Comparison of metabolic rates of ropivacaine in cerebrospinal fluid as inferred from plasma concentrations between elderly patients and young patients.

BACKGROUND: With the aging of human society, more and more elderly patients have to undergo surgery and anesthesia. Clinical observations have indicated from time to time that spinal anesthesia in the elderly appears to last longer than in young people, although there is limited research in this area and the mechanism is unclear at present time. This research work is expected to help understand the decline of local anesthetic metabolism in cerebrospinal fluid of elderly patients so as to help them with precise anesthesia and rapid rehabilitation. METHODS: Twenty patients with spinal anesthesia in orthopedic lower limb surgery were selected to study the rate of drug metabolism in cerebrospinal fluid in two age groups, i.e.,18-30 years old and 75-90 years old. Ropivacaine in peripheral blood is used as a probe to reflect the speed of drug metabolism in cerebrospinal fluid. The contents of total Aß protein and hyaluronic acid in the cerebrospinal fluid were investigated as well. RESULTS: The equivalent dose of ropivacaine anesthetizes the elderly group for a longer time. The metabolism rate of ropivacaine in an elderly patient was slower than that of a young patient. No significant difference in total Aß protein between the two groups was observed while hyaluronic acid in the elderly group was significantly higher than that in the young group. CONCLUSIONS: This study shows that the dose of ropivacaine should be reduced when used for anesthesia in elderly patients. The cumulation of ropivacaine and HA appears to imitate the degeneration of central lymphatic circulation metabolism in elderly people.

Two chromosome-level genome assemblies of Rhodiola shed new light on genome evolution in rapid radiation and evolution of the biosynthetic pathway of salidroside.

Rhodiola L. is a genus that has undergone rapid radiation in the mid-Miocene and may represent a typic case of adaptive radiation. Many species of Rhodiola have also been widely used as an important adaptogen in traditional medicines for centuries. However, a lack of high-quality chromosome-level genomes hinders in-depth study of its evolution and biosynthetic pathway of secondary metabolites. Here, we assembled two chromosome-level genomes for two Rhodiola species with different chromosome number and sexual system. The assembled genome size of R. chrysanthemifolia (2n = 14; hermaphrodite) and R. kirilowii (2n = 22; dioecious) were of 402.67 and 653.62 Mb, respectively, with approximately 57.60% and 69.22% of transposable elements (TEs). The size difference between the two genomes was mostly due to proliferation of long terminal repeat-retrotransposons (LTR-RTs) in the R. kirilowii genome. Comparative genomic analysis revealed possible gene families responsible for high-altitude adaptation of Rhodiola, including a homolog of plant cysteine oxidase 2 gene of Arabidopsis thaliana (AtPCO2), which is part of the core molecular reaction to hypoxia and contributes to the stability of Group VII ethylene response factors (ERF-VII). We found extensive chromosome fusion/fission events and structural variations between the two genomes, which might have facilitated the initial rapid radiation of Rhodiola. We also identified candidate genes in the biosynthetic pathway of salidroside. Overall, our results provide important insights into genome evolution in plant rapid radiations, and possible roles of chromosome fusion/fission and structure variation played in rapid speciation.

Genomic data provide a robust phylogeny backbone for Rhodiola L. (Crassulaceae) and reveal extensive reticulate evolution during its rapid radiation.

The Tibetan Plateau and adjacent mountain regions (TP; including the Tibetan Plateau, Himalaya, Hengduan Mountains and Mountains of Central Asia) harbor great biodiversity, some lineages on which may have undergone rapid radiations. However, only a few studies have investigated the evolutionary pattern of such diversification in depth using genomic data. In this study, we reconstructed a robust phylogeny backbone of Rhodiola, a lineage that may have undergone rapid radiation in the TP, using Genotyping-by-sequencing data, and conducted a series of gene flow and diversification analyses. The concatenation and coalescent-based methods yield similar tree topologies, and five well-supported clades were revealed. Potential gene flow and introgression events were detected, both between species from different major clades and closely related species, suggesting pervasive hybridization and introgression. An initial rapid and later slowdown of the diversification rate was revealed, indicating niche filling. Molecular dating and correlation analyses showed that the uplift of TP and global cooling in the mid-Miocene might have played an important role in promoting the rapid radiation of Rhodiola. Our work demonstrates that gene flow and introgression might be an important contributor to rapid radiation possibly by quickly reassembling old genetic variation into new combinations.

