Use of cholecystostomy tubes in the management of patients with primary diagnosis of acute cholecystitis
BACKGROUND: Management of patients with severe acute cholecystitis (AC) remains controversial. In settings where laparoscopic cholecystectomy (LC) can be technically challenging or medical risks are exceedingly high, surgeons can choose between different options, including LC conversion to open cholecystectomy or surgical cholecystostomy tube (CCT) placement, or initial percutaneous CCT. We reviewed our experience treating complicated AC with CCT at a tertiary-care academic medical center.
STUDY DESIGN: All adult patients (n = 185) admitted with a primary diagnosis of AC and who received CCT from 2002 to 2010 were identified retrospectively through billing and diagnosis codes.
RESULTS: Mean patient age was 71 years and 80% had >/=1 comorbidity (mean 2.6). Seventy-eight percent of CCTs were percutaneous CCT placement and 22% were surgical CCT placement. Median length of stay from CCT insertion to discharge was 4 days. The majority (57%) of patients eventually underwent cholecystectomy performed by 20 different surgeons in a median of 63 days post-CCT (range 3 to 1,055 days); of these, 86% underwent LC and 13% underwent open conversion or open cholecystectomy. In the radiology and surgical group, 50% and 80% underwent subsequent cholecystectomy, respectively, at a median of 63 and 60 days post-CCT. Whether surgical or percutaneous CCT placement, approximately the same proportion of patients (85% to 86%) underwent LC as definitive treatment.
CONCLUSIONS: This 9-year experience shows that use of CCT in complicated AC can be a desirable alternative to open cholecystectomy that allows most patients to subsequently undergo LC. Additional studies are underway to determine the differences in cost, training paradigms, and quality of life in this increasingly high-risk surgical population. rights reserved.
OBJECTIVES: Many medical schools are currently undergoing curriculum reform. When considering the means by which students will be evaluated in a revised curriculum, the need to reduce the prevalences of depression and anxiety associated with academic stress must be weighed against the importance of academic outcomes. Pass/fail evaluation, as compared with tiered grading, is commonly presented as a means to adequately assess student performance while minimising stress and anxiety. The purpose of this literature review was to determine the impact of pass/fail grading on medical student well-being and academic outcomes.
METHODS: A systematic search was performed of the available literature published between January 1980 and August 2010, using the PubMed, Ovid Medline, Ovid PsycINFO and ERIC databases. Eligible papers assessed the impact of pass/fail grading on medical student well-being, academic outcomes or both. Academic outcomes included but were not limited to objective measures, such as performance on the US Medical Licensing Examination, and subjective measures, such as student desirability by residency programmes. Reference lists in identified papers were searched and all identified papers were run through a citation index.
RESULTS: Four papers met the inclusion criteria for both well-being and academic outcomes. An additional five papers met the inclusion criteria for academic outcomes only. The four papers that focused on well-being reported improvement in specified areas. No significant difference was identified in any of the five papers examining objective academic outcomes or in those papers that examined the quality of residency programmes attained. Results from two studies suggested that some programme directors believe pass/fail grading creates disadvantages for students in attaining a residency, whereas a third study yielded mixed results about its impact on residency attainment.
CONCLUSIONS: Student well-being is enhanced and objective academic performance is not adversely affected by a pass/fail evaluation system, but students' ability to obtain a desired residency programme may be hindered by individual programme directors' preferences for tiered grading systems. There is an overall paucity of literature on this topic and additional study is needed.
OBJECT: Massachusetts' health insurance mandate and subsidized insurance program, Commonwealth Care, have been active for 2 years.
METHODS: The financial impact on the neurosurgery division and demographics of the relevant patient groups were assessed. The billing records of neurosurgical patients from January 2007 to September 2008 were collected and analyzed.
