Waste Not,Want Not: Why Rarefying Microbiome Data Is Inadmissible

Current practice in the normalization of microbiome count data is inefficient in the statistical sense. For apparently historical reasons, the common approach is either to use simple proportions (which does not address heteroscedasticity) or to use rarefying of counts, even though both of these approaches are inappropriate for detection of differentially abundant species. Well-established statistical theory is available that simultaneously accounts for library size differences and biological variability using an appropriate mixture model. Moreover, specific implementations for DNA sequencing read count data (based on a Negative Binomial model for instance) are already available in RNA-Seq focused R packages such as edgeR and DESeq. Here we summarize the supporting statistical theory and use simulations and empirical data to demonstrate substantial improvements provided by a relevant mixture model framework over simple proportions or rarefying. We show how both proportions and rarefied counts result in a high rate of false positives in tests for species that are differentially abundant across sample classes. Regarding microbiome sample-wise clustering, we also show that the rarefying procedure often discards samples that can be accurately clustered by alternative methods. We further compare different Negative Binomial methods with a recently-described zero-inflated Gaussian mixture, implemented in a package called metagenomeSeq. We find that metagenomeSeq performs well when there is an adequate number of biological replicates, but it nevertheless tends toward a higher false positive rate. Based on these results and well-established statistical theory, we advocate that investigators avoid rarefying altogether. We have provided microbiome-specific extensions to these tools in the R package, phyloseq.     

Homology: Why We Know a Whale Is Not a Fish

Homology is a fundamental concept in comparative and evolutionary biology and yet often the focus of antievolution challenges. In describing structural similarity that is the result of common ancestry, hypotheses about homology require rigorous testing and form the basis for making predictions about anatomy and physiology as well as the fossil record. Communicating the basics of homology to students is essential for a high school biology curriculum.     

Inhospitable Healthcare Spaces: Why Diversity Training on LGBTQIA Issues Is Not Enough

In an effort to address healthcare disparities in lesbian, gay, bisexual, transgender, and queer (LGBTQ) populations, many hospitals and clinics institute diversity training meant to increase providers’ awareness of and sensitivity to this patient population. Despite these efforts, many healthcare spaces remain inhospitable to LGBTQ patients and their loved ones. Even in the absence of overt forms of discrimination, LGBTQ patients report feeling anxious, unwelcome, ashamed, and distrustful in healthcare encounters. We argue that these negative experiences are produced by a variety of subtle, ostensibly insignificant features of healthcare spaces and interpersonal interactions called microaggressions. Healthcare spaces and providers often convey heteronormative microaggressions, which communicate to LGBTQ—and, we suggest, intersex and asexual (IA)—people that their identities, experiences, and relationships are abnormal, pathological, unexpected, unwelcome, or shameful. We identify heteronormative microaggressions common to healthcare settings and specify how they negatively impact LGBTQIA patients. We argue that standard diversity training cannot sufficiently address heteronormative microaggressions. Despite these challenges, healthcare institutions and providers must take responsibility for heteronormative microaggressions and take steps to reduce their frequency and mitigate their effects on LGBTQIA care. We conclude by offering strategies for problem-solving at the level of medical education, institutional culture and policy, and individual awareness.     

Deconstructing Community for Conservation: Why Simple Assumptions are Not Sufficient

Many conservation policies advocate engagement with local people, but conservation practice has sometimes been criticised for a simplistic understanding of communities and social context. To counter this, this paper explores social structuring and its influences on conservation-related behaviours at the site of a conservation intervention near Pipar forest, within the Seti Khola valley, Nepal. Qualitative and quantitative data from questionnaires and Rapid Rural Appraisal demonstrate how links between groups directly and indirectly influence behaviours of conservation relevance (including existing and potential resource-use and proconservation activities). For low-status groups the harvesting of resources can be driven by others’ preference for wild foods, whilst perceptions of elite benefit-capture may cause reluctance to engage with future conservation interventions. The findings reiterate the need to avoid relying on simple assumptions about ‘community’ in conservation, and particularly the relevance of understanding relationships between groups, in order to understand natural resource use and implications for conservation.     

Prehypertension Is Not Associated with All-Cause Mortality: A Systematic Review and Meta-Analysis of Prospective Studies

Objectives

Quantitative associations between prehypertension or its two separate blood pressure (BP) ranges and cardiovascular disease (CVD) or all-cause mortality have not been reliably documented. In this study, we performed a comprehensive systematic review and meta-analysis to assess these relationships from prospective cohort studies.

Methods

We conducted a comprehensive search of PubMed (1966-June 2012) and the Cochrane Library (1988-June 2012) without language restrictions. This was supplemented by review of the references in the included studies and relevant reviews identified in the search. Prospective studies were included if they reported multivariate-adjusted relative risks (RRs) and corresponding 95% confidence intervals (CIs) of CVD or all-cause mortality with respect to prehypertension or its two BP ranges (low range: 120–129/80–84 mmHg; high range: 130–139/85–89 mmHg) at baseline. Pooled RRs were estimated using a random-effects model or a fixed-effects model depending on the between-study heterogeneity.

