The WHO guidelines for the management of advanced HIV disease recommend a package of care consisting of rapid initiation of antiretroviral therapy (ART), enhanced screening and diagnosis of tuberculosis (TB) and cryptococcal meningitis, co-trimoxazole prophylaxis, isoniazid preventive therapy (IPT), fluconazole pre-emptive therapy, and adherence support. The goals of this study were to determine the prevalence of advanced HIV disease among individuals initiating ART in Senegal, to identify predictors of advanced disease, and to evaluate adherence to the WHO guidelines.This study was conducted among HIV-positive individuals initiating ART in Dakar and Ziguinchor, Senegal. Clinical evaluations, laboratory analyses, questionnaires and chart review were conducted. Logistic regression was used to identify predictors of advanced disease.3 or clinical stage 3 or 4. The median CD4 count was 185 cells/mm3. The strongest predictors of advanced disease were age ≥ 35  and having sought care from a traditional healer . Approximately one third of subjects initiated ART within 7 days of diagnosis. Co-trimoxazole prophylaxis was provided to 65% of subjects with CD4 counts ≤350 cells/mm3 or stage 3 or 4 disease. TB symptom screening was available for 166 subjects; 54% reported TB symptoms. Among those with TB symptoms, 39% underwent diagnostic evaluation. Among those eligible for IPT, one subject received isoniazid. No subjects underwent CrAg screening or received fluconazole to prevent cryptococcal meningitis.A total of 198 subjects were enrolled; 70% were female. The majority of subjects (71%) had advanced HIV disease, defined by the WHO as a CD4 count < 200 cells/mmThis is the first study to report an association between seeking care from a traditional healer and presentation with WHO defined advanced disease in sub-Saharan Africa. Given the widespread use of traditional healers in sub-Saharan Africa, future studies to further explore this finding are indicated. Although the majority of individuals in this study presented with advanced disease and warranted management according to WHO guidelines, there were numerous missed opportunities to prevent HIV-associated morbidity and mortality. Programmatic evaluation is needed to identify barriers to implementation of the WHO guidelines and enhanced funding for operational research is indicated. Progress towards reducing AIDS-related deaths in Western and Central Africa lags behind other regions. Nearly one third of global HIV-related deaths occur in Western and Central Africa, and although globally HIV-related deaths have declined by 34%, deaths in Western and Central Africa have declined by only 24%, from 370,000 in 2010 to 280,000 in 2017 [3, which is lower than other regions on the continent and is indicative of severe immunodeficiency and susceptibility to opportunistic infections [Despite efforts to improve access to HIV testing and treatment, approximately one third of individuals with HIV in sub-Saharan Africa (SSA) present with advanced disease –5. In WeIn 2017, the WHO published guidelines recommending a package of targeted interventions to reduce morbidity and mortality among people presenting with advanced HIV . The guiThis study was conducted at HIV testing and treatment sites located at the Centre Hospitalier National Universitaire de Fann  in Dakar and the Centre de Santé de Ziguinchor, located in Ziguinchor. Dakar is the urban capital of the country, while Ziguinchor is located in the southern Casamance region. All HIV-positive individuals initiating ART through the Senegalese National AIDS program (ISAARV) who provided written informed consent were eligible for enrollment. For subjects < 18 years of age, consent was obtained from their legal guardian. Subjects were enrolled sequentially from April 2017 to April 2018. Study procedures were approved by the University of Washington Institutional Review Board and the Senegal Comité National d’Ethique pour la Recherche en Santé (CNERS).Study encounters were conducted in the participant’s preferred language, including French, Wolof, Diola, Peular, Mandinka, or Creole. Sociodemographic characteristics were determined using interviewer administered questionnaires. Food insecurity was determined using the Household Food Insecurity Access Scale . WHO cliP-values < 0.05 were considered significant.Data were analyzed using SPSS Statistics 23 . Descriptive analysis was performed for all variables. Logistic regression was used to identify predictors of presentation with advanced disease. Variables identified in the literature as associated with advanced disease were evaluated using simple regression, Variables which were predictive using simple regression were included in the multiple regression model. Missing data were excluded from analysis. A total of 198 subjects were enrolled, 83 (42%) in Dakar and 115 (58%) in Ziguinchor . Nearly three quarters of subjects presented with a CD4 count ≤350 cells/mm3, 55% presented with < 200 cells/mm3, 36% had < 100 cells/mm3, and 20% had < 50 cells/mm3. WHO clinical staging was available for 167 subjects, of which 53% had WHO stage 3 or 4 disease. The most common WHO stage 3 conditions were severe weight loss, chronic diarrhea, oral candidiasis, and pulmonary TB. The most common WHO stage 4 condition was extra-pulmonary TB. The majority of subjects (71%) had advanced HIV disease, defined by the WHO as a CD4 count < 200 cells/mm3 or stage 3 or 4 disease.CD4 cell counts were available for 185 subjects Table . The medVariables which were predictive of advanced disease using simple regression included male sex, age ≥ 35, and having sought care from a traditional healer prior to presentation  did not report symptoms consistent with TB  and 89 (54%) reported symptoms consistent with TB. Among those without TB symptoms, 4 had extra-pulmonary TB, resulting in 73 (44%) of subjects eligible for IPT. Among those eligible for IPT, one subject (1%) received a 2 week prescription for INH. Among those with TB symptoms, only 35 (39%) underwent diagnostic evaluation, 31 were evaluated by microscopy for acid-fast bacilli using Ziehl-Neelsen staining and 10 were evaluated by Xpert® MTB/RIF (Cepheid). There was no rifampicin resistance detected. The diagnostic evaluation was negative for 25 (71%) of the subjects who were evaluated. Among non-pregnant subjects ≥18 years of age, a review of symptoms and CD4 counts were available for 133. There were 46 subjects (35%) with CD4 cell counts < 100 and no neurological symptoms reported. No subjects underwent cryptococcal antigen (CrAg) screening and none received fluconazole to prevent CM.The most frequently prescribed antiretrovirals were tenofovir (TDF) (95%), lamivudine or emtricitabine (3TC or FTC) (100%), and efavirenz (EFV) 84%) .Provision of a package of care for people with advanced HIV disease is supported by data from two randomized trials of intervention packages for individuals with advanced disease in SSA , 11. TheAccording to the WHO guidelines, “rapid initiation of ART should be offered to all PLHIV following a confirmed diagnosis and clinical assessment” with the exception of individuals suspected of having TB or CM . Rapid iRapid initiation of ART has the potential to improve ART uptake, retention in care and viral suppression, and to decrease disease progression, HIV transmission, and mortality , 12–14. Notably, we did not find an association between advanced disease and clinic site, residence, education, number of children, marital status, employment, or food insecurity. The strongest predictors of advanced disease were age ≥ 35 years and having sought care from a traditional healer. To our knowledge, this is the first study to report an association between seeking care from a traditional healer and presentation with WHO defined advanced disease in SSA. Given the widespread use of traditional healers in SSA , 21, thi3 or clinical stage 3 or 4, or lifelong co-trimoxazole for any CD4 count in settings with a high prevalence of malaria or bacterial infections. An area of high malaria transmission is defined as > 1 case/1000 population per year [The WHO criteria for co-trimoxazole prophylaxis among PLHIV are CD4 count ≤350 cells/mmper year . Malariaper year . The risper year –28. Nearper year .Tuberculosis remains the leading cause of death among PLHIV, responsible for one third of deaths among PLHIV globally , 30. StrApproximately half of the subjects in this study reported symptoms consistent with TB. Among those who were symptomatic, only 39% underwent diagnostic evaluation and for the majority, traditional microscopy was utilized. These findings indicate a need to identify barriers to diagnostic evaluation and to improve access to WHO recommended diagnostic tests. Importantly, among those who did not have TB symptoms, only one individual was provided with isoniazid (INH). That individual received a 2 week prescription for INH, although the WHO recommends a minimum of 6 months of IPT. The provision of IPT is recommended by the Senegal National AIDS program. Although the reasons for poor IPT uptake in Senegal have not been evaluated, barriers may include provider fear of undiagnosed TB, concerns about drug interactions, adverse effects, or medication adherence, uncertainty about eligibility criteria, or lack of awareness of national and international policies. A survey of physicians and healthcare workers involved in the care of PLHIV in Senegal could serve to elucidate these factors.3 and that screening should be considered at a CD4 count threshold of < 200 cells/mm3 [3  and may be considered at a higher CD4 cell count threshold of < 200 cells/mm3 . Neither CrAg screening combined with PET nor fluconazole prophylaxis were provided to any of the subjects evaluated in our study. CrAg screening is not routinely available in Senegal and the recommendation for prophylaxis in the absence of screening was only recently released. If implemented, these strategies could provide important opportunities to decrease morbidity and mortality due to CM among PLHIV in Senegal.Cryptococcal meningitis (CM) is a leading cause of death among individuals with advanced HIV, accounting for 15–20% of global AIDS-related deaths , 31. CrAThe primary limitation of our study is sample size. Turnover of healthcare personnel and regional strikes involving healthcare personnel limited our ability to enroll all individuals who initiated ART during the study period. This was not a population-based study and may not be representative of PLHIV or those presenting for care overall in Dakar or Ziguinchor or more broadly in Senegal. Our study is further limited by incomplete data; completion of standardized national forms for the evaluation and management of HIV was inconsistent. A strength of our study is that we sequentially enrolled HIV-positive individuals initiating ART at two study sites in Senegal to capture a sample that is representative of the HIV-positive population and provide an evaluation of the management of advanced HIV disease that represents national practices.Senegal was an early leader in responding to the HIV epidemic through the implementation of the first government ART treatment program in Africa in 1998. In the current era, proactive efforts to ensure adherence to WHO and Senegalese guidelines and access to evidence-based interventions for individuals with advanced HIV disease are similarly warranted.We evaluated the implementation of the WHO guidelines for the management of advanced HIV disease among individuals initiating ART in Senegal, West Africa. We found that although the majority of individuals presented with advanced disease and warranted management according to WHO guidelines, there were numerous missed opportunities to prevent HIV-associated morbidity and mortality. Programmatic evaluation is needed to identify barriers to implementation and enhanced funding for operational research is indicated.


Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for  Drosophila species [Anopheles [Exciting efforts to sequence the diversity of life are building momentum  but one  species ), but lenopheles ). Therefnopheles .Aedes aegypti assembly used DNA extracted from the offspring of a single cross, thus reducing the maximum number of haplotypes for any given locus to four, thereby improving the assembly process and achieving a contig N50 of 1.3 Mb [Another way to overcome DNA input requirements, while also reducing the number of haplotypes present in a DNA pool, is to limit the number of haplotypes in the pool of individuals by using offspring from a single cross. This is easier than multiple generations of inbreeding, and can be successful. For example, a recent PacBio f 1.3 Mb .However, for an initiative like the Earth BioGenome Project  that aimAnopheles coluzzii female from the Ngousso colony. This colony was created in 2006 from the broods of approximately 100 wild-caught pure An. coluzzii females in Cameroon (pers. comm. Anna Cohuet). Although the colony has been typically held at >100 breeding individuals, given the long time since colonization, there is undoubtedly inbreeding. A single female was ground in 200 µl PBS using a pestle with several up and down strokes , and DNA extraction was carried out using a Qiagen MagAttract HMW kit (PN-67653) following the manufacturer’s instructions, with the following modifications: 200 ul 1X PBS was used in lieu of Buffer ATL; PBS was mixed simultaneously with RNAse A, Proteinase K, and Buffer AL prior to tissue homogenisation and incubation; incubation time was shortened to 2 h; solutions were mixed by gently flicking the tube rather than pipetting; and subsequent wash steps were performed for one minute. Any time DNA was transferred, wide-bore tips were used. These modifications were in accordance with recommendations from 10X Genomics HMW protocols that aim to achieve >50 kb molecules. The resulting sample contained ~250 ng of DNA, and we used the FEMTO Pulse  to examine the molecular weight of the resulting DNA. This revealed a relatively sharp band at ~150 kb . Because the genomic DNA was already fragmented with the majority of DNA fragments above 20 kb, shearing was not necessary. 100 ng of the genomic DNA was carried into the first enzymatic reaction to remove single-stranded overhangs followed by treatment with repair enzymes to repair any damage that may be present on the DNA backbone. After DNA damage repair, ends of the double stranded fragments were polished and subsequently tailed with an A-overhang. Ligation with T-overhang SMRTbell adapters was performed at 20 °C for 60 min. Following ligation, the SMRTbell library was purified with two AMPure PB bead clean up steps , first with 0.45X followed by 0.80X AMPure. The size and concentration of the final library  were assSequencing primer v4 and Sequel DNA Polymerase 3.0 were annealed and bound, respectively, to the SMRTbell library. The library was loaded at an on-plate concentration of 5–6 pM using diffusion loading. SMRT sequencing was performed on the Sequel System with Sequel Sequencing Kit 3.0, 1200 min movies with 120 min pre-extension and Software v6.0 (PacBio). A total of 3 SMRT Cells were run.Anopheles gambiae (GenBank assembly accession GCA_000005575.2) [The genome was assembled using FALCON-Unzip, a diploid assembler that captures haplotype variation in the sample  ,9. We reWe explored the performance as a function of the number of SMRT Cells used for the assembly , and fouThe contigs were screened by the Sanger Institute and NCBI to identify contaminants and mitochondrial sequence. Windowmasker was used to mask repeats and the MegaBLAST algorithm was run  on the masked genome versus all complete bacterial genomes to find hits with ≥98% homology. In addition, we screened the primary assembly for duplicate haplotypes using Purge Haplotigs [In the process of using PEST to order and orient the PacBio contigs, we found one large potential heterozygous interchromosomal rearrangement between 2L and 3R . Upon fuAn. gambiae PEST assembly, using a previously described script [To assess the completeness of the curated assembly, we searched for conserved, single copy genes using BUSCO  v3.0.2  with thed script .Anopheles gambiae reference genome [To assess the quality of contig assembly and concordance with existing assemblies, the curated primary contigs were aligned to the PEST e genome ,9 using e genome . For thee genome ). Confide genome .High molecular weight DNA was extracted from a single female mosquito. Given that the genomic DNA had a suitable size range for long-insert PacBio sequencing , the seqde novo assembly consisted of 372 contigs totaling 266 Mb in length, with half of the assembly in contigs (contig N50) of 3.5 Mb or longer  gene set), resulting in 14,972 alignments (99.5%) and an average alignment length of 96.6%, and with >96% of alignments showing no frame shift-inducing indels.To evaluate genome completeness and sequence accuracy of the assembly, we performed alignment analyses to a set of conserved genes. Using the ‘diptera’ set of the BUSCO gene collection , we obseAn. gambiae genome, published in 2002, was created using BACs and Sanger sequencing [Anopheles genome among the 21 that have now been sequenced [An. gambiae sensu stricto, and the “M” form has acquired species status and a new name, An. coluzzii [An. gambiae genome  designated as the “UNKN” (unknown) chromosome. We compared the alignments of contigs from the PEST chromosomes  versus the contigs from the UNKN to the new assembly. Any regions with mapq60 alignments of both UNKN and chromosomal contigs are likely to be haplotigs in the UNKN. In total, we find that 7.27 Mb are haplotigs  and another 10.9 Mb are newly placed sequence that do not overlap with PEST chromosomal alignments. The UNKN bin also contains 737 annotated genes. Remarkably, our single-insect assembly now places 667 (>90%) of these formerly unplaced genes into their appropriate chromosomal contexts  of PacBio sequence corresponding to several pieces in the UNKN section of PEST that can now be assigned to the X chromosome.We also identified several potential rearrangements in the 20–22 Mb region of the X chromosome . PEST hade novo genome assemblies, but because of the relatively large DNA input requirements, it has not been used to its full potential for small organisms, requiring time-consuming inbreeding or pooling strategies to generate enough DNA for library preparation and sequencing. Here we present, to our knowledge, the first example of a high-quality de novo assembly from a single insect. This assembly, using only one individual and one sequencing technology, exhibits a higher level of contiguity, completeness, accuracy, and degree of haplotype separation than any previous Anopheles assembly, demonstrating the impact of long reads on assembly statistics. While the assembly did not achieve independent full chromosomal scale assignment of contigs, its mega-base scale contiguity without gaps immediately provides insights into gene structure and larger-scale genomic architecture, such as promoters, enhancers, repeat elements, large-scale structural variation relative to other species, resolution of tandem repeats , or aliWe noted the importance of the initial DNA size distribution in conjunction with this protocol. Since neither shearing prior to library construction nor size-selection thereafter were employed, the starting high-molecular weight DNA should contain fragments at greater than ~20 kb on average, and without the significant presence of short  DNA fragments. Further research into suitable DNA extraction, storage and transportation methodologies is needed to fulfill these requirements for a broader spectrum of different species and environments, in order to allow for the preparation of suitable DNA samples from wild-caught samples originating in sometimes remote areas with limited sample preparation infrastructure.We anticipate that the new workflow described here will facilitate the sequencing and high-quality assembly of many more species of small organisms, as well as groups of individuals within a species for population-scale analyses, representing an important prerequisite in view of large-scale initiatives such as i5K and the Earth BioGenome Project ,5. In ad


