Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Teachers' and adult students' attitudes and beliefs about the use of codeswitching in teaching and learning English

Due to globalization, immigration, and mobility, people are more concerned with languages of contact and diversity than ever. By definition, the term codeswitching (CS) was termed as the language alternation involving a bilingual’s use of both languages in the learning of English. Throughout history, codeswitching has been a topic of biases and arguments presenting gaps in research. Recent studies have provided support to the argument that switching assists in problem solving which influences language performance. This research study was conducted to examine the teachers’ and adult ESL students’ attitudes and beliefs about the use of CS in teaching and learning English. A self-report online survey was launched to collect the data. A multivariate analysis of variance (MANOVA) was used to assess if there were differences between the teachers’ and students’ groups on their attitudes and beliefs. In reviewing the descriptive statistics, the teachers in the SW region of the U.S. had higher mean scores on both attitudes and beliefs than those in the WC region, but the differences were not statistically significant. The teachers who had fewer than five years of teaching experience had higher mean scores in both attitudes and beliefs than those with more than five years of teaching. The students had slightly lower mean scores in attitudes in the 18-30 years of age bracket; they had higher mean scores in beliefs than those in the 31-60 age brackets, but the differences were not statistically significant. The students in the SW region of Texas had higher mean scores in both attitudes and beliefs than the SE region of Texas, but the differences were not statistically significant. The inferential statistics disclosed a statistical significance found in the difference in the students’ attitudes and beliefs about the use of CS in relation to age (p < .05). The difference in the students’ attitudes and beliefs in relation to location was not significant. Also, the differences in the teachers’ attitudes and beliefs about the use of CS were not statistically significant in their regional locations and years of teaching. Key words: Codeswitching, attitudes of codeswitching, beliefs of codeswitching, second language teaching and learning

Palliative Care on Caregiver Health Outcomes: A Scoping Review

This scoping review aims to investigate existing research that examines the effect of palliative care interventions provided for terminally ill patients on caregivers

Laser Scanning Confocal Microscopy Was Used to Validate the Presence of Burkholderia pseudomallei or B. mallei in Formalin-Fixed Paraffin Embedded Tissues

Burkholderia pseudomallei and B. mallei are Gram-negative, facultative intracellular bacteria that cause melioidosis and glanders, respectively. Currently, there are no vaccines for these two diseases. Animal models have been developed to evaluate vaccines and therapeutics. Tissues from infected animals, however, must be fixed in formalin and embedded in paraffin (FFPE) before analysis. A brownish staining material in infected tissues that represents the exopolysaccharide of the pathogen was seen by bright field microscopy but not the actual microorganism. Because of these results, FFPE tissue was examined by laser scanning confocal microscopy (LSCM) in an attempt to see the microorganism. Archival FFPE tissues were examined from ten mice, and five nonhuman primates after exposure to B. pseudomallei or B. mallei by LSCM. Additionally, a historical spleen biopsy from a human suspected of exposure to B. mallei was examined. B. pseudomallei was seen in many of the infected tissues from mice. Four out of five nonhuman primates were positive for the pathogen. In the human sample, B. mallei was seen in pyogranulomas in the spleen biopsy. Thus, the presence of the pathogen was validated by LSCM in murine, nonhuman primate, and human FFPE tissues

Characterization of the plasma proteome of nonhuman primates during Ebola virus disease or melioidosis: a host response comparison

Abstract Background In-depth examination of the plasma proteomic response to infection with a wide variety of pathogens can assist in the development of new diagnostic paradigms, while providing insight into the interdependent pathogenic processes which encompass a host’s immunological and physiological responses. Ebola virus (EBOV) causes a highly lethal infection termed Ebola virus disease (EVD) in primates and humans. The Gram negative non-spore forming bacillus Burkholderia pseudomallei (Bp) causes melioidosis in primates and humans, characterized by severe pneumonia with high mortality. We sought to examine the host response to infection with these two bio-threat pathogens using established animal models to provide information on the feasibility of pre-symptomatic diagnosis, since the induction of host molecular signaling networks can occur before clinical presentation and pathogen detection. Methods Herein we report the quantitative proteomic analysis of plasma collected at various times of disease progression from 10 EBOV-infected and 5 Bp-infected nonhuman primates (NHP). Our strategy employed high resolution LC–MS/MS and a peptide-tagging approach for relative protein quantitation. In each infection type, for all proteins with > 1.3 fold abundance change at any post-infection time point, a direct comparison was made with levels obtained from plasma collected daily from 5 naïve rhesus macaques, to determine the fold changes that were significant, and establish the natural variability of abundance for endogenous plasma proteins. Results A total of 41 plasma proteins displayed significant alterations in abundance during EBOV infection, and 28 proteins had altered levels during Bp infection, when compared to naïve NHPs. Many major acute phase proteins quantitated displayed similar fold-changes between the two infection types but exhibited different temporal dynamics. Proteins related to the clotting cascade, immune signaling and complement system exhibited significant differential abundance during infection with EBOV or Bp, indicating a specificity of the response. Conclusions These results advance our understanding of the global plasma proteomic response to EBOV and Bp infection in relevant primate models for human disease and provide insight into potential innate immune response differences between viral and bacterial infections

Comparative virulence of three different strains of Burkholderia pseudomallei in an aerosol non-human primate model.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1β, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1β at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis

Proteomic Analysis of Non-human Primate Peripheral Blood Mononuclear Cells During Burkholderia mallei Infection Reveals a Role of Ezrin in Glanders Pathogenesis

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1β (IL-1β). On the contrary, the expression of IL-1β receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1β production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model

<i>B</i>. <i>pseudomallei</i> LPS profiles.

<p><b>A.</b> Purified LPS samples from 11 <i>B</i>. <i>pseudomallei</i> strains were separated by SDS-PAGE. The gel was electroblotted, and the western blot was developed using a monoclonal antibody directed against <i>B</i>. <i>pseudomallei</i> LPS (11G3-1). <b>B.</b> Results were confirmed by silver staining. <i>B</i>. <i>pseudomallei</i> 576 displaying the LPS type B banding pattern is shown for comparison. M = molecular weight marker.</p

Statistical analyses of the LD₅₀ determinations.

<p>Kernel density plot of the posterior distributions of the LD_<b>50</b> values of each <i>B</i>. <i>pseudomallei</i> isolate in BALB/c mice through <b>A.</b> 21 day survival challenge and <b>B.</b> 60 day survival challenge.</p

Several noteworthy clinical/gross observations and their histopathological correlates.

<p><b>A.</b> Swelling of or around the eye (white arrow). Histologic evaluation revealed abscesses and pyogranulomatous inflammation filling the retro-orbital space causing the globe of the eye (E) to protrude from the socket (BALB/c mouse challenged with 4.2 x 10⁴ CFU 1106a, H&E 2X). The nasal cavity (N) is also marked for orientation. <b>B.</b> Large swellings on the tail (often seen in conjunction with hind limb swelling and/or paralysis). Histologic evaluation revealed pyogranulomatous inflammation from the haired skin of the tail extending into adjacent bone, skeletal muscle, and adipose tissue (BALB/c mouse challenged with 1.4 x 10⁴ CFU K96243, H&E 20X). The tail vertebra (V) is denoted for orientation. <b>C.</b> Firm white nodules in the spleen. Histologic evaluation revealed marked pyogranulomatous inflammation and necrosis (identified with an *) in the spleen which effaces normal white and red pulp (BALB/c mouse challenged with 9.0 x 10² MSHR668, H&E 20X).</p