Indeterminate pulmonary subsolid nodules in patients with no history of cancer: growing prediction, CT pattern, and pathological diagnosis.

PURPOSE We aimed to evaluate and compare the growth patterns among pathological types of inde- terminate subsolid nodules in patients without a history of cancer as observed on computed tomography (CT). METHODS This retrospective study included 77 consecutive patients with 80 indeterminate subsolid nod- ules on unenhanced thin-section CT. Subsolid nodules were classified into 2 growth pattern groups based on volume: growth (n = 35) and non-growth (n = 42). According to the pathologi- cal diagnosis, subsolid nodules were further subdivided into 3 groups: adenocarcinoma in situ (growth, n = 8 vs. non-growth, n = 22), minimally invasive adenocarcinoma (n = 14 vs. n = 15), and invasive adenocarcinoma (n=13 vs. n=5). Kaplan-Meier and Cox proportional hazards regres- sion analyses were performed to identify the risk factors for subsolid nodules growth. The CT findings of the 35 subsolid nodules in the growth group were compared among the 3 pathologi- cal groups. RESULTS In the growth group, the overall mean volume doubling time and mass doubling time (MDT) were 811.5 days and 616.5 days, respectively. Patient's age (odds ratio=1.041, P=.045) and CT subtype of non-solid nodule and part-solid nodule (odds ratio=3.430, P=.002) could predict subsolid nodule growth. The baseline volume, mass, and mean CT value were larger in the inva- sive adenocarcinoma group than in the adenocarcinoma in situ group (all P < .01). The shortest volume doubling time was observed in the invasive adenocarcinoma group, followed by the minimally invasive adenocarcinoma group and the adenocarcinoma in situ group. A shorter mass doubling time was observed in the minimally invasive adenocarcinoma group than in the adenocarcinoma in situ group (all P < .02). CONCLUSION As age increases, the risk of pulmonary subsolid nodule growth increases by 4% each year, and part-solid nodules have a 3 times higher risk of growth compared to non-solid nodules in patients with no history of cancer. Subsolid nodules with more aggressive pathological charac- teristics grow at a faster rate.

Large cystic-solid pulmonary hamartoma: A case report.

BACKGROUND: It now seems that all pulmonary hamartomas (PHs) are large cystic-solid lesions that are difficult to diagnose. However, few cases of large cystic-solid PHs have been reported. The present case report presents a large cystic-solid PH and provides a literature review of the imaging features, formation mechanism and histopathological basis of PHs. CASE SUMMARY: A 53-year-old woman with no clinical symptoms underwent a chest computed tomography (CT) examination at our hospital. Nonenhanced CT images revealed a large, flat tumor with multiple air-containing cysts in the left thoracic cavity and a cystic part confined to the medial side of the tumor; the solid part of the tumor showed abundant fat and lamellar soft tissue components. Multiple small blood vessels were detected in the solid part of the tumor on contrast-enhanced CT images. Given the large size of the lesion, the patient elected to undergo surgery. Histological examination revealed PH. A detailed review of the patient's CT imaging showed that the lesion had a small vascular pedicle to the left lower lobe, which was a clue to its lung tissue histological origin. According to immunohistochemical staining, the confined multiple air-containing cysts were caused by the entrapment of respiratory/alveolar epithelium. CONCLUSION: This case shows the imaging manifestations of a large PH. Heightened awareness of its formation mechanism and histopathological basis may alert radiologists to consider this diagnosis in their daily workflow.

Evaluation of ultralow-dose computed tomography on detection of pulmonary nodules in overweight or obese adult patients.

