Previously we have shown that trityl and diphenyl deoxyuridine derivatives and their acyclic analogues can inhibit Plasmodium falciparum dUTPase (PfdUTPase). We report the synthesis of conformationally restrained amide derivatives as inhibitors PfdUTPase, including both acyclic and cyclic examples. Activity was dependent on the orientation and location of the amide constraining group. In the case of the acyclic series, we were able to obtain amide-constrained analogues which showed similar or greater potency than the unconstrained analogues. Unfortunately these compounds showed lower selectivity in cellular assays.
We report the palladium-catalyzed, pyrrolidine-mediated α-benzylation of enamines generated from aldehydes and ketones. The method allows for direct coupling of medicinally relevant coumarin moieties with aldehydes and ketones in good yield under mild conditions. The reaction is believed to proceed via a Pd-π-benzyl complex generated from (coumarinyl)methyl acetates.
Predicting the effects of mutations on the kinetic rate constants of protein-protein interactions is central to both the modeling of complex diseases and the design of effective peptide drug inhibitors. However, while most studies have concentrated on the determination of association rate constants, dissociation rates have received less attention. In this work we take a novel approach by relating the changes in dissociation rates upon mutation to the energetics and architecture of hotspots and hotregions, by performing alanine scans pre- and post-mutation. From these scans, we design a set of descriptors that capture the change in hotspot energy and distribution. The method is benchmarked on 713 kinetically characterized mutations from the SKEMPI database. Our investigations show that, with the use of hotspot descriptors, energies from single-point alanine mutations may be used for the estimation of off-rate mutations to any residue type and also multi-point mutations. A number of machine learning models are built from a combination of molecular and hotspot descriptors, with the best models achieving a Pearson's Correlation Coefficient of 0.79 with experimental off-rates and a Matthew's Correlation Coefficient of 0.6 in the detection of rare stabilizing mutations. Using specialized feature selection models we identify descriptors that are highly specific and, conversely, broadly important to predicting the effects of different classes of mutations, interface regions and complexes. Our results also indicate that the distribution of the critical stability regions across protein-protein interfaces is a function of complex size more strongly than interface area. In addition, mutations at the rim are critical for the stability of small complexes, but consistently harder to characterize. The relationship between hotregion size and the dissociation rate is also investigated and, using hotspot descriptors which model cooperative effects within hotregions, we show how the contribution of hotregions of different sizes, changes under different cooperative effects.
Within a cell, protein-protein interactions vary considerably in their degree of stickiness. Mutations at protein interfaces can alter the interaction between protein pairs, causing them to dissociate faster or slower. This may lead to an alteration in the dynamics of the cellular networks in which these proteins are involved. Therefore, the calculation and interpretation of mutants, which affect the rate of dissociation, is critical to our understanding of complex networks and disease. A key characteristic of protein–protein interfaces is that a subset of residues are responsible for most of the binding energy, such residues are called hotspots and effectively represent the sticky points of the interaction. In this work, we exploit both hotspot energies and organization and use them for the calculation of off-rate changes upon mutations. The insights gained provide us with a clearer understanding of the critical regions of stability and how they change for complexes of different sizes. Moreover, we provide a comprehensive map of the key determinants responsible for the accurate characterization of different classes of mutations, complexes and interface regions. This paves the way for more intelligent computational-interface-design algorithms and provides new insight into the interpretation of destabilizing mutations involved in complex diseases.
Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAFV600E) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAFV600E-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAFV600E oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAFV600E leads to lung adenoma development. Importantly, expression of BRAFV600E concomitant with the loss of only a single-copy of Lkb1, overcomes senencence–like features of BRAFV600E-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAFV600E mutated lung adenomas in human cancer patients.
Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis.
The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.
