The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.

Nanoscale Interaction of Endonuclease APE1 with DNA.

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA-APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.

Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons.

A homozygous mutation of the DNAJC6 gene causes autosomal recessive familial type 19 of Parkinson's disease (PARK19). To test the hypothesis that PARK19 DNAJC6 mutations induce the neurodegeneration of dopaminergic cells by reducing the protein expression of functional DNAJC6 and causing DNAJC6 paucity, an in vitro PARK19 model was constructed by using shRNA-mediated gene silencing of endogenous DANJC6 in differentiated human SH-SY5Y dopaminergic neurons. shRNA targeting DNAJC6 induced the neurodegeneration of dopaminergic cells. DNAJC6 paucity reduced the level of cytosolic clathrin heavy chain and the number of lysosomes in dopaminergic neurons. A DNAJC6 paucity-induced reduction in the lysosomal number downregulated the protein level of lysosomal protease cathepsin D and impaired macroautophagy, resulting in the upregulation of pathologic α-synuclein or phospho-α-synucleinSer129 in the endoplasmic reticulum (ER) and mitochondria. The expression of α-synuclein shRNA or cathepsin D blocked the DNAJC6 deficiency-evoked degeneration of dopaminergic cells. An increase in ER α-synuclein or phospho-α-synucleinSer129 caused by DNAJC6 paucity activated ER stress, the unfolded protein response and ER stress-triggered apoptotic signaling. The lack of DNAJC6-induced upregulation of mitochondrial α-synuclein depolarized the mitochondrial membrane potential and elevated the mitochondrial level of superoxide. The DNAJC6 paucity-evoked ER stress-related apoptotic cascade, mitochondrial malfunction and oxidative stress induced the degeneration of dopaminergic neurons via activating mitochondrial pro-apoptotic signaling. In contrast with the neuroprotective function of WT DNAJC6, the PARK19 DNAJC6 mutants (Q789X or R927G) failed to attenuate the tunicamycin- or rotenone-induced upregulation of pathologic α-synuclein and stimulation of apoptotic signaling. Our data suggest that PARK19 mutation-induced DNAJC6 paucity causes the degeneration of dopaminergic neurons via downregulating protease cathepsin D and upregulating neurotoxic α-synuclein. Our results also indicate that PARK19 mutation (Q789X or R927G) impairs the DNAJC6-mediated neuroprotective function.

(G2019S) LRRK2 causes early-phase dysfunction of SNpc dopaminergic neurons and impairment of corticostriatal long-term depression in the PD transgenic mouse.

Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive neuronal death of substantia nigra pars compacta (SNpc) dopaminergic cells. In the present study, we hypothesized that prior to a late-phase death of SNpc dopaminergic neurons, (G2019S) LRRK2 also causes an early-phase neuronal dysfunction of SNpc dopaminergic cells in the (G2019S) LRRK2 mouse. Eight to nine-month-old (G2019S) LRRK2 transgenic mice exhibited the symptom of hypoactivity in the absence of the degeneration of SNpc dopaminergic neurons or nigrostriatal dopaminergic terminals. Whole-cell current-clamp recordings of SNpc dopaminergic cells in brain slices demonstrated a significant decrease in spontaneous firing frequency of SNpc dopaminergic neurons of 8-month-old (G2019S) LRRK2 mice. Carbon fiber electrode amperometry recording using striatal slices showed that (G2019S) LRRK2 transgenic mice at the age of 8 to 9months display an impaired evoked dopamine release in the dorsolateral striatum. Normal nigrostriatal dopaminergic transmission is required for the induction of long-term synaptic plasticity expressed at corticostriatal glutamatergic synapses of striatal medium spiny neurons. Whole-cell voltage-clamp recordings showed that in contrast to medium spiny neurons of 8 to 9-month-old wild-type mice, high-frequency stimulation of corticostriatal afferents failed to induce long-term depression (LTD) of corticostriatal EPSCs in medium spiny neurons of (G2019S) LRRK2 mice at the same age. Our study provides the evidence that mutant (G2019S) LRRK2 causes early-phase dysfunctions of SNpc dopaminergic neurons, including a decrease in spontaneous firing rate and a reduction in evoked dopamine release, and impairment of corticostriatal LTD in the (G2019S) LRRK2 transgenic mouse.

Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons.

BACKGROUND: Several lines of evidence suggest that CCL2 could initiate the hyperalgesia of neuropathic pain by causing central sensitization of spinal dorsal horn neurons and facilitating nociceptive transmission in the spinal dorsal horn. The cellular and molecular mechanisms by which CCL2 enhances spinal pain transmission and causes hyperalgesia remain unknown. The substantia gelatinosa (lamina II) of the spinal dorsal horn plays a critical role in nociceptive transmission. An activated spinal microglia, which is believed to release pro-inflammatory cytokines including TNF-α, plays an important role in the development of neuropathic pain, and CCL2 is a key mediator for spinal microglia activation. In the present study, we tested the hypothesis that spinal CCL2 causes the central sensitization of substantia gelatinosa neurons and enhances spinal nociceptive transmission by activating the spinal microglia and augmenting glutamatergic transmission in lamina II neurons. METHODS: CCL2 was intrathecally administered to 2-month-old male rats. An intrathecal injection of CCL2 induced heat hyperalgesia, which was assessed using the hot plate test. Whole-cell voltage-clamp recordings substantia gelatinosa neurons in spinal cord slices were performed to record glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs). RESULTS: The hot plate test showed that 1 day after the intrathecal injection of CCL2 (1 µg), the latency of hind-paw withdrawal caused by a heat stimulus was significantly reduced in rats. One day after the intrathecal administration of CCL2, the amplitude of the evoked glutamatergic EPSCs and the frequency of spontaneous glutamatergic miniature EPSCs (mEPSCs) were significantly increased in outer lamina II neurons. Intrathecal co-injection of minocycline, a specific inhibitor of microglial activation, and CCL2 blocked the CCL2-induced reduction in the latency of hind-paw withdrawal and thermal hyperalgesia. Following intrathecal co-administration of CCL2 and minocycline, CCL2 failed to increase the frequency of glutamatergic mEPSCs and failed to promote glutamine release in lamina II neurons. Intrathecal co-injection of WP9QY, a selective TNF-α antagonist, and CCL2 completely inhibited CCL2-induced heat hyperalgesia and inhibited the increase in the frequency of glutamatergic mEPSCs in substantia gelatinosa neurons. CONCLUSION: In summary, our results suggest that an intrathecal injection of CCL2 causes thermal hyperalgesia by augmenting the excitatory glutamatergic transmission in substantia gelatinosa neurons through a presynaptic mechanism and facilitating nociceptive transmission in the spinal dorsal horn. Further studies show that intrathecal co-administration of minocycline, a specific inhibitor of microglial activation, or WP9QY, a selective TNF-α antagonist, completely inhibited CCL2 potentiation of glutamatergic transmission in substantia gelatinosa neurons and CCL2-induced heat hyperalgesia. The results of the present study suggest that peripheral nerve injury-induced upregulation of the spinal CCL2 level causes the central sensitization of substantia gelatinosa neurons by activating spinal microglia and that TNF-α mediates CCL2-induced thermal hyperalgesia and augmentation of glutamatergic transmission in lamina II neurons.

Tumor-stroma ratio is an independent predictor for survival in early cervical carcinoma.

OBJECTIVE: Tumor-stroma ratio (TSR) has recently been identified as an independent prognostic parameter for several solid tumors. The aim of the present study was to evaluate the prognostic role of TSR in early cervical cancer. METHODS: A cohort of 184 patients who had surgery for early stage cervical cancer (FIGO [International Federation of Gynecology and Obstetrics] stages IA2-IIA) were enrolled in this study. TSR was estimated on hematoxylin-eosin-stained tissue sections from the most invasive part of the primary tumor. Patients with less than 50% stroma were classified as stroma-poor and patients with ≥ 50% stroma were classified as stroma-rich. The relationship between TSR and survival time was statistically analyzed. RESULTS: The disease-free survival and overall survival rates were 88.44% and 92.52%, respectively, in the stroma-poor group, and 62.16% and 70.27%, respectively, in the stroma-rich group. Both the disease-free and overall survival rates in the stroma-poor group were significantly better than those in the stroma-rich group (p=0.001). In a multivariate analysis, TSR was further confirmed as a significant prognostic factor for disease-free survival (hazard ratio 3.125; p=0.005) and overall survival (hazard ratio 3.464; p=0.003), independent of tumor size, FIGO stage and lymph node metastasis. CONCLUSION: Our study identified that TSR was an independent prognostic factor of early cervical cancer. Patients with stroma-rich tumors had worse prognosis and higher risk of relapse compared with those with stroma-poor tumors. Considering its simplicity and availability for conventional clinical pathology, TSR may serve as a new prognostic histological characteristic in early cervical cancer.