PURPOSE: To evaluate the accuracy of pulmonary nodule (PN) detection in overweight or obese adult patients using ultralow-dose computed tomography (ULDCT) with tin filtration at 100 kV and advanced model-based iterative reconstruction (ADMIRE). METHODS: Eighty-one patients with body mass indices of ≥25 kg/m2 were enrolled. All patients underwent low-dose chest CT (LDCT), followed by ULDCT. Two radiologists experienced in LDCT established the standard of reference (SOR) for PNs. The number, type, size, and location of PNs were identified in the SOR. Effective dose, objective image quality (IQ), and subjective IQ based on two radiologists' scores were compared between ULDCT and LDCT. The detection performances of radiologists based on ULDCT were calculated according to the nodule analyses. Logistic regression was used to test for independent predictors of PN detection sensitivity. RESULTS: Both the effective dose and objective IQ were lower for ULDCT than for LDCT (both p < 0.001). Both radiologists rated the subjective IQ of the overall IQ on ULDCT to be diagnostically sufficient. In total, 234 nodules (mean diameter, 3.4 ± 1.9 mm) were classified into 32 subsolid, 149 solid, and 53 calcified nodules according to the SOR. The overall sensitivity of ULDCT for nodule detection was 93.6%. Based on multivariate analyses, the nodule types (p = 0.015) and sizes (p = 0.013) were independent predictors of nodule detection. CONCLUSIONS: Compared with LDCT, ULDCT with tin filtration at 100 kV and ADMIRE could significantly reduce the radiation dose in overweight or obese patients while maintaining good sensitivity for nodule detection.

The incidence, risk factors and outcomes of acute kidney injury in critically ill patients undergoing emergency surgery: a prospective observational study.

BACKGROUND: Without sufficient evidence in postoperative acute kidney injury (AKI) in critically ill patients undergoing emergency surgery, it is meaningful to explore the incidence, risk factors, and prognosis of postoperative AKI. METHODS: A prospective observational study was conducted in the general intensive care units (ICUs) from January 2014 to March 2018. Variables about preoperation, intraoperation and postoperation were collected. AKI was diagnosed using the Kidney Disease: Improving Global Outcomes criteria. RESULTS: Among 383 critically ill patients undergoing emergency surgery, 151 (39.4%) patients developed postoperative AKI. Postoperative reoperation, postoperative Acute Physiology and Chronic Health Evaluation (APACHE II) score, and postoperative serum lactic acid (LAC) were independent risk factors for postoperative AKI, with the adjusted odds ratio (ORadj) of 1.854 (95% confidence interval [CI], 1.091-3.152), 1.059 (95%CI, 1.018-1.102), and 1.239 (95%CI, 1.047-1.467), respectively. Compared with the non-AKI group, duration of mechanical ventilation, renal replacement therapy, ICU and hospital mortality, ICU and hospital length of stay, total ICU and hospital costs were higher in the AKI group. CONCLUSIONS: Postoperative reoperation, postoperative APACHE II score, and postoperative LAC were independent risk factors of postoperative AKI in critically ill patients undergoing emergency surgery.

Design and synthesis of new indole containing biaryl derivatives as potent antiproliferative agents.

A new series of indole containing biaryl derivatives were designed and synthesized, and further biological evaluations of their antiproliferative activity against cancer cell lines (MGC-803 and TE-1 cells) were also conducted. Of these synthesized biaryls, compound 4-methyl-2-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)methyl)quinazoline (23) performed as the most potent antiproliferative agent that inhibited cell viability of MGC-803 cells with an IC50 value of 8.28 µM. In addition, investigation of mechanism exhibited that the compound 4-methyl-2-((5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)methyl)quinazoline (23) could inhibit the expression of c-Myc and glycolysis related proteins, decrease the ATP and lactate production, and further induce apoptosis by activating the AMP-activated protein kinase (AMPK) and p53 signaling pathways.