RESULTS: Commonwealth Care comprised 2.2% of neurosurgical inpatients, and these patients did not have significantly different acuity or lengths of stay from the average. Length of stay of MassHealth patients was significantly greater, although acuity was significantly lower than the average. Increased free care reimbursement and increased MassHealth/Commonwealth Care enrollment resulted in a net gain in reimbursement of hospital charges.
CONCLUSIONS: The increased insurance rates have resulted in increased reimbursement for the neurosurgical division.
Limbic and motor function comparison of deep brain stimulation of the zona incerta and subthalamic nucleus
BACKGROUND: Psychiatric and neuropsychological side effects of subthalamic nucleus (STN) stimulation have been increasingly recognized. Most programming regimens focus on contacts 0 and 1, whereas contact 3, which often is located near or in the zona incerta (ZI), is usually not used. The question of whether ZI stimulation may limit limbic effects has not been answered.
OBJECTIVE: To examine the effects of short-term stimulation near or in the ZI (contact 3) compared with stimulation of the STN using standard trajectories and targeting as measured by limbic and motor functions.
METHODS: Motor and limbic functions of 11 patients with STN DBS were assessed with the Unified Parkinson Disease Rating Scale-3, structured gait video analysis, Visual Analog Scale mood scales, task testing of impulsivity, and facial recognition under routine STN programming and under stimulation in or near the ZI. Postoperative magnetic resonance imaging confirmed the location of contact 3 near or in the ZI.
RESULTS: Data analysis with repeated-measures analysis of variance revealed that motor scores remained stable with both stimulation settings, with specific improvements in finger taps (P = .02) and rapid alternating movements (P = .03) in ZI stimulation. Stimulation near or in the ZI led to a decrease in self- reported anxiety and depression (P = .03 for both) and an improvement in fear recognition (P = .02).
CONCLUSION: We provide preliminary evidence that stimulation in or near the ZI results in maintained motor function while improving self-reported depression and anxiety in patients with bilateral STN DBS. Stimulation in or near the ZI may provide a useful programming setting for patients prone to psychiatric side effects.
The paramedian forehead flap has become the standard of care for major nasal reconstruction. The classic procedure involves a second-stage operation to divide and inset the external pedicle. We present our experience in a clinical series using single-stage forehead flap reconstruction. Our indications include elderly patients, pediatric patients treated during mission trips, and any patient in whom an external pedicle or two-stage procedure is problematic. From 2008 to 2009, 9 patients underwent a single-stage forehead flap. The majority had defects after excision of skin cancer. Our modification involves removal of radix and proximal nasal skin and fat and deepithelialization of the proximal pedicle to allow inset without excess compression or kinking. This modification avoids the sequelae of an external pedicle, which include bleeding, dressings, the inability to wear eyeglasses, and the patient's reluctance to appear in public. It safely provides acceptable results and avoids a mandatory secondary procedure.
STUDY DESIGN: This is a prospective study.
OBJECTIVE: The aim of our study is to identify whether vertebral arteries (VA), normal or aberrant, are routinely described in cervical spine magnetic resonance imaging (MRI) interpretations.
SUMMARY OF BACKGROUND DATA: VA injury is a serious complication of anterior cervical spine surgery. Aberrant VA anatomy is a potential cause of such complications. Therefore, VA anatomy should be evaluated in cervical MRIs.
METHODS: Six neuroradiologists were blinded to the study design and were asked to interpret 79 cervical MRIs. Of these, 39 had aberrant VAs, whereas 40 had normal VAs. Initially, the indications for the study included only a description of patient's symptoms. The radiologists were then given the same MRIs with different indications. This time, the indications included the patient's symptoms, a request for annotations on the VA, and a definition of VA anomaly. All of the MRI interpretations were then evaluated for the frequency and accuracy of VA description.
RESULTS: When the indications for the study did not specifically request a comment on VAs, the VA was never described (0%). When the indications included the specific request and definition, all 6 commented on the VA (100%). Three of the 6 radiologists were 100% accurate in identifying all 40 normal and 39 aberrant VAs, whereas the other 3 identified all 40 normal and 38 of 39 aberrant VAs.
