A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO‐DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO‐DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA‐based CHO‐DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self‐organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up‐regulating NCK1 and down‐regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial‐ and endoplasmic reticulum‐mediated cell death pathways by up‐regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1026–1038, 2015     

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.     

GATE Validation of Standard Dual Energy Corrections in Small Animal SPECT-CT

This paper addresses ¹²³I and ¹²⁵I dual isotope SPECT imaging, which can be challenging because of spectrum overlap in the low energy spectrums of these isotopes. We first quantify the contribution of low-energy photons from each isotope using GATE-based Monte Carlo simulations for the MOBY mouse phantom. We then describe and analyze a simple, but effective method that uses the ratio of detected low and high energy ¹²³I activity to separate the mixed low energy ¹²³I and ¹²⁵I activities. Performance is compared with correction methods used in conventional tissue biodistribution techniques. The results indicate that the spectrum overlap effects can be significantly reduced, if not entirely eliminated, when attenuation and scatter is either absent or corrected for using standard methods. In particular, we show that relative activity levels of the two isotopes can be accurately estimated for a wide range of organs and provide quantitative validation that standard methods for spectrum overlap correction provide reasonable estimates for reasonable corrections in small-animal SPECT/CT imaging.     

The Critical Periphery in the Growth of Social Protests

Social media have provided instrumental means of communication in many recent political protests. The efficiency of online networks in disseminating timely information has been praised by many commentators; at the same time, users are often derided as “slacktivists” because of the shallow commitment involved in clicking a forwarding button. Here we consider the role of these peripheral online participants, the immense majority of users who surround the small epicenter of protests, representing layers of diminishing online activity around the committed minority. We analyze three datasets tracking protest communication in different languages and political contexts through the social media platform Twitter and employ a network decomposition technique to examine their hierarchical structure. We provide consistent evidence that peripheral participants are critical in increasing the reach of protest messages and generating online content at levels that are comparable to core participants. Although committed minorities may constitute the heart of protest movements, our results suggest that their success in maximizing the number of online citizens exposed to protest messages depends, at least in part, on activating the critical periphery. Peripheral users are less active on a per capita basis, but their power lies in their numbers: their aggregate contribution to the spread of protest messages is comparable in magnitude to that of core participants. An analysis of two other datasets unrelated to mass protests strengthens our interpretation that core-periphery dynamics are characteristically important in the context of collective action events. Theoretical models of diffusion in social networks would benefit from increased attention to the role of peripheral nodes in the propagation of information and behavior.     

Clinicopathologic Predictors of Survival in Patients with Desmoplastic Melanoma

Background and Objectives

Desmoplastic melanoma is a unique subtype of melanoma which typically affects older patients who often have comorbidities that can adversely affect survival. We sought to identify melanoma-specific factors influencing survival in patients with desmoplastic melanoma.

Methods

Retrospective review from 1993 to 2011 identified 316 patients with primary desmoplastic melanoma. Clinicopathologic characteristics were correlated with nodal status and outcome.

Results

Fifty-five patients (17.4%) had nodal disease: 33 had a positive sentinel lymph node biopsy and 22 developed nodal recurrences (no sentinel lymph node biopsy or false-negative sentinel lymph node biopsy). Nodal disease occurred more often in younger patients and in cases with mixed compared with pure histology (26.7% vs. 14.6%); both of these variables significantly predicted nodal status on multivariable analysis (p<0.05). After a median follow-up of 5.3 years, recurrence developed in 87 patients (27.5%), and 111 deaths occurred. The cause of death was known in 79 cases, with 47 deaths (59.5%) being melanoma-related. On multivariable analysis, Breslow thickness, mitotic rate ≥1/mm² and nodal status significantly predicted melanoma-specific survival (p<0.05).

Conclusions

Nodal status predicts melanoma-specific survival in patients with desmoplastic melanoma. However, since patients with desmoplastic melanoma represent an older population, and a considerable proportion of deaths are not melanoma-related (40.5%), comorbidities should be carefully considered in making staging and treatment decisions in this population.     

