Protein Kinase C Life Cycle: Explained Through Systems Biology Approach.

Protein kinase C (PKC) enzymes are a family of kinases that mediate signal transduction originating at the cell surface. Most cell membranes can contain functional PKC enzymes. Aberrations in the PKC life cycle may result in cellular damage and dysfunction. For example, some cancerous cells exhibit alterations in PKC activity. Here, we use a systems biology approach to describe a molecular model of the PKC life cycle. Understanding the PKC life cycle is necessary to identify new drug targets. The PKC life cycle is composed of three key regulatory processes: maturation, activation, and termination. These processes precisely control PKC enzyme levels. This model describes the fate of PKC during de novo synthesis and PKC's lipid-mediated activation cycle. We utilize a systems biology approach to show the PKC life cycle is controlled by multiple phosphorylation and dephosphorylation events. PKC processing events can be divided into two types: maturation via processing of newly synthesized enzyme and secondary messenger-dependent activation of dormant, but catalytically competent enzyme. Newly synthesized PKC enzyme is constitutively processed through three ordered phosphorylations and stored in the cytosol as a stable, signaling-competent inactive and autoinhibited molecule. Upon extracellular stimulation, diacylglycerol (DAG) and calcium ion (Ca2+) generated at the membrane bind PKC. PKC then undergoes cytosol-to-membrane translocation and subsequent activation. Our model shows that, once activated, PKC is prone to dephosphorylation and subsequent degradation. This model also describes the role of HSP70 in stabilization and re-phosphorylation of dephosphorylated PKC, replenishing the PKC pool. Our model shows how the PKC pool responds to different intensities of extracellular stimuli? We show that blocking PHLPP dephosphorylation replenishes the PKC pool in a dose-dependent manner. This model provides a comprehensive understanding of PKC life cycle regulation.

TRPC3 Channel Activity and Viability of Purkinje Neurons can be Regulated by a Local Signalosome.

Canonical transient receptor potential channels (TRPC3) may play a pivotal role in the development and viability of dendritic arbor in Purkinje neurons. This is a novel postsynaptic channel for glutamatergic synaptic transmission. In the cerebellum, TRPC3 appears to regulate functions relating to motor coordination in a highly specific manner. Gain of TRPC3 function is linked to significant alterations in the density and connectivity of dendritic arbor in Purkinje neurons. TRPC3 signals downstream of class I metabotropic glutamate receptors (mGluR1). Moreover, diacylglycerol (DAG) can directly bind and activate TRPC3 molecules. Here, we investigate a key question: How can the activity of the TRPC3 channel be regulated in Purkinje neurons? We also explore how mGluR1 activation, Ca2+ influx, and DAG homeostasis in Purkinje neurons can be linked to TRPC3 activity modulation. Through systems biology approach, we show that TRPC3 activity can be modulated by a Purkinje cell (PC)-specific local signalosome. The assembly of this signalosome is coordinated by DAG generation after mGluR1 activation. Our results also suggest that purinergic receptor activation leads to the spatial and temporal organization of the TRPC3 signaling module and integration of its key effector molecules such as DAG, PKCγ, DGKγ, and Ca2+ into an organized local signalosome. This signaling machine can regulate the TRPC3 cycling between active, inactive, and desensitized states. Precise activity of the TRPC3 channel is essential for tightly regulating the Ca2+ entry into PCs and thus the balance of lipid and Ca2+ signaling in Purkinje neurons and hence their viability. Cell-type-specific understanding of mechanisms regulating TRPC3 channel activity could be key in identifying therapeutic targeting opportunities.

Developing sulfur-doped titanium oxide nanoparticles loaded chitosan/cellulose-based proangiogenic dressings for chronic ulcer and burn wounds healing.