A cloud system for mobile medical services of traditional Chinese medicine.

Many medical centers in Taiwan have started to provide Traditional Chinese Medicine (TCM) services for hospitalized patients. Due to the complexity of TCM modality and the increasing need for providing TCM services for patients in different wards at distantly separate locations within the hospital, it is getting difficult to manage the situation in the traditional way. A computerized system with mobile ability can therefore provide a practical solution to the challenge presented. The study tries to develop a cloud system equipped with mobile devices to integrate electronic medical records, facilitate communication between medical workers, and improve the quality of TCM services for the hospitalized patients in a medical center. The system developed in the study includes mobile devices carrying Android operation system and a PC as a cloud server. All the devices use the same TCM management system developed by the study. A website of database is set up for information sharing. The cloud system allows users to access and update patients' medical information, which is of great help to medical workers for verifying patients' identification and giving proper treatments to patients. The information then can be wirelessly transmitted between medical personnel through the cloud system. Several quantitative and qualitative evaluation indexes are developed to measure the effectiveness of the cloud system on the quality of the TCM service. The cloud system is tested and verified based on a sample of hospitalized patients receiving the acupuncture treatment at the Lukang Branch of Changhua Christian Hospital (CCH) in Taiwan. The result shows a great improvement in operating efficiency of the TCM service in that a significant saving in labor time can be attributable to the cloud system. In addition, the cloud system makes it easy to confirm patients' identity through taking a picture of the patient upon receiving any medical treatment. The result also shows that the cloud system achieves significant improvement in the acupuncture treatment. All the acupuncture needles now can be removed at the time they are expected to be removed. Furthermore, through the cloud system, medical workers can access and update patients' medical information on-site, which provides a means of effective communication between medical workers. These functions allow us to make the most use of the portability feature of the acupuncture service. The result shows that the contribution made by the cloud system to the TCM service is multi-dimensional: cost-effective, environment-protective, performance-enhancing etc. Developing and implementing such a cloud system for the TCM service in Taiwan symbolizes a pioneering effort. We believe that the work we have done here can serve as a stepping-stone toward advancing the TCM service quality in the future.

Genome-Wide Binding Analysis of DNA Repair Protein APE1 in Tumor Cells by ChIP-Seq.

The base excision repair (BER) is the primary damage repair pathway for repairing most of the endogenous DNA damage including oxidative base lesions, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs) in the genome. Repair of these damages in cells relies on sequential recruitment and coordinated actions of multiple DNA repair enzymes, which include DNA glycosylases (such as OGG1), AP-endonucleases (APE1), DNA polymerases, and DNA ligases. APE1 plays a key role in the BER pathway by repairing the AP sites and SSBs in the genome. Several methods have been developed to generate a map of endogenous AP sites or SSBs in the genome and the binding of DNA repair proteins. In this chapter, we describe detailed approaches to map genome-wide occupancy or enrichment of APE1 in human cells using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). Further, we discuss standard bioinformatics approaches for analyzing ChIP-seq data to identify APE1 enrichment or binding peaks in the genome.

Using a Flipped Classroom to Compare 2 Ultrasonography Operating Methods to Improve Practice in Ultrasound Detection Acupuncture.