The increase in antibiotic resistant bacteria has raised global concern regarding the future effectiveness of antibiotics. Human activities that influence microbial communities and environmental resistomes can generate additional risks to human health. In this work, we characterized aquatic microbial communities and their resistomes in samples collected at three sites along the Bogotá River and from wastewaters at three city hospitals, and investigated community profiles and antibiotic resistance genes (ARGs) as a function of anthropogenic contamination. The presence of antibiotics and other commonly used drugs increased in locations highly impacted by human activities, while the diverse microbial communities varied among sites and sampling times, separating upstream river samples from more contaminated hospital and river samples. Clinically relevant antibiotic resistant pathogens and ARGs were more abundant in contaminated water samples. Tracking of resistant determinants to upstream river waters and city sources suggested that human activities foster the spread of ARGs, some of which were co-localized with mobile genetic elements in assembled metagenomic contigs. Human contamination of this water ecosystem changed both community structure and environmental resistomes that can pose a risk to human health. Bacterial pathogens resistant to one or more antibiotics, and even to all known drug regimens, as is the case of XDR-TB2, can undermine treatments and lead to increased health care costs due to prolonged illness and hospital stays3. Resistant strains are responsible for a great number of deaths worldwide, with an annual death toll estimated to rise to 10 million people by 20504. The current situation is aggravated by the slow rate at which new antimicrobial drugs are being developed and by the spread of antibiotic resistance genes (ARGs) both in the community and in settings where microorganisms are exposed to the selective pressure of antibiotics, such as hospitals.It is now well recognized that the medical use of an antibiotic is inevitably followed by the appearance of resistant bacterial strains. The rise in antibiotic resistance worldwide and the menace posed by antibiotic resistant pathogens to human health have led to recent global action plans to tackle antimicrobial resistance7. These environmental resistomes are not necessarily linked to human activities and the modern use of antibiotics; rather, they originate from years of evolution as microbes interact and adapt to survive in variable environmental conditions. Microorganisms also have the unique capacity to exchange genetic material by means of mobile genetic elements, which give them greater plasticity and the possibility of accessing the genetic pool of other microbes8. Environmental resistomes are extensive and diverse and have gained interest because they may serve as reservoirs for ARGs that could be potentially transferred to clinically relevant bacterial pathogens.Recent studies using mostly metagenomic approaches have shown that ARGs are present in various natural environments, even those considered pristine or ancient9, have transformed various environments by introducing contaminants and compounds that can adversely affect ecosystems and microbial communities10. Chemicals, toxic metals and antibiotics can accumulate in the environment due to overuse or unregulated waste management practices, and can inadvertently promote the appearance of resistant bacteria11. Certain activities, such as agricultural spread of manure and sewage disposal can introduce ARGs and resistant microorganisms into the environment and create new opportunities for interaction among bacteria of human, animal and environmental origin8. Although aquatic environments are particularly important because they provide a basic resource, they receive effluents from industrial and human activities, and thus they represent a unique setting for the acquisition and spread of ARGs12, as well as for the proliferation of resistant bacteria14.Human activities, recognized as one of the main drivers of evolution in the planet16, perhaps even at concentrations that can fall below minimal inhibitory doses17. Wastewater treatment plants, which can reduce bacterial and ARG load, can still serve as reservoirs of resistant bacteria and ARGs that may impact bacterial water communities20. Other human activities and anthropogenic pollutants, such as animal feeding and industrial and pharmaceutical wastes, have also been shown to contribute to ARG abundance and dissemination in rivers21 and in estuary sediments22. The recent use of shotgun metagenomics showed that wastewater contamination of river waters resulted in increased abundances of ARGs, mobile genetic elements and bacterial genera known to harbor pathogenic species23. A study of several lakes also found that ARGs were present even in less impacted locations, suggesting a role of these freshwater reservoirs in the spread of resistance markers24. In fact, a recent meta-analysis of lakes indicated that contamination with antibiotics and ARGs occurs on a global scale14. Overall these studies show that human activities directly affect both microbial communities and resistance genes in freshwater ecosystems, stressing the importance of efficient waste and pollutant management to limit inadvertent effects on the environment and humans.Several studies have looked at the influence of human activities on the presence of antimicrobial agents, resistant bacteria and ARGs in freshwater ecosystems. Antibiotics released into the environment can persist and continue to exert selective pressure for resistance determinants10. Knowledge of these environmental resistomes and their possible role in human health can raise awareness and help to catalyze actions to control contamination and potential risks. In this work, we studied aquatic microbial communities and antibiotic resistance markers in the Bogotá River, a vital freshwater supply for the region that is greatly deteriorated by industrial, agricultural and municipal discharges before, during and after its passage through the city of Bogotá25. A variety of pharmaceutical compounds, including antibiotics, are also commonly found in effluent wastewater and surface waters from Bogotá26. However, work is lacking regarding the effect of pollutants on the microbial communities and the ARGs present in this river ecosystem. This work shows that anthropogenic contamination alters the structure and diversity of aquatic microbial communities that in turn can provide a setting for microbial interactions and dispersion of resistance determinants in the environment.The surveillance and study of microorganisms in impacted environments can provide information on how microbial communities and ARGs change due to human activities. ARGs have also been proposed as a proxy for understanding anthropogenic influence on dispersion of microorganisms and their genes in the environmentSamples with distinct levels of anthropogenic influence were collected from the Bogotá River (river source (RS), middle section (RM) and lower section (RL), in increasing order of anthropogenic influence) and from wastewater effluents at three hospitals in Bogotá , B (HB) and C (HC)) Fig. . TriplicEscherichia coli cells, common indicators of fecal contamination. There was low recovery from the RS samples, consistent with previous reports for the Bogotá River27, while the RM, RL and hospital samples had similar E. coli counts (between 106 and 108 CFU/L) and total counts (between 107 to 108 CFU/L)  and one K. pneumoniae on CARBA agar. No counts were conducted since these microorganisms were expected to be present at very low levels.The microbiological quality of the sampled water was assessed by estimating total coliforms and 25, and confirmed that samples were collected at sites with different levels of contamination.Both physicochemical and microbiological water quality results, as well as the presence of pharmaceutical compounds, indicated increasing contamination in rural and urban areas , consistent with previous reports of water quality deteriorationp = 0.03762; Faith’s PD, p = 0.01672) (Table p = 0.8275).Metagenomic DNA isolated from the water samples was used to determine community taxonomic profiles. Although both RS samples yielded low amounts of DNA (<1 ng/µl), in agreement with the low bacterial recoveries upon plating, it was impossible to PCR-amplify the 16S rRNA gene from the sample collected in 2015 (RS15) and it was therefore not further used. Amplicon libraries done with the remaining 11 samples were sequenced and, after quality filtering and merging, the number of clean reads