Perioperative Opioid Counseling Reduces Opioid Use Following Primary Total Joint Arthroplasty

BackgroundPreoperative counseling may reduce postoperative opioid requirements; however, there is a paucity of randomized controlled trials (RCTs) demonstrating efficacy. The purpose of this study was to perform an interventional, telehealth-based RCT evaluating the effect of peri-operative counseling on quantity and duration of opioid consumption following primary total joint arthroplasty (TJA).MethodsParticipants were randomized into three groups: 1. Control group, no perioperative counseling; 2. Intervention group, preoperative educational video; 3. Intervention group, preoperative educational video and postoperative acceptance and commitment therapy (ACT). Opioid consumption was evaluated daily for 14 days and at 6 weeks postoperatively. Best-case and worse-case intention to treat analyses were performed to account for non-responses. Bonferroni corrections were applied.Results183 participants were analyzed (63 in Group 1, 55 in Group 2, and 65 in Group 3). At 2 weeks postoperatively, there was no difference in opioid consumption between Groups 1, 2, and 3 (p>0.05 for all). At 6 weeks postoperatively, Groups 2 and 3 had consumed significantly less opioids than Group 1 (p=0.04, p<0.001) (

Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation

Cefoperazone/sulbactam (CSL) and piperacillin/tazobactam (TZP) are commonly used in clinical practice in China because of their excellent antimicrobial activity. CSL and TZP-nonsusceptible Enterobacteriaceae are typically resistant to extended-spectrum cephalosporins such as ceftriaxone (CRO). However, 11 nonrepetitive Enterobacteriaceae strains, which were resistant to CSL and TZP yet susceptible to CRO, were collected from January to December 2020. Antibiotic susceptibility tests and whole-genome sequencing were conducted to elucidate the mechanism for this rare phenotype. Antibiotic susceptibility tests showed that all isolates were amoxicillin/clavulanic-acid resistant and sensitive to ceftazidime, cefepime, cefepime/tazobactam, cefepime/zidebactam, ceftazidime/avibactam, and ceftolozane/tazobactam. Whole-genome sequencing revealed three of seven Klebsiella pneumoniae strains harbored bla_SHV-1 only, and four harbored bla_SHV-1 and bla_TEM-1B. Two Escherichia coli strains carried bla_TEM-1B only, while two Klebsiella oxytoca isolates harbored bla_OXY-1-3 and bla_OXY-1-1, respectively. No mutation in the β-lactamase gene and promoter sequence was found. Outer membrane protein (Omp) gene detection revealed that numerous missense mutations of OmpK36 and OmpK37 were found in all strains of K. pneumoniae. Numerous missense mutations of OmpK36 and OmpK35 and OmpK37 deficiency were found in one K. oxytoca strain, and no OmpK gene was found in the other. No Omp mutations were found in E. coli isolates. These results indicated that narrow spectrum β-lactamases, TEM-1, SHV-1, and OXY-1, alone or in combination with Omp mutation, contributed to the resistance to CSL and TZP in CRO-susceptible Enterobacteriaceae.Antibiotic susceptibility tests

Does an Eye-Hand Coordination Test Have Added Value as Part of Talent Identification in Table Tennis? A Validity and Reproducibility Study

This study investigated the added value, i.e. discriminative and concurrent validity and reproducibility, of an eye-hand coordination test relevant to table tennis as part of talent identification. Forty-three table tennis players (7–12 years) from national (n = 13), regional (n = 11) and local training centres (n = 19) participated. During the eye-hand coordination test, children needed to throw a ball against a vertical positioned table tennis table with one hand and to catch the ball correctly with the other hand as frequently as possible in 30 seconds. Four different test versions were assessed varying the distance to the TotalNationalRegionalLocalTotal43131119Boys268810Girls17539Age (years)10.4±1.410.9±1.510.4±1.510.1±1.47 year olds1--18 year olds51139 year olds3-3-10 year olds1232711 year olds1151512 year olds11443Length (cm)149±11150±12150±12148±10Weight (kg )38±837±737±738±9Right-handed359917Left-handed8422Training (hours*week-1)6 (0–20)11 (7–20)7 (4–11)2 (0–3)Competition (points)173 (−52–430)297 (144–430)188 (72–317)36 (−52–130)Open in a separate windowData are frequencies, except for age, length and weight (years±SD), and training and competition (mean (range)).     