The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. While AMPylases from various pathogenic microorganisms have recently been characterized, the only virulence protein with de-AMPylation activity known to date is the Legionella pneumophila effector SidD which catalyzes AMP removal from the host GTPase Rab1. Thus, both AMPylation and de-AMPylation constitute a novel catalytic mechanism to precisely control the function and membrane dynamics of a host Rab GTPase. In spite of this pivotal role, the molecular mechanism of AMP removal and the structural determinants for Rab1 recognition by SidD have remained largely unexplored. Here, we present the crystal structure of the de-adenylylation domain of SidD and reveal the catalytic mechanism of Rab1 de-adenylylation. Surprisingly, the structure of SidD is not related to the other known enzyme with de-AMPylation activity, the Escherichia coli GS-ATase. Instead, the catalitic domain of SidD is remarkably similar to that of the metal-dependent protein phosphatases (PPMs), however with distinctive structural features to distinguish AMPylated Rab1 from similarly modified substrates. Importantly, we provide a model for the SidD-Rab1 complex which sheds light into the specific details of substrate recognition and catalysis by this virulence factor.
Diagnosis of invasive aspergillosis (IA) requires increasingly rapid molecular methods that enable sensitive detection and discrimination between species. We designed and evaluated a real-time PCR-based method that combined melting temperature (Tm) calling analysis of a specific probe with high-resolution melting analysis of the full amplicon. The test correctly identified 78 isolates of Aspergillus section Fumigati and non-Fumigati sections of Aspergillus with a limit of detection of 102 conidia/ml (102 fg/ml). No cross-reactivity with other fungi was found. The assay was further validated on lower respiratory tract specimens containing Aspergillus or not. It successfully identified Aspergillus to section level in 56 of 59 specimens. With culture as the gold standard, our assay shows 100% sensitivity and specificity and constitutes an efficient alternative for identification of Aspergillus in lower respiratory tract samples.
This paper presents a methodology for high resolution radar image generation and automatic target recognition emphasizing the computational cost involved in the process. In order to obtain focused inverse synthetic aperture radar (ISAR) images certain signal processing algorithms must be applied to the information sensed by the radar. From actual data collected by radar the stages and algorithms needed to obtain ISAR images are revised, including high resolution range profile generation, motion compensation and ISAR formation. Target recognition is achieved by comparing the generated set of actual ISAR images with a database of ISAR images generated by electromagnetic software. High resolution radar image generation and target recognition processes are burdensome and time consuming, so to determine the most suitable implementation platform the analysis of the computational complexity is of great interest. To this end and since target identification must be completed in real time, computational burden of both processes the generation and comparison with a database is explained separately. Conclusions are drawn about implementation platforms and calculation efficiency in order to reduce time consumption in a possible future implementation.
high resolution radar; radar imagery; ISAR; target recognition; computational burden; NCC
Basilar-membrane responses to clicks were measured, using laser velocimetry, at a site of the chinchilla cochlea located about 3.5 mm from the oval window (characteristic frequency or CF: typically 8–10 kHz). They consisted of relatively undamped oscillations with instantaneous frequency that increased rapidly (time constant: 200 µs) from a few kHz to CF. Such frequency modulation was evident regardless of stimulus level and was also present post-mortem. Responses grew linearly at low stimulus levels, but exhibited a compressive nonlinearity at higher levels. Velocity-intensity functions were almost linear near response onset but became nonlinear within 100 µs. Slopes could be as low as 0.1–0.2 dB/dB at later times. Hence, the response envelopes became increasingly skewed at higher stimulus levels, with their center of gravity shifting to earlier times. The phases of near-CF response components changed by nearly 180 degrees as a function of time. At high stimulus levels, this generated cancellation notches and phase jumps in the frequency spectra. With increases in click level, sharpness of tuning deteriorated and the spectral maximum shifted to lower frequencies. Response phases also changed as a function of increasing stimulus intensity, exhibiting relative lags and leads at frequencies somewhat lower and higher than CF, respectively. In most respects, the magnitude and phase frequency spectra of responses to clicks closely resembled those of responses to tones. Post-mortem responses were similar to in vivo responses to very intense clicks.
