Many broadly neutralizing antibodies (bnAbs) against HIV-1 recognize and/or penetrate the glycan shield on native, virion-associated envelope glycoprotein (Env) spikes. The same bnAbs also bind to ...recombinant, soluble trimeric immunogens based on the SOSIP design. While SOSIP trimers are close structural and antigenic mimics of virion Env, the extent to which their glycan structures resemble ones on infectious viruses is undefined. Here, we compare the overall glycosylation of gp120 and gp41 subunits from BG505 (clade A) virions produced in a lymphoid cell line with those from recombinant BG505 SOSIP trimers, including CHO-derived clinical grade material. We also performed detailed site-specific analyses of gp120. Glycans relevant to key bnAb epitopes are generally similar on the recombinant SOSIP and virion-derived Env proteins, although the latter do contain hotspots of elevated glycan processing. Knowledge of native versus recombinant Env glycosylation will guide vaccine design and manufacturing programs.
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•HIV envelope glycans are central features of broadly neutralizing antibody epitopes•Recombinant mimetics of the HIV virus trimer are vaccine candidates•Glycosylation of a leading trimer immunogen is similar to that of infectious virus
Struwe et al. present site-specific analyses of N-glycosylation sites on HIV-1 envelope glycoproteins from an infectious virus and a recombinant trimer mimic. The structural and antigenic details of the glycan shield will be valuable for designing next-generation immunogens and understanding virus neutralization by broadly active antibodies.
Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins ...and DNA. Although a number nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Herein, we used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. We thus characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, we identified sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work constitutes the most extensive structural characterization of a viral lncRNA and interactions with its protein partners in discrete biological contexts, providing a broad framework for understanding the roles of PAN RNA in KSHV infection.
Genetically barcoded viral populations are powerful tools for evaluating the overall viral population structure as well as assessing the dynamics and evolution of individual lineages
over time. ...Barcoded viruses are generated by inserting a small, genetically unique tag into the viral genome, which is retained in progeny virus. We recently reported barcoding the well-characterized molecular clone simian immunodeficiency virus (SIV) SIVmac239, resulting in a synthetic swarm (SIVmac239M) containing approximately 10,000 distinct viral clonotypes for which all genetic differences were within a 34-base barcode that could be tracked using next-generation deep sequencing. Here, we assessed the population size, distribution, and authenticity of individual viral clonotypes within this synthetic swarm using samples from 120 rhesus macaques infected intravenously. The number of replicating barcodes in plasma correlated with the infectious inoculum dose, and the primary viral growth rate was similar in all infected animals regardless of the inoculum size. Overall, 97% of detectable clonotypes in the viral stock were identified in the plasma of at least one infected animal. Additionally, we prepared a second-generation barcoded SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and an additional barcoded stock with suboptimal nucleotides corrected (SIVmac239Opt5M). We also generated four barcoded stocks from subtype B and C simian-human immunodeficiency virus (SHIV) clones. These new SHIV clones may be particularly valuable models to evaluate Env-targeting approaches to study viral transmission or viral reservoir clearance. Overall, this work further establishes the reliability of the barcoded virus approach and highlights the feasibility of adapting this technique to other viral clones.
We recently developed and published a description of a barcoded simian immunodeficiency virus that has a short random sequence inserted directly into the viral genome. This allows for the tracking of individual viral lineages with high fidelity and ultradeep sensitivity. This virus was used to infect 120 rhesus macaques, and we report here the analysis of the barcodes of these animals during primary infection. We found that the vast majority of barcodes were functional
We then expanded the barcoding approach in a second-generation SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and a barcoded stock of SIVmac239Opt5M whose sequence had 5 changes from the wild-type SIVmac239 sequence. We also generated 4 barcoded stocks from subtype B and C SHIV clones each containing a human immunodeficiency virus (HIV) type 1 envelope. These virus models are functional and can be useful for studying viral transmission and HIV cure/reservoir research.