Results

Thirteen studies met our inclusion criteria, with 870,678 participants. Prehypertension was not associated with an increased risk of all-cause mortality either in the whole prehypertension group (RR: 1.03; 95% CI: 0.91 to 1.15, P = 0.667) or in its two separate BP ranges (low-range: RR: 0.91; 95% CI: 0.81 to 1.02, P = 0.107; high range: RR: 1.00; 95% CI: 0.95 to 1.06, P = 0.951). Prehypertension was significantly associated with a greater risk of CVD mortality (RR: 1.32; 95% CI: 1.16 to 1.50, P<0.001). When analyzed separately by two BP ranges, only high range prehypertension was related to an increased risk of CVD mortality (low-range: RR: 1.10; 95% CI: 0.92 to 1.30, P = 0.287; high range: RR: 1.26; 95% CI: 1.13 to 1.41, P<0.001).

Conclusions

From the best available prospective data, prehypertension was not associated with all-cause mortality. More high quality cohort studies stratified by BP range are needed.     

Why Threonine Is an Essential Amino Acid in Mammals and Birds: Studies at the Enzyme Level

The only pathway for the synthesis of essential amino acids in vertebrates is reversible transamination of their keto analogs with glutamic acid. At the same time, it is commonly accepted that such essential amino acids as lysine and threonine are not involved in transamination and, therefore, cannot be synthesized from their keto analogs. However, using radio-labeled isotopes, synthesis of threonine was demonstrated in rat liver and in a reaction mixture containing chicken liver threonine dehydrogenase. In the review, we discuss why threonine is an essential amino acid in mammals and birds based on the pathways of threonine biosynthesis in these two classes of vertebrates.     

Systemic Negligence: Why It Is Morally Important for Developing World Bioethics

In the context of clinical and non‐clinical biomedical practices, negligence is usually understood as a lapse of a specific professional duty by a healthcare worker or by a medical facility. This paper tries to delineate systemic negligence as another kind of negligence in the context of health systems, particularly in developing countries, that needs to be recognized and addressed. Systemic negligence is not just a mere collection of stray incidences of medical errors and system failures in a health system, but is proposed in this paper as a more pervasive kind of neglect. Several non‐medical factors, such as lack of social and political will, also contribute to it and hence is more difficult to address in a health system. This paper argues that recognizing systemic negligence and including it research agenda have special moral importance for researchers in developing world bioethics, public health ethics and for health activists in the developing world. For, it can be a potent health system barrier, and can seriously impair efforts to ensure patient safety, particularly in the weaker health systems. As it erodes accountability in a health system, addressing it is also important for the twin goals of ensuring patient safety and improving health system performance. Above all, it needs to be addressed because the tolerance of its persistence in a health system seems to undervalue health as a social good.     

Mass Drug Administration for Trachoma: How Long Is Not Long Enough?

BackgroundBlinding trachoma is targeted for elimination by 2020 using the SAFE strategy (Surgery, Antibiotics, Facial cleanliness, and Environmental improvements). Annual mass drug administration (MDA) with azithromycin is a cornerstone of this strategy. If baseline prevalence of clinical signs of trachomatous inflammation – follicular among 1-9 year-olds (TF1-9) is ≥10% but <30%, the World Health Organization guidelines are for at least 3 annual MDAs; if ≥30%, 5. We assessed the likelihood of achieving the global elimination target of TF1-9 <5% at 3 and 5 year evaluations using program reports.ConclusionsNumber of annual MDAs alone appears insufficient to predict program progress; more information on the effects of baseline prevalence, coverage, and underlying environmental and hygienic conditions is needed. Programs should not skip MDAs, and at prevalences >30%, 7 or more annual MDAs may be required to achieve the target. There are five years left before the 2020 deadline to eliminate blinding trachoma. Low endemic settings are poised to succeed in their elimination goals. However, newly-identified high prevalence districts warrant immediate inclusion in the global program. Intensified application of the SAFE strategy is needed in order to guarantee blinding trachoma elimination by 2020.     

Why Therapeutic Response May Not Prolong the Life of a Cancer Patient: Selection for Oncogenic Resistance

Most cancers do not respond to chemotherapy. Disappointedly, even objective clinical responses to anticancer therapy often do not translate into improvements in overall survival. To explain the response-survival paradox, it has been pointed out that effective therapy is ineffective against cancer stem cells, which replenish the tumor (causing relapse). In contrast, I discuss that, according to this scenario, patient survival will be prolonged at least by the duration of remission. Furthermore, stem-cell-based relapses will be sensitive to the initial therapy, and in theory cancer could be controlled indefinitely. To explain the paradox, I discuss that effective therapy selects for resistance among proliferating cancer cells. Importantly, mechanisms of resistance can be divided into non-oncogenic (e.g., drug transporters and mutation in drug-targets) and oncogenic (apoptosis and cell cycle dysregulation). The latter is associated with highly aggressive cancer phenotype and, therefore, there is no increase in overall survival. I further suggest that (a) therapeutic response is a prerequisite for successful therapy, (b) resistance can be exploited for therapeutic advantage, (c) each response can be translated into increased survival, and (e) in slow growing tumors, cancer can be stopped without tumor shrinkage. Therapy will control cancer if it can selectively suppress proliferating cancer cells and will improve survival as long as acquired resistance can be exploited.     

