Noninvasive graft monitoring using donor-derived cell-free DNA in Japanese liver transplantation.

AIM: To evaluate the use of donor-derived cell-free DNA (dd-cfDNA) in diagnosing graft injuries in Japanese liver transplantation (LTx), including family-related living donors. METHODS: A total of 321 samples from 10 newly operated LTx recipients were collected to monitor the early dynamics of dd-cfDNA levels after LTx. Fifty-five samples from 55 recipients were collected during protocol biopsies (PB), whereas 36 samples from 27 recipients were collected during event biopsies, consisting of 11 biopsy-proven acute rejection (AR), 20 acute dysfunctions without rejection (ADWR), and 5 chronic rejections. The levels of dd-cfDNA were quantified using a next-generation sequencer based on single nucleotide polymorphisms. RESULTS: The dd-cfDNA levels were elevated significantly after LTx, followed by a rapid decline to the baseline in patients without graft injury within 30 days post-LTx. The dd-cfDNA levels were significantly higher in the 11 samples obtained during AR than those obtained during PB (p < 0.0001), which decreased promptly after treatment. The receiver operator characteristic curve analysis of diagnostic ability yielded areas under the curve of 0.975 and 0.897 for AR (rejection activity index [RAI] ≥3) versus PB and versus non-AR (ADWR + PB). The dd-cfDNA levels during AR were elevated earlier and correlated more strongly with the RAI (r = 0.740) than aspartate aminotransferase/alanine aminotransferase. The dd-cfDNA levels were neither associated with graft fibrosis based on histology nor the status of donor-specific antibodies in PB samples. CONCLUSIONS: Donor-derived cell-free DNA serves as a sensitive biomarker for detecting graft injuries in LTx. Further large-scale cohort studies are warranted to optimize its use in differentiating various post-LTx etiologies.

Comprehensive discovery of CRISPR-targeted terminally redundant sequences in the human gut metagenome: Viruses, plasmids, and more.

Viruses are the most numerous biological entity, existing in all environments and infecting all cellular organisms. Compared with cellular life, the evolution and origin of viruses are poorly understood; viruses are enormously diverse, and most lack sequence similarity to cellular genes. To uncover viral sequences without relying on either reference viral sequences from databases or marker genes that characterize specific viral taxa, we developed an analysis pipeline for virus inference based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR is a prokaryotic nucleic acid restriction system that stores the memory of previous exposure. Our protocol can infer CRISPR-targeted sequences, including viruses, plasmids, and previously uncharacterized elements, and predict their hosts using unassembled short-read metagenomic sequencing data. By analyzing human gut metagenomic data, we extracted 11,391 terminally redundant CRISPR-targeted sequences, which are likely complete circular genomes. The sequences included 2,154 tailed-phage genomes, together with 257 complete crAssphage genomes, 11 genomes larger than 200 kilobases, 766 genomes of Microviridae species, 56 genomes of Inoviridae species, and 95 previously uncharacterized circular small genomes that have no reliably predicted protein-coding gene. We predicted the host(s) of approximately 70% of the discovered genomes at the taxonomic level of phylum by linking protospacers to taxonomically assigned CRISPR direct repeats. These results demonstrate that our protocol is efficient for de novo inference of CRISPR-targeted sequences and their host prediction.

Influenza A Viruses of Swine (IAV-S) in Vietnam from 2010 to 2015: Multiple Introductions of A(H1N1)pdm09 Viruses into the Pig Population and Diversifying Genetic Constellations of Enzootic IAV-S.

Active surveillance of influenza A viruses of swine (IAV-S) involving 262 farms and 10 slaughterhouses in seven provinces in northern and southern Vietnam from 2010 to 2015 yielded 388 isolates from 32 farms; these viruses were classified into H1N1, H1N2, and H3N2 subtypes. Whole-genome sequencing followed by phylogenetic analysis revealed that the isolates represented 15 genotypes, according to the genetic constellation of the eight segments. All of the H1N1 viruses were entirely A(H1N1)pdm09 viruses, whereas all of the H1N2 and H3N2 viruses were reassortants among 5 distinct ancestral viruses: H1 and H3 triple-reassortant (TR) IAV-S that originated from North American pre-2009 human seasonal H1, human seasonal H3N2, and A(H1N1)pdm09 viruses. Notably, 93% of the reassortant IAV-S retained M genes that were derived from A(H1N1)pdm09, suggesting some advantage in terms of their host adaptation. Bayesian Markov chain Monte Carlo analysis revealed that multiple introductions of A(H1N1)pdm09 and TR IAV-S into the Vietnamese pig population have driven the genetic diversity of currently circulating Vietnamese IAV-S. In addition, our results indicate that a reassortant IAV-S with human-like H3 and N2 genes and an A(H1N1)pdm09 origin M gene likely caused a human case in Ho Chi Minh City in 2010. Our current findings indicate that human-to-pig transmission as well as cocirculation of different IAV-S have contributed to diversifying the gene constellations of IAV-S in Vietnam. IMPORTANCE: This comprehensive genetic characterization of 388 influenza A viruses of swine (IAV-S) isolated through active surveillance of Vietnamese pig farms from 2010 through 2015 provides molecular epidemiological insight into the genetic diversification of IAV-S in Vietnam after the emergence of A(H1N1)pdm09 viruses. Multiple reassortments among A(H1N1)pdm09 viruses and enzootic IAV-S yielded 14 genotypes, 9 of which carried novel gene combinations. The reassortants that carried M genes derived from A(H1N1)pdm09 viruses became predominant, replacing those of the IAV-S that had been endemic in Vietnam since 2011. Notably, one of the novel reassortants likely caused a human case in Vietnam. Given that Vietnam is the second-largest pig-producing country in Asia, continued monitoring of IAV-S is highly important from the viewpoints of both the swine industry and human public health.