Hepatitis C Virus (HCV) Sequence Variation Induces an HCV-Specific T-Cell Phenotype Analogous to Spontaneous Resolution

Hepatitis C virus (HCV)-specific CD8⁺ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127⁺ T cells is driven by viral variation.Hepatitis C virus (HCV) persists in the majority of acutely infected individuals, potentially leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The cellular immune response has been shown to play a significant role in viral control and protection from liver disease. Phenotypic and functional studies of virus-specific T cells have attempted to define the determinants of a successful versus an unsuccessful T-cell response in viral infections (10). So far these studies have failed to identify consistent distinguishing features between a T-cell response that results in self-limiting versus chronic HCV infection; similarly, the impact of viral persistence on HCV-specific memory T-cell formation is poorly understood.Interleukin-7 (IL-7) receptor alpha chain (CD127) is a key molecule associated with the maintenance of memory T-cell populations. Expression of CD127 on CD8 T cells is typically only observed when the respective antigen is controlled and in the presence of significant CD4⁺ T-cell help (9). Accordingly, cells specific for persistent viruses (e.g., HIV, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) have been shown to express low levels of CD127 (6, 12, 14) and to be dependent on antigen restimulation for their maintenance. In contrast, T cells specific for acute resolving virus infections, such as influenza virus, respiratory syncytial virus (RSV), hepatitis B virus (HBV), and vaccinia virus typically acquire expression of CD127 rapidly with the control of viremia (5, 12, 14). Results for HCV have been inconclusive. The expected increase in CD127 levels in acute resolving but not acute persisting infection has been found, while a substantial proportion of cells with high CD127 expression have been observed in long-established chronic infection (2). We tried to reconcile these observations by studying both subjects with acute and chronic HCV infection and identified the presence of antigen as the determinant of CD127 expression.Using HLA-peptide multimers we analyzed CD8⁺ HCV-specific T-cell responses and CD127 expression levels in acute and chronic HCV infection. We assessed a cohort of 18 chronically infected subjects as well as 9 individuals with previously resolved infection. In addition, we longitudinally studied 9 acutely infected subjects (5 individuals who resolved infection spontaneously and 4 individuals who remain chronically infected) (Tables ​(Tables11 and ​and2).2). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval from the local institutional review boards. HLA-multimeric complexes were obtained commercially from Proimmune (Oxford, United Kingdom) and Beckman Coulter (CA). The staining and analysis procedure was as described previously (10). Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies: CD3 from Caltag; CD8, CD27, CCR7, CD127, and CD38 from BD Pharmingen; and PD-1 (kindly provided by Gordon Freeman). Primer sets were designed for different genotypes based on alignments of all available sequences from the public HCV database (http://hcvpub.ibcp.fr). Sequence analysis was performed as previously described (8).

TABLE 1.

Patient information and autologous sequence analysis for patients with chronic and resolved HCV infection

A conifer genome spruces up plant phylogenomics

The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure ​(Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure ​Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table ​(Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.

Table 1

Genome sizes in land plants

Economic Benefits of Investing in Women’s Health: A Systematic Review

Background

Globally, the status of women’s health falls short of its potential. In addition to the deleterious ethical and human rights implications of this deficit, the negative economic impact may also be consequential, but these mechanisms are poorly understood. Building on the literature that highlights health as a driver of economic growth and poverty alleviation, we aim to systematically investigate the broader economic benefits of investing in women’s health.

Methods

Using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, we systematically reviewed health, gender, and economic literature to identify studies that investigate the impact of women’s health on micro- and macroeconomic outcomes. We developed an extensive search algorithm and conducted searches using 10 unique databases spanning the timeframe 01/01/1970 to 01/04/2013. Articles were included if they reported on economic impacts stemming from changes in women’s health (table of outcome measures included in full review, Outcome measures   FertilityIntergenerational Health SpilloverEducationProductivitySavingsMicroeconomic level    Total fertility rateChild survivalEnrollment in schoolIncomeMoneyChange in fertilityChild wellbeing and behaviorYears of schoolingPurchasing powerAssetsAge at first birth/ teenage pregnanciesAnthropometryEarly drop outPerformanceBirth spacingImproved cognitive developmentPerformance in school Life expectancyHigher education  Adult health outcomesLiteracy  Nutrition   Intrauterine growth  Macroeconomic level    Open in a separate windowGross domestic product/gross national product, gross domestic product/gross national product growth, income per capita, labor force participation, per capita income.