The responses to sound of mammalian cochlear neurons exhibit many nonlinearities, some of which (such as two-tone rate suppression and intermodulation distortion) are highly frequency specific, being strongly tuned to the characteristic frequency (cf) of the neuron. With the goal of establishing the cochlear origin of these auditory-nerve nonlinearities, mechanical responses to clicks and to pairs of tones were studied in relatively healthy chinchilla cochleae at a basal site of the basilar membrane with cf of 8–10 kHz. Responses were also obtained in cochleae in which hair cell receptor potentials were reduced by systemic furosemide injection. Vibrations were recorded using either the Mössbauer technique or laser Doppler-shift velocimetry. Responses to tone pairs contained intermodulation distortion products whose magnitudes as a function of stimulus frequency and intensity were comparable to those of distortion products in cochlear afferent responses. Responses to cf tones could be selectively suppressed by tones with frequency either higher or lower than cf; in most respects, mechanical two-tone suppression resembled rate suppression in cochlear afferents. Responses to clicks displayed a cf-specific compressive nonlinearity, similar to that present in responses to single tones, which could be profoundly and selectively reduced by furosemide. The present findings firmly support the hypothesis that all cf-specific nonlinearities present in the auditory nerve originate in analogous phenomena of basilar membrane vibration. However, because of their lability, it is almost certain that the mechanical nonlinearities themselves originate in outer hair cells.
Basilar-membrane responses to single tones were measured, using laser velocimetry, at a site of the chinchilla cochlea located 3.5 mm from its basal end. Responses to low-level (<10–20 dB SPL) characteristic-frequency (CF) tones (9–10 kHz) grow linearly with stimulus intensity and exhibit gains of 66–76 dB relative to stapes motion. At higher levels, CF responses grow monotonically at compressive rates, with input–output slopes as low as 0.2 dB/dB in the intensity range 40–80 dB. Compressive growth, which is significantly correlated with response sensitivity, is evident even at stimulus levels higher than 100 dB. Responses become rapidly linear as stimulus frequency departs from CF. As a result, at stimulus levels >80 dB the largest responses are elicited by tones with frequency about 0.4–0.5 octave below CF. For stimulus frequencies well above CF, responses stop decreasing with increasing frequency: A plateau is reached. The compressive growth of responses to tones with frequency near CF is accompanied by intensity-dependent phase shifts. Death abolishes all nonlinearities, reduces sensitivity at CF by as much as 60–81 dB, and causes a relative phase lead at CF.
Responses to tones of a basilar membrane site and of auditory nerve fibers innervating neighboring inner hair cells were recorded in the same cochleae in chinchillas. At near-threshold stimulus levels, the frequency tuning of auditory nerve fibers closely paralleled that of basilar membrane displacement modified by high-pass filtering, indicating that only relatively minor signal transformations intervene between mechanical vibration and auditory nerve excitation. This finding establishes that cochlear frequency selectivity in chinchillas (and probably in mammals in general) is fully expressed in the vibrations of the basilar membrane and renders unnecessary additional (“second”) filters, such as those present in the hair cells of the cochleae of reptiles.
Basilar-membrane responses to white Gaussian noise were recorded using laser velocimetry at basal sites of the chinchilla cochlea with characteristic frequencies near 10 kHz and first-order Wiener kernels were computed by cross correlation of the stimuli and the responses. The presence or absence of minimum-phase behavior was explored by fitting the kernels with discrete linear filters with rational transfer functions. Excellent fits to the kernels were obtained with filters with transfer functions including zeroes located outside the unit circle, implying nonminimum-phase behavior. These filters accurately predicted basilar-membrane responses to other noise stimuli presented at the same level as the stimulus for the kernel computation. Fits with all-pole and other minimum-phase discrete filters were inferior to fits with nonminimum-phase filters. Minimum-phase functions predicted from the amplitude functions of the Wiener kernels by Hilbert transforms were different from the measured phase curves. These results, which suggest that basilar-membrane responses do not have the minimum-phase property, challenge the validity of models of cochlear processing, which incorporate minimum-phase behavior.