The complete mitochondrial genome sequence of the indigenous I pig (Sus scrofa) in Vietnam.

OBJECTIVE: The I pig is a long nurtured longstanding breed in Vietnam, and contains excellent indigenous genetic resources. However, after 1970s, I pig breeds have become a small population because of decreasing farming areas and increasing pressure from foreign breeds with a high growth rate. Thus, there is now the risk of the disappearance of the I pigs breed. The aim of this study was to focus on classifying and identifying the I pig genetic origin and supplying molecular makers for conservation activities. METHODS: This study sequenced the complete mitochondrial genome and used the sequencing result to analyze the phylogenetic relationship of I pig with Asian and European domestic pigs and wild boars. The full sequence was annotated and predicted the secondary tRNA. RESULTS: The total length of I pig mitochondrial genome (accession number KX094894) was 16,731 base pairs, comprised two rRNA (12S and 16S), 22 tRNA and 13 mRNA genes. The annotation structures were not different from other pig breeds. Some component indexes as AT content, GC, and AT skew were counted, in which AT content (60.09%) was smaller than other pigs. We built the phylogenetic trees from full sequence and D loop sequence using Bayesian method. The result showed that I pig, Banna mini, wild boar (WB) Vietnam and WB Hainan or WB Korea, WB Japan were a cluster. They were a group within the Asian clade distinct from Chinese pigs and other Asian breeds in both phylogenetic trees (0.0004 and 0.0057, respectively). CONCLUSION: These results were similar to previous phylogenic study in Vietnamese pig and showed the genetic distinctness of I pig with other Asian domestic pigs.

Effect of herd size on subclinical infection of swine in Vietnam with influenza A viruses.

BACKGROUND: Influenza A viruses of swine (IAV-S) cause acute and subclinical respiratory disease. To increase our understanding of the etiology of the subclinical form and thus help prevent the persistence of IAV-S in pig populations, we conducted active virologic surveillance in Vietnam, the second-largest pig-producing country in Asia, from February 2010 to December 2013. RESULTS: From a total of 7034 nasal swabs collected from clinically healthy pigs at 250 farms and 10 slaughterhouses, we isolated 172 IAV-S from swine at the weaning and early-fattening stages. The isolation rate of IAV-S was significantly higher among pigs aged 3 weeks to 4.5 months than in older and younger animals. IAV-S were isolated from 16 large, corporate farms and 6 family-operated farms from among the 250 farms evaluated. Multivariate logistic regression analysis revealed that "having more than 1,000 pigs" was the most influential risk factor for IAV-S positivity. Farms affected by reassortant IAV-S had significantly larger pig populations than did those where A(H1N1)pdm09 viruses were isolated, thus suggesting that large, corporate farms serve as sites of reassortment events. CONCLUSIONS: We demonstrate the asymptomatic circulation of IAV-S in the Vietnamese pig population. Raising a large number of pigs on a farm has the strongest impact on the incidence of subclinical IAV-S infection. Given that only some of the corporate farms surveyed were IAV-S positive, further active monitoring is necessary to identify additional risk factors important in subclinical infection of pigs with IAV-S in Vietnam.

Methods Used to Control the Reproductive Choices of Women Who Are Sex Trafficked: Considerations for Health Care Providers.

Reproductive control or reproductive coercion has negative health consequences but has not been systematically studied within the context of sex trafficking. Our goal is to identify the range of methods used by sex traffickers and buyers to control the reproductive choices of trafficked women and to provide specific examples of these methods. We searched PubMed, Embase, and PsycInfo using the terms "reproductive control" or "reproductive coercion" and "human trafficking" or "sex trafficking," including papers that contained original, specific examples of reproductive control occurring within the context of sex trafficking. These reports were described and categorized into established domains of reproductive control. Eight articles were located that met our inclusion criteria, of which 6 described outcomes of birth control sabotage, 2 described pressuring into pregnancy, 5 described controlling the outcome of a pregnancy, and 2 described forced birth control or sterilization. Our findings have implications for how to take sexual histories and for identifying and assisting trafficked persons.