Development of biomaterials supporting angiogenesis are highly desired in medical applications. In current work, chitosan and cellulose were cross-linked by using triethyl orthoformate and loaded with sulfur-doped titanium oxide nanoparticles. A readily available and inexpensive titanium oxide was added as a potential proangiogenic agent based on our group findings and other reports on metal oxide nanoparticles activity to stimulate angiogenesis. A simple freeze gelation method led to the development of flexible, foldable, and porous membranes. To investigate the chemical characteristics of the synthesized membranes, Fourier-transform infrared spectroscopy was used. Scanning electron microscopy equipped with energy-dispersive X-ray microanalysis was employed for surface morphological investigations. The cross-linked membranes showed higher degree of swelling capacity compared to the same material with titania-loaded nanoparticles in vitro. The synthesized materials showed higher degree of degradation in H2 O2 as compared to phosphate-buffered saline and lysozyme. Chorioallantoic membrane assay was done to investigate the angiogenic potential. Titanium oxide nanoparticles loaded membranes (CLHTS-5 wt%) exhibited the best degree of angiogenesis in comparison to the other tested materials. In CLHTS-5 wt% experimental group, a good level of attachment and ingrowth of several blood vessels was observed. Interestingly, the same tested group (CLHTS-5 wt%) had shown the increasing trend of cellular metabolic rate of the seeded cells from Day 0 to Day 7 in vitro. These findings were further confirmed by the decline in lactate dehydrogenase enzyme release which was monitored until 72 h, indicating the promising ability of this biomaterial in wound healing applications.

Amino acids loaded chitosan/collagen based new membranes stimulate angiogenesis in chorioallantoic membrane assay.

Chitosan/collagen-based hydrogels were studied for their promising role in skin tissue engineering applications due to their unique biocompatibility and biodegradation properties. Amino acids are not only the mean of protein building units but also support endothelial cells proliferation and trigger angiogenesis during wound healing. The purpose of this study was to prepare amino acid based pro-angiogenic materials. Three structurally closed amino acids (AA) (arginine, alanine and phenylalanine) were loaded into chitosan/collagen hydrogels (ACC hydrogels) to study their effect on angiogenesis. In this study the ACC hydrogels were prepared through freeze drying procedure and their angiogenic potential was studied by chorioallantoic membrane assay (CAM assay). FTIR analysis was performed to confirm that there was no chemical change took place in polymeric materials during synthesis procedures. Results revealed that, arginine-loaded hydrogels were the most porous, with more interconnected pores and also the maximum growth of blood vessels were found around and inside the arginine loaded scaffold. The qualitative analysis for blood vessels showed the significant difference between control, chitosan/collagen alanine loaded hydrogel (CH-Ala), chitosan/collagen phenylalanine loaded hydrogel (CH-Phe) and chitosan/collagen arginine loaded hydrogel (CH-Arg) materials. Among these studied materials the CH-Arg was found more capable for angiogenesis.

Simplified Model of PKCγ Signaling Dysregulation and Cytosol-to-Membrane Translocation Kinetics During Neurodegenerative Spinocerebellar Ataxia Type 14 (SCA14).

Spinocerebellar ataxia type 14 (SCA14) is an autosomal neurodegenerative disease clinically characterized by progressive ataxia in the patient's gait, accompanied by slurred speech and abnormal eye movements. These symptoms are linked to the loss of Purkinje cells (PCs), which leads to cerebellar neurodegeneration. PC observations link the mutations in PRKCG gene encoding protein kinase C γ (PKCγ) to SCA14. Observations also show that the link between PKCγ and SCA14 relies on a gain-of-function mechanism, and, in fact, both positive and negative regulation of PKCγ expression and activity may result in changes in cellular number, size, and complexity of the dendritic arbors in PCs. Here, through a systems biology approach, we investigate a key question relating to this system: why is PKCγ membrane residence time reduced in SCA14 mutant PCs compared to wild-type (WT) PCs? In this study, we investigate this question through two contrasting PKCγ signaling models in PCs. The first model proposed in this study describes the mechanism through which PKCγ signaling activity may be regulated in WT PCs. In contrast, the second model explores how mutations in PKCγ signaling affect the state of SCA14 in PCs. Numerical simulations of both models show that, in response to extracellular stimuli-induced depolarization of the membrane compartment, PKCγ and diacylglycerol kinase γ (DGKγ) translocate to the membrane. Results from our computational approach indicate that, for the same set of parameters, PKCγ membrane residence time is shorter in the SCA14 mutant model compared to the WT model. These results show how PKCγ membrane residence time is regulated by diacylglycerol (DAG), causing translocated PKCγ to return to the cytosol as DAG levels drop. This study shows that, when the strength of the extracellular signal is held constant, the membrane lifetime of mutant PKCγ is reduced. This reduction is due to the presence of constitutively active mutant PKCγ in the cytosol. Cytosolic PKCγ, in turn, leads to phosphorylation and activation of DGKγ while it is still residing in the cytosol. This effect occurs even during the resting conditions. Thus, the SCA14 mutant model explains that, when both DAG effector molecules are active in the cytosol, their interactions in the membrane compartment are reduced, critically influencing PKCγ membrane residence time.