Autoregressive moving-average (ARMA) modeling; basilar membrane (BM); cochlea; Hilbert transform; minimum phase; Wiener kernels
Spatial magnitude and phase profiles for inner hair cell depolarization throughout the chinchilla cochlea were inferred from responses of auditory-nerve fibers to threshold- and moderate-level tones and tone complexes. Firing-rate profiles for frequencies ≤ 2 kHz are bimodal, with the major peak at the characteristic place and a secondary peak at 3–5 mm from the extreme base. Response-phase trajectories are synchronous with peak outward stapes displacement at the extreme cochlear base and accumulate 1.5-period lags at the characteristic places. High-frequency phase trajectories are very similar to the trajectories of basilar-membrane peak velocity toward scala tympani. Low-frequency phase trajectories undergo a polarity flip in a region, 6.5–9 mm from the cochlear base, where traveling-wave phase velocity attains a local minimum and a local maximum and where the onset latencies of near-threshold impulse responses computed from responses to near-threshold white noise exhibit a local minimum. That region is the same where frequency-threshold tuning curves of auditory-nerve fibers undergo a shape transition. Since depolarization of inner hair cells presumably indicates the mechanical stimulus to their stereocilia, the present results suggest that distinct low-frequency forward waves of organ of Corti vibration are launched simultaneously at the extreme base of the cochlea and at the 6.5–9 mm transition region, from where antiphasic reflections arise.
The T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky) with that of normal controls.
Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/− individuals, whereas CD3δ+/− and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.
The results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.
T cells; CD3; Haploinsufficiency
This report describes the baseline characteristics of patients in the Reduction of Events with Darbepoetin alfa in Heart Failure trial (RED-HF) which is testing the hypothesis that anaemia correction with darbepoetin alfa will reduce the composite endpoint of death from any cause or hospital admission for worsening heart failure, and improve other outcomes.
Methods and results
Key demographic, clinical, and laboratory findings, along with baseline treatment, are reported and compared with those of patients in other recent clinical trials in heart failure. Compared with other recent trials, RED-HF enrolled more elderly [mean age 70 (SD 11.4) years], female (41%), and black (9%) patients. RED-HF patients more often had diabetes (46%) and renal impairment (72% had an estimated glomerular filtration rate <60 mL/min/1.73 m2). Patients in RED-HF had heart failure of longer duration [5.3 (5.4) years], worse NYHA class (35% II, 63% III, and 2% IV), and more signs of congestion. Mean EF was 30% (6.8%). RED-HF patients were well treated at randomization, and pharmacological therapy at baseline was broadly similar to that of other recent trials, taking account of study-specific inclusion/exclusion criteria. Median (interquartile range) haemoglobin at baseline was 112 (106–117) g/L.
The anaemic patients enrolled in RED-HF were older, moderately to markedly symptomatic, and had extensive co-morbidity.
Heart failure; Anaemia
Patients with spinal cord injury (SCI) have many factors that are associated with pressure ulcer formation, including paralysis, loss of sensation, poor nutrition, anemia, and skin maceration related to incontinence. Treatment of these ulcers involves relieving pressure, improving nutrition and skin hygiene, treating infections, removing necrotic tissues, and applying the appropriate dressings. However, some cases are not responsive to the above treatment. Electrical stimulation (ES) is thought to enhance soft tissue healing through promotion of protein synthesis, inhibition of bacterial growth, facilitation of epithelial tissue migration, improvement of blood flow, and tensile strength. This data is mainly based on evidence from animal studies and very few rigorously controlled studies conducted in humans.
To demonstrate the effectiveness of ES in the treatment of recalcitrant pressure ulcers.