Results

The existing literature indicates that healthier women and their children contribute to more productive and better-educated societies. This study documents an extensive literature confirming that women’s health is tied to long-term productivity: the development and economic performance of nations depends, in part, upon how each country protects and promotes the health of women. Providing opportunities for deliberate family planning; healthy mothers before, during, and after childbirth, and the health and productivity of subsequent generations can catalyze a cycle of positive societal development.

Conclusions

This review highlights the untapped potential of initiatives that aim to address women’s health. Societies that prioritize women’s health will likely have better population health overall, and will remain more productive for generations to come.     

Cognitive Manic Symptoms in Bipolar Disorder Associated with Polymorphisms in the DAOA and COMT Genes

Introduction

Bipolar disorder is characterized by severe mood symptoms including major depressive and manic episodes. During manic episodes, many patients show cognitive dysfunction. Dopamine and glutamate are important for cognitive processing, thus the COMT and DAOA genes that modulate the expression of these neurotransmitters are of interest for studies of cognitive function.

Methodology

Focusing on the most severe episode of mania, a factor was found with the combined symptoms of talkativeness, distractibility, and thought disorder, considered a cognitive manic symptoms (CMS) factor. 488 patients were genotyped, out of which 373 (76%) had talkativeness, 269 (55%) distractibility, and 372 (76%) thought disorder. 215 (44%) patients were positive for all three symptoms, thus showing CMS (Bipolar disorder type 1 [n]488Men [n (%)]209 (43)Talkativeness [n (%)]373 (76)Distracibility [n (%)]269 (55)Thought disorder [n (%)]372 (76)Cognitive manic symptoms^* [n (%)]215 (44)Men [n (%)]81 (39)Non-Cognitive manic symptoms [n (%)]248 (51)Men [n (%)]117 (56)Unknown [n (%)]25 (5)Men [n (%)]11 (44)Anonymous blood donors (ABD)1044Men [n (%)]616 (59)Open in a separate window^*having all three symptoms: talkativeness, distractibility, and tought disorder.

Results

The finding of this study was that cognitive manic symptoms in patients with bipolar 1 disorder was associated with genetic variants in the DAOA and COMT genes. Nominal association for DAOA SNPs and COMT SNPs to cognitive symptoms factor in bipolar 1 disorder was found in both allelic (BP1 CMSBP1 non-CMSABDBP1 CMS vs. non-CMS^b BP1 CMS vs. ABD controls^b GeneSNP^a aa/ab/bbaa/ab/bbaa/ab/bbpEMP1^c EMP2^d OR [95% CI] ^e pEMP1^c EMP2^d OR [95% CI] ^e DAOA rs3916967 (C/T)32/88/8950/118/77177/494/3610.0180.0180.210.72 [0.55–0.93]0.0290.0260.280.78 [0.66–1.0] DAOA rs2391191 (A/C)28/75/7939/111/70179/487/3570.0550.0390.500.75 [0.57–1.0]0.0200.0190.210.75 [0.63–1.0] DAOA rs1935062 (C/A)26/67/8935/102/86146/460/4050.120.120.780.80 [0.58–1.0]0.0690.0660.520.80 [0.65–1.0] COMT rs5993883 (T/G)33/120/5371/112/57269/510/2230.0250.0300.270.73 [0.56–0.95]0.0017^* 1.0E^{−4 *} 0.021^* 0.68 [0.91–1.4] COMT rs165599 (G/A)29/94/8725/93/12687/443/5010.0930.0940.691.27 [1.0–1.8]0.0140.0170.161.34 [1.1–1.7]Open in a separate window^aSNP (minor allele(a)/major allele(b)).^bgender and rs1718119 as covariate.^cpoint-wise p-value from 10,000 pemutations with no covarite (EMP1).^dcorrected empirical p-value by max (T) permutation.^eodds ratio (OR), the proportion of minor versus major allele affected (cognitive manic symptoms factor)/proportion of minor versus major allele unaffected (non-cognitive manic symptoms factor or ABD controls).^*significant after correction for multiple testing by max (T) permutation.

Table 3

Haplotype association of haplotype group 1 in bipolar 1 patients with cognitive manic symptoms (CMS) compared with non-CMS patients or ABD controls in the DAOA gene.

Big Data: Astronomical or Genomical?