BMI and Risk Factors for Suicide: Why Is BMI Inversely Related to Suicide?

Higher BMI has been associated with a lower risk of suicide in large prospective studies, but the mechanisms for this link require elucidation. In the 2002 and 2004 iterations of the Behavioral Risk Factor Surveillance System (BRFSS), a large, population‐based telephone survey of US adults conducted by the Centers for Disease Control and Prevention, participants reported their height, weight, and several potential risk factors for suicide, including alcohol use, mental health, marital status, firearm ownership, and risk‐taking behaviors. We assessed whether BMI was associated with these risk factors among 224,247 eligible respondents in 2002 and 275,194 in 2004 after sample‐weighted adjustment for age, race, region, smoking, and education. Alcohol‐related risk factors tended to be lower with heavier BMI among women, while firearm‐related risk factors tended to be higher with heavier BMI among men. Heavier BMI also tended to be associated with unmarried status and poor mental health, especially among women, and with infrequent seat belt use in men and women. No potential risk factors were consistently inversely associated with BMI in both sexes and years. In summary, in these samples of the US population, conventional risk factors for suicide were inconsistently associated with BMI, making them unlikely mediators for the observed relationship of BMI with lower risk of suicide. In some cases, risk factors were actually greater with heavier BMI. Further study of the relationship of BMI and suicide may yield novel modifiable risk factors that could cause or prevent this important cause of death.     

Endoproteolytic Processing of the Ebola Virus Envelope Glycoprotein: Cleavage Is Not Required for Function

Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP₁ and GP₂, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.     

Exposure to Ionizing Radiation during Dental X-Rays Is Not Associated with Risk of Developing Meningioma: A Meta-Analysis Based on Seven Case-Control Studies

BackgroundMany observational studies have found that exposure to dental X-rays is associated with the risk of development of meningioma. However, these findings are inconsistent. We conducted a meta-analysis to assess the relationship between exposure to dental X-rays and the risk of development of meningioma.MethodsThe PubMed and EMBASE databases were searched to identify eligible studies. Summary odds ratio (OR) estimates and 95% confidence intervals (95% CIs) were used to compute the risk of meningioma development according to heterogeneity. Subgroup and sensitivity analyses were performed to further explore the potential heterogeneity. Finally, publication bias was assessed.ResultsSeven case-control studies involving 6,174 patients and 19,459 controls were included in the meta-analysis. Neither exposure to dental X-rays nor performance of full-mouth panorex X-rays was associated with an increased risk of development of meningioma (overall: OR, 0.97; 95% CI, 0.70–1.32; dental X-rays: OR, 1.05; 95% CI, 0.89–1.25; panorex X-rays: OR, 1.01; 95% CI, 0.76–1.34). However, exposure to bitewing X-rays was associated with a slightly increased risk of development of meningioma (OR, 1.73; 95% CI, 1.28–2.34). Similar results were obtained in the subgroup and sensitivity analyses. Little evidence of publication bias was observed.ConclusionBased on the currently limited data, there is no association between exposure to dental X-rays and the risk of development of meningioma. However, these results should be cautiously interpreted because of the heterogeneity among studies. Additional large, high-quality clinical trials are needed to evaluate the association between exposure to dental X-rays and the risk of development of meningioma.     

Studies on differentiation without cleavage in Chaetopterus variopedatus: requirements for nucleic acid and protein synthesis

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species.     

Molecular Characterization of Three PRORP Proteins in the Moss Physcomitrella patens: Nuclear PRORP Protein Is Not Essential for Moss Viability

RNase P is a ubiquitous endonuclease that removes the 5′ leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5′-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5′-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNA^Asp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNA^Arg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNA^Arg (CCG) and pre-tRNA^Arg (UCU) but they cleaved pre-tRNA^Arg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.     

Two Years Later: Journals Are Not Yet Enforcing the ARRIVE Guidelines on Reporting Standards for Pre-Clinical Animal Studies

There is growing concern that poor experimental design and lack of transparent reporting contribute to the frequent failure of pre-clinical animal studies to translate into treatments for human disease. In 2010, the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were introduced to help improve reporting standards. They were published in PLOS Biology and endorsed by funding agencies and publishers and their journals, including PLOS, Nature research journals, and other top-tier journals. Yet our analysis of papers published in PLOS and Nature journals indicates that there has been very little improvement in reporting standards since then. This suggests that authors, referees, and editors generally are ignoring guidelines, and the editorial endorsement is yet to be effectively implemented.     

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.     

Accumulation of Extracellular Glutamate by Inhibition of Its Uptake Is Not Sufficient for Inducing Neuronal Damage: An In Vivo Microdialysis Study

Abstract: It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high-affinity sodium-dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (l -trans-pyrrolidine-2,4-dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, dl -threo-β-hydroxyaspartate and l -aspartate-β-hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate or by intrastriatal coinjection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used.