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1.  A proliferation saturation index to predict radiation response and personalize radiotherapy fractionation 
Although altered protocols that challenge conventional radiation fractionation have been tested in prospective clinical trials, we still have limited understanding of how to select the most appropriate fractionation schedule for individual patients. Currently, the prescription of definitive radiotherapy is based on the primary site and stage, without regard to patient-specific tumor or host factors that may influence outcome. We hypothesize that the proportion of radiosensitive proliferating cells is dependent on the saturation of the tumor carrying capacity. This may serve as a prognostic factor for personalized radiotherapy (RT) fractionation.
We introduce a proliferation saturation index (PSI), which is defined as the ratio of tumor volume to the host-influenced tumor carrying capacity. Carrying capacity is as a conceptual measure of the maximum volume that can be supported by the current tumor environment including oxygen and nutrient availability, immune surveillance and acidity. PSI is estimated from two temporally separated routine pre-radiotherapy computed tomography scans and a deterministic logistic tumor growth model. We introduce the patient-specific pre-treatment PSI into a model of tumor growth and radiotherapy response, and fit the model to retrospective data of four non-small cell lung cancer patients treated exclusively with standard fractionation. We then simulate both a clinical trial hyperfractionation protocol and daily fractionations, with equal biologically effective dose, to compare tumor volume reduction as a function of pretreatment PSI.
With tumor doubling time and radiosensitivity assumed constant across patients, a patient-specific pretreatment PSI is sufficient to fit individual patient response data (R2 = 0.98). PSI varies greatly between patients (coefficient of variation >128 %) and correlates inversely with radiotherapy response. For this study, our simulations suggest that only patients with intermediate PSI (0.45–0.9) are likely to truly benefit from hyperfractionation. For up to 20 % uncertainties in tumor growth rate, radiosensitivity, and noise in radiological data, the absolute estimation error of pretreatment PSI is <10 % for more than 75 % of patients.
Routine radiological images can be used to calculate individual PSI, which may serve as a prognostic factor for radiation response. This provides a new paradigm and rationale to select personalized RT dose-fractionation.
PMCID: PMC4521490  PMID: 26227259
Proliferation saturation index; Personalized radiotherapy; Logistic tumor growth; Mathematical model; Hyperfractionation
2.  The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells 
PLoS ONE  2015;10(7):e0133263.
Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.
PMCID: PMC4507941  PMID: 26191799
3.  An Extended Polyanion Activation Surface in Insulin Degrading Enzyme 
PLoS ONE  2015;10(7):e0133114.
Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898) within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity.
PMCID: PMC4506039  PMID: 26186535
4.  Automatic detection of beating cilia with frequencies estimations 
Cilia  2015;4(Suppl 1):P85.
PMCID: PMC4518898
5.  Phenotypic differentiation in love song traits among sibling species of the Lutzomyia longipalpis complex in Brazil 
Parasites & Vectors  2015;8:290.
Brazilian populations of Lutzomyia longipalpis may constitute a complex of cryptic species, and this report investigates the distribution and number of potential sibling species. One of the main differences observed among Brazilian populations is the type of acoustic signal produced by males during copulation. These copulation song differences seem to be evolving faster than neutral molecular markers and have been suggested to contribute to insemination failure observed in crosses between these sibling species. In previous studies, two main types of copulation songs were found, burst-type and pulse-type. The latter type can, in turn, be further subdivided into five different patterns.
We recorded male song from 13 new populations of the L. longipalpis complex from Brazil and compared the songs with 12 already available.
Out of these 25 populations, 16 produce burst-type and 9 produce pulse-type songs. We performed a principal component analysis in these two main groups separately and an additional discriminant analysis in the pulse-type group. The pulse-type populations showed a clear separation between the five known patterns with a high correspondence of individuals to their correct group, confirming the differentiation between them. The distinctiveness of the burst-type subgroups was much lower than that observed among the pulse-type groups and no clear population structure was observed. This suggests that the burst-type populations represent a single species.