Interferon-gamma (IFN-gamma) is a specific activator of macrophage function and plays a critical role in the host immune defense to bacterial infection. In this study we examined the role of ...IFN-gamma in the regulation of bacterial load in the cecal ligation and puncture (CLP) model of intra-abdominal sepsis in the rat. In initial studies, levels of IL-12, MCP-1, and IFN-gamma were measured in the peritoneal lavage fluid 24 and 48 h after CLP. IL-12 and MCP-1 levels were both significantly increased at 24 h after CLP compared to sham controls and this difference was maintained at 48 h after CLP. Interestingly, IFN-gamma levels were not significantly increased 24 h after CLP, but were increased at 48 h after CLP. These results clearly suggest that although an inflammatory response had occurred 24 h post-surgery, with increases in the proinflammatory cytokine IL-12 and the potent chemotactic agent MCP-1, levels of IFN-gamma in CLP rats were similar to sham controls. To further investigate the role of IFN-gamma on the development of sepsis we examined the effect(s) of administering anti-IFN-gamma antibody on bacterial load after CLP. We show that use of anti-IFN-gamma antibody can significantly decrease bacterial load in the peritoneum. The mechanism of the effect(s) of anti-IFN-gamma is probably by increasing intestinal adhesions to seal the cecum and reduce bacterial movement into the peritoneum.
Diet is key to understanding the past, present, and future of our species. Much of human evolutionary success can be attributed to our ability to consume a wide range of foods. On the other hand, ...recent changes in the types of foods we eat may lie at the root of many of the health problems we face today. To deal with these problems, we must understand the evolution of the human diet.Studies of traditional peoples, non-human primates, human fossil and archaeological remains, nutritional chemistry, and evolutionary medicine, to name just a few, all contribute to our understanding of the evolution of the human diet. Still, as analyses become more specialized, researchers become more narrowly focused and isolated. This volume attempts to bring together authors schooled in a variety of academic disciplines so that we might begin to build a more cohesive view of the evolution of the human diet. The book demonstrates how past diets are reconstructed using both direct analogies with living traditional peoples and non-human primates, and studies of the bones and teeth of fossils. An understanding of our ancestral diets reveals how health relates to nutrition, and conclusions can be drawn as to how we may alter our current diets to further our health.
Ketogenic diets are low in carbohydrates and high in fat, which forces cells to rely more heavily upon mitochondrial oxidation of fatty acids for energy. Relative to normal cells, cancer cells are ...believed to exist under a condition of chronic mitochondrial oxidative stress that is compensated for by increases in glucose metabolism to generate reducing equivalents. In this study we tested the hypothesis that a ketogenic diet concurrent with radiation and chemotherapy would be clinically tolerable in locally advanced non-small cell lung cancer (NSCLC) and pancreatic cancer and could potentially exploit cancer cell oxidative metabolism to improve therapeutic outcomes. Mice bearing MIA PaCa-2 pancreatic cancer xenografts were fed either a ketogenic diet or standard rodent chow, treated with conventionally fractionated radiation (2 Gy/fraction), and tumor growth rates were assessed daily. Tumors were assessed for immunoreactive 4-hydroxy-2-nonenal-(4HNE)-modfied proteins as a marker of oxidative stress. Based on this and another previously published preclinical study, phase 1 clinical trials in locally advanced NSCLC and pancreatic cancer were initiated, combining standard radiation and chemotherapy with a ketogenic diet for six weeks (NSCLC) or five weeks (pancreatic cancer). The xenograft experiments demonstrated prolonged survival and increased 4HNE-modfied proteins in animals consuming a ketogenic diet combined with radiation compared to radiation alone. In the phase 1 clinical trial, over a period of three years, seven NSCLC patients enrolled in the study. Of these, four were unable to comply with the diet and withdrew, two completed the study and one was withdrawn due to a dose-limiting toxicity. Over the same time period, two pancreatic cancer patients enrolled in the trial. Of these, one completed the study and the other was withdrawn due to a dose-limiting toxicity. The preclinical experiments demonstrate that a ketogenic diet increases radiation sensitivity in a pancreatic cancer xenograft model. However, patients with locally advanced NSCLC and pancreatic cancer receiving concurrent radiotherapy and chemotherapy had suboptimal compliance to the oral ketogenic diet and thus, poor tolerance.
John Rankin Laboratory of Pulmonary Medicine, Department of Population Health Sciences, School of Medicine, University of Wisconsin, Madison, Wisconsin
Submitted 29 July 2005
; accepted in final form ...7 September 2005
We assessed the speed of the ventilatory response to square-wave changes in alveolar P CO 2 and the relative gains of the steady-state ventilatory response to CO 2 of the central chemoreceptors vs. the carotid body chemoreceptors in intact, unanesthetized dogs. We used extracorporeal perfusion of the reversibly isolated carotid sinus to maintain normal tonic activity of the carotid body chemoreceptor while preventing it from sensing systemic changes in CO 2 , thereby allowing us to determine the response of the central chemoreceptors alone. We found the following. 1 ) The ventilatory response of the central chemoreceptors alone is 11.2 (SD = 3.6) s slower than when carotid bodies are allowed to sense CO 2 changes. 2 ) On average, the central chemoreceptors contribute 63% of the gain to steady-state increases in CO 2 . There was wide dog-to-dog variability in the relative contributions of central vs. carotid body chemoreceptors; the central exceeded the carotid body gain in four of six dogs, but in two dogs carotid body gain exceeded central CO 2 gain. If humans respond similarly to dogs, we propose that the slower response of the central chemoreceptors vs. the carotid chemoreceptors prevents the central chemoreceptors from contributing significantly to ventilatory responses to rapid, transient changes in arterial P CO 2 such as those after periods of hypoventilation or hyperventilation ("ventilatory undershoots or overshoots") observed during sleep-disordered breathing. However, the greater average responsiveness of the central chemoreceptors to brain hypercapnia in the steady-state suggests that these receptors may contribute significantly to ventilatory overshoots once unstable/periodic breathing is fully established.