Genomics is a Big Data science and is going to get much bigger, very soon, but it is not known whether the needs of genomics will exceed other Big Data domains. Projecting to the year 2025, we compared genomics with three other major generators of Big Data: astronomy, YouTube, and Twitter. Our estimates show that genomics is a “four-headed beast”—it is either on par with or the most demanding of the domains analyzed here in terms of data acquisition, storage, distribution, and analysis. We discuss aspects of new technologies that will need to be developed to rise up and meet the computational challenges that genomics poses for the near future. Now is the time for concerted, community-wide planning for the “genomical” challenges of the next decade.We compared genomics with three other major generators of Big Data: astronomy, YouTube, and Twitter. Astronomy has faced the challenges of Big Data for over 20 years and continues with ever-more ambitious studies of the universe. YouTube burst on the scene in 2005 and has sparked extraordinary worldwide interest in creating and sharing huge numbers of videos. Twitter, created in 2006, has become the poster child of the burgeoning movement in computational social science [6], with unprecedented opportunities for new insights by mining the enormous and ever-growing amount of textual data [7]. Particle physics also produces massive quantities of raw data, although the footprint is surprisingly limited since the vast majority of data are discarded soon after acquisition using the processing power that is coupled to the sensors [8]. Consequently, we do not include the domain in full detail here, although that model of rapid filtering and analysis will surely play an increasingly important role in genomics as the field matures.To compare these four disparate domains, we considered the four components that comprise the “life cycle” of a dataset: acquisition, storage, distribution, and analysis ( Data Phase Astronomy Twitter YouTube Genomics Acquisition 25 zetta-bytes/year0.5–15 billion tweets/year500–900 million hours/year1 zetta-bases/year Storage 1 EB/year1–17 PB/year1–2 EB/year2–40 EB/year Analysis In situ data reductionTopic and sentiment miningLimited requirementsHeterogeneous data and analysisReal-time processingMetadata analysisVariant calling, ~2 trillion central processing unit (CPU) hoursMassive volumesAll-pairs genome alignments, ~10,000 trillion CPU hours Distribution Dedicated lines from antennae to server (600 TB/s)Small units of distributionMajor component of modern user’s bandwidth (10 MB/s)Many small (10 MB/s) and fewer massive (10 TB/s) data movementOpen in a separate window     

The Russian invasion of Ukraine: a humanitarian tragedy and a tragedy for science

The Invasion of Ukraine prompts us to support our Ukranian colleagues but also to keep open communication with the Russian scientists who oppose the war.