Amniotic band syndrome leading to severe malformations of the newborn: a case report at Tu Du Hospital, Vietnam, and literature review.

Amniotic band syndrome (ABS) refers to fibrous bands that appear to entangle various fetal parts in utero, leading to deformation, malformation, or disruption. To alleviate implementation of this diverse malformation, an early diagnosis on ultrasound is necessary to explain to the patient, thus, avoiding psychological shock and requiring timely intervention. Case Presentation: In the present case report, the authors describe a case of ABS that was diagnosed at the time of delivery at full term. Although the male newborn was alive, the infant underwent the distal deformity of amputated limbs and clubfoot. He has currently been followed up for the reconstruction treatment. Clinical Discussion: ABS remains a challenging diagnosis for obstetricians following the onset timepoint. A prenatal ultrasound scan is carefully required to detect the morphologic abnormalities of the fetus. Postnatal management should be integrated by a multidisciplinary team in order to improve the infant's outcome. Conclusion: ABS is an extremely dangerous entity during pregnancy, which leads to poor outcomes for the infant. An early detection on ultrasound helps in preparing better for the acceptance of the mother and the family as well as the prognosis afterwards.

Extraction of CRISPR-targeted sequences from the metagenome.

Homology-based search is commonly used to uncover mobile genetic elements (MGEs) from metagenomes, but it heavily relies on reference genomes in the database. Here we introduce a protocol to extract CRISPR-targeted sequences from the assembled human gut metagenomic sequences without using a reference database. We describe the assembling of metagenome contigs, the extraction of CRISPR direct repeats and spacers, the discovery of protospacers, and the extraction of protospacer-enriched regions using the graph-based approach. This protocol could extract numerous characterized/uncharacterized MGEs. For complete details on the use and execution of this protocol, please refer to Sugimoto et al. (2021).

Oreocharisphuongii (Gesneriaceae), a new species from central Vietnam.

Oreocharisphuongii, a new species of Gesneriaceae from central Vietnam, is described and illustrated here. The new species is most similar to O.longifolia by sharing peduncles up to 22 cm long, bracts 2, zygomorphic, yellow flowers with tubular corolla, stamens 4 with two pairs of coherent anthers and capsules up to 6 cm long. It mainly differs from the latter by the combination of some morphological characters of leaves (shape, base, apex and margin), size of calyx lobes, indumentum of corolla tube and inner surface of three lower corolla lobes. Detailed morphological description together with colour illustration, information on phenology, distribution, ecology, preliminarily conservation status of the new species and comparison with its similar species are also presented.

Determination of Pharmaceutical Residues by UPLC-MS/MS Method: Validation and Application on Surface Water and Hospital Wastewater.

In this study, an analytical method for the simultaneous determination of 7 major pharmaceutical residues in Vietnam, namely, carbamazepine, ciprofloxacin, ofloxacin, ketoprofen, paracetamol, sulfamethoxazole, and trimethoprim, in surface water and hospital wastewater has been developed. The method includes enrichment and clean-up steps by solid phase extraction using mix-mode cation exchange, followed by identification and quantification using an ultrahigh-performance liquid chromatography and tandem mass spectrometry and employing electrospray ionization (UPLC-ESI-MS/MS). Seven target compounds were separated on the reversed phase column and detected in multiple reaction monitoring (MRM) mode within 6 minutes. The present study also optimized the operating parameters of the mass spectrometer to achieve the highest analytical signals for all target compounds. All characteristic parameters of the analytical method were investigated, including linearity range, limit of detection, limit of quantification, precision, and accuracy. The important parameter in UPLC-ESI-MS/MS, matrix effect, was assessed and implemented via preextraction and postextraction spiking experiments. The overall recoveries of all target compounds were in the ranges from 55% to 109% and 56 % to 115% for surface water and hospital wastewater, respectively. Detection limits for surface water and hospital wastewater were 0.005-0.015 µg L-1 and 0.014-0.123 µg L-1, respectively. The sensitivity of the developed method was allowed for determination of target compounds at trace level in environmental water samples. The in-house validation of the developed method was performed by spiking experiment in both the surface water and hospital wastewater matrix. The method was then applied to analyze several surface water and hospital wastewater samples taken from West Lake and some hospitals in Vietnam, where the level of these pharmaceutical product residues was still missed. Sulfamethoxazole was present at a high detection frequency in both surface water (33% of analyzed samples) and hospital wastewater (81% of analyzed samples) samples.