carotid body; chemosensitivity; control of breathing; sleep apnea
Address for reprint requests and other correspondence: C. A. Smith, Rm. 4245 MSC, Univ. of Wisconsin, 1300 Univ. Ave., Madison, WI 53706 (e-mail: casmith4{at}wisc.edu )
Commercially available ferrochrome lignosulfonate drilling mud additives were studied by x-ray K-absorption edge fine structure spectroscopy and by ion exchange. The materials were found to be ...essentially identical with regard to the chromium and iron species present. Neither the chromium nor the iron spectrum of Q-Broxin, a commercial ferro- chrome lignosulfonate, was found to change over the pH range of 1 to 11 or over the temperature range of 70° to 400° F.A method for detecting chromium(VI) in the presence of chromium(III) was established. No chromium(VI) was found in any of the chromium lignosulfonates.It was found that chromium from Q-Broxin was not exchangeable onto a strong cation exchange resin over the pH range of 1.5 to 11. Possible modes of interaction between a chromium lignosulfonate and a clay are discussed.
The use of electron probe X-ray microanalysis (EPXMA) in association with appropriate cell and tissue preparative procedures has allowed several laboratories to measure the intracellular levels of a ...number of ions during mitogenesis. It has been possible to study changes in the ionic concentration of normal, preneoplastic, and overtly neoplastic cells for determining the stage of involvement of ionic changes during carcinogenesis. A demonstration of such preneoplastic changes in ionic concentration could provide the basis for differential diagnosis of cancer by EPXMA and could ultimately provide a basis for rational cancer chemotherapeutic intervention. The use of EPXMA in association with appropriate cell and tissue preparative procedures has allowed several laboratories to measure and to test Cone's prediction that the intracellular levels of Na+ are changed in rapidly dividing and overt tumor cells as compared to slowly dividing or nondividing normal cells. An advantage of such a microprobe procedure is that quantitative data can be simultaneously obtained on several elements in a single analysis.
To determine reproductive urologists’ (RU) practice patterns for microdissection testicular sperm extraction (microTESE) and factors associated with use of fresh vs frozen microTESE for ...non-obstructive azoospermia.
We electronically surveyed Society for Study of Male Reproduction members with a 21-item questionnaire. Our primary outcomes were to determine RU preference for fresh or frozen microTESE and to understand barriers to performing microTESE. Pearson's chi-square and Fisher's exact tests were used to analyze categorical outcomes and candidate predictor variables. Firth logistic regression was performed to identify the predictors for preferring and performing fresh vs frozen microTESE.
A total of 208 surveys were sent with 76 responses. Most (63.0%) primarily perform frozen microTESE for non-obstructive azoospermia, while 37.0% primarily perform fresh. However, in an ideal practice, 59.3% prefer fresh microTESE, 22.2% prefer frozen microTESE, and 18.5% had no preference. MicroTESE is performed most often (61.1%) at surgical centers not affiliated with a fertility practice. The most commonly reported barriers for both fresh and frozen microTESE are cost (42.6%), scheduling (33.3%), and andrologist unavailability (16.7%). There are no statistically significant differences between these barriers and performing fresh vs frozen microTESE. On multivariable analysis, reproductive endocrinology and infertility-based surgical center (OR 22.9; 95% CI 1.1-467.2; P = 0.04) and professional fee $2,500-$4,999 (OR 20.7; 95% CI 1.27-337.9; P = 0.03) are significant predictors of performing fresh microTESE.
Frozen microTESE is performed more commonly than fresh, despite most RU preferring fresh microTESE in an ideal setting. Both fresh and frozen microTESE have a role in reproductive care. Barriers to performing fresh microTESE include cost, scheduling and andrologist availability.
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