In the eyes of the civilized world, Russia has already lost the war: politically, it is becoming ever more isolated; economically as the sanctions take an enormous toll; militarily as the losses of the Russian army mount. In contrast, the courage of Ukrainian people fighting for their independence has united the Western world that is providing enormous support for those Ukrainians who fight the Russian invasion and those who have fled their war‐torn country. Once this war is over, Ukraine will have to heal the wounds of war, reunite families, restore its economy, reestablish infrastructure, and rebuild science and education. Russia will have to restore its dignity and overcome its self‐inflicted isolation.Europe’s unity in condemning Russia’s war of aggression and showing its solidarity with Ukraine has been impressive. This includes not the least welcoming and accommodating millions of refugees. We, the scientific community in Europe, have a moral obligation to help Ukrainian students and colleagues by providing safe space to study and to continue their research. First, European research organizations and funding agencies should develop strategies to support them in the years to come. Second, efforts by EMBO, research funders, universities, and research institutions to support Ukrainian students and scientists are necessary. As a first priority, dedicated and unbureaucratic short‐term scholarship and grant programs are required to accommodate Ukrainian scientists; such programs have been already initiated by many organizations, for example, by EMBO, Volkswagen Stiftung, Max Planck Society, and the ERC among others. These help Ukrainian scientists to stay connected to research and become integrated into the European research landscape. In the long‐term and after the war, this aid should be complemented by funding for research centers of excellence in Ukraine, to which scientists could then return.Even though the priority must be to help Ukrainians, we must also think of students and colleagues in Russia who oppose the war and are affected by the sanctions. As the Iron Curtain closes again, we have to think differently about our ongoing and future collaborations. Although freezing most, if not all, research collaborations with official Russian organizations is justified, it would be a mistake to extend these sanctions to all scientists and students. There is already an exodus of Russian and Belarusian scholars, which will only accelerate in the next months and years, and accepting scientists who ask for political asylum will be beneficial for Europe.The fraction of Russian society in open opposition to the war is, unfortunately, smaller than that officially in support of it. At the beginning of the war, a number of Russian scientists published an open letter on the internet, in which they condemn this war (https://t‐invariant.org/2022/02/we‐are‐against‐war/). They clearly state that "The responsibility for unleashing a new war in Europe lies entirely with Russia. There is no rational justification for this war”, and “demand an immediate halt to all military operations directed against Ukraine". At the same time, other prominent Russian science and education officials signed the “Statement of the Russian Union of University Rectors (Provosts)”, which expressed unwavering support for Russia, its president and its Army and their goal to “to achieve demilitarization and denazification of Ukraine and thus to defend ourselves from the ever‐growing military threat” (https://www.rsr‐online.ru/news/2022‐god/obrashchenie‐rossiyskogo‐soyuza‐rektorov1/).Inevitably, Russian scientists must decide themselves how to live and continue their scientific work under the increasingly tight surveillance of the Kremlin regime. History is repeating itself. Not long ago, during the Cold War, Soviet scientists were largely isolated from the international research community and worked in government‐controlled research. In some fields, no one knew what they were working on or where. However, even in those dark times, courageous individuals such as Andrei Sakharov spoke out against the regime and tried to educate the next generation about the importance of free will. Many Soviet geneticists had been arrested under Stalin’s regime of terror and as a result of Lysenkoism and were executed or sent to the Gulag or had to emigrate, such as Nikolaj Timofeev‐Resovskij, one of the great geneticists of his time and an opponent of communism. As a result of sending dissident scientists to Siberia, great educational institutions were created in the region, which trained many famous scientists. History tells us that it is impossible to kill free will and the search for truth.The Russian invasion of Ukraine is a major humanitarian tragedy and a tragedy for science at many levels. Our hope is that the European science community, policymakers, and funders will be prepared to continue and expand support for our colleagues from Ukraine and eventually help to rebuild the bridges with Russian science that have been torn down.This commentary has been endorsed and signed by the EMBO Young Investigators and former Young Investigators listed below.

All signatories are current and former EMBO Young Investigators and endorse the statements in this article.

Conventional transmission electron microscopy

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.Transmission electron microscopy (TEM) has been an important technology in cell biology ever since it was first used in the early 1940s. The most frequently used TEM application in cell biology entails imaging stained thin sections of plastic-embedded cells by passage of an electron beam through the sample such that the beam will be absorbed and scattered, producing contrast and an image (see TermDefinitionBeem capsulePlastic forms that hold samples in resin during polymerizationBlocks (bullets)Polymerized samples in plastic removed from the Beem capsule and ready to sectionBlock faceSmall surface trimmed on a block before sectioningBoatWater reservoir in which sections float after being cut by a knifeCLEMCorrelative light and electron microscopyDehydrationRemoval of water from a sample by replacement with solventElectron tomography (ET)A method to image thick sections (200–300 nm) and produce three-dimensional imagesEmbeddingProcess of infiltrating the sample with resinFixationSample preservation with low temperature and/or chemicals to maintain sample integrityGridSmall metal support that holds the sections for viewing in the electron microscopeHPF/FSHigh-pressure freezing/freeze substitution sample preparation techniqueImmuno-EMDetection of proteins in EM samples using antibodiesIn-FXXKing credible!!!!Actual user quote in response to particularly beautiful sample. You may embellish with your own words.KnifeA very sharp edge, either glass or diamond, used to slice off resin-embedded samples into sectionsPre-embedding labelingApplication of antibodies before fixation and embeddingPost-embedding labelingApplication of antibodies to sections on the gridPoststainingStaining with heavy metals of sections on a gridResinLiquid form of the plastics used for embeddingRibbonCollection of serial sections placed on the gridSerials sectionsOne-after-the-other thin sections in a ribbonTEMTransmission electron microscopyThin sectionsThe 60- to 70-nm sections cut from the samples in blocksTrimmingProcess of cutting away excess resin to create a block faceUltramicrotomeInstrument used to cut sectionsVitrification/vitreous iceUnordered ice in which samples can be viewed without fix or stainOpen in a separate windowTEM has proven valuable in the analysis of nearly every cellular component, including the cytoskeleton, membrane systems, organelles, and cilia, as well as specialized structures in differentiated cells, such as microvilli and the synaptonemal complex. There is simply no way to visualize the complexity of cells and see cellular structures without TEM. Despite its power, the use of TEM does have limitations. Among the limitations are the relatively small data set of cells that can be imaged in detail, the obligate use of fixed—therefore deceased—cells, and the ever-present potential for fixation and staining artifacts. However, many of these artifacts are well known and have been catalogued (e.g., Bozzola and Russell, 1999 ; Maunsbach and Afzelius, 1999) .A typical TEM experiment consists of two phases: the live-cell experiment, in which a cell type, possibly a mutant, is grown under given conditions for analysis, followed by preparation of the specimen and imaging by TEM. Specimen preparation for conventional TEM is comprehensively reviewed in Hayat (1970) and briefly described here (Figure 1).Open in a separate windowFIGURE 1:A brief flowchart showing the work to be done with different types of sample preparation for conventional electron microscopy (yellow background). The advanced cryo-EM techniques are shown with a blue background. For immuno-EM, the samples can be stained before embedding (pre-embedding staining) or the sections can be stained (post-embedding staining).     