Objective: Ultrasound (US) detection acupuncture (UDA) is an innovative acupuncture technique that uses ultrasonography (USG) to detect the depth of the lung before performing acupuncture on the points around the chest to avoid puncturing the lungs. For acupuncturists to use UDA appropriately, it is crucial to have a good operating method to identify the pleura with USG. This study compared 2 US operating methods through active learning in a "flipped classroom" setting for acupuncture students. Materials and Methods: Students and interns were recruited to complete the UDA flipped classroom course and evaluate the operations of 2 US methods on either of 2 simulation models: (1) a single B-mode or (2) a combined M-mode + B-mode. Participants were interviewed and satisfaction surveys were administered to obtain feedback. Results: A total of 37 participants completed the course and evaluations. The combined mode had better measurement accuracy, acupuncture safety, and operating time (P < 0.05), and no pneumothoraxes occurred. Among both participant groups, the combined mode allowed the student group to learn quickly and the intern group to become more proficient. Both interviews and satisfaction surveys yielded positive feedback. Conclusions: Using a combined mode for UDA can improve its performance greatly. The combined mode is definitely helpful for learning and promotion of UDA.

Reflex Auriculo-Cardiac (RAC) Induced by Auricular Laser and Needle Acupuncture: New Case Results Using a Smartphone.

The reflex auriculo-cardiac (RAC), dynamic pulse reaction (Nogier reflex), or vascular autonomic signal was proposed by Nogier. It refers to the pulse changes that can occur in the radial artery immediately after auricular acupuncture is performed. RAC is helpful for the clinical practice of auricular acupuncture, but there is a lack of objective verification methods. Photoplethysmography (PPG) has been used to objectively calculate radial artery blood flow. This study used PPG via a smartphone to measure RAC induced by auricular acupuncture. Thirty subjects without major diseases were recruited to receive traditional needle and laser acupuncture. The Shen Men ear point and control points were stimulated for 20 s. PPG was continuously measured during the acupuncture. The PPG data were tested for differences with a paired t-test. The results showed that there were no statistical differences in the frequency and amplitude of PPG obtained before and after acupuncture, either with a traditional needle or laser acupuncture. However, interestingly, it was found that one patient with insomnia, one patient with viral respiratory symptoms, and two menstruating females exhibited changes in PPG within five seconds of needle placement. We hypothesized that RAC might be induced by auricular acupuncture and could be quantified by PPG, even among subjects suffering from mild diseases; however, auricular acupuncture might not induce a measurable RAC in totally healthy subjects.

Radial arterial waves for chemotherapy- and radiotherapy-related myocardial damage identification in patients with breast cancer.

Introduction: Chemotherapy and radiation therapy for breast cancer cause side effects, such as cardiovascular changes, which can be monitored with echocardiography. However, more convenient methods are always encouraged. Radial arterial waves that are used to detect cardiovascular changes can be used to assist in confirming cardiovascular changes. Aim: This retrospective study aimed to analyze the frequency and time domains of the radial artery pulse wave in patients with breast cancer to understand its effectiveness in identifying cardiovascular changes. Methods: Patients with breast cancer were screened from the pulse examination records in Changhua Christian Hospital and divided into the treatment and remission groups. After unlinking the data, the pulse data were analyzed for the breast cancer treatment and remission group, including the average value of the parameters of four consecutive pulse diagnosis records in four consecutive months to test the difference in pulse waves due to breast cancer treatment between the two groups. Additionally, the pulse wave stability of the two groups was compared using the coefficient of variation. Results and conclusion: The comparison of the pulse wave data between 19 patients in the treatment group and 40 patients in the remission group revealed 45 parameters in time and 50 in frequency domains. D3, ND3, NA1, and NT1 are the four parameters with significant differences (p < 0.05), which are all related to heart function, and mainly related to cardiac output and peripheral resistance, indicating that patients in the treatment period have poor heart function. No difference was found in the degree of data dispersion between the two groups. Cardiovascular side effects caused by breast cancer treatment can mainly be shown in the pulse wave time domain.

Multi-organ hereditary hemorrhagic telangiectasia: A case report.

