians accurately. In this study, we developed a hybrid collaborative filtering model to recommend search terms for a specific patient to a clinician. The model draws on information from patients' clinical encounters and the searches that were performed during them. To generate recommendations, the model uses search terms which are (1) frequently co-occurring with the ICD codes recorded for the patient and (2) highly relevant to the most recent search terms. In one variation of the model (Hybrid Collaborative Filtering Method for Healthcare, or HCFMH), we use only the most recent ICD codes assigned to the patient, and in the other (Co-occurrence Pattern based HCFMH, or cpHCFMH), all ICD codes. We have conducted comprehensive experiments to evaluate the proposed model. These experiments demonstrate that our model outperforms state-of-the-art baseline methods for top-N search term recommendation on different data sets.

Effect of miR-132 on bupivacaine-induced neurotoxicity in human neuroblastoma cell line.

BACKGROUND: Local anesthetics (LAs) may generate neurotoxicity in neurons. In the current study, we explored the mechanisms by which microRNA-132 (miR-132) regulated the neurotoxicity of human neuroblastoma cells (SH-SY5Y) induced by bupivacaine (BUP). METHODS: CCK-8, flow cytometry, EdU detection, qRT-PCR and western blotting were used to explore the cell viability, apoptosis and gene expression, respectively. RESULTS: In this study, we found that 600 µM BUP dramatically inhibited SH-SY5Y cells viability. In addition, BUP induced cell apoptosis and neurotoxicity via increasing active caspase-3 and cleaved PARP1 levels. More importantly, the level of miR-132 was significantly up-regulated in BUP-treated cells, which was significantly reversed by miR-132 inhibitor. In addition, dual-luciferase assay indicated IGF1R was the directly binding target of miR-132 in cells. Our study further indicated that the level of IGF1R was markedly decreased by BUP interference, while miR-132 inhibitor exerted the opposite effect. Furthermore, BUP induced apoptosis and neurotoxicity in SH-SY5Y cells were attenuated by IGF1, which further confirmed IGF1R was the downstream target of BUP in SH-SY5Y cells. CONCLUSION: In the present study, miR-132 played important roles in regulating BUP-induced neurotoxicity through IGF1R and may act as a promising molecular target for the treatment of human neurotoxicity induced by BUP.

Galectin-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin ß1-RhoA-JNK signaling activation.

Oxidized low-density lipoprotein (Ox-LDL)-induced endothelial cell injury plays a crucial role in the pathogenesis of atherosclerosis (AS). Plasma galectin-3 (Gal-3) is elevated inside and drives diverse systemic inflammatory disorders, including cardiovascular diseases. However, the exact role of Gal-3 in ox-LDL-mediated endothelial injury remains unclear. This study explores the effects of Gal-3 on ox-LDL-induced endothelial dysfunction and the underlying molecular mechanisms. In this study, Gal-3, integrin ß1, and GTP-RhoA in the blood and plaques of AS patients were examined by ELISA and western blot respectively. Their levels were found to be obviously upregulated compared with non-AS control group. CCK8 assay and flow cytometry analysis showed that Gal-3 significantly decreased cell viability and promoted apoptosis in ox-LDL-treated human umbilical vascular endothelial cells (HUVECs). The upregulation of integrinß1, GTP-RhoA, p-JNK, p-p65, p-IKKα, and p-IKKß induced by ox-LDL was further enhanced by treatment with Gal-3. Pretreatment with Gal-3 increased expression of inflammatory factors (interleukin [IL]-6, IL-8, and IL-1ß), chemokines(CXCL-1 and CCL-2) and adhesion molecules (VCAM-1 and ICAM-1). Furthermore, the promotional effects of Gal-3 on NF-κB activation and inflammatory factors in ox-LDL-treated HUVECs were reversed by the treatments with integrinß1-siRNA or the JNK inhibitor. We also found that integrinß1-siRNA decreased the protein expression of GTP-RhoA and p-JNK, while RhoA inhibitor partially reduced the upregulated expression of p-JNK induced by Gal-3. In conclusion, our finding suggests that Gal-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin ß1-RhoA-JNK signaling activation.

Effect of intraperitoneal or intracerebroventricular injection of streptozotocin on learning and memory in mice.