ranged from 41,207 to 65,614 per sample. Diversity analysis of these samples was performed by randomly subsampling each dataset to 41,000 sequences. The estimated richness by direct OTU count (observed OTUs) was highest for RL15, but after being corrected by phylogenetic relatedness (Faith’s PD) it was highest for the upstream river samples (source and middle section), followed by the lower section (RL) waters and the hospitals , Thiocapsa (sulfur-reducing), Rhodobacter (photosynthetic), Limnohabitans (planktonic), Dechloromonas (aromatic-compound-oxidizing) and Polynucleobacter (planktonic), were enriched in the upstream samples; while genera associated with the human gut microbiota, such as Faecalibacterium, Roseburia, Bacteroides and Ruminococcus were more abundant in hospitals , and showed that the two samples from the river’s lower section were different from each other, one featuring more taxa enriched in hospitals and the other more taxa enriched in upstream river locations.Taxonomic assignment was done at various levels using the naïve Bayesian RDP Classifierion Fig.  indicateion Fig. . The timion Fig. . Genera 29. The broad taxonomic patterns obtained were consistent with those observed by doing 16S rRNA gene profiles, though Metaxa2 failed to identify phyla of low abundance 32, and to the bacterial proportion of 16S copies found by Metaxa2  (Table p = 0.002).In addition to having greater ARG counts, hospitals and the river’s lower section also had higher ARG diversity  Table . An LDA 7) Table . Further7) Table . ARGs wi7) Table . A princ7) Table . These rp = 0.008, M2 = 0.554), suggesting that some of the resistances could be associated with certain taxa observed at specific locations. Given that the river’s lower section (RL) is influenced by both upstream waters and urban effluents, including hospital wastewaters, SourceTracker35 was used to identify the potential source of both microbial taxa and ARGs present in the downstream river samples (RL). In the case of taxa, the source of the bacterial genera detected in the RL samples from different years, RL15 and RL16, had different origins. RL15 genera originated solely from unknown sources  and hospitals , while the composition of RL16 was derived mainly from the upstream RS and RM sources  , followed by unknown sources  and upstream river sources ; while in RL16, most genes could be traced to upstream river sources , followed by hospital wastewaters  and unknown sources . The significant contributions from unknown sources were expected because contaminants released into the river have numerous additional origins, like agricultural activities and household discharges. When considering only the known sources, again RL15 had a larger ARG contribution from hospitals and RL16 from upstream river locations (RS and RM) . This strategy generated a total of 57 contigs, for all samples, 10 with a size larger than 1,000 bp. In four of these contigs we found co-occurrence of these markers: two cases in sample HC15 , and single occurrences for HC16 (Sul1 protein and an integrase family protein) and RL15 (streptomycin adenylyltransferase gene). The SPAdes global assembly was also searched for contigs with co-occurrence of ARGs and mobile genetic elements. In this case, we identified four such contigs: in samples HB16 (AAC(6′)-Ib gene with IntI1 integrase), HC16 (qacH gene with IS6100 transposase), RL16 (AAC(6′)-Ib gene with IntI1 integrase) and RM15 (mphD gene with IS26 transposase). Of these 8 contigs with evidence of co-localization for both an ARG and a mobilization gene, only one was from the river middle section and none were from the river source, suggesting a higher potential for horizontal gene transfer in densely populated communities as those of the hospitals and the lower section of the river.To evaluate possible associations between ARGs and mobile genetic elements that could facilitate horizontal gene transfer, we assembled contigs using GenSeed-HMM25. The presence of drugs of common clinical use in these water samples reflects both drug usage26 and contamination39. Antibiotics were present in low amounts that, even if not effective for selection of resistant bacteria33, particularly in the upstream river samples, may nonetheless alter microbial signaling and affect interactions40. It is important to keep in mind that, given the limited sampling scheme and the variable drug concentrations obtained, these measurements are not meant as a comprehensive analysis and are thus difficult to correlate with microbial populations. Importantly, however, the detection of these drugs provides evidence that they were present, even though their concentration and effects at other locations or microenvironments, such as biofilms, are unknown. Concentrations in hospital effluents showed variability, possibly due to usage and stability, and were similar to those found in other hospital wastewaters from Colombia41. These locations may serve as sites for gene exchange and selection of resistant microorganisms20, even at sub-inhibitory concentrations17.In this work, we characterized aquatic microbial community composition and the resistome in water samples taken at sites with varying degrees of contamination: three locations along the Bogotá River and the wastewaters from three hospitals in the city of Bogotá. Physicochemical measures, bacterial counts and antibiotic and pharmaceutical quantifications indicated that human activities had a negative impact on water quality, consistent with previous reports42; or (ii) rural and urban discharges into the river at the different sampling times. Additional sampling would be required to define possible causation and specific correlations with environmental variables.The changes observed in water quality were accompanied by alterations in microbial community composition and ARG profiles. The taxa differentially enriched in the upstream (RS and RM) and hospital samples were indicative of different ecological contexts. Environmental microbes, including methanotrophic, sulfur-reducing, photosynthetic and aromatic-compound-oxidizing bacteria, were enriched in the upstream samples, while taxa associated with the human gut microbiota were more abundant in hospitals, consistent with human fecal contamination. Both PCoA and source tracking analyses showed that communities from downstream samples (RL) could be derived from upstream river and hospital wastewater sources. For example, the abundance of Firmicutes in the RL15 sample could be explained by the presence of this discriminant taxon in hospital samples Fig. , Fig. 6.43. The drop in abundance of these genes in the river mid-section (RM samples) in both years was intriguing and could be due to multiple urban, agricultural and industrial discharges25 that change microbial communities and dilute the microbial resistances. This location can also constitute a transition state from a non-contaminated to a more contaminated environment. More closely spaced samplings could be done to address this issue. The intermediate abundance of ARGs in downstream river samples (RL) is consistent with this location being a sink that combines water and communities from upstream river and urban sources. Clinically relevant ARGs were also detected in all sites studied and follow the overall trend seen for ARGs in general, with greater abundance in hospital and more contaminated water samples. On the other hand, many environmental ARGs could have been missed given our limited knowledge of environmental ARG diversity and ability to detect them, and the bias of public databases towards the most frequently encountered genes. These issues highlight the importance of thoroughly curating databases and doing functional evaluation of resistant phenotypes. In this study, the limited amount of DNA recovered made it impossible to carry out functional metagenomics to detect expressed resistance markers44. Future studies would require collecting larger amounts of water per site and/or improving extraction protocols in order to obtain sufficient high-quality DNA.The resistome varied both in abundance and diversity. ARG abundance followed a gradient that correlated with microbial community shifts and was greater in the downstream sites than in the upstream river samples, with a marked increase in hospital samples that also had greater presence of antibiotics. The higher ARG abundance and diversity in the river source sample RS16, when compared to the river’s middle section, confirmed the natural occurrence of these