CONCLUSION: This study demonstrates that the VA is not a standard component of cervical spine MRI interpretations. Because of the significant complications related to its injury, VA anatomy, whether normal or variant, needs to be evaluated in cervical MRIs. When ordering a cervical MRI, surgeons should request a description of the VA and any anomalies.
In a previous study, intraforaminal anomalies were found to occur at a rate of 7.6%. This increases the risk of injury to this vessel if the surgeon is unaware of such abnormalities preoperatively. The aim of our retrospective study was to identify patient factors that may predict anomalous intraforaminal vertebral arteries. Patient records were obtained from a previous study. In that study, the records of each consecutive patient who underwent cervical spine magnetic resonance imaging (MRI) for axial neck pain, radiculopathy, or myelopathy between January 2007 and January 2008 were reviewed. The social and medical histories of each patient were evaluated with respect to the presence or absence of an aberrant vertebral artery. We reviewed the medical records of the 250 patients whose MRIs were reviewed in the previous study. Seven patients were excluded for incomplete records. Chi-square and Fisher's exact tests were performed to compare the normal vertebral artery anatomy patients to the aberrant patients. The medical records of 19 patients with aberrant vertebral arteries and 224 patients with normal vertebral arteries were reviewed. The aberrant group was significantly older than the normal group (P=.00015). The only diagnostic condition that represented a statistically significant difference between the 2 groups was incidence of cancer. A relationship may exist between patient age, cancer, and medialization of the vertebral artery. The mechanism of this possible relationship is unclear. Although aberrant vertebral arteries are rare, a surgeon should have raised suspicion of this possibility in patients with a history of cancer.
Vitreoretinal disorders constitute a significant portion of treatable ocular disease. Advances in vitreoretinal surgery have included the development and characterization of suitable substitutes for the vitreous. Air, balanced salt solutions, perfluorocarbons, expansile gases, and silicone oil serve integral roles in modern vitreoretinal surgery. Vitreous substitutes vary widely in their properties, serve different clinical functions, and present different shortcomings. Permanent vitreous replacement has been attempted with collagen, hyaluronic acid, hydroxypropylmethylcellulose, and natural hydrogel polymers. None, however, have proven to be clinically viable. A long-term vitreous substitute remains to be found, and recent research suggests promise in the area of synthetic polymers. Here we review the currently available vitreous substitutes, as well those in the experimental phase. We classify these compounds based on their functionality, composition, and properties. We also discuss the clinical use, advantages, and shortcomings of the various substitutes. In addition we define the ideal vitreous substitute and highlight the need for a permanent substitute with long-term viability and compatibility. Finally, we attempt to define the future role of biomaterials research and the various functions they may serve in the area of vitreous substitutes.
Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile infection (CDI)
BACKGROUND: Previous studies have demonstrated a correlation between Clostridium difficile anti-toxin A serum antibodies and protection against symptomatic disease and recurrence.
METHODS: A neutralizing monoclonal antibody to C. difficile toxin A (CDA1) developed by MBL and Medarex, Inc. was studied in a phase II, randomized, double-blind, placebo-controlled trial in patients receiving standard of care treatment for C. difficile infection (CDI). Twenty-nine subjects received a single intravenous infusion of 10mg/kg CDA1 and 17 subjects received placebo and were evaluated for recurrence of CDI during the 56-day study period. Serum antibodies against C. difficile toxin A and B were measured by ELISA and cytotoxicity assay at various time points before and after infusion.