Targeted Antagonism of Vascular Endothelial Growth Factor Reduces Mortality of Mice with Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is associated with a mortality of 45%. Our previous research indicated that anti-vascular endothelial growth factor (VEGF) could maintain the normal structure and function of the respiratory barrier. However, systemic application of VEGF antagonists would lead to animal death. This study attempts to study the targeted drug delivery for ARDS. In this study, we used soluble fms-like tyrosine kinase-1 (sFlt)-targeted ultrasound microbubbles to antagonize the effect of VEGF on lung tissue. Ninety male BALB/c mice were randomly assigned to 6 groups: phosphate buffer saline (PBS) group (PBS+PBS); blank group (PBS+empty microbubbles); lipopolysaccharide (LPS) group (LPS+PBS); ARDS group (LPS+empty microbubbles); control group (PBS+sFlt microbubbles); and treatment group (LPS+sFlt microbubbles). After administration of LPS or PBS in the corresponding groups, the sFlt-targeted microbubbles or empty microbubbles were injected into the blood circulation. Then the lungs were irradiated with ultrasound, which ruptured the drug-loaded microbubbles and helped release drugs to the lung tissues targeted. The lung injury score, lung wet/dry ratio (W/D), liver and kidney functions, and the mortality of the mice in all groups were investigated at the predetermined time point. The difference in mortality between groups was examined by Fisher test. Other data were analyzed by one-way analysis of variance (ANOVA). A value of P<0.05 indicates that the difference was significant. The results showed that the PaO2 levels were normal in the PBS group, the blank group, and the control group. The LPS group and ARDS group showed significant hypoxia. PaO2 was improved significantly in the treatment group. The lung injury score and W/D were normal in the PBS group, the blank group, and the control group. The lung injury score and W/D increased significantly in the LPS group and ARDS group and decreased in the treatment group (P<0.05). The mortality rate of the ARDS model was 60% (95% confidence interval 47.5%-72.5%), and that with sFlt-targeted microbubbles was significantly lower at only 40% (95% confidence interval 27.5%-52.5%, P<0.05). It was concluded that anti-VEGF with sFlt targeted ultrasound microbubbles attenuated the lung injury and ultimately reduced the 7-day mortality effectively. It might be a suitable therapeutic tool for the treatment of ARDS.

2,4-Disubstituted quinazolines targeting breast cancer cells via EGFR-PI3K.

A series of novel 2,4-disubstituted quinazolines were synthesized and evaluated for their anti-tumor activity against five human cancer cells (MDA-MB-231, MCF-7, PC-3, HGC-27 and MGC-803) using MTT assay. Among them, compound 9n showed the most potent cytotoxicity against breast cancer cells. Compound 9n also significantly inhibited the colony formation and migration of MDA-MB-231 and MCF-7 cells. Meanwhile, compound 9n induced cell cycle arrest at G1 phase and cell apoptosis, as well as increased accumulation of intracellular ROS. Furthermore, compound 9n exerted anti-tumor effects in vitro via decreasing the expression of anti-apoptotic protein Bcl-2 and increasing the pro-apoptotic protein Bax and p53. Mechanistically, compound 9n markedly decreased p-EGFR and p-PI3K expression, which revealed that compound 9n targeted breast cancer cells via interfering with EGFR-PI3K signaling pathway. Molecular docking suggested that compound 9n could indeed bind into the active pocket of EGFR. All the findings suggest that compound 9n might be a valuable lead compound for anti-tumor agents targeting breast cancer cells.

The influence of glycemic status on the performance of cystatin C for acute kidney injury detection in the critically ill.