Overall, our results are consistent with the existence in Brazil of at least six species of the L. longipalpis complex, one with a wide distribution comprising all the populations with burst-type songs, and five more closely related allopatric siblings with different pulse-type song patterns and more restricted distribution ranges.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0900-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4456791  PMID: 26017472
Sexual behaviour; Sand fly; Copulation song; Copulatory courtship; Insect vector; Species complex
The New England journal of medicine  2000;343(26):1925-1932.
Colonization and infection with vancomycin-resistant enterococci have been associated with exposure to antibiotics that are active against anaerobes. In mice that have intestinal colonization with vancomycin-resistant enterococci, these agents promote high-density colonization, whereas antibiotics with minimal antianaerobic activity do not.
We conducted a seven-month prospective study of 51 patients who were colonized with vancomycin-resistant enterococci, as evidenced by the presence of the bacteria in stool. We examined the density of vancomycin-resistant enterococci in stool during and after therapy with antibiotic regimens and compared the effect on this density of antianaerobic agents and agents with minimal antianaerobic activity. In a subgroup of 10 patients, cultures of environmental specimens (e.g., from bedding and clothing) were obtained.
During treatment with 40 of 42 antianaerobic-antibiotic regimens (95 percent), high-density colonization with vancomycin-resistant enterococci was maintained (mean [±SD] number of organisms, 7.8± 1.5 log per gram of stool). The density of colonization decreased after these regimens were discontinued. Among patients who had not received antianaerobic antibiotics for at least one week, 10 of 13 patients who began such regimens had an increase in the number of organisms of more than 1.0 log per gram (mean increase, 2.2 log per gram), whereas among 10 patients who began regimens of antibiotics with minimal antianaerobic activity, there was a mean decrease in the number of enterococci of 0.6 log per gram (P= 0.006 for the difference between groups). When the density of vancomycin-resistant enterococci in stool was at least 4 log per gram, 10 of 12 sets of cultures of environmental specimens had at least one positive sample, as compared with 1 of 9 sets from patients with a mean number of organisms in stool of less than 4 log per gram (P=0.002).
For patients with vancomycin-resistant enterococci in stool, treatment with antianaerobic antibiotics promotes high-density colonization. Limiting the use of such agents in these patients may help decrease the spread of vancomycin-resistant enterococci.
PMCID: PMC4370337  PMID: 11136263
8.  Utility of EBUS-TBNA for diagnosis of mediastinal tuberculous lymphadenitis: a multicentre Australian experience 
Journal of Thoracic Disease  2015;7(3):439-448.
Endobronchial ultrasound (EBUS) transbronchial needle aspiration (TBNA) is an important diagnostic procedure for the interrogation of mediastinal lymph nodes. There is limited data describing the accuracy & safety of this technique for the diagnosis of tuberculous mediastinal lymphadenitis.
A multi-centre retrospective study of all EBUS-guided TBNA procedures that referred samples for mycobacteriology was performed. Results were correlated with post-procedural diagnoses after a period of surveillance and cross-checked against relevant statewide tuberculosis (TB) registries, and sensitivity and specificity was calculated. In addition, nucleic acid amplification techniques (NAAT) were assessed, and sensitivity and specificity calculated using positive mycobacterial culture as the reference gold standard.
One hundred and fifty-nine patients underwent EBUS-TBNA and had tissue referred for mycobacterial culture, of which 158 were included in the final analysis. Thirty-nine were ultimately diagnosed with TB (25%). Sensitivity of EBUS-TBNA for microbiologically confirmed tuberculous mediastinal lymphadenitis was 62% (24/39 cases). Specificity was 100%. Negative predictive value (NPV) and diagnostic accuracy for microbiologic diagnosis was 89% [95% confidence intervals (CI), 82-93%] and 91% (95% CI, 84-94%) respectively. For a composite clinicopathologic diagnosis of TB NPV and accuracy were 98% (95% CI, 93-99%) and 98% (95% CI, 95-99%) respectively. Sensitivity for NAAT was 38% (95% CI, 18-65%).