Trichoderma songyi sp. nov., a new species associated with the pine mushroom (Tricholoma matsutake)

A new species, Trichoderma songyi, was found to be associated with the pine mushroom (Tricholoma matsutake) in Korea. This species was isolated from three different substrates: Tricholoma matsutake basidiomata, as well as roots of Pinus densiflora and soil in the fairy ring. Based on its molecular and phenotypic characteristics, we demonstrate that Trichoderma songyi is unique and distinguishable from closely related species. We performed phylogenetic analyses based on two molecular markers, the genes for both translation elongation factor 1-alpha and the second largest subunit of RNA polymerase II. Phylogenetic analyses showed that Trichoderma songyi is closely related to Trichoderma koningii aggregate and Trichoderma caerulescens. Morphologically, Trichoderma songyi can be distinguished from these closely related taxa by its growth rates, colony morphology on PDA in darkness, and coconut-like odour. Due to the economic importance of the pine mushroom, the relationship between Trichoderma songyi and Tricholoma matsutake should be studied further.     

fastSTRUCTURE: Variational Inference of Population Structure in Large SNP Data Sets

Tools for estimating population structure from genetic data are now used in a wide variety of applications in population genetics. However, inferring population structure in large modern data sets imposes severe computational challenges. Here, we develop efficient algorithms for approximate inference of the model underlying the STRUCTURE program using a variational Bayesian framework. Variational methods pose the problem of computing relevant posterior distributions as an optimization problem, allowing us to build on recent advances in optimization theory to develop fast inference tools. In addition, we propose useful heuristic scores to identify the number of populations represented in a data set and a new hierarchical prior to detect weak population structure in the data. We test the variational algorithms on simulated data and illustrate using genotype data from the CEPH–Human Genome Diversity Panel. The variational algorithms are almost two orders of magnitude faster than STRUCTURE and achieve accuracies comparable to those of ADMIXTURE. Furthermore, our results show that the heuristic scores for choosing model complexity provide a reasonable range of values for the number of populations represented in the data, with minimal bias toward detecting structure when it is very weak. Our algorithm, fastSTRUCTURE, is freely available online at http://pritchardlab.stanford.edu/structure.html.     

Operons Are a Conserved Feature of Nematode Genomes

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.     

Time to reconsider the role of ribavirin in Lassa fever

Ribavirin is the only available Lassa fever treatment. The rationale for using ribavirin is based on one clinical study conducted in the early 1980s. However, reanalysis of previous unpublished data reveals that ribavirin may actually be harmful in some Lassa fever patients. An urgent reevaluation of ribavirin is therefore needed.