OBJECTIVE: Serum cystatin C (sCysC) used clinically for detecting early acute kidney injury (AKI) was reported to be independently associated with hemoglobin (HbA1c) levels, diabetes, and prediabetes. We aimed to assess the influence of HbA1c levels, diabetes, or prediabetes on the performance of sCysC for AKI detection in critically ill adults. METHODS: A prospective observational study was conducted in a mixed medical-surgical intensive care unit (ICU). Patients were divided into four quartiles based on levels of HbA1c or serum glucose at ICU admission, respectively. Additionally, patients were stratified into four subgroups according to HbA1c levels and history of diabetes, namely recognized diabetes (previous diagnosis of diabetes), unrecognized diabetes, prediabetes, and normal glycemic status. Comparisons were made using the area under the receiver operator characteristic curve (AUC) for AKI detection, and reassessed after patient stratification by above-mentioned glycemic status. RESULTS: Multivariable linear regression revealed that HbA1c levels and history of diabetes were positively related with sCysC (all p < .05). Although stratification for above-mentioned glycemic status displayed no significant difference between AUC of sCysC (all p > .05), sCysC yielded the highest AUCs for detecting AKI in diabetic patients. Moreover, higher optimal cutoff values of sCysC to detect AKI were observed in patients with versus without diabetes. CONCLUSION: Glycemic status has no significant impact on the accuracy of sCysC for AKI detection in critically ill adults and a higher optimal cutoff value of sCysC for AKI detection should be considered in diabetic patients.

Impact of thyroid function on cystatin C in detecting acute kidney injury: a prospective, observational study.

BACKGROUND: Cystatin C (Cys C) used clinically for detecting early acute kidney injury (AKI) was reported to be associated with thyroid function. Therefore, whether the performance of Cys C is affected by thyroid hormones has raised concern in critically ill patients. This study aimed to investigate the impact of thyroid hormones on the diagnostic and predictive accuracy of Cys C for AKI, and hence optimize the clinical application of Cys C. METHODS: A prospective observational study was conducted in the general intensive care units (ICUs). Serum creatinine (SCr), Cys C, and thyroid function were documented for all patients at ICU admission. Patients were separated into five quintiles based on free triiodothyronine (FT3) and total triiodothyronine (TT3), and two categories according to the presence of low T3 syndrome or not. The impact of thyroid function on the performance of Cys C in diagnosing and predicting AKI was assessed by area under the receiver operating characteristic curve (AUC). RESULTS: The AKI incidence was 30.0% (402/1339); 225 patients had AKI upon entry, and 177 patients developed AKI during the subsequent 7 days. The AUCs for Cys C in detecting total AKI, established AKI, and later-onset AKI was 0.753, 0.797, and 0.669, respectively. The multiple linear regression analysis demonstrated that TT3 and FT3 were independently associated with Cys C. Overall, although Cys C did not yield any significant difference in AUCs for detecting AKI among patients with different thyroid hormones, the optimal cut-off value of Cys C to detect AKI was markedly different between patients with and without low T3 syndrome. CONCLUSIONS: The thyroid function had no significant impact on the diagnostic and predictive accuracy of Cys C in detecting AKI in ICU patients. However, the optimal cut-off value of Cys C to detect AKI could be affected by thyroid function.

The Incidence, Risk Factors and Outcomes of Postoperative Acute Kidney Injury in Neurosurgical Critically Ill Patients.

We investigated the incidence, perioperative risk factors, and outcomes of postoperative acute kidney injury (AKI) in neurosurgical critically ill patients. A prospective multicenter cohort study was conducted, enrolling adult patients who underwent neurosurgical procedure and admitted to the neurosurgical intensive care units (ICU). Postoperative AKI was diagnosed within 7 days after surgery based on the Kidney Disease Improving Global Outcomes criteria. Of 624 enrolled patients, postoperative AKI occurred in 84 patients. AKI was associated with increased rates of ICU and in-hospital mortality, postoperative renal replacement therapy, postoperative tracheotomy, and postoperative tracheal reintubation. Patients who developed AKI had higher total ICU costs, prolonged length of hospital and ICU stay, and longer duration of postoperative mechanical ventilation. Multivariate analysis identified postoperative reoperation (adjusted odds ratio [OR] 5.70 [95% CI, 1.61-20.14]), postoperative concentration of serum cystatin C (adjusted OR 4.53 [95% CI, 1.98-10.39]), use of mannitol during operation (adjusted OR 1.97 [95% CI, 1.13-3.43]), postoperative APACHE II score (adjusted OR 1.11 [95% CI, 1.06-1.16]), and intraoperative estimated blood loss (adjusted OR 1.04 [95% CI, 1.00-1.08]) as independent risk factors for postoperative AKI. Postoperative AKI in neurosurgical critically ill cohort is prevalent and associated with adverse in-hospital outcomes.