EBUS-TBNA is a safe and well tolerated procedure in the assessment of patients with suspected isolated mediastinal lymphadenitis and demonstrates good sensitivity for a microbiologic diagnosis of isolated mediastinal lymphadenitis. When culture and histological results are combined with high clinical suspicion, EBUS-TBNA demonstrates excellent diagnostic accuracy and NPV for the diagnosis of mediastinal TB lymphadenitis. We suggest EBUS-TBNA should be considered the procedure of choice for patients in whom TB is suspected.
PMCID: PMC4387413  PMID: 25922723
Endobronchial ultrasound (EBUS); minimally invasive; bronchoscopy; tuberculosis (TB); mycobacteria; culture; PCR
9.  In Memoriam: Robert C. Moellering, Jr. 
PMCID: PMC4068561  PMID: 24798286
10.  The Impact of Economic Recession on the Incidence and Treatment of Cancer 
Journal of Cancer  2015;6(8):727-733.
Purpose: The impact of economic recessions on the incidence and treatment of cancer is unknown. We test the hypothesis that cancer incidence and treatment rates decrease during a recession, and that this relationship is more pronounced in cancers that present with mild, more easily ignored symptoms.
Methods and Materials: Data on incidence and treatment for all cancers, and breast and pancreatic cancers specifically, from 1973-2008, were collected using Surveillance Epidemiology and End Results (SEER). The data was adjusted for race, income, and education. Unemployment rate was used as the measure of economic recession. Data was log-transformed, and multivariate linear mixed regression was used.
Results: Adjusting for socioeconomic factors, the data revealed a significant inverse correlation between unemployment and rates of cancer incidence and treatment. Every 1% increase in unemployment was associated with a 2.2% (95% CI: 1.6-2.8%, p<0.001) reduction in cancer incidence, a 2.0% (1.2-2.8%, p=0.0157) decrease in surgery, and a 9.1% (8.2-10.0% p<0.001) decrease in radiation therapy (RT). Breast cancer incidence and treatment had a dramatic inverse relationship - 7.2% (6.3-8.1%), 6.7% (5.7-7.6%), and 19.0% (18.1-19.8%), respectively (p<0.001 for all). The decrease in incidence was only significant for in situ and localized tumors, but not in regional or distant breast cancer. Compared to breast cancer, pancreatic cancer had a weaker relationship between unemployment and incidence: 2.6% (1.8-3.3%, p=0.0005), surgery: 2.4% (2.0-2.7%, p<0.001), and RT: 1.9% (1.5-2.2% p<0.001).
Conclusions: Increasing unemployment rates are associated with a decrease in the incidence and treatment of all cancers. This effect is exaggerated in breast cancer, where symptoms can more easily be ignored and where there are widely used screening tests relative to pancreatic cancer.
PMCID: PMC4504108  PMID: 26185534
economic recessions; cancer; treatment
11.  Genomic Characterisation of Small Cell Lung Cancer Patient-Derived Xenografts Generated from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Specimens 
PLoS ONE  2014;9(9):e106862.
Patient-derived xenograft (PDX) models generated from surgical specimens are gaining popularity as preclinical models of cancer. However, establishment of PDX lines from small cell lung cancer (SCLC) patients is difficult due to very limited amount of available biopsy material. We asked whether SCLC cells obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) could generate PDX lines that maintained the phenotypic and genetic characteristics of the primary tumor. Following successful EBUS-TBNA sampling for diagnostic purposes, we obtained an extra sample for cytologic analysis and implantation into the flanks of immunodeficient mice. Animals were monitored for engraftment for up to 6 months. Histopathologic and immunohistochemical analysis, and targeted next-generation re-sequencing, were then performed in both the primary sample and the derivative PDX line. A total of 12 patients were enrolled in the study. EBUS-TBNA aspirates yielded large numbers of viable tumor cells sufficient to inject between 18,750 and 1,487,000 cells per flank, and to yield microgram quantities of high-quality DNA. Of these, samples from 10 patients generated xenografts (engraftment rate 83%) with a mean latency of 104 days (range 63–188). All but one maintained a typical SCLC phenotype that closely matched the original sample. Identical mutations that are characteristic of SCLC were identified in both the primary sample and xenograft line. EBUS-TBNA has the potential to be a powerful tool in the development of new targeting strategies for SCLC patients by providing large numbers of viable tumor cells suitable for both xenografting and complex genomic analysis.