BACKGROUND: Type 2 hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant disease and is associated with ALK1 gene mutations. Type 2 HHT patients primarily suffer from recurrent bleeding. There is currently no promising treatment. CASE SUMMARY: A 5-year-old Chinese patient (III23) was admitted to Zhongshan Hospital for recurrent melena occurring over 2 mo. She had been experiencing epistaxis for years and had been diagnosed with idiopathic pulmonary hypertension 4 mo before presentation. Abdominal computed tomography examination showed hepatic arteriovenous malformation. Gene testing revealed a c.1121G>A mutation on the ALK1 gene. According to the international diagnostic criteria, this patient was diagnosed with HHT. In addition, 8 more family members exhibited HHT symptoms to varying degrees. Gene testing in 5 family members (2 with HHT symptoms and 3 without HHT symptoms) revealed the ALK1 c.1121G>A mutation in the 2 family members with HHT symptoms. This missense mutation results in the substitution of arginine for glutamine at amino acid position 374 (R374Q) in the conserved functional kinase domain of ALK1. Biological studies revealed that this mutation decreased the kinase activity of ALK1 and impeded the phosphorylation of its substrate Smad1. Moreover, the R374Q mutant downregulated the protein level of collagen-1, a fibrogenic factor, indicating abnormal fiber generation during vascular formation. CONCLUSION: The R374Q mutant of ALK1 and its subsequent influence on fiber generation highly indicated its pathogenic role in this family with type 2 HHT. Detection of this gene mutation will facilitate early diagnosis of suspected type 2 HHT patients, and mechanistic studies will provide insights for future therapy.

Deficiency of RAB39B Activates ER Stress-Induced Pro-apoptotic Pathway and Causes Mitochondrial Dysfunction and Oxidative Stress in Dopaminergic Neurons by Impairing Autophagy and Upregulating α-Synuclein.

Deletion and missense or nonsense mutation of RAB39B gene cause familial Parkinson's disease (PD). We hypothesized that deletion and mutation of RAB39B gene induce degeneration of dopaminergic neurons by decreasing protein level of functional RAB39B and causing RAB39B deficiency. Cellular model of deletion or mutation of RAB39B gene-induced PD was prepared by knocking down endogenous RAB39B in human SH-SY5Y dopaminergic cells. Transfection of shRNA-induced 90% reduction in RAB39B level significantly decreased viability of SH-SY5Y dopaminergic neurons. Deficiency of RAB39B caused impairment of macroautophagy/autophagy, which led to increased protein levels of α-synuclein and phospho-α-synucleinSer129 within endoplasmic reticulum (ER) and mitochondria. RAB39B deficiency-induced increase of ER α-synuclein and phospho-α-synucleinSer129 caused activation of ER stress, unfolded protein response, and ER stress-induced pro-apoptotic cascade. Deficiency of RAB39B-induced increase of mitochondrial α-synuclein decreased mitochondrial membrane potential and increased mitochondrial superoxide. RAB39B deficiency-induced activation of ER stress pro-apoptotic pathway, mitochondrial dysfunction, and oxidative stress caused apoptotic death of SH-SY5Y dopaminergic cells by activating mitochondrial apoptotic cascade. In contrast to neuroprotective effect of wild-type RAB39B, PD mutant (T168K), (W186X), or (G192R) RAB39B did not prevent tunicamycin- or rotenone-induced increase of neurotoxic α-synuclein and activation of pro-apoptotic pathway. Our results suggest that RAB39B is required for survival and macroautophagy function of dopaminergic neurons and that deletion or PD mutation of RAB39B gene-induced RAB39B deficiency induces apoptotic death of dopaminergic neurons via impairing autophagy function and upregulating α-synuclein.

STAT3 Inhibition Attenuates MYC Expression by Modulating Co-Activator Recruitment and Suppresses Medulloblastoma Tumor Growth by Augmenting Cisplatin Efficacy In Vivo.

MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.

CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Nav1.8 sodium channels in dorsal root ganglion neurons.