Alteration of behavior and PSD proteins in cerebral cortex and hippocampal synaptosome in the Alzheimer's disease (AD) mouse model were determined. AD was established by intraperitoneal injection of streptozotocin (STZ) in neonatal mice (intraperitoneal AD group) or intracerebroventricular injection of STZ in adult mice (intracerebroventricular AD group). Body weight and blood sugar level were measured. Following Morris water maze (MWM) test and fear-conditioning test, cerebral cortex and hippocampus tissues were collected and the levels of PSD95 and shank3 proteins in these tissues were measured by western blot analysis. The body weight was reduced and the blood sugar concentration was increased in the intraperitoneal AD group compared with the control group. In contrast, the body weight was reduced, while the blood sugar concentration was not increased in the intracerebroventricular AD group compared with the control group. Escape latency in both AD groups was extended compared with the control group. The freezing time in the intraperitoneal AD group was increased, while in the intracerebroventricular AD group, the freezing time was reduced. PSD95 and shank3 proteins in the cerebral cortex in both AD groups were decreased compared with the control group. PSD95 in the hippocampus was reduced in both AD groups compared with the control group. Shank3 in the hippocampus in the intracerebroventricular AD group was significantly reduced compared with the control group. Intraperitoneal injection of STZ in neonatal mice led to elevated blood sugar, impaired spatial memory and enhanced emotional memory when they become adults. In contrast, intracerebroventricular injection of STZ in adults directly led to deteriorated spatial and emotional memory without alteration of blood sugar content, which could be associated with the changes of PSD95 and shank3 proteins in hippocampus.

Efficacy of gefitinib­celecoxib combination therapy in docetaxel­resistant prostate cancer.

Resistance to docetaxel is a major clinical problem in castration­resistant prostate cancer (CRPC). We have previously reported that the combined inhibition of epidermal growth factor receptor (EGFR) and cyclooxygenase­2 (COX­2) led to an increased antitumor activity of docetaxel in CRPC. In the present study, we explored the efficacy of the combination of EGFR inhibition (by gefitinib) and COX­2 inhibition (by celecoxib) as a potential treatment for docetaxel­resistant CRPC. We established two docetaxel­resistant prostate cancer cell lines, PC3/DR and DU145/DR, by culturing PC3 and DU145 cells in docetaxel in a dose­escalating manner. The EGFR and COX­2 protein expression levels were determined. The effects of gefitinib and celecoxib on cell proliferation, apoptosis and invasion in vitro and in vivo were evaluated. In vitro changes in Bcl­2, FOXM1 and ABCB1 expression were analyzed. The expression of Ki­67 and cleaved­caspase­3 was also examined in DU145/DR tumor tissue. The enhanced expression of EGFR and COX­2 was observed in docetaxel­resistant CRPC relative to the parental cell lines. MTT, clone formation and fluorescence­activated cell sorting (FACS) analyses demonstrated that gefitinib and celecoxib in combination decreased cell viability and enhanced the rate of apoptosis when compared with either drug used alone. Additionally, the combination treatment was superior in inhibiting cell invasion and induced significant decreases in Bcl­2, FOXM1 and ABCB1 expression levels. Furthermore, the gefitinib­celecoxib combination inhibited DU145/DR tumor growth to a greater extent than either treatment used individually. The expression of Ki­67 was reduced, whereas cleaved­caspase­3 protein expression was increased in the tumors from the combination therapy group. In conclusion, the combined inhibition of EGFR and COX­2 by gefitinib and celecoxib may overcome docetaxel resistance in human CRPC. These findings provided a molecular basis for the clinical application of a novel combination therapy for docetaxel­resistant CRPC.

Pathological Role of Peptidyl-Prolyl Isomerase Pin1 in the Disruption of Synaptic Plasticity in Alzheimer's Disease.

Synaptic loss is the structural basis for memory impairment in Alzheimer's disease (AD). While the underlying pathological mechanism remains elusive, it is known that misfolded proteins accumulate as ß-amyloid (Aß) plaques and hyperphosphorylated Tau tangles decades before the onset of clinical disease. The loss of Pin1 facilitates the formation of these misfolded proteins in AD. Pin1 protein controls cell-cycle progression and determines the fate of proteins by the ubiquitin proteasome system. The activity of the ubiquitin proteasome system directly affects the functional and structural plasticity of the synapse. We localized Pin1 to dendritic rafts and postsynaptic density (PSD) and found the pathological loss of Pin1 within the synapses of AD brain cortical tissues. The loss of Pin1 activity may alter the ubiquitin-regulated modification of PSD proteins and decrease levels of Shank protein, resulting in aberrant synaptic structure. The loss of Pin1 activity, induced by oxidative stress, may also render neurons more susceptible to the toxicity of oligomers of Aß and to excitation, thereby inhibiting NMDA receptor-mediated synaptic plasticity and exacerbating NMDA receptor-mediated synaptic degeneration. These results suggest that loss of Pin1 activity could lead to the loss of synaptic plasticity in the development of AD.