Phage Display Selection of Cyclic Peptides That Inhibit Andes Virus Infection

Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β₃ (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table ​(Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).

TABLE 1.

Peptide-bearing phage eluted from ANDV

Sequences from Ancestral Single-Stranded DNA Viruses in Vertebrate Genomes: the Parvoviridae and Circoviridae Are More than 40 to 50 Million Years Old

Vertebrate genomic assemblies were analyzed for endogenous sequences related to any known viruses with single-stranded DNA genomes. Numerous high-confidence examples related to the Circoviridae and two genera in the family Parvoviridae, the parvoviruses and dependoviruses, were found and were broadly distributed among 31 of the 49 vertebrate species tested. Our analyses indicate that the ages of both virus families may exceed 40 to 50 million years. Shared features of the replication strategies of these viruses may explain the high incidence of the integrations.It has long been appreciated that retroviruses can contribute significantly to the genetic makeup of host organisms. Genes related to certain other viruses with single-stranded RNA genomes, formerly considered to be most unlikely candidates for such contribution, have recently been detected throughout the vertebrate phylogenetic tree (1, 6, 13). Here, we report that viruses with single-stranded DNA (ssDNA) genomes have also contributed to the genetic makeup of many organisms, stretching back as far as the Paleocene period and possibly the late Cretaceous period of evolution.Determining the evolutionary ages of viruses can be problematic, as their mutation rates may be high and their replication may be rapid but also sporadic. To establish a lower age limit for currently circulating ssDNA viruses, we analyzed 49 published vertebrate genomic assemblies for the presence of sequences derived from the NCBI RefSeq database of 2,382 proteins from known viruses in this category, representing a total of 23 classified genera from 7 virus families. Our survey uncovered numerous high-confidence examples of endogenous sequences related to the Circoviridae and to two genera in the family Parvoviridae: the parvoviruses and dependoviruses (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree of vertebrate organisms and history of ssDNA virus integrations. Times of integration of ancestral dependoviruses (yellow icosahedrons), parvoviruses (blue icosahedrons), and circoviruses (triangles) are approximate.The Dependovirus and Parvovirus genomes are typically 4 to 6 kb in length, include 2 major open reading frames (encoding replicase proteins [Rep and NS1, respectively] and capsid proteins [Cap and VP1, respectively]), and have characteristic hairpin structures at both ends (Fig. ​(Fig.2).2). For replication, these viruses depend on host enzymes that are recruited by the viral replicase proteins to the hairpin regions, where self-primed viral DNA synthesis is initiated (2). Circovirus genomes are typically ∼2-kb circles. DNA of the type species, porcine circovirus 1 (PCV-1), contains a stem-loop structure within the origin of replication (Fig. ​(Fig.2),2), and the largest open reading frame includes sequences that are homologous to the Parvovirus replicase open reading frame (9, 11). The circoviruses also depend on host enzymes for replication, and DNA synthesis is self-primed from a 3′-OH end formed by endonucleolytic cleavage of the stem-loop structure (4). The frequency of Dependovirus infection is estimated to be as high as 90% within an individual''s lifetime. None of the dependoviruses have been associated with human disease, but related viruses in the family Parvoviridae (e.g., erythrovirus B19 and possibly human bocavirus) are pathogenic for humans, and members of both the Parvoviridae and the Circoviridae can cause a variety of animal diseases (2, 4).Open in a separate windowFIG. 2.Schematics illustrating the structure and organization of Parvoviridae and Circoviridae genomes and origins of several of the longest-integrated ancestral viral sequences found in vertebrates. Integrations were aligned to the Dependovirus adeno-associated virus 2 (AAV2), the Parvovirus minute virus of mice (MVM), and the Circovirus porcine circovirus 1 (PCV-1). The inverted terminal repeat (ITR) sequences in the Dependovirus and Parvovirus genomes are depicted on an expanded scale. A linear representation of the circular genome of PCV-1 is shown with the 10-bp stem-loop structure on an expanded scale. Horizontal lines beneath the maps indicate the lengths of similar sequences that could be identified by BLAST. The numbers indicate the locations of amino acids in the viral proteins where the sequence similarities in the endogenous insertions start and end. The actual ancestral virus-derived integrated sequences may extend beyond the indicated regions.With some ancestral endogenous sequences that we identified, phylogenetic comparisons can be used to estimate age. For example, as a Dependovirus-like sequence is present at the same location in the genomes of mice and rats, the ancestral virus must have existed before their divergence, more than 20 million years ago. Some Circovirus- and Dependovirus-related integrations also predate the split between dog and panda, about 42 million years ago. However, in most other cases, we rely on an indirect method for estimating age (1). As genomic sequences evolve, they accumulate new stop codons and insertion/deletion-induced frameshifts. The rates of these events can be tied directly to the rates of neutral sequence drift and, therefore, the time of evolution. To apply this method, we first performed a BLAST search of vertebrate genomes for all known ssDNA virus proteins (BLAST options, -p tblastn -M BLOSUM62 -e 1e−4). Candidate sequences were then recorded, along with 5 kb of flanking regions, and then again aligned against the database of ssDNA viruses to find the most complete alignment (BLAST options, -t blastx -F F -w 15 -t 1500 -Z 150 -G 13 -E 1 -e 1e−2). Detected alignments were then compared with a neutral model of genome evolution, as described in the supplemental material, and the numbers of stop codons and frameshifts were converted into the expected genomic drift undergone by the sequences. The age of integration was then estimated from the known phylogeny of vertebrates (7, 10). Using these methods, we discovered that as many as 110 ssDNA virus-related sequences have been integrated into the 49 vertebrate genomes considered, during a time period ranging from the present to over 40 to 60 million years ago (Table ​(Table1;1; see also Tables S1 to S3 in the supplemental material).