Evaluation of clinically available renal biomarkers in critically ill adults: a prospective multicenter observational study.

BACKGROUND: Although serum cystatin C (sCysC), urinary N-acetyl-ß-D-glucosaminidase (uNAG), and urinary albumin/creatinine ratio (uACR) are clinically available, their optimal combination for acute kidney injury (AKI) detection and prognosis prediction remains unclear. We aimed to assess the discriminative abilities of these biomarkers and their possible combinations for AKI detection and intensive care unit (ICU) mortality prediction in critically ill adults. METHODS: A multicenter, prospective observational study was conducted in mixed medical-surgical ICUs at three tertiary care hospitals. One thousand eighty-four adult critically ill patients admitted to the ICUs were studied. We assessed the use of individual biomarkers (sCysC, uNAG, and uACR) measured at ICU admission and their combinations with regard to AKI detection and prognosis prediction. RESULTS: AUC-ROCs for sCysC, uNAG, and uACR were calculated for total AKI (0.738, 0.650, and 0.683, respectively), severe AKI (0.839, 0.706, and 0.771, respectively), and ICU mortality (0.727, 0.793, and 0.777, respectively). The panel of sCysC plus uNAG detected total and severe AKI with significantly higher accuracy than either individual biomarkers or the other two panels (uNAG plus uACR or sCysC plus uACR). For detecting total AKI, severe AKI, and ICU mortality at ICU admission, this panel yielded AUC-ROCs of 0.756, 0.863, and 0.811, respectively; positive predictive values of 0.71, 0.31, and 0.17, respectively; and negative predictive values of 0.81, 0.97, and 0.98, respectively. Moreover, this panel significantly contributed to the accuracy of the clinical models for AKI detection and ICU mortality prediction, as measured by the AUC-ROC, continuous net reclassification index, and incremental discrimination improvement index. The comparable performance of this panel was further confirmed with bootstrap internal validation. CONCLUSIONS: The combination of a functional marker (sCysC) and a tubular damage marker (uNAG) revealed significantly superior discriminative performance for AKI detection and yielded additional prognostic information on ICU mortality.

Tuberin activates and controls the distribution of Rac1 via association with p62 and ubiquitin through the mTORC1 signaling pathway.

Recent studies indicated that the tuberous sclerosis 2 (TSC2) gene product, tuberin, regulates Rac1 activity. However, the underlying mechanism by which tuberin regulates Rac1 activity has not been clearly elucidated to date. To better understand the molecular link between tuberin function and Rac1, we characterized the activity and distribution of Rac1 in mouse Tsc2-deficient renal tumor cells using restoration experiments with wild-type tuberin. Rac1 activity was significantly higher in tuberin-expressing cells compared with control Tsc2-deficient cells. Further, Rac1 activation was induced by rapamycin treatment or knockdown of raptor, but not rictor, in Tsc2-deficient cells, indicating that mTORC1 is an upstream negative regulator of Rac1. Intriguingly, Rac1 appeared to form cytoplasmic dots in Tsc2-deficient cells, but not in tuberin-expressing and since rapamycin treatment dispersed these dots, involvement of aberrant mTOR complex 1 (mTORC1) activation in the dot formation was suspected. Moreover, the dots were co-localized with p62/sequestosome-1 and ubiquitin. These findings imply that Rac1 distribution and/or its degradation may be regulated by tuberin through the mTORC1 signaling pathway.

Deficiency of the Erc/mesothelin gene ameliorates renal carcinogenesis in Tsc2 knockout mice.