FINDINGS: CDI recurrence occurred in 5 of 29 (17%) in the CDA1 group and 3 of 17 (18%) (p=NS) in the placebo group with a trend toward delay in time to recurrence in the group treated with CDA1. The geometric mean concentration of antibody to an epitope of the receptor-binding domain of toxin B (0.300 and 1.20microg/ml, respectively; p=0.02) and geometric mean titer of neutralizing B antibody (8.00 and 100, respectively; p=0.02) at study day 28 were lower for those subjects with recurrence compared to those who did not recur. In addition, a significantly greater proportion of subjects who recurred were infected with the epidemic BI/NAP1/027 strain compared with those that did not recur (88% vs. 22%; p=0.002). Finally, in a multiple logistic regression analysis neutralizing anti-toxin B at day 14 (p<0.001), anti-toxin A at day 28 (p<0.001) and infection with the BI/NAP1/027 strain at enrollment (p=0.002) were all predictive of CDI recurrence.
INTERPRETATION: In this prospective study, lower concentrations of neutralizing anti-toxin B and anti-toxin A antibody and infection with the BI/NAP1/027 strain of C. difficile were significantly associated with recurrence of CDI.
Epidemiological and clinical changes in American tegumentary leishmaniasis in an area of Leishmania (Viannia) braziliensis transmission over a 20-year period
The Health Post of Corte de Pedra is located in a region endemic for American tegumentary leishmaniasis (ATL) in the Brazilian state of Bahia, and it treats 500-1,300 patients annually. To describe temporal changes in the epidemiology of ATL, we reviewed a random sample of 10% of patient charts (N = 1,209) from 1988 to 2008. There was a twofold increase in the number of cases over the 20-year period, with fluctuations in 10-year cycles. Patients were most frequently male, between the ages of 10 and 30 years, and engaged in agricultural labor; 4.3% of patients had mucosal disease, and 2.4% of patients had disseminated disease. Over the study period, the number of disseminated cases increased threefold, the proportion of cases in younger patients and agricultural workers decreased, and the proportion of patients residing in coastal areas increased. ATL is on the rise in Bahia, with a 10-year periodicity and evolving changes in epidemiology and manifestations of disease.
Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells
The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro-interleukin-1beta (pro-IL-1beta) into secreted IL-1beta, is induced by endogenous and exogenous danger signals. Lipopolysaccharide (LPS), a toll-like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals.
METHODS: Using mouse models of methionine choline-deficient (MCD) diet-induced NASH and high-fat diet-induced NASH, we found up-regulation of the inflammasome [including NACHT, LRR, and PYD domains-containing protein 3 (NALP3; cryopyrin), apoptosis-associated speck-like CARD-domain containing protein, pannexin-1, and pro-caspase-1] at the messenger RNA (mRNA) level increased caspase-1 activity, and mature IL-1beta protein levels in mice with steatohepatitis in comparison with control livers. There was no inflammasome activation in mice with only steatosis. The MCD diet sensitized mice to LPS-induced increases in NALP3, pannexin-1, IL-1beta mRNA, and mature IL-1beta protein levels in the liver. We demonstrate for the first time that inflammasome activation occurs in isolated hepatocytes in steatohepatitis. Our novel data show that the saturated fatty acid (FA) palmitic acid (PA) activates the inflammasome and induces sensitization to LPS-induced IL-1beta release in hepatocytes. Furthermore, PA triggers the release of danger signals from hepatocytes in a caspase-dependent manner. These hepatocyte-derived danger signals, in turn, activate inflammasome, IL-1beta, and tumor necrosis factor alpha release in liver mononuclear cells.
CONCLUSION: Our novel findings indicate that saturated FAs represent an endogenous danger in the form of a first hit, up-regulate the inflammasome in NASH, and induce sensitization to a second hit with LPS for IL-beta release in hepatocytes. Furthermore, hepatocytes exposed to saturated FAs release danger signals that trigger inflammasome activation in immune cells. Thus, hepatocytes play a key role in orchestrating tissue responses to danger signals in NASH.
AIM: To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide (LPS)-induced stimulation in the liver.