markers in the environment45, as hospital effluents released to the environment impact and alter microbial communities. The RL16 sample, which clustered close to upstream river samples when based on taxonomy and to hospital samples when based on resistances, can also reflect differences in terms of the relative contributions of microbes and their resistance genes in the incoming contaminating effluents and how these affect the resulting community. Given the low-depth Illumina sequencing and the limited number of contigs obtained, the co-occurrence of mobilization genes and ARGs in contigs mainly from hospital and RL samples suggests potential for horizontal gene transfer. Isolation of microorganisms or deeper sequencing would be needed to identify specific markers, mobile elements and genetic backgrounds that could help to accurately trace the genes and assess the risks posed by these ARGs46.The similarity of ARG profiles in contaminated sites  and their differentiation from upstream river locations are also indicative of composition being constrained by ecology46 in downstream river samples and by the similarity between these and hospital samples. The identification of known clinical pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and resistant enterobacteria also exposes the risk associated with use of this water supply for diverse activities such as animal farming and crop irrigation47 that can promote dissemination of undesired microorganisms and their genes into the environment and the food chain48. The close proximity between genes associated with mobile genetic elements and ARGs, especially those considered to be of high risk to human health such as sul1 and AAC(6′)-Ib33, raises the possibility that these genes may be mobilized among microorganisms in these new settings. These results emphasize the need for surveillance of microbiological risks in this river ecosystem, which can be done with metagenomics50, and implementation of effective strategies for water management aimed at avoiding undesired spread of microorganisms and resistance determinants due to the cumulative effect of small-scale contaminating discharges39.Despite the limited sampling scheme, this study shows that ARGs and pathogens are present in this freshwater ecosystem and are influenced by anthropogenic contamination, an important driver of micro-scale environmental changes. This influence is demonstrated by the presence of antibiotics, drugs and high-risk ARGs39.This study provides a snapshot and brief survey of microbial communities and their resistance genes at two points in time and at geographically close sites along the Bogotá River. Sampling at additional locations, periodic sampling at the same sites, as well as greater sample volumes and depth of sequencing, would provide a broader picture of the dynamics of the microbial communities and their resistomes and allow correlation with particular environmental variables. However, our metagenomic data shows that microbial communities and the resistomes of this river ecosystem are heavily impacted by anthropogenic activities and change as a consequence of contamination. These results also illustrate how human contamination can drive the spread of ARGs in this river ecosystem and generate conditions for microbial communities and antibiotic resistance genes of diverse origins to come together, mix and disseminate, posing risk to the local population. Further work is needed, however, to understand the risk of dissemination of these determinants from contaminated sites, such as hospitals, into environmental water supplies42. Samples were obtained from three different locations along the Bogotá River , and three hospitals  in the city of Bogotá, Colombia  and 2016 (between May and June), both during the rainy season, but with slightly higher rainfall in 201651.Physicochemical analyses , biological oxygen demand (BOD), total nitrogen (TN) and total suspended solids (TSS)) were performed on the 2015 samples at Biopolab Ltda. , following standard protocols52 (Supplementary Methods).The samples were stored at −18 °C until they were analyzed for the presence of the antibiotics metronidazole, sulfamethoxazole, trimethoprim, norfloxacin, ciprofloxacin, clindamycin, clarithromycin, erythromycin and azithromycin; and the drugs carbamazepine, venlafaxine, losartan, irbesartan, valsartan, naproxen and diclofenac. On the day of the analysis, samples were thawed, centrifuged and an aliquot was used for direct analysis by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry with triple quadrupole analyzer (UHPLC-MS/MS), using electrospray ionizationE. coli (blue colonies), total coliforms  and other Gram-negative bacteria (transparent colonies). Resistant bacteria were recovered by plating on chromogenic media selective for β-lactamase producing Enterobacteriaceae  and extended-spectrum β-lactamases , methicillin-resistant S. aureus , and vancomycin-resistant enterococci . Recovered colonies were subjected to previously reported PCR assays for species identification and resistance detection60.Water samples (400 mL) were serially diluted in sterile distilled water and filtered through 0.45 µm cellulose nitrate filters (Sartorius) and GN-6 Metricel MCE Membrane Disc Filters , which were placed on Chromocult® Coliform Agar (Merck Millipore) for 24 h at 37 °C for detection of http://www.earthmicrobiome.org/protocols-and-standards/16s/)61. PCR amplifications were done by triplicate in a 96-well plate in a 25 µL volume containing 10.2 µL H2O, 12.8 µL Takara Premix Taq DNA Polymerase , 1 µL 515F forward primer (10 µM), 0.5 µL 806R reverse primer (10 µM) with the unique identifier and 0.5 µL DNA (1 ng/µL). PCR reactions were done as follows: 98 °C for 30 s, 35 cycles of 98 °C for 10 s, 50 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 120 s. PCR products were purified using the Agencourt AMPure XP PCR Purification kit , quantified using the Picogreen kit , and loaded with a concentration of 8 pM with 25% PhiX spike-in to an Illumina MiSeq platform, where they were sequenced using 250 bp paired ends62.The V4 region of the 16S rRNA gene was amplified using primers 515F and 806R that include adaptors for Illumina flow cells (63 script split_libraries_fastq.py (-p 0.1 -n 500) and operational taxonomic units (OTUs) were generated using UPARSE64 (usearch8.1), which included merging of forward and reverse reads , quality filtration , de-replication (-derep_fulllength) and sorting , clustering (-cluster_otus), checking for chimeric sequences against the Gold database , mapping back of reads to OTUs at 97% identity ; and generation of the final OTU table using the script uc2otutab.py65. Taxonomy was assigned using the RDP Classifier28 against the Greengenes database66 .Sequence data was de-multiplexed by sample with the Qiime 167, and libraries were sequenced using an Illumina NextSeq . Reads were de-multiplexed by the sequencing center, analyzed for quality using FastQC (Babraham Bioinformatics), and quality filtered with Trimmomatic68 . Filtered reads were used for microbial community and antibiotic resistance gene analyses. Metaxa229 was used to assess microbial community composition from metagenomic reads69.DNA samples were processed using the Nextera DNA Library Prep Kit (Illumina) with modifications70. ShortBRED markers were generated based on the Comprehensive Antibiotic Resistance Database (CARD) 31 and the Lahey Clinic β-lactamase database provided by Erica Pehrsson , clustered at 100% identity70. As a reference database to remove constitutive bacterial genes, we used the UniRef90 protein dataset, as suggested30 . ShortBRED generated 2,937 markers that it used to quantify ARGs in each water sample, producing files containing each marker family and its abundance, normalized to reads per gene kilobase per million reads (RPKM). These marker families were classified according to target using the hand-curated metadata file of the CARD and Lahey Clinic β-lactamase databases, provided by Erica Pehrsson , downstream  and hospital (n3 = 6) samples with Kruskal-Wallis tests (α = 0.05). For beta diversity analyses, the community composition of each sample was compared using a weighted UniFrac distance matrix calculated with the Qiime script beta_diversity.py, and the same script was used with the ARG table to calculate a Bray-Curtis distance matrix. PCoA plots were generated from the distance matrices using the script principal_coordinates.py. Permutational analysis of variance (PERMANOVA) was