PMCID: PMC4156408  PMID: 25191746
12.  An Experimental Evaluation of the Force Requirements for Robotic Mastoidectomy 
During robotic milling of the temporal bone, forces on the cutting burr may be lowered by choice of cutting parameters.
Robotic bone removal systems are used in orthopedic procedures but they are currently not accurate enough for safe use in otologic surgery. We propose the use of a bone attached milling robot to achieve the required accuracy and speed. To design such a robot and plan its milling trajectories, it is necessary to predict the forces that the robot must exert and withstand under likely cutting conditions.
Materials and Methods
We measured forces during bone removal for several surgical burr types, drill angles, depths of cut, cutting velocities, and bone types (cortical/surface bone and mastoid) on human temporal bone specimens.
Lower forces were observed for 5 mm diameter burrs compared to 3 mm burrs for a given bone removal rate. Higher linear cutting velocities and greater cutting depths independently resulted in higher forces. For combinations of velocities and depths that resulted in the same overall bone removal rate, lower forces were observed in parameter sets that combined higher cutting velocities and shallower depths. Lower mean forces and higher variability were observed in the mastoid compared with cortical/surface bone.
Forces during robotic milling of the temporal bone can be predicted from the parameter sets tested in this study. This information can be used to guide the design of a sufficiently rigid and powerful bone-attached milling robot and to plan efficient milling trajectories. To reduce the time of the surgical intervention without creating very large forces, high linear cutting velocities may be combined with shallow depths of cut. Faster and deeper cuts may be used in mastoid bone compared to cortical bone for a chosen force threshold.
PMCID: PMC3761064  PMID: 23787968
13.  Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales 
Synthesis of peptidoglycan precursors ending in d-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d-Lac into cytoplasmic precursors. This was due to a d,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d-Lac for d-Ala and Gly. The contribution of l,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d,d-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l,d-transpeptidases merely act as a tolerance mechanism in this bacterium.
PMCID: PMC3957865  PMID: 24395229
14.  A Case of Spontaneous Systemic Immunity to Melanoma Associated with Cure after Amputation For Extensive Regional Recurrence 
Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation.
A 71 year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELIspot assay, and antibody responses to a panel of tumor antigens were assayed by ELISA.
The patient's tumor had minimal lymphocytic infiltrate (Immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2-6 years after surgery.
The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.
PMCID: PMC4082724  PMID: 23666534
Melanoma; amputation for melanoma; NY-ESO-1; tumor immunity; antibody; T-cell
15.  Design of a Bone-Attached Parallel Robot for Percutaneous Cochlear Implantation 
Access to the cochlea requires drilling in close proximity to bone-embedded nerves, blood vessels, and other structures, the violation of which can result in complications for the patient. It has recently been shown that microstereotactic frames can enable an image-guided percutaneous approach, removing reliance on human experience and hand–eye coordination, and reducing trauma. However, constructing current microstereotactic frames disrupts the clinical workflow, requiring multiday intrasurgical manufacturing delays, or an on-call machine shop in or near the hospital. In this paper, we describe a new kind of microsterotactic frame that obviates these delay and infrastructure issues by being repositionable. Inspired by the prior success of bone-attached parallel robots in knee and spinal procedures, we present an automated image-guided microstereotactic frame. Experiments demonstrate a mean accuracy at the cochlea of 0.20 ± 0.07 mm in phantom testing with trajectories taken from a human clinical dataset. We also describe a cadaver experiment evaluating the entire image-guided surgery pipeline, where we achieved an accuracy of 0.38 mm at the cochlea.