METHODS: Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5 mug/g bodyweight LPS and sacrificed 2, 4, 6, 18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase (ALT) levels. To determine if LPS stimulation in the liver led to activation of the inflammasome, real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome. Enzyme-linked immunosorbent assays were used to determine the protein expression levels of several downstream targets of the Nalp3 inflammasome, including caspase-1 and two cytokine targets of caspase-1, interleukin (IL)-1beta and IL-18.
RESULTS: We found that LPS injection resulted in liver damage as indicated by elevated ALT levels. This was associated with a significant increase in both mRNA and protein levels of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha in the liver, as well as increased levels of TNFs in serum. We showed that LPS stimulation led to upregulation of mRNA levels in the liver for all the receptor components of the inflammasome, including Nalp3, Nalp1, pannexin-1 and the adaptor molecule apoptosis-associated speck-like, caspase recruitment domain-domain containing protein. We also found increased levels of mRNA and protein for caspase-1, a downstream target of the inflammasome. In addition, LPS challenge led to increased levels of both mRNA and protein in the liver for two cytokine targets of caspase-1, IL-1beta and IL-18. Interestingly, substantial baseline expression of pre-IL-1beta and pre-IL-18 was found in the liver. Inflammasome and caspase-1 activation was indicated by the significant increase in the active forms of IL-1beta and IL-18 after LPS stimulation.
CONCLUSION: Our results show that the Nalp3 inflammasome is upregulated and activated in the liver in response to LPS stimulation.
Carotid artery brain aneurysm model: in vivo molecular enzyme-specific MR imaging of active inflammation in a pilot study
PURPOSE: To demonstrate the feasibility of using a myeloperoxidase (MPO)-specific paramagnetic magnetic resonance (MR) contrast agent to identify active inflammation in an animal model of common carotid artery (CCA) aneurysm.
MATERIALS AND METHODS: All animal experiments were approved by the institutional animal care and use committee. Elastase-induced saccular aneurysms were created at the root of the right CCA in 16 New Zealand white rabbits. Intramural and perivascular injection of Escherichia coli lipopolysaccharide (LPS) was performed with an endovascular approach to induce aneurysm inflammation. After intraarterial injection of an MPO-specific (di-5-hydroxytryptamide of gadopentetate dimeglumine, 0.1 mmol per kilogram of bodyweight) or a non-MPO-specific (di-tyrosine of gadopentetate dimeglumine, 0.1 mmol/kg) contrast agent, animals underwent 3-T MR imaging. Intramural presence of MPO in aneurysms in which LPS had been injected was confirmed at immunohistologic analysis. Active MPO activity was verified by measuring the spectrophotometric oxidation of guaiacol.
RESULTS: Endovascular injection of LPS resulted in inflammatory cell infiltration into the aneurysm wall, and there was a difference in active MPO expression between aneurysms in which LPS had been injected and control aneurysms (20.3 ng of MPO per milligram of tissue vs 0.12 ng of MPO per milligram of tissue, respectively; P < .002). MR imaging with di-5-hydroxytryptamide of gadopentetate dimeglumine revealed a difference in enhancement ratio between inflamed aneurysms in which LPS had been injected and control aneurysms (1.55 +/- 0.05 vs 1.16 +/- 0.10, respectively; P < .02). In inflamed aneurysms, di-5-hydroxytryptamide of gadopentetate dimeglumine exhibited delayed washout kinetics compared with the kinetics of di-tyrosine of gadopentetate dimeglumine. This finding enabled the verification of MPO specificity.
CONCLUSION: The findings of this pilot study established the feasibility of an animal model of saccular aneurysm inflammation that can be seen with clinical-field-strength MR imaging and use of the enzyme-sensitive MR contrast agent di-5-hydroxytryptamide of gadopentetate dimeglumine, which is a paramagnetic MPO substrate that specifically enhances MR signal.