carried out independently with both distance matrices to compare upstream, downstream and hospital samples using the script compare_categories.py and 999 permutations (α = 0.05), while using the year of sampling as a covariable. A Bray-Curtis distance matrix was also calculated for community composition and was used along the resistome matrix for a Procrustes analysis using the script transform_coordinate_matrices.py and 999 permutations (α = 0.05).Alpha and beta diversity analyses were carried out under the Python 2.7 environment for both community composition and ARG profiles. OTU tables were rarefied to 41,000 sequences per sample using Qiime 171, which performs non-parametrical statistical tests, to determine differentially enriched OTUs and ARGs in upstream and hospital samples. OTU tables for phyla and genera were used as inputs for biomarker taxa discovery in LEfSe. The ARG table was also used as input for LEfSe after removing undetected ARGs and classifying the remaining according to the type of antibiotic to which they confer resistance. For both OTU and ARG comparisons, alpha was set at 0.05 for the Kruskal-Wallis and the pairwise Wilcoxon rank-sum tests. No multiple-testing correction is necessary as significant results are reported only if all the pairwise Wilcoxon rank-sum tests are significant. The linear discriminant analysis effect size threshold was set at 3.0 and the program performed all-against-all comparisons.Given our small sample size and the lack of a normal distribution for the data, we chose LEfSe35 was used to determine the proportion of OTUs and ARGs of each sample from the river’s lower section that were associated predominantly with either hospitals or the river upstream of the city (river source and middle section).SourceTracker36 was used to assemble the metagenomic reads using hidden Markov models (HMMs) of integrase genes and high-risk ARGs. Fifty-eight integrase HMMs were downloaded from the Gypsy Database 2.0 (http://gydb.org/index.php/Collection_HMM)72 and 37 HMMs were generated using HMMER 3.173, after alignment of high-risk indicator proteins and their closest sequences obtained from the NCBI. These relevant proteins were obtained from the risk classification done by Martínez, Coque & Baquero46, from the risk indicator ARGs outlined by Berendonk et al.33 and from the contigs assembled by Forsberg et al.74. All HMMs were used as seeds for the GenSeed-HMM assembly and contigs were searched for ARGs and mobile genetic elements using blastx38 against the nr database .GenSeed-HMM37. Blastx  was used to search for ARGs and mobile genetic elements using a custom database containing all entries from the ARG database generated in this study and the ACLAME database of mobile genetic elements (downloaded 12 August 2017)75.The metagenomic reads were assembled into contigs with SPAdesSupplementary MaterialTable S7


Refractory gastroparesis and chronic constipation can lead to severe gastric and small bowel dilatation, which can be life threatening. Here, we present a case of a 21-year-old male with a gigantic stomach secondary to DMD resolved with conservative management and no surgical intervention.Duchenne muscular dystrophy (DMD) is characterized by degeneration and atrophy of skeletal, cardiac, and smooth muscles after a latent period of apparently normal development and function. The gastrointestinal manifestations start in the second decade of life and are mainly due to atrophy of smooth muscle layers. Duchenne muscular dystrophy (DMD) is characterized by degeneration and atrophy of skeletal, cardiac, and smooth muscles after a latent period of apparently normal development and function. The gastrointestinal manifestations start in the second decade of life and are mainly due to atrophy of smooth muscle layers . GastroiA 21-year-old male with a past medical history of DMD, scoliosis with multiple back surgeries, failure to thrive and atrial fibrillation presented with abdominal pain and vomiting for two days. He was chronically constipated since the age of 15 years and had one to two bowel movements per month with no use of laxatives. On admission, the patient was afebrile, tachycardic with a heart rate of 148 beats per minute, hypotensive with a blood pressure of 89/55 mmHg, and tachypneic with a respiratory rate of 36/min. He was cachectic with severe muscle wasting and had dry mucous membranes. His body mass index (BMI) was 14.8 kg/m2. Physical exam showed a soft, non-tender, distended abdomen, with no guarding and rigidity. The patient presented with hypoactive bowel sounds and chronic muscle contractions in all the four extremities. Laboratory values showed leukocytosis 20300/µL  with left shift (bands 22), hemoglobin 16.4 g/dL  and hematocrit 51.1% , blood urea nitrogen (BUN) 31 mg/dL , creatinine 0.40 mg/dL , mild elevation of aspartate aminotransferase (AST) 54 IU/L , prothrombin time (PT) 14s , international normalized ratio (INR) 1.3 , partial thromboplastin time (PTT) 32.3  and normal albumin 4.7 g/dL . He was severely intravascular volume-depleted as per physical exam and laboratory values. He received aggressive fluid resuscitation with Ringer’s lactate.Computed tomography (CT) scan of the abdomen and pelvis with contrast showed massive gastric distention with the stomach extending down to the low pelvis, and dilatation of the proximal duodenum to the level of the midline/superior mesenteric artery (SMA) Figures -3.Since aorto-mesenteric distance (AMD) was less than 1 cm, a working diagnosis of SMA syndrome was made. Upon placement of a nasogastric tube, three liters of gastric bilious contents were evacuated. He was started on lactulose and was given a fleet of enemas with manual disimpaction to help him have bowel movements. Esophagogastroduodenoscopy showed ulcers in the anterior wall and greater curvature of the stomach body, with a normal duodenal bulb and the second/third part of the duodenum. No strictures were visualized during the procedure. CT angiography of the abdomen showed interval resolution of gastric and duodenal distention, ruling out SMA syndrome. The patient’s gastric distention resolved with conservative measures without any need of surgical intervention Figure .BackgroundDMD is a progressive, X-linked recessive disorder with an incidence of approximately 30 per 100,000 live-born males. Patients can become symptomatic at 3-5 years of age and become fully wheelchair dependent by 12 years of age . DystropIn patients with DMD, the gastrointestinal tract, which primarily consists of smooth muscle, degenerates and atrophies while the connective tissue infiltrates . Animal Gastrointestinal manifestationsGastrointestinal symptoms that manifest as a result of DMD have garnered more interest in order to more optimally diagnose and treat. Many patients’ life spans do not reach adulthood, leading to a lack of experience in treating adults with DMD. Since smooth muscle is located throughout the gastrointestinal tract, its degeneration affects multiple organs. Dysphagia (36% in 1 series) due to oropharyngeal dysfunction is one of the most predominant symptoms in DMD patients . Barohn Treatment of gastrointestinal manifestationsThe gastric manifestations in patients with DMD have been poorly understood and thus, under-treated. This holds especially true in the adult population. Because DMD is a progressive disease, adult patients have more extensive muscular deterioration and therefore present with more severe symptoms. Proton pump inhibitors are effective, with few side effects in patients with GERD and esophagitis. Constipation may be treated conservatively with increased fluid and fiber intake or medically using stool softeners, laxatives, and fleet enemas . StudiesRuling out other differential diagnosesThe severe gastric and small bowel dilatation in our patient reached the level of the SMA and was found to have a narrowed AMD of less than 1 cm, leading to the inclusion of SMA syndrome. Normal AMD is 1 to 2.8 cm . HoweverThis was a unique case of acute gastric and small bowel dilatation, which is a rare manifestation of DMD most likely secondary to gastroparesis. Our patient’s symptoms were due to gastric and small bowel dilatation, which resolved with the conservative management with no need of surgery. Based on our experience, internists should be further educated on gastrointestinal manifestations in adult patients with DMD in order to improve outcomes, as it is a rare entity encountered in adults due to the short lifespan of these patients.