Genetic crossing experiments were performed between tuberous sclerosis-2 (Tsc2) KO and expressed in renal carcinoma (Erc) KO mice to analyze the function of the Erc/mesothelin gene in renal carcinogenesis. We found the number and size of renal tumors were significantly less in Tsc2+/-;Erc-/- mice than in Tsc2+/-;Erc+/+ and Tsc2+/-;Erc+/- mice. Tumors from Tsc2+/-;Erc-/- mice exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen (Ki67) and TUNEL analysis, respectively. Adhesion to collagen-coated plates in vitro was enhanced in Erc-restored cells and decreased in Erc-suppressed cells with siRNA. Tumor formation by Tsc2-deficient cells in nude mice was remarkably suppressed by stable knockdown of Erc with shRNA. Western blot analysis showed that the phosphorylation of focal adhesion kinase, Akt and signal transducer and activator of transcription protein 3 were weaker in Erc-deficient/suppressed cells compared with Erc-expressed cells. These results indicate that deficiency of the Erc/mesothelin gene ameliorates renal carcinogenesis in Tsc2 KO mice and inhibits the phosphorylation of several kinases of cell adhesion mechanism. This suggests that Erc/mesothelin may have an important role in the promotion and/or maintenance of carcinogenesis by influencing cell-substrate adhesion via the integrin-related signal pathway.

Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product.

Recently, it was reported that the product of Birt-Hogg-Dubé syndrome gene (folliculin, FLCN) is directly phosphorylated by 5'-AMP-activated protein kinase (AMPK). In this study, we identified serine 62 (Ser62) as a phosphorylation site in FLCN and generated an anti-phospho-Ser62-FLCN antibody. Our analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK. By binding with FLCN-interacting proteins (FNIP1 and FNIP2/FNIPL), Ser62 phosphorylation is increased. A phospho-mimic mutation at Ser62 enhanced the formation of the FLCN-AMPK complex. These results suggest that function(s) of FLCN-AMPK-FNIP complex is regulated by Ser62 phosphorylation.

Regulation of folliculin (the BHD gene product) phosphorylation by Tsc2-mTOR pathway.

The Birt-Hogg-Dubé gene (BHD) encodes the tumor suppressor protein folliculin (FLCN). The function of FLCN has recently been implicated in the regulation of rapamycin-sensitive mTOR complex (mTORC1). Reciprocally, the mTORC1-dependent phosphorylation of FLCN was reported. However, precise mechanism of FLCN phosphorylation and functional interaction of FLCN with tuberin, the product of tuberous sclerosis 2 gene (TSC2) which is a negative regulator of mTORC1, are unclear. Here we report that multiple phosphorylation in FLCN are evoked by downregulation of tuberin as well as by Rheb expression. We found that phosphorylation at Ser62 and Ser302 are differently regulated by mTORC1-dependent pathway. Some unknown kinases downstream of tuberin-mTORC1 are thought to directly phosphorylate FLCN. Interestingly, our results also suggest that the complex formation of FLCN with AMPK is modulated by FLCN phosphorylation. These results suggest that FLCN is involved in a novel mechanism of signal transduction downstream of tuberin.

siRNA-mediated Erc gene silencing suppresses tumor growth in Tsc2 mutant renal carcinoma model.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery of siRNAs for stable treatment except short hairpin RNAs (shRNAs). On the other hand, there are many reports of systemic delivery of siRNAs for transient treatment using liposome carriers and others. With regard to shRNAs, a report showed fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Therefore, we decided to use original siRNA microspheres instead of shRNA for stable treatment of disease. In this study, we designed rat-specific siRNA sequences for Erc/mesothelin, which is a tumor-specific gene expressed in the Eker (Tsc2 mutant) rat model of hereditary renal cancer and confirmed the efficacy of gene silencing in vitro. Then, by using siRNA microspheres, we found that the suppression of Erc/mesothelin caused growth inhibition of Tsc2 mutant renal carcinoma cells in tumor implantation experiments in mice.