Retrospective case series describing the care of adults with SCI and recalcitrant pressure ulcers. ES was applied directly into the wound bed: 60 minutes per session, 3–5 times per week; with an intensity of 100 milliamperes and a frequency of 100 pulses per second. Polarity was negative initially and was switched weekly. The amplitude and wave form were maintained throughout.
The long-standing (11–14 months) pressure ulcers were completely healed after 7 to 22 weeks of treatment with high-voltage ES.
This case series demonstrates the effectiveness of ES for enhanced healing of Stage III–IV ulcers otherwise unresponsive to standard wound care. Further study is needed to identify the most effective protocol for ES therapy in the treatment of recalcitrant pressure ulcers.
Electrical stimulation; Spinal cord injuries; Pressure ulcer; Wound healing
Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs.
Neuronal differentiation; ERK1/2; Akt; PLCγ; progenitors; spinal cord
The naphthoquinone shikonin, a major component of the root of Lithospermum erythrorhizon, now is studied as an anti-inflammatory agent in the treatment of ulcerative colitis (UC). Acute UC was induced in Balb/C mice by oral administration of 5% dextran sodium sulfate (DSS). The disease activity index was evaluated, and a histologic study was carried out. Orally administered shikonin reduces induced UC in a dose-dependent manner, preventing the shortening of the colorectum and decreasing weight loss by 5% while improving the appearance of feces and preventing bloody stools. The disease activity index score was much lower in shikonin-treated mice than in the colitic group, as well as the myeloperoxidase activity. The expression of cyclooxygenase-2 was reduced by 75%, activation of NF-κB was reduced by 44%, and that of pSTAT-3 by 47%, as well as TNF-α, IL-1β, and IL-6 production. Similar results were obtained in primary macrophages culture. This is the first report of shikonin's ability to attenuate acute UC induced by DSS. Shikonin acts by blocking the activation of two major targets: NF-κB and STAT-3, and thus constitutes a promising potential therapeutic agent for the management of the inflammatory bowel disease.
Epigenetic regulation in mammals begins in the first stages of embryogenesis. This prenatal programming determines, in part, phenotype expression in adult life. Some species, particularly dairy cattle, are conceived during the maternal lactation, which is a period of large energy and nutrient needs. Under these circumstances, embryo and fetal development compete for nutrients with the mammary gland, which may affect prenatal programming and predetermine phenotype at adulthood. Data from a specialized dairy breed were used to determine the transgenerational effect when embryo development coincides with maternal lactation. Longitudinal phenotypic data for milk yield (kg), ratio of fat-protein content in milk during first lactation, and lifespan (d) from 40,065 cows were adjusted for environmental and genetic effects using a Bayesian framework. Then, the effect of different maternal circumstances was determined on the residuals. The maternal-related circumstances were 1) presence of lactation, 2) maternal milk yield level, and 3) occurrence of mastitis during embryogenesis. Females born to mothers that were lactating while pregnant produced 52 kg (MonteCarlo standard error; MCs.e. = 0.009) less milk, lived 16 d (MCs.e. = 0.002) shorter and were metabolically less efficient (+0.42% milk fat/protein ratio; MCs.e.<0.001) than females whose fetal life developed in the absence of maternal lactation. The greater the maternal milk yield during embryogenesis, the larger the negative effects of prenatal programming, precluding the offspring born to the most productive cows to fully express their potential additive genetic merit during their adult life. Our data provide substantial evidence of transgenerational effect when pregnancy and lactation coincide. Although this effect is relatively low, it should not be ignored when formulating rations for lactating and pregnant cows. Furthermore, breeding, replacement, and management strategies should also take into account whether the individuals were conceived during maternal lactation because, otherwise, their performance may deviate from what it could be expected.