In 2008 we witnessed a rapid advancement in stent technology, which is reflected in the high number of case reports, publications of case series, and randomized trials. Stents not only served for a combined intrasaccular and extrasaccular treatment of challenging aneurysms but also assisted the revascularization in acute and chronic ischemic conditions of the neurovascular system. Although a self-expanding nitinol semiopen cell stent is currently used for intracranial occlusive disease, a new retrievable closed-cell designed stent is widely used for aneurysms because of its easy delivery through a microcatheter in frequently tortuous head and neck as well as cerebrovascular circulation (Figure 1). However, despite numerous publications in the field, the widespread acceptance of the use of stents to routinely treat carotid stenosis awaits the results of the multicenter randomized clinical trials that should be available in 2009. The role of interventional neuroradiology in the treatment of acute ischemic stroke continues to expand and excite interest.
Targeted signal-amplifying enzymes enhance MRI of EGFR expression in an orthotopic model of human glioma
Epidermal growth factor receptor (EGFR) imaging in brain tumors is essential to visualize overexpression of EGFRvIII variants as a signature of highly aggressive gliomas and to identify patients that would benefit from anti-EGFR therapy. Seeking imaging improvements, we tested a novel pretargeting approach that relies on initial administration of enzyme-linked anti-EGFR monoclonal antibodies (mAb; EMD72000) followed by administration of a low-molecular-weight paramagnetic molecule (diTyr-GdDTPA) retained at the site of EGFR mAb accumulation. We hypothesized that diTyr-GdDTPA would become enzyme activated and retained on cells due to binding to tissue proteins. In support of this hypothesis, mAb-enzyme conjugates reacted with both membrane-isolated wild-type (wt) EGFR and EGFRvIII, but they bound primarily to EGFRvIII-expressing cells and not to EGFRwt-expressing cells. In vivo analysis of magnetic resonance (MR) tumor signal revealed differences in MR signal decay following diTyr-GdDTPA substrate administration. These differences were significant in that they suggested differences in substrate elimination from the tissue which relied on the specificity of the initial mAb binding: a biexponential signal decay was observed in tumors only upon preinjection with EGFR-targeted conjugates. Endpoint MRI in this setting revealed detailed images of tumors which correlated with immunohistochemical detection of EGFR expression. Together, our findings suggest an improved method to identify EGFRvIII-expressing gliomas in vivo that are best suited for treatment with therapeutic EGFR antibodies.
Intra-arterial abciximab for the treatment of thrombus formation during coil embolization of intracranial aneurysms
BACKGROUND AND PURPOSE: Thrombus formation during endovascular embolization of intracranial aneurysms occurs in 2.9%-6% of patients. Use of IIb/IIIA inhibitors such as abciximab or eptifibatide intravenously has been reported in management of this complication. Because the intra-arterial infusion of IIb/IIIA inhibitors may require lower doses to achieve thrombolysis, it may reduce the risk of haemorrhage. Therefore, we retrospectively analyze our database and review the literature.
METHODS: This is a retrospective analysis of a prospectively acquired database of patients with ruptured or unruptured aneurysm treated intra-arterially for thrombus formation during endovascular coil embolization between July 2005 and August 2008. Patient demographics, aneurysmal characteristics, procedural, clinical outcome and complications were recorded.
RESULTS: From July 2005 to August 2008, out of 184 patients who underwent coil embolization, 19 patients (15 smokers, 14 female, mean age 52) developed intraprocedural thrombus formation and received intra-arterial abciximab treatment. Mean aneurysm size was 6.6 mm+/-4.9 mm; neck size was 3.8 mm+/-2.1 mm. Eight (42.1%) aneurysms were ruptured. Most aneurysms (63.1%) were in anterior communicating and middle cerebral arteries. Thrombus was visualized in all cases by angiogram and treated intra-arterially with a mean dose of 10.5 mg+/-4.2. There were no periprocedural hemorrhagic complications. No deaths or other complications occurred during follow-up.