Development of K-doped ZnO nanoparticles encapsulated crosslinked chitosan based new membranes to stimulate angiogenesis in tissue engineered skin grafts.

Nanoparticles are well recognized for their biological applications including tissue-regeneration due to large surface area and chemical properties. In this study, K-doped zinc oxide (ZnO) nanoparticles containing porous hydrogels were synthesized via freeze gelation. The morphology and pore dimensions were studied by scanning electron microscopy (SEM). The chemical structural analysis of the synthesized hydrogels was investigated by Fourier Transform Infrared (FTIR) spectroscopy. In swelling studies, material containing ZnO nanoparticles with 2% potassium dopant concentration CLH-K2.0) showed greater degree of swelling as compared to all other materials. The degradation studied was tested in three different degradation media, i.e. phosphate buffer saline (PBS), lysozyme and hydrogen peroxide and relatively higher degradation was seen in hydrogen peroxide. The synthesized hydrogels were implanted on the chick chorioallantoic membrane (CAM) to investigate their angiogenic potential. The CLH-K2.0 hydrogel stimulated angiogenesis greater than all other materials; blood vessels were attached and grown inside this scaffold, showing its strong angiogenic potential.

Laparoscopic Major Vascular Injury Simulation Using a Synthetic Compared With Porcine Model.

BACKGROUND: Major vascular injury training may improve clinical skills and reduce patient morbidity during gynecologic laparoscopy; thus, reliable models for simulation should be identified. METHOD: Two laparoscopic major vascular injury simulations using synthetic or live porcine models were constructed. The primary surgeon was given the opportunity to complete both simulations. After obtaining peritoneal access, the surgeon quickly encountered a major vascular injury. Degrading vital signs and estimated blood loss coupled with the replay of a human heartbeat that increased in volume and intensity were provided to heighten tension during the synthetic simulation. EXPERIENCE: Twenty-two gynecologic surgery educators evaluated the simulations. Educators considered the porcine model superior to the synthetic model with regard to tissue handling. The synthetic model simulation was found to be equivalent to the porcine model on how likely the simulation would be able to improve performance in a clinical setting. Educators were more likely to implement the synthetic simulation over the porcine simulation. CONCLUSION: The synthetic model was found to be more feasible and as effective as the porcine model to simulate and teach the initial management steps of major vascular injury at laparoscopy by gynecologic educators.

New paradigms in the conservative surgical and interventional management of adenomyosis.

PURPOSE OF REVIEW: Adenomyosis is commonly diagnosed in women of reproductive age. Interest in conservative interventions has grown as more women desire fertility preservation or avoidance of hysterectomy. This review discusses surgical and interventional methods for treatment of symptomatic adenomyosis. The technique, evidence, and utility of each method are described. RECENT FINDINGS: Hysteroscopic ablative techniques are associated with lower morbidity than with hysterectomy but may result in an unacceptable risk of treatment failure. Surgical adenomyomectomy may provide good symptomatic improvement, especially when combined with preoperative gonadotropin-releasing hormone agonist treatment. Laparoscopic myometrial coagulation is associated with high rates of future pregnancy complications. Uterine artery ligation has limited value as an isolated approach but, coupled with other techniques, provides adequate therapeutic control. Bilateral uterine artery embolization may improve symptoms, without significantly compromising fertility. Focused ultrasonic surgical methods also show promise in alleviating symptoms without compromising reproductive outcomes. SUMMARY: A multitude of surgical and interventional options are available for young women with symptomatic adenomyosis. These treatment methods have unique associated risks and benefits, and may have varying impacts on long-term symptom control, fertility, and reproductive outcomes.