PMCID: PMC4104131  PMID: 21788181
Bone-attached robot; cochlear implant; Gough–Stewart platform; microtable; minimally invasive surgery (MIS); parallel robot
16.  Serum Amyloid A Promotes Lung Neutrophilia by Increasing IL-17A Levels in the Mucosa and γδ T Cells 
Rationale: Neutrophilic inflammation is an important pathologic feature of chronic obstructive pulmonary disease (COPD) and infectious exacerbations of COPD. Serum amyloid A (SAA) promotes neutrophilic inflammation by its interaction with lung mucosal ALX/FPR2 receptors. However, little is known about how this endogenous mediator regulates IL-17A immunity.
Objectives: To determine whether SAA causes neutrophilic inflammation by IL-17A–dependent mechanisms.
Methods: The relationship between SAA and neutrophils was investigated in lung sections from patients with COPD and a chronic mouse model of SAA exposure. A neutralizing antibody to IL-17A was used to block SAA responses in vivo, and a cell-sorting strategy was used to identify cellular sources.
Measurements and Main Results: SAA mRNA expression was positively associated with tissue neutrophils in COPD (P < 0.05). SAA predominately promoted expression of the TH17 polarizing cytokine IL-6, which was opposed by 15-epi-lipoxin A4, a counter-regulatory mediator, and ALX/FPR2 ligand. SAA-induced inflammation was markedly reduced by a neutralizing antibody to IL-17A in vivo. Cellular sources of IL-17A induced by SAA include CD4+ T cells, γδ T cells, and an Epcam+CD45− population enriched for epithelial cells. SAA promotes expression of IL-17A in γδ T cells and this innate cell proportionally expressed higher levels of IL-17A transcript than CD4+ T cells or epithelial cells.
Conclusions: The SAA–IL-17A axis represents an important innate defense network that may underlie persistent neutrophilic airway inflammation in COPD and modulating the ALX/FPR2 receptor represents a novel approach to targeting aberrant IL-17A–mediated lung immunity.
PMCID: PMC3778755  PMID: 23627303
inflammation; neutrophils; chronic obstructive pulmonary disease; innate immunity
17.  Serine/Threonine Protein Phosphatase-Mediated Control of the Peptidoglycan Cross-Linking l,d-Transpeptidase Pathway in Enterococcus faecium 
mBio  2014;5(4):e01446-14.
The last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of β-lactam antibiotics. d,d-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, peptidoglycan cross-linking is performed mainly by active-site cysteine l,d-transpeptidases that use tetrapeptides in Mycobacterium tuberculosis, Clostridium difficile, and β-lactam-resistant mutants of Enterococcus faecium. We have investigated reprogramming of the E. faecium peptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs by l,d-transpeptidase Ldtfm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of the l,d-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldtfm at the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of the d,d-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldtfm. Here we show that full bypass of PBPs by Ldtfm also requires increased Ser/Thr protein phosphorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of the l,d-transpeptidation pathway in the absence of mutational alteration of peptidoglycan synthesis enzymes.
The mechanism of acquisition of high-level ampicillin resistance involving bypass of the penicillin-binding proteins (PBPs) by l,d-transpeptidase Ldtfm was incompletely understood, as production of tetrapeptide precursors following transcriptional activation of the ddc locus by the DdcRS two-component regulatory system was necessary but not sufficient for full activation of the l,d-transpeptidation pathway. Here, we identified the release of a negative control of Ser/Thr protein phosphorylation mediated by phosphatase StpA as the additional factor essential for ampicillin resistance. Thus, bypass of PBPs by Ldtfm requires the modification of signal transduction regulatory systems without any gain of function by mutational alteration of peptidoglycan biosynthetic enzymes. In contrast, previously characterized mechanisms of antibiotic resistance involve horizontal gene transfer and mutational alteration of drug targets. Activation of the l,d-transpeptidation pathway reported in this study is an unprecedented mechanism of emergence of a new metabolic pathway since it involved the recruitment of preexisting functions following modifications of regulatory circuits.