CONCLUSION: Thrombus formation during coil embolization of intracranial aneurysms occurred more in women and smokers. Low doses of intra-arterial abciximab may be effective in the thromboembolic complications occurring during endovascular embolization of intracranial aneurysms.
Emerging evidence in both vertebrates and invertebrates is redefining glia as active and mobile players in synapse formation, maturation and function. However, the molecular mechanisms through which neurons and glia interact with each other to regulate these processes is not well known. My thesis work begins to understand how glia use secreted factors to modulate synaptic function. We use Drosophila melanogaster, a simple and genetically tractable model system, to understand the molecular mechanisms by which glia communicate with neurons at glutamatergic neuromuscular junctions (NMJs). We previously showed that a specific subtype of glia, subperineurial peripheral glia cells (SPGs), establish dynamic transient interactions with synaptic boutons of the NMJ and is required for synaptic growth. I identified a number of potential functional targets of the glial transcription factor, reverse polarity (repo) using ChIP-chip. I found that one novel target of Repo, Wg, is expressed in SPGs and is regulated by repo in vivo. Wnt/Wg signaling plays a pivotal role during synapse development and plasticity, including the coordinated development of the molecular architecture of the synapse. While previous studies demonstrated that Wg is secreted by motor neurons, herein I provide evidence that a significant amount of Wg at the NMJ is additionally provided by glia. I found that Wg derived from SPGs is required for proper GluR distribution and electrophysiological responses at the NMJ. In summary, my results show that Wg expression is regulated by Repo in SPGs and that glial-derived Wg, together with motor neuron-derived Wg, orchestrate different aspects of synapse development. My thesis work identifies synapse stabilization and/or assembly as a new role for SPGs and demonstrates that glial secreted factors such as Wg regulate synaptic function at the Drosophila NMJ.
Replication of HIV-1 requires the assembly and release of mature and infectious viral particles. In order to accomplish this goal, HIV-1 has evolved multiple methods to interact with the host cell. HIV-1 recruits the host cell ESCRT machinery to facilitate the release of nascent viral particles from the host cell membrane. Recruitment of these cellular factors is dependent on the presence of short motifs in Gag referred to as Late-domains. Deletion or mutation of these domains results in substantial decrease in the release of infectious virions. However, previously published work has indicated that over-expression of the E3 ubiquitin ligase, NEDD4.2s is able to robustly rescue release of otherwise budding-defective HIV-1 particles. This rescue is specific to the NEDD4.2s isoform as related E3 ubiquitin ligases display no ability to rescue particle release. In addition, rescue of particle release is dependent on the presence of the partial C2 domain and a catalytically active HECT domain of NEDD4.2s. Here I provide evidence supporting the hypothesis that a partial C2 domain of NEDD4.2s constitutes a Gag interacting module capable of targeting the HECT domains of other E3 ubiquitin ligases to HIV-1 Gag. Also, by generating chimeras between HECT domains shown to form poly-ubiquitin chains linked through either K48 or K63 of ubiquitin, I demonstrate that the ability of NEDD4.2s to catalyze the formation of K63-polyubiquitin chains is required for its stimulation of HIV-1 L-domain mutant particle release. In addition, I present findings from on-going research into the role of the HIV-1 accessory protein Nef during viral replication using the culture T-cell line, MOLT3. My current findings indicate that downregulation of CD4 from the host cell membrane does not solely account for the dramatic dependence of HIV-1 replication on Nef expression in this system. In addition, I present evidence indicating that Nef proteins from diverse HIV-1 Groups and strains are capable of enhancing HIV-1 replication in this system. Analysis of a range of mutations in Nef known to impact interaction with cellular proteins suggest that the observed replication enhancement requires Nef targeting to the host cell membrane and may also require the ability to interact with select Src-kinases. Lastly, we find that the ability of Nef to enhance replication in this system is separate from any increase in viral particle infectivity, in agreement with current literature.