Trends in malpractice claims for obstetric and gynecologic procedures, 2005 through 2014.

BACKGROUND: Interest in medical malpractice and areas of medicolegal vulnerability for practicing obstetricians and gynecologists has grown substantially, and many providers report changing surgical practice out of fear of litigation. Furthermore, education on medical malpractice and risk management is lacking for obstetrics and gynecology trainees. Recent obstetric and gynecologic malpractice claims data are lacking. We report on recent trends in malpractice claims for obstetrics and gynecology procedures, and compare these trends to those of other medical specialties. OBJECTIVE: We sought to evaluate recent trends in malpractice claims for obstetrics and gynecology procedures and compare these to other medical specialties. STUDY DESIGN: A search was performed on all medicolegal claims data for obstetrics and gynecology procedures from Jan. 1, 2005, through Dec. 31, 2014, using the Physician Insurers' Association of America data-sharing project, which was created to identify medical professional liability trends. Data from 20 insurance carriers were reviewed based on a search using International Classification of Diseases, Ninth Revision codes and unique database-specific codes. RESULTS: Of the 10,915 total claims closed from 2005 through 2014, the majority (59.5%) were dropped, withdrawn, or dismissed. The average indemnity of the remaining paid claims (31.1%) was $423,250. The most frequently litigated procedure was operative procedures on the uterus; 27.8% of cases were paid with an average indemnity of $279,384. The procedure associated with the highest proportion of paid claims was vacuum extraction. The average indemnity for paid obstetrics and gynecology procedural claims was 27% higher than that for all medical specialties combined. Obstetrics and gynecology procedural claims had the second highest average indemnity payment and the fifth highest paid-to-closed ratio of all medical specialties. CONCLUSION: Litigation claims for obstetrics and gynecology procedures have higher average indemnity payments and higher paid-to-closed ratios than most other medical specialties. Claims most frequently relate to gynecologic surgery, but obstetric procedures are more expensive. Possible factors may include procedural experience and unique perioperative complications. We encourage efforts addressing procedures, litigation, and quality interventions to improve outcomes, mitigate risk, and potentially lower indemnity payments.

Neoadjuvant chemotherapy and primary debulking surgery utilization for advanced-stage ovarian cancer at a comprehensive cancer center.

OBJECTIVE: The aim of this study was to evaluate the use of neoadjuvant chemotherapy (NACT) and primary debulking surgery (PDS) before and after results from a randomized trial were published and showed non-inferiority between NACT and PDS in the management of advanced-stage ovarian carcinoma. METHODS: We evaluated consecutive patients with advanced-stage ovarian cancer treated at our institution from 1/1/08-5/1/13, which encompassed 32 months before and 32 months after the randomized trial results were published. We included all newly diagnosed patients with high-grade histology and stage III/IV disease. Associations between the use of NACT and clinical variables over time were evaluated. RESULTS: Our study included 586 patients. Median age was 62 years (range, 30-90); 406 patients (69%) had stage III disease, and 570 (97%) had disease of serous histology. Twenty-six percent (154/586) were treated with NACT and 74% (432/586) with PDS. NACT use increased significantly from 22% (56/256) before 2010 (at which point the results of the randomized trial were published) to 30% (98/330) after 2010 (p=0.037). Although patients who underwent PDS were more likely to experience grade 3/4 surgical complications than those who underwent NACT, those selected for PDS had a median OS of 71.7 months (CI, 59.8-not reached) compared with 42.9 months (CI 37.1-56.3) for those selected for NACT. CONCLUSIONS: In this single-institution analysis, the best survival outcomes were observed in patients who were deemed eligible for PDS followed by platinum-based chemotherapy. Selection criteria for NACT require further definition and should take institutional surgical strategy into account.