This study was conducted to investigate the correlation between blood components and carcass traits, and to find the biomarkers related to meat quality and quantity in Hanwoo steers.One hundred twenty-six Hanwoo steers  were used to find the correlation between blood compositions and carcass traits. The steers were fed concentrate and rice straw (30 steers) or total mixed rations (96 steers) during the whole experimental period. Blood samples were collected from the jugular vein at the growing (8 to 12 months), fattening (13 to 23 months) and finishing phases (24 to 30 months). Steers were slaughtered at 30 to 31 months of age (body weight of 701.6±60.45 kg) and the carcass traits were evaluated. Blood metabolites and hormones were analyzed and the correlation coefficients and regression equations with carcass traits were determined.longissimus muscle area of 13th rib, and the following regression equations were obtained: Marbling score (1–9) = −0.009×retinol (IU/100 mL)+ 9.125 (R2 = 0.643), Longissimus muscle area (cm2) = −0.250× AST(U/L)+112.498 (R2 = 0.450).Average concentrations of retinol, leptin and insulin were 1.10 IU, 30.34 ng, and 35.35 ng per mL of blood plasma, respectively. Retinol has negative correlations (p<0.01) with insulin and leptin. Blood insulin and total protein decreased with the age of steers, but retinol, aspartic acid transaminase (AST), glucose, cholesterol and triglyceride increased. In the finishing phase, significant (p<0.01) negative correlations occurred between blood retinol content and marbling score, and between blood AST content and It is possible to make highly marbled beef by controlling the blood retinol content during the fattening and finishing phases of Hanwoo steers. Accordingly, blood retinol and AST could be biomarkers for determining beef quality and quantity, respectively, prior to slaughter. The beef industry is developing to meet consumer demand for higher quality production. So far, beef quality is associated with increasing the amount of intramuscular fat, or marbling. Because beef quality judgement depends exceedingly on the marbling score, the Korean government is trying to reform the beef quality criteria. Intramuscular fat, however, is still the most important index for beef quality in Korean food culture, which roasts meat directly over a charcoal fire.Changes of blood components in ruminants have been used as a criterion of nutrient status  and as aOne hundred twenty-six Hanwoo steers aged 8 months  were fed formula feed with rice straw or total mixed rations until 30 months of age . The experimental period was divided into growing (8 to 12 months of age), fattening (13 to 23 months of age) and finishing phases (24 to 30 months of age).ad libitum during the growing phase, fattening phase and finishing phase, respectively, and fed rice straw freely.Thirty steers were fed formula feed twice daily (at 0900 and 1700) at a body weight (BW) of 1.8%, 1.5%, and Ninety-six steers were fed total mixed rations according to feeding programs. All the steers were allowed free access to water and mineral blocks. The steers were allotted to pens  with feeders divided by stanchion installed and were weighed monthly and feed intake was recorded weekly per pen.The animal handling procedures of the present study followed the guidelines of the institutional animal care and use committee.longissimus dorsi (rib eye) muscle was then taken and used for grading the yield and quality of the carcass according to the standard criteria guided by [Blood samples were collected at the end of the growing, fattening and finishing phases from the jugular vein at 3 h after morning feeding (1100 h). Blood plasma was obtained by centrifugation for 20 min at 3,000 rpm and stored at −70°C until analyzed. Carcass samples were harvested from steers (30 to 31 months of age) slaughtered at a commercial meat abattoir after fasting for 24 h. Carcasses were chilled for 24 h at 4°C, after which the left side was opened between the 13th rib and first lumbar and the uided by .A meat yield grade index (Y) was determined as 1, 2, 3 when the results of the following equation were below 63.3, 63.3 to 67.5, and above 67.5, respectively.Meat quality grade (1 for lowest to 5 for highest) was estimated by regarding meat color (1 for very light red to 9 for very dark red), fat color (1 for white to 9 for yellow), texture (1 for soft to 3 for rough), and maturity (1 for youthful to 9 for mature) based on the marbling score (1 for lowest to 9 for highest).2 injection using a rotary vacuum evaporator attached to a spider tube set, and the residue was dissolved in 50 μL of iso-propanol. For the quantification of plasma retinol, 20 μL of aliquot was injected into the HPLC operating with the following conditions  by duplication. A calibration curve was prepared using retinol standard .High-performance liquid chromatography  was used for the analysis of retinol in the blood of Hanwoo steers according to the modified methods of the Vitamin Society of Japan . Blood p125 radioactivity was monitored using a γ-counter .Blood biochemical components including aspartic acid transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), glucose, total protein, albumin, cholesterol and triglyceride were analyzed with the procedure and corresponding kit supplied by the manufacturer using an Automatic Biochemical Analyzer . Plasma insulin and leptin were assayed by enzyme-immunoassay using a multi-species insulin RIA kit and multi-species leptin RIA kit , respectively. IThe differences in blood component concentration according to each phase  were evaluated using analysis of variance . The significance of differences was tested at p<0.05 using Duncan’s multiple range test . From thThe concentrations of blood retinol, insulin, leptin and biochemical components according to growing, fattening and finishing phases in Hanwoo steers are shown in AST is commonly measured clinically to determine liver health; however, the reason for increased AST may come from other tissues such as muscle . In thisThe minimum, maximum and mean values of the carcass traits of steers fed concentrates with rice straw or total mixed rations until slaughter at 30 to 31 months of age are shown in The carcass characteristics of Hanwoo steers used in this study were higher in average marbling score and ribeye area, and similar in backfat thickness compared to those of KAPE .The correlations among blood biochemical components, including hormones, are shown in Ruminants are less sensitive to insulin than monogastric animals , since tFinding blood composition bound up with meat quality and quantity in carcass traits would surely be important to make a proper feeding program for beef cattle production, since blood composition might be manipulated by feeding control. The correlation coefficients between blood biochemical components and carcass traits in Hanwoo steers are shown in Blood retinol concentration had a significant (p<0.01) negative correlation with marbling score and meat quality grade. Gorocica-Buenfil et al  reportedBlood leptin, which is secreted from adipocytes, had a positive correlation (p<0.05) with back fat thickness, whereas it had a negative correlation (p<0.01) with meat quality grade.Geary et al  demonstrlongissimus dorsi muscle.However, Yonekura et al  reportedAST concentration had a negative correlation (p<0.01) with rib-eye muscle area, yield index, marbling score and meat quality grade.Carcass weight had a negative relationship with total protein concentration, whereas it was positive with total cholesterol and triglyceride concentrations of blood. And total cholesterol concentration had a negative correlation with rib-eye muscle area, yield index, marbling score and meat quality grade (p< 0.01). But Otani et al  reportedTriglyceride concentration in blood was negatively correlated with marbling score and meat quality grade, whereas it was positive with meat yield grade (p<0.01). The triglyceride circulating in blood is derived from the mobilization of the deposited fat content . In agreIn the components with a high correlation, we searched for blood biomarkers related to beef quality and quantity in Hanwoo production and obtained regression equations for estimating the quality and quantity of meat. The results of regression analysis based on the relationship between blood components and carcass traits are shown in 2) for meat quantity and a marbling score (1 to 9) for meat quality could be estimated by the equations Y = 112.498– and Y = 9.125–, respectively. Moreover, blood AST might be a biomarker of not only the quantity but also the quality of Hanwoo beef production. Blood retinol, however, is affected by many factors including infection, inflammation and recent dietary intake, and some biomarkers are more sensitive to changes in liver vitamin A reserves than others [Regarding the r square value of the equation, a rib-eye muscle area (cmn others .Finally, we can conclude that it might be possible to make highly marbled beef by controlling blood retinol during the fattening and finishing phases of Hanwoo steers. Accordingly, blood retinol and AST could be biomarkers for determining beef quality and quantity prior to slaughter, respectively.