BACKGROUND: Inflammation or nerve injury-induced upregulation and release of chemokine CC chemokine ligand 2 (CCL2) within the dorsal root ganglion (DRG) is believed to enhance the activity of DRG nociceptive neurons and cause hyperalgesia. Transient receptor potential vanilloid receptor 1 (TRPV1) and tetrodotoxin (TTX)-resistant Na(v)1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons. We therefore tested the hypothesis that CCL2 causes peripheral sensitization of nociceptive DRG neurons by upregulating the function and expression of TRPV1 and Nav1.8 channels. METHODS: DRG neuronal culture was prepared from 3-week-old Sprague-Dawley rats and incubated with various concentrations of CCL2 for 24 to 36 hours. Whole-cell voltage-clamp recordings were performed to record TRPV1 agonist capsaicin-evoked inward currents or TTX-insensitive Na(+) currents from control or CCL2-treated small DRG sensory neurons. The CCL2 effect on the mRNA expression of TRPV1 or Na(v)1.8 was measured by real-time quantitative RT-PCR assay. RESULTS: Pretreatment of CCL2 for 24 to 36 hours dose-dependently (EC(50) value = 0.6 ± 0.05 nM) increased the density of capsaicin-induced currents in small putative DRG nociceptive neurons. TRPV1 mRNA expression was greatly upregulated in DRG neurons preincubated with 5 nM CCL2. Pretreating small DRG sensory neurons with CCL2 also increased the density of TTX-resistant Na(+) currents with a concentration-dependent manner (EC(50) value = 0.7 ± 0.06 nM). The Na(v)1.8 mRNA level was significantly increased in DRG neurons pretreated with CCL2. In contrast, CCL2 preincubation failed to affect the mRNA level of TTX-resistant Nav1.9. In the presence of the specific phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 or Akt inhibitor IV, CCL2 pretreatment failed to increase the current density of capsaicin-evoked inward currents or TTX-insensitive Na(+) currents and the mRNA level of TRPV1 or Na(v)1.8. CONCLUSIONS: Our results showed that CCL2 increased the function and mRNA level of TRPV1 channels and Na(v)1.8 sodium channels in small DRG sensory neurons via activating the PI3K/Akt signaling pathway. These findings suggest that following tissue inflammation or peripheral nerve injury, upregulation and release of CCL2 within the DRG could facilitate pain transmission mediated by nociceptive DRG neurons and could induce hyperalgesia by upregulating the expression and function of TRPV1 and Na(v)1.8 channels in DRG nociceptive neurons.

Normal and diseased personal eye modeling using age-appropriate lens parameters.

Personalized eye modeling of normal and diseased eye conditions is attractive due to the recent availability of detailed ocular measurements in clinic environments and the promise of its medical and industrial applications. In the customized modeling, the optical properties of the crystalline lens including the gradient refractive index, the lens bio-geometry and orientation are typically assigned with average lens parameters from literature since typically they are not clinically available. Although, through the optical optimization by assigning lens parameters as variables, the clinical measured wavefront aberration can be achieved, the optimized lens biometry and orientation often end up at edges of the statistical distribution. Without an effective validation of these models today, the fidelity of the final lens (and therefore the model) remains questionable. To develop a more reliable customized model without detailed lens information, we incorporate age-appropriate lens parameters as the initial condition of optical optimization. A biconic lens optimization was first performed to provide a correct lens profile for accurate lower order aberration and then followed by the wavefront optimization. Clinical subjects were selected from all ages with both normal and diseased corneal and refractive conditions. 19 ammetropic eyes ( + 4D to -11D), and 16 keratoconus eyes (mild to moderate with cylinder 0.25 to 6D) were modeled. Age- and gender-corrected refractive index was evaluated. Final models attained the lens shapes comparable to the statistical distribution in their age.

TDP-43 regulates the mammalian spinogenesis through translational repression of Rac1.

Impairment of learning and memory is a significant pathological feature of many neurodegenerative diseases including FTLD-TDP. Appropriate regulation and fine tuning of spinogenesis of the dendrites, which is an integral part of the learning/memory program of the mammalian brain, are essential for the normal function of the hippocampal neurons. TDP-43 is a nucleic acid-binding protein implicated in multi-cellular functions and in the pathogenesis of a range of neurodegenerative diseases including FTLD-TDP and ALS. We have combined the use of single-cell dye injection, shRNA knockdown, plasmid rescue, immunofluorescence staining, Western blot analysis and patch clamp electrophysiological measurement of primary mouse hippocampal neurons in culture to study the functional role of TDP-43 in mammalian spinogenesis. We found that depletion of TDP-43 leads to an increase in the number of protrusions/spines as well as the percentage of matured spines among the protrusions. Significantly, the knockdown of TDP-43 also increases the level of Rac1 and its activated form GTP-Rac1, a known positive regulator of spinogenesis. Clustering of the AMPA receptors on the dendritic surface and neuronal firing are also induced by depletion of TDP-43. Furthermore, use of an inhibitor of Rac1 activation negatively regulated spinogenesis of control hippocampal neurons as well as TDP-43-depleted hippocampal neurons. Mechanistically, RT-PCR assay and cycloheximide chase experiments have indicated that increases in Rac1 protein upon TDP-43 depletion is regulated at the translational level. These data together establish that TDP-43 is an upstream regulator of spinogenesis in part through its action on the Rac1 â†’ GTP-Rac1 â†’ AMPAR pathway. This study provides the first evidence connecting TDP-43 with the GTP-Rac1 â†’ AMPAR regulatory pathway of spinogenesis. It establishes that mis-metabolism of TDP-43, as occurs in neurodegenerative diseases with TDP-43 proteinopathies, e.g., FTLD-TDP, would alter its homeostatic cellular concentration, thus leading to impairment of hippocampal plasticity.