TABLE 1.

Selected endogenous sequences in vertebrate genomes related to single-stranded DNA viruses

The Diagnosis and Management of Hereditary Haemochromatosis

Hereditary haemochromatosis (HH) is a common genetic disorder of iron metabolism in individuals of Northern European ancestry which leads to inappropriate iron absorption from the intestine and iron overload in susceptible individuals. Iron overload is suggested by elevations in serum ferritin and transferrin saturation. The majority of patients with clinically significant iron overload are homozygous for the C282Y mutation of the HFE gene, however only a minority of C282Y homozygotes fully express the disease clinically. Those with a high serum ferritin (>1000 μg/L) and additional hepatic insults from cofactors are more likely to develop cirrhosis and its complications. The mainstay of treatment is venesection. Those without cirrhosis who undergo appropriate venesection have a normal life expectancy. Family screening is recommended for all first degree relatives of an individual with the disease.HH refers to a group of inherited disorders that result in progressive iron overload. Mutations of the HFE gene are responsible for the majority of cases of HH,¹ although disease expression is highly variable.² The ready availability of testing for the two clinically relevant mutations: C282Y and H63D, has substantially altered the approach to suspected iron overload in clinical practice. A number of rare but important forms of non-HFE related HH have also been described.³ The other main causes of iron overload are outlined in the HH HFE related HH (C282Y/C282Y, C282Y/H63D) Non-HFE related HH  Juvenile Haemochromatosis   Hemojuvelin related   Hepcidin related  Transferrin receptor-2 related HH Ferroportin related HHSecondary Iron Overload Iron loading anaemia  Thalassaemia major  Sideroblastic anaemia  Chronic haemolytic anaemia Parenteral iron overload (multiple transfusions)Others Metabolic syndrome Chronic liver disease  Hepatitis C  Alcoholic liver disease  Non-alcoholic steatohepatitis  Porphyria cutanea tarda African Iron Overload Acaeruloplasminaemia Atransferrinaemia Neonatal iron overloadOpen in a separate window