Analysis of Polycyclic Aromatic Hydrocarbon in Airborne Particulate Matter Samples by Gas Chromatography in Combination with Tandem Mass Spectrometry (GC-MS/MS).

Polycyclic aromatic hydrocarbons (PAHs), the family of organic contaminations, have been shown to have negative effects on human health. However, until now, the comprehension on occurrence, distribution, and risk assessment of human exposure to PAHs has been limited in Vietnam. In this work, a capillary gas chromatography coupled with electron impact ionization tandem mass spectrometry (GC-EI-MS/MS) has been introduced for analysis of 16 PAHs in some particulate matter samples. PAHs have been separated on the TG 5 ms capillary gas chromatographic column and detected by tandem mass spectrometry in multiple reaction monitoring mode. The PAHs in the particulate matter (PM 2.5 and PM 10) samples were extracted by ultrasonic-assisted liquid extraction and cleaned up by an acidic silica gel solid phase extraction. The linearity range of all analyzed PAHs was from 5 to 2000 ng mL-1 with R 2 ≥0.9990. Limit of detection (LOD) of PAHs in particulate matter sample was from 0.001 ng m-3 (Br-Naph) to 0.276 ng m-3 (Fln). The recovery of PAHs was investigated by international proficiency testing samples. The recoveries of PAHs in proficiency testing sample ranged from 79.3% (Chr) to 109.8% (IcdP). The in-house validated GC-EI-MS/MS method was then applied to analysis of some particulate matter samples that were collected in the Hanoi areas. The total concentrations of PAHs in several brands of samples collected from Hanoi were found in the range of 226.3 ng m-3-706.43 ng m-3. Among the studied compounds, naphthalene was found at high frequency and ranged from 106.5 ng m-3 to 631.1 ng m-3. The main distribution of the PAHs in particulate matter samples was two-ring and three-ring compounds.

Isolation of highly pathogenic H5N6 avian influenza virus in Southern Vietnam with genetic similarity to those infecting humans in China.

Since 2013, H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for outbreaks in poultry and wild birds around Asia. H5N6 HPAIV is also a public concern due to sporadic human infections being reported in China. In the current study, we isolated an H5N6 HPAIV strain (A/Muscovy duck/Long An/AI470/2018; AI470) from an outbreak at a Muscovy duck farm in Long An Province in Southern Vietnam in July 2018 and genetically characterized it. Basic Local Alignment Search Tool (BLAST) analysis revealed that the eight genomic segments of AI470 were most closely related (99.6%-99.9%) to A/common gull/Saratov/1676/2018 (H5N6), which was isolated in October 2018 in Russia. Furthermore, AI470 also shared 99.4%-99.9% homology with A/Guangxi/32797/2018, an H5N6 HPAIV strain that infected humans in China in 2018. Phylogenetic analyses of the entire genome showed that AI470 was directly derived from H5N6 HPAIVs that were in South China from 2015 to 2018 and clustered with four H5N6 HPAIV strains of human origin in South China from 2017 to 2018. This indicated that AI470 was introduced into Vietnam from China. In addition, molecular characteristics related to mammalian adaptation among the recent human H5N6 HPAIV viruses, except PB2 E627K, were shared by AI470. These findings are cause for concern since H5N6 HPAIV strains that possess a risk of human infection have crossed the Chinese border.

Impacts of Dams and Global Warming on Fish Biodiversity in the Indo-Burma Hotspot.

Both hydropower dams and global warming pose threats to freshwater fish diversity. While the extent of global warming may be reduced by a shift towards energy generation by large dams in order to reduce fossil-fuel use, such dams profoundly modify riverine habitats. Furthermore, the threats posed by dams and global warming will interact: for example, dams constrain range adjustments by fishes that might compensate for warming temperatures. Evaluation of their combined or synergistic effects is thus essential for adequate assessment of the consequences of planned water-resource developments. We made projections of the responses of 363 fish species within the Indo-Burma global biodiversity hotspot to the separate and joint impacts of dams and global warming. The hotspot encompasses the Lower Mekong Basin, which is the world's largest freshwater capture fishery. Projections for 81 dam-building scenarios revealed progressive impacts upon projected species richness, habitable area, and the proportion of threatened species as generating capacity increased. Projections from 126 global-warming scenarios included a rise in species richness, a reduction in habitable area, and an increase in the proportion of threatened species; however, there was substantial variation in the extent of these changes among warming projections. Projections from scenarios that combined the effects of dams and global warming were derived either by simply adding the two threats, or by combining them in a synergistic manner that took account of the likelihood that habitat shifts under global warming would be constrained by river fragmentation. Impacts on fish diversity under the synergistic projections were 10-20% higher than those attributable to additive scenarios, and were exacerbated as generating capacity increased-particularly if CO2 emissions remained high. The impacts