New therapeutic strategies are needed for malignant pleural mesothelioma (MPM). We conducted a single-center, open-label, nonrandomized, pilot and feasibility trial using two intrapleural doses of an adenoviral vector encoding human IFN-α (Ad.IFN-α2b). Nine subjects were enrolled at two dose levels. The first three subjects had very high pleural and systemic IFN-α concentrations resulting in severe “flu-like” symptoms necessitating dose de-escalation. The next six patients had reduced (but still significant) pleural and serum IFN-α levels, but with tolerable symptoms. Repeated vector administration appeared to prolong IFN-α expression levels. Anti-tumor humoral immune responses against mesothelioma cell lines were seen in seven of the eight subjects evaluated. No clinical responses were seen in the four subjects with advanced disease. However, evidence of disease stability or tumor regression was seen in the remaining five patients, including one dramatic example of partial tumor regression at sites not in contiguity with vector infusion. These data show that Ad.IFN-α2b has potential therapeutic benefit in MPM and that it generates anti-tumor immune responses that may induce anatomic and/or metabolic reductions in distant tumor.
Clinical trial registered with www.clinicaltrials.gov (NCT 01212367).
clinical trials; immunotherapy; gene therapy
Styrene–acrylonitrile Trimer (SAN Trimer), a by-product in production of acrylonitrile styrene plastics, was identified at a Superfund site in Dover Township, NJ, where childhood cancer incidence rates were elevated for a period of several years. SAN Trimer was therefore tested by the National Toxicology Program in a 2-year perinatal carcinogenicity study in F344/N rats and a bacterial mutagenicity assay; both studies gave negative results. To further characterize its genotoxicity, SAN Trimer was subsequently evaluated in a combined micronucleus (MN)/Comet assay in juvenile male and female F344 rats. SAN Trimer (37.5, 75, 150, or 300 mg/kg/day) was administered by gavage once daily for 4 days. Micronucleated reticulocyte (MN-RET) frequencies in blood were determined by flow cytometry, and DNA damage in blood, liver, and brain cells was assessed using the Comet assay. Highly significant dose-related increases (P < 0.0001) in MN-RET were measured in both male and female rats administered SAN Trimer. The RET population was reduced in high dose male rats, suggesting chemical-related bone marrow toxicity. Results of the Comet assay showed significant, dose-related increases in DNA damage in brain cells of male (P < 0.0074) and female (P < 0.0001) rats; increased levels of DNA damage were also measured in liver cells and leukocytes of treated rats. Chemical-related cytotoxicity was not indicated in any of the tissues examined for DNA damage. The results of this subacute MN/Comet assay indicate induction of significant genetic damage in multiple tissues of weanling F344 male and female rats after oral exposure to SAN Trimer.
chromosomal damage; DNA damage; Comet assay; micronuclei; superfund; childhood cancer
The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH>13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hour intervals (VS was administered to rats for 3 days); animals were euthanized 4 hours after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.
DNA damage; Comet assay; acrylamide; ethyl methanesulfonate; cyclophosphamide; vincristine sulfate
Patients with chronic lymphocytic leukemia and 13q deletion as their only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus, cases with a high number of 13q- cells (13q-H) had both shorter overall survival and time to first therapy. The goal of the study was to analyze the genetic profile of 13q-H patients.
Design and Methods:
A total of 102 samples were studied, 32 of which served as a validation cohort and five were healthy donors.
Chronic lymphocytic leukemia patients with higher percentages of 13q- cells (>80%) showed a different level of gene expression as compared to patients with lower percentages (<80%, 13q-L). This deregulation affected genes involved in apoptosis and proliferation (BCR and NFkB signaling), leading to increased proliferation and decreased apoptosis in 13q-H patients. Deregulation of several microRNAs, such as miR-15a, miR-155, miR-29a and miR-223, was also observed in these patients. In addition, our study also suggests that the gene expression pattern of 13q-H cases could be similar to the patients with 11q- or 17p-.
This study provides new evidence regarding the heterogeneity of 13q deletion in chronic lymphocytic leukemia patients, showing that apoptosis, proliferation as well as miRNA regulation are involved in cases with higher percentages of 13q- cells.