PMCID: PMC4161250  PMID: 25006233
18.  A Manually Operated, Advance Off-Stylet Insertion Tool for Minimally Invasive Cochlear Implantation Surgery 
The current technique for cochlear implantation (CI) surgery requires a mastoidectomy to gain access to the cochlea for electrode array insertion. It has been shown that microstereotactic frames can enable an image-guided, minimally invasive approach to CI surgery called percutaneous cochlear implantation (PCI) that uses a single drill hole for electrode array insertion, avoiding a more invasive mastoidectomy. Current clinical methods for electrode array insertion are not compatible with PCI surgery because they require a mastoidectomy to access the cochlea; thus, we have developed a manually operated electrode array insertion tool that can be deployed through a PCI drill hole. The tool can be adjusted using a preoperative CT scan for accurate execution of the advance off-stylet (AOS) insertion technique and requires less skill to operate than is currently required to implant electrode arrays. We performed three cadaver insertion experiments using the AOS technique and determined that all insertions were successful using CT and microdissection.
PMCID: PMC4081037  PMID: 22851233
Advance off-stylet (AOS); cochlear implant; image-guided surgery; minimally invasive surgery; percutaneous cochlear implantation (PCI)
19.  Obesity and diabetes cause cognitive dysfunction in the absence of accelerated β-amyloid deposition in a novel murine model of mixed or vascular dementia 
Mid-life obesity and type 2 diabetes mellitus (T2DM) confer a modest, increased risk for Alzheimer’s disease (AD), though the underlying mechanisms are unknown. We have created a novel mouse model that recapitulates features of T2DM and AD by crossing morbidly obese and diabetic db/db mice with APPΔNL/ΔNLx PS1P264L/P264L knock-in mice. These mice (db/AD) retain many features of the parental lines (e.g. extreme obesity, diabetes, and parenchymal deposition of β-amyloid (Aβ)). The combination of the two diseases led to additional pathologies-perhaps most striking of which was the presence of severe cerebrovascular pathology, including aneurysms and small strokes. Cortical Aβ deposition was not significantly increased in the diabetic mice, though overall expression of presenilin was elevated. Surprisingly, Aβ was not deposited in the vasculature or removed to the plasma, and there was no stimulation of activity or expression of major Aβ-clearing enzymes (neprilysin, insulin degrading enzyme, or endothelin-converting enzyme). The db/AD mice displayed marked cognitive impairment in the Morris Water Maze, compared to either db/db or APPΔNLx PS1P264L mice. We conclude that the diabetes and/or obesity in these mice leads to a destabilization of the vasculature, leading to strokes and that this, in turn, leads to a profound cognitive impairment and that this is unlikely to be directly dependent on Aβ deposition. This model of mixed or vascular dementia provides an exciting new avenue of research into the mechanisms underlying the obesity-related risk for age-related dementia, and will provide a useful tool for the future development of therapeutics.
PMCID: PMC4229778  PMID: 24916066
Dementia; Diabetes; Obesity; Stroke; Alzheimer’s disease
Hypertension  2013;61(4):921-930.
Reduced neprilysin (NEP), a cell surface metallopeptidase, which cleaves and inactivates pro-inflammatory and vasoactive peptides, predisposes the lung vasculature to exaggerated remodeling in response to hypoxia. We hypothesize that loss of NEP in pulmonary artery smooth muscle cells (PASMCs) results in increased migration and proliferation.
PASMCs isolated from NEP−/− mice exhibited enhanced migration and proliferation in response to serum and PDGF, which was attenuated by NEP replacement. Inhibition of NEP by overexpression of a peptidase dead mutant or knockdown by siRNA in NEP+/+ cells increased migration and proliferation. Loss of NEP led to an increase in Src kinase activity and phosphorylation of PTEN resulting in activation of the PDGF receptor (PDGFR). Knockdown of Src kinase with siRNA or inhibition with PP2 a src kinase inhibitor decreased PDGFRY751 phosphorylation and attenuated migration and proliferation in NEP−/− SMCs.