Fifty years after its discovery, Lassa fever remains uncontrolled, and mortality remains unacceptably high. Since 2015, Nigeria has been experiencing increasingly large outbreaks of Lassa fever, with new peaks reached in 2016, 2017, and 2018. In 1987, McCormick and colleagues reported a case fatality rate (CFR) of 16.5% among 441 patients hospitalized in Sierra Leone [1]. In Nigeria in 2019, 124 deaths were recorded among 554 laboratory-confirmed cases for a CFR of 22% [2].Ribavirin is the only available Lassa fever–specific treatment and has been used routinely for over 25 years. However, intravenous ribavirin is not licensed for Lassa fever. Its mechanism of action is unclear, it is expensive and hard to source, and it has well-known toxicities [3]. Therefore, the evidence for using ribavirin in Lassa fever deserves careful scrutiny. The emergence of potential new therapeutics for Lassa fever, such as favipiravir and monoclonal antibodies, adds further weight to the case for reconsidering the role of ribavirin since the evaluation of new drugs in clinical trials requires a comparison against existing treatments with a known efficacy and safety profile [4,5].The rationale for using ribavirin in Lassa fever is primarily based on one clinical study conducted in Sierra Leone in the late 1970s and early 1980s. McCormick and colleagues [6] reported that in Lassa fever patients with a serum aspartate aminotransferase (AST) level of ≥150 IU/L, the use of intravenous ribavirin within the first 6 days of illness reduced the fatality rate from 61% (11/18) with no ribavirin to 5% (1/20) (p = 0.002). These authors concluded that ribavirin is effective in the treatment of Lassa fever. However, there are long-standing concerns about the methods used in this study. Although randomization was used to assign patients to treatment groups, the comparisons presented were not according to original randomized groups, and we have reconstructed their derivation (Fig 1). Serious limitations to the comparisons presented include the use of historic controls, inclusion of pregnant women in the control group but their exclusion from the ribavirin group (case fatality is around 2-fold higher in pregnant women than nonpregnant patients), and post hoc merging of treatment groups. Despite this and the fact that the results only supported the use of ribavirin in nonpregnant adult patients with AST ≥150 IU/L, this study is the basis upon which ribavirin is now used in all patients with Lassa fever, including children, pregnant women, and people with normal liver function.Open in a separate windowFig 1Reconstruction of the McCormick et al. data.AST, aspartate aminotransferase; PW, pregnant women. † Discrepancy within McCormick et al, with 39 patients reported treated with oral ribavirin but only 38 (14+24) outcomes reported. ‡ Discrepancy within McCormick et al, with table 1 reporting 12/63 but text reporting 13/62.It has been well known among Lassa specialists that the McCormick study reports a subset of a much larger dataset assembled by the Lassa treatment unit in Sierra Leone and that a report on the full dataset was commissioned by the United States Army Medical Research and Development Command. One of us (PH) therefore submitted a freedom of information (FOI) request to access this report. The full report and an accompanying memo are available, and we encourage readers to access and read the materials [7,8]. The memo states that some of the original trial records were unavailable, and the data should be “interpreted with extreme caution.” Nonetheless, the report presents data from 1977 through to 1991 on 807 Lassa fever patients with a known outcome that were assigned to different ribavirin treatment regimens. These newly available data raise important questions about the safety and efficacy of ribavirin for the treatment of Lassa fever.The original data were lost during the civil war in Sierra Leone, but the report contains tables showing the distribution of characteristics of the whole population according to treatment group, an appendix showing individual data for the 405 patients who died, and results of a logistic regression analysis comparing the effect of ribavirin with no treatment for some of the ribavirin regimens, after adjusting for patient characteristics. Based on these data, we derived aggregated datasets containing the number of deaths according to treatment groups and individual characteristics. We combined groups I (“No treatment given”) and X (“Drugs were not available”) as no treatment and all groups in which ribavirin was administered (II, III, and V to IX) as ribavirin. Exhibit III-8 in the FOI report presented case fatality by treatment group and AST, from which we derived crude odds ratios (ORs) comparing ribavirin with no treatment. The logistic regression reported in Exhibit III-9 was restricted to “those treatment groups that yielded the lowest case fatality rates with respect to untreated patients in the high severity patient illness category” (groups II, III, V, and VII). It was adjusted for age, gender, time to admission, time to treatment, length of stay, and log(AST). We also reconstructed analyses by digitizing the data on individuals who died in Appendix D, calculating the number of deaths according to treatment group and AST, and subtracting these numbers from the totals presented in Exhibit III-2. These allowed us to estimate overall mortality ORs before and after adjusting for ribavirin, although the numbers did not entirely match, and so the number of deaths was reduced in some small groups.Estimates of the effect of oral and intravenous ribavirin from the McCormick study and of all ribavirin from the full report are shown in Fig 2. Based on the crude ORs derived from Exhibit III-8, ribavirin reduced mortality only in patients with serum AST ≥150 IU/L, with less benefit (OR 0.48 [95% CI 0.30 to 0.78]) than reported by McCormick and colleagues. However, ribavirin appeared to increase mortality in patients with serum AST <150 IU/L (2.90 [1.42 to 5.95]). In fact, in our analysis, the only stratum in which ribavirin appeared protective (0.38 [0.21 to 0.70]) was serum AST >300 IU/L (Table H in S1 Text). The logistic regression reported in the FOI report suggested a modest reduction in mortality, but the reasons for the choice of treatment groups compared were unclear. In the reconstructed analyses, ribavirin was associated with overall increased mortality (2.12 [1.67, 2.68]), although this was attenuated after adjustment for AST (1.48 [1.05, 2.08]).Open in a separate windowFig 2Forest plot of the OR of death in treatment and risk subgroups.AST, aspartate aminotransferase; FOI, freedom of information; OR, odds ratio.In our view, there is a compelling case to reevaluate the role of ribavirin in the care of patients with Lassa fever. The data suggest that ribavirin treatment may harm Lassa fever patients with AST <150 IU/L. The limitations revealed by the US Army report, such as large amounts of missing data, unclear treatment allocation practices, imbalances in treatment groups, and errors in coding serology results, cast further doubt on the conclusions of the McCormick study. This aligns with 2 recent systematic reviews by Eberhardt and colleagues and Cheng and colleagues, which concluded that the efficacy of ribavirin in Lassa fever was uncertain because of critical risk of bias in existing studies [9,10].Challenging a quarter of century of clinical practice is difficult. The first step is to acknowledge inadequacies in our knowledge and to ensure that treatment recommendations for Lassa fever better reflect the (weak) strength of evidence for ribavirin in different patient populations. Vigorous efforts should be made to engage clinicians and patients in designing a placebo-controlled trial to assess the safety and efficacy of ribavirin treatment in Lassa fever patients, particularly in those with milder disease (as may be indicated by an admission AST <150 IU/L) in whom the available evidence is compatible with ribavirin causing more harm than good.In conclusion, Lassa fever patients are receiving a drug that may lack efficacy or cause harm. It is incumbent on us to ensure that the next 25 years of Lassa fever treatment are built on more solid foundations.     

Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.