Paramecium bursaria Chlorella virus-1 (PBCV-1) is one of the most intensively studied giant viruses. Here, we implemented a multi-scale approach to investigate the interactions among PBCV-1 capsid building units called capsomers. Three binding modes with different strengths are found between capsomers around the relatively flat area of the virion surface at the icosahedral 2-fold axis. Furthermore, a capsomer structure manipulation package is developed to simulate the capsid assembly process. Using these tools, binding forces among capsomers were investigated and binding funnels were observed that were consistent with the final assembled capsid. In addition, total binding free energies of each binding mode were calculated. The results helped to explain previous experimental observations. Results and tools generated in this work established an initial computational approach to answer current unresolved questions regarding giant virus assembly mechanisms. Results will pave the way for studying more complicated process in other biomolecular structures.In the last three decades, many giant DNA viruses have been discovered. Giant viruses present a unique and essential research frontier for studies of self-assembly and regulation of supramolecular assemblies. The question on how these giant DNA viruses assemble thousands of proteins so accurately to form their protein shells, the capsids, remains largely unanswered. Revealing the mechanisms of giant virus assembly will help to discover the mysteries of many self-assembly biology problems.  Paramecium bursaria Chlorella virus 1 (PBCV-1) [Chilo iridescent virus [Phaeocystis pouchetii virus [Mimivirus [Megavirus [Cafeteria roenbergensis virus [Faustovirus [Sambavirus [Since the early 1990s, many giant DNA viruses that infect eukaryotic cells have been discovered and studied [(PBCV-1) ,3, Chilont virus , Phaeocyii virus , Mimivirimivirus , Megaviregavirus , Cafeteris virus , Faustovmbavirus , as wellmbavirus . Cellulambavirus ,13 as wembavirus . The sizmbavirus . The “jembavirus , varyingSericesthis iridescent virus (SIV) [In 1969, Wrigley described large triangular and pentagonal arrays of capsomers resulting from broken us (SIV) . He defius (SIV) . Based ous (SIV) . Recentlus (SIV) . ThirteeComputational simulations are widely used in viral capsid assembly research. Some of the simulations helped to resolve capsid structures , others Electrostatic interaction has been recognized as an important factor for protein–protein recognition and assembly ,30,31,32Three binding modes were found between capsomers with different strengths. To mimic the assembly process, we manipulated capsomer pairs in four different operations. Binding force calculation showed that electrostatic interaction was critical to guide the assembly of the capsomers. Calculated by the MM/PBSA (molecular mechanics with Poisson–Boltzmann and surface area solvation) method, the total binding energies of these three binding modes were all negative, indicating their contribution to stabilize the capsid. Furthermore, formulas were derived to calculate total numbers of different binding modes within a single capsid. One of the binding modes provided the dominating stabilization interactions, which were consistent with experimental observations.In this study we focused on the dominant binding modes, which are located in intra- and inter-trisymmetrons regions. However, the interactions among capsomers within the pentasymmetrons are also important because they are essential for initializing the assembly of the viral capsid. Structural manipulation tools with four operations have been developed to explore the electrostatic binding forces between two capsomers in each binding mode, which pave the way for future investigation of capsomer interactions among pentasymmetrons of giant viruses. The MM/PBSA approach is based on 20 ns molecular dynamics (MD) simulations on three capsomer complexes and is the first step to investigate capsomer–capsomer interactions. The spiral pattern of capsomer orientations and the protruding shape of the 5-fold vertices of the pentasymmetron require more complicated calculations and more detailed examinations, which will be our future work.In summary, our results demonstrated that the electrostatic interactions played crucial roles in driving the assembly as well as stabilizing the final capsid after the assembly process of PBCV-1. It is noteworthy that our work establishes the initial computational studies for the interactions among certain capsomers on relatively flat areas of the capsid. Further computations are needed before expanding to other capsomers where curvature needs to be considered. Nevertheless, these results shed the first light on the mechanisms of giant virus assembly and are a solid stepping stone for addressing more complicated assembly interactions such as those between mCPs and MCPs . FurtherThe charge distribution on a single capsomer showed an interesting pattern. Calculations in Delphi demonstrated that the net charge of the capsomer was −3. The charge distribution was visualized in Chimera (Movie S1). Note that negatively charged residues were predominant in between the two jelly-rolls within each MCP monomer; positively charged residues were mostly located at the interface between the neighboring MCP monomers, and the rest of the positively charged residues were located on the outside edge of each jelly-roll e. NegatiWithin the three capsomers, an attractive electrostatic interaction was observed in binding mode 1 between capsomers A and B as well as in binding mode 2 between capsomers B and C. In binding mode 3, capsomers A and C were shown to have repulsive electrostatic interactions, as expected, since their interfaces were dominated with positively charged residues e. FurtheAt the interfaces of binding modes 1, 2, and 3, a total of four, two, and two salt bridges were found during MD simulations, respectively . For eacTo compare the strengths of the binding forces in all three binding modes, the calculated total forces versus the distance from 5 to 40 Å for each binding mode were plotted a. This cFor perpendicular shifting operations from −60 to 60 Å for all three binding modes, global maximum attractive forces for modes 1 and 2 and repulsive force for mode 3 were all located near the native position b. The diFor spinning operations of binding mode 1, one of the strongest forces exerted toward the fixed capsomer was observed in the native orientation. The range of attractive forces funnel was from −30° to 10° c1. For bThe binding forces of rotating capsomer B around A in binding mode 1 showed the binding funnel width was 25°, from −10° to 15°. The center of the binding funnel was located near the native position d1. For bFor each binding mode, binding funnels were investigated in various perspectives. These calculations of the effects of distance and orientation to the binding force showed that when the manipulated capsomer was placed at a relatively close distance, within 35 Å to the fixed one, the electrostatic forces not only attracted them together but also adjusted their orientations to achieve optimal binding for the assembly.Based on the capsomer orientations d,e, withBinding free energy was calculated to be −60.85 kcal/mol for binding mode 1, −98.43 kcal/mol for binding mode 2, and −23.81 kcal/mol for binding mode 3 . Overallh), and k steps along the other hexagonal axis (k). The T-number can then be calculated using the equation: T = h2 + h∙k + k2. For example, the T-number of PBCV-1 is 169   pseudohexameric capsomers [h number includes three steps in the pentasymmetron, three steps in the trisymmetron, and one more step to align with the pentameric capsomers , to describe the assembly geometry of icosahedral viruses . The T-n 8 + 82) c. It is apsomers c. The sik plus 3 .N of each mode of interactions within one virion.Capsomers inside a trisymmetron with the same orientation interacted with each other in binding mode 1, while capsomers at the boundary of trisymmetrons interacted with each other in binding mode 2 or 3. Using PBCV-1 trisymmetron as an example , we can n. For binding modes 2 and 3 at each edge of trisymmetron, the number of their interaction per edge is n = k + 3, formulas of total number of each mode for one intact virion can be derived as:Because each icosahedron giant virus has 20 trisymmetron, 30 trisymmetron edges, and as aforementioned, Based on these formulas, the total number of binding in modes 1, 2, and 3 in PBCV-1 were 12,600, 540, and 540, respectively. Clearly, binding mode 1 was the dominant interaction that stabilized the final assembled virion.The capsomer structure was downloaded from the Protein data Bank (PDB ID 5TIP ). The hiThe electrostatic potential maps (phimap) of capsomers were generated by Delphi ,38. Seee. In ordxy-plane  shixy-plane d from −3To investigate the roles of electrostatic interactions in guiding the assembly of the viral capsid, electrostatic binding forces in all three binding modes were calculated using DelphiForce ,41. As mTo predict the dynamic interactions between capsomers in all the three binding modes, MD simulations using an implicit generalized Born (GB) model were carried out using the CHARMM force field See . SimulatFor the energy calculations of each binding mode, the well-developed MM/PBSA method was utilized to calculate the average binding energy from 2000 frames, which were extracted from the last 10 ns trajectory of each MD simulation (See In this work, we investigated capsomer–capsomer interactions at the icosahedral 2-fold region of PBCV-1 capsid. We demonstrated that because of the geometrical symmetry of the capsomer and charge distribution, there were three binding modes: binding mode 1, the dominant binding mode in the whole capsid, which resided inside the trisymmetron where capsomers were packed in the same orientation, and binding modes 2 and 3 were formed at the boundary between two trisymmetrons where the capsomers had different orientations. The electrostatic interactions in all the binding modes were quite different. For binding mode 1, the electrostatic interaction was attractive. However, binding modes 2 and 3, which were at the boundary of two trisymmetrons, resulted in attractive and repulsive electrostatic interactions, respectively. The salt bridge analyses demonstrated that several key residues from the binding interfaces might have played significant roles for the capsomer interactions in PBCV-1 by forming stable inter-capsomer salt bridges. Simulations showed binding mode 1 formed the highest average number of stable salt bridges . For insIn order to verify the importance of these residues for capsomer intermolecular interactions, these residues can be mutated and further studied by simulations and experiments in the future. Combining our novel structure manipulation tool with DelPhiForce, the net forces between two capsomers in each binding mode are studied and it is shown that in binding modes 1 and 2, the electrostatic attractive binding forces are responsible for capsomer recognition. Binding funnels are found at the binding interfaces when manipulating capsomer pairs in four different ways. Such binding funnels guide and rotate two capsomers towards the final assembled positions. The results demonstrate that electrostatic forces play vital roles in viral capsid assembly by guiding the capsomers to interact with each other at the favored distance and orientation to maintain stable assembling.Overall, when considering total binding energies, all three binding modes were shown to have attractive binding interactions, among which binding mode 2 was the strongest . Even inIn this study we focused on dominant binding modes, which are located in intra- and inter-trisymmetrons regions. However, the interactions among capsomers within the pentasymmetrons are also important because they are essential for initializing the assembly of the viral capsid. Structural manipulation tools with four operations have been developed to explore the electrostatic binding forces between two capsomers in each binding mode , which p