NEP substrates, endothelin-1(ET-1) or fibroblast growth factor-2 (FGF2), increased activation of Src and PDGFR in NEP+/+ cells, which was decreased by an ETAR antagonist, neutralizing antibody to FGF2 and Src inhibitor.
Similar to the observations in PASMCs levels of p-PDGFR, p-Src and p-PTEN were elevated in NEP−/− lungs. ETAR antagonist also attenuated the enhanced responses in NEP−/−PASMCs and lungs. Taken together our results suggest a novel mechanism for regulation of PDGFR signaling by NEP substrates involving Src and PTEN. Strategies that increase lung NEP activity/expression or target key downstream effectors, like Src, PTEN or PDGFR, may be of therapeutic benefit in pulmonary vascular disease.
PMCID: PMC3667616  PMID: 23381789
Neprilysin; smooth muscle cell; migration; PDGFR; Src; PTEN
21.  Putting PhDs to Work: Career Planning for Today's Scientist 
CBE Life Sciences Education  2014;13(1):49-53.
The authors examined individual development plan (IDP) awareness and use, the benefits of creating an IDP, and ways to facilitate IDP use by administering surveys to postdoctoral researchers, mentors, and administrators.
Individual development plans (IDPs) have been promoted nationally as a tool to help research trainees explore career opportunities and set career goals. Despite the interest in IDPs from a policy perspective, there is little information about how they have been used. The authors examined IDP awareness and use, the benefits of creating an IDP, and ways to facilitate its use by administering a survey to current or former postdoctoral researchers via the National Postdoctoral Association (NPA) and University of Alabama at Birmingham email lists; individuals belonging to Federation of American Societies for Experimental Biology member societies who mentored postdocs; and postdoctoral administrators at member institutions of the Association of American Medical Colleges and the NPA. Although most postdoctoral administrators (>80%) were familiar with IDPs, less than 50% of postdocs and only 20% of mentors were aware of IDPs. For those postdocs and mentors who reported creating an IDP, the process helped postdocs to identify the skills and abilities necessary for career success and facilitated communication between postdocs and their mentors. Despite the fact that creating an IDP benefits postdocs and mentors, IDP use will likely remain low unless institutions and research mentors encourage trainees to engage in this process.
PMCID: PMC3940462  PMID: 24591503
23.  Lymphotoxin α1β2 Expression on B Cells Is Required for Follicular Dendritic Cell Activation During the Germinal Center Response 
European journal of immunology  2012;43(2):348-359.
CD19-deficient mice were used as a model to study FDC activation because these mice have normal numbers of FDC-containing primary follicles, but lack the ability to activate FDC or form GC. It was hypothesized that CD19 expression is necessary for B cell activation and upregulation of membrane-lymphotoxin (mLT) expression, which promotes FDC activation. Using VCAM-1 and FcγRII/III as FDC activation markers, it was determined that the adoptive transfer of CD19+ wild-type B cells into CD19-deficient hosts rescued GC formation and FDC activation, demonstrating that CD19 expression on B cells is required for FDC activation. In contrast, CD19+ donor B cells lacking mLT were unable to induce VCAM-1 expression on FDC and FcγRII/III upregulation was impaired. VCAM-1 expression on FDC, but not FcγRII/III, was rescued when CD19-deficient B cells expressing transgenic mLT were cotransferred into recipient mice with CD19+, mLT-deficient B cells, suggesting that FDC activation requires the CD19-dependent upregulation of mLT on activated B cells. Collectively, these data demonstrate that activated B cells are responsible for the initiation of FDC activation resulting in a microenvironment supportive of GC development and maintenance.
PMCID: PMC3753018  PMID: 23112125
Follicular Dendritic Cell; Germinal Center; CD19; Membrane Lymphotoxin
24.  Structural Analysis of Muscles Elevating the Hyolaryngeal Complex 
Dysphagia  2012;27(4):445-451.