Analysis of agreement on traditional chinese medical diagnostics for many practitioners.

In Traditional Chinese Medicine (TCM) diagnostics, it is an important issue to study the degree of agreement among several distinct practitioners. In order to study the reliability of TCM diagnostics, we have to design an experiment to simultaneously deal with both of the cases when the data is ordinal and when there are many TCM practitioners. In this study, we consider a reliability measure called "Krippendorff's alpha" to investigate the agreement of tongue diagnostics in TCM. Besides, since it is not easy to obtain a large data set with patients rated simultaneously by many TCM practitioners, we use the renowned "bootstrapping" to obtain a 95% confidence interval for the Krippendorff's alpha. The estimated Krippendorff's alpha for the agreement among ten physicians that discerned fifteen randomly chosen patients is 0.7343, and the 95% bootstrapping confidence interval for the true alpha coefficient is [0.6570, 0.7349]. The data was collected and analyzed at the Department of Traditional Chinese Medicine, Changhua Christian Hospital (CCH) in Taiwan.

Laser Acupuncture and Dynamic Pulse Reaction Quantified by Smartphone:Two Experimental Examples.

The dynamic pulse reaction (Nogier reflex), Reflex-Auriculo-Cardiac (RAC), or vascular autonomic signal is a physiologic phenomenon that is not fully accepted and widely understood in contemporary medical practice. In order to provide appropriate scientific evidence for better understanding, qualitative and-above all-quantitative research in this area is necessary. In this short report, 2 recordings of dynamic pulse reactions based on an analysis performed on a modified smartphone are demonstrated during laser acupuncture experiments using stimulation at ear acupuncture points.

Ubiquitylation of cyclin C by HACE1 regulates cisplatin-associated sensitivity in gastric cancer.

BACKGROUND: Cyclin C (CCNC) was reported to take part in regulating mitochondria-derived oxidative stress under cisplatin stimulation. However, its effect in gastric cancer is unknown. This study aimed to investigate the role of cyclin C and its ubiquitylation in regulating cisplatin resistance in gastric cancer. METHODS: The interaction between HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 (HACE1) and cyclin C was investigated by GST pull-down assay, co-immunoprecipitation and ubiquitylation assay. Mitochondria-derived oxidative stress was studied by MitoSOX Red assay, seahorse assay and mitochondrial membrane potential measurement. Cyclin C-associated cisplatin resistance was studied in vivo via xenograft. RESULTS: HACE1 catalysed the ubiquitylation of cyclin C by adding Lys11-linked ubiquitin chains when cyclin C translocates to cytoplasm induced by cisplatin treatment. The ubiquitin-modified cyclin C then anchor at mitochondira, which induced mitochondrial fission and ROS synthesis. Depleting CCNC or mutation on the ubiquitylation sites decreased mitochondrial ROS production and reduced cell apoptosis under cisplatin treatment. Xenograft study showed that disrupting cyclin C ubiquitylation by HACE1 conferred impairing cell apoptosis response upon cisplatin administration. CONCLUSIONS: Cyclin C is a newly identified substrate of HACE1 E3 ligase. HACE1-mediated ubiquitylation of cyclin C sheds light on a better understanding of cisplatin-associated resistance in gastric cancer patients. Ubiquitylation of cyclin C by HACE1 regulates cisplatin-associated sensitivity in gastric cancer. With cisplatin-induced nuclear-mitochondrial translocation of cyclin C, its ubiquitylation by HACE1 increased mitochondrial fission and mitochondrial-derived oxidative stress, leading to cell apoptosis.