A critical event of pharyngeal swallowing is the elevation of the hyolaryngeal complex to open the upper esophageal sphincter. Current swallowing theory assigns this function to the submental and thyrohyoid muscles. However, the attachments of the long pharyngeal muscles indicate that they could contribute to this function, yet their role is uninvestigated in humans. In addition, there is evidence the posterior digastric and stylohyoid contribute to hyoid elevation. A cadaver model was used to document the structural properties of muscles. These properties were used to model muscle groups as force vectors and analyze their potential for hyolaryngeal elevation. Vector magnitude was determined using physiological cross-sectional areas (PCSAs) of muscles calculated from structural properties of muscle taken from 12 hemisected cadaver specimens. Vector direction (lines of action) was calculated from the three-dimensional coordinates of muscle attachment sites. Unit force vectors in the superior direction of submental, suprahyoid (which includes the submental muscles), long pharyngeal, and thyrohyoid muscles were derived and compared by an analysis of variance (ANOVA) to document each muscle’s potential contribution to hyolaryngeal elevation. An ANOVA with Tukey HSD post hoc analysis of unit force vectors showed no statistically significant difference between the submental (0.92 ± 0.24 cm2) and long pharyngeal (0.73 ± 0.20 cm2) muscles. Both demonstrated greater potential to elevate the hyolaryngeal complex than the thyrohyoid (0.49 ± 0.18 cm2), with P < 0.01 and P < 0.05, respectively. The suprahyoid muscles (1.52 ± 0.35 cm2) demonstrated the greatest potential to elevate the hyolaryngeal complex: greater than both the long pharyngeal muscles (P < 0.01) and the thyrohyoid (P < 0.01). The submental and thyrohyoid muscles by convention are thought to elevate the hyolaryngeal complex. This study demonstrates that structurally the long pharyngeal muscles have similar potential to contribute to this critical function, with the suprahyoid muscles having the greatest potential. If verified by functional data, these findings would amend current swallowing theory.
PMCID: PMC3350616  PMID: 22278076
Deglutition; Laryngeal elevation; Physiological cross-sectional area; Structural properties; Hyolaryngeal complex; Deglutition disorders
25.  Impact of Oncology Drug Shortages on Patient Therapy: Unplanned Treatment Changes 
Journal of Oncology Practice  2013;9(4):e122-e128.
Drug shortages have substantial economic costs and mandate treatment changes that may affect efficacy and toxicity.
Cancer drug shortages have increased considerably over the past 5 years, but quantitative analyses of the scope and effects are limited. We assessed the effects of drug shortages on outpatient medication use in a single New York City university hospital.
We examined pharmacy records for drug shortages, as defined by the American Society of Health-System Pharmacists. We assessed outpatient records for all patients with cancer treated with infusional antineoplastic medications from April 2010 to September 2010 and April 2011 to September 2011.
Twelve medications were in shortage in 2010 and 22 in 2011. Drugs in shortage were used for 170 patients (50.8%) in 2010 and 241 patients (63.6%) in 2011 (P < .001). Of 235 patients treated in August-September 2011, there were 23(9.8%) documented therapy changes due to shortages, compared with zero changes in August-September 2010 (P < .001). Among patients treated in August-September 2010, 24 (11.4%) received paclitaxel and 19 (9.0%) received docetaxel. Among patients treated in August-September 2011, 11 (4.7%) received paclitaxel and 38 (16.2%) received docetaxel, a 69% decrease for paclitaxel and 80% increase for docetaxel from 1 year prior (P = .009, and P = .024, respectively). The estimated cost of a single treatment with paclitaxel for one patient with body-surface area 1.75 was $47.59 versus $858.39 for docetaxel, a 1,704% increase. Surveyed physicians frequently reported lower level evidence (30.4%) and increased risk of toxicity (34.8%) with alternative therapy in drug shortage cases.
Oncology drug shortages affected the majority of patients in our center and increased at an alarming rate. Drug shortages have substantial economic costs and mandate treatment changes that may affect efficacy and toxicity.
PMCID: PMC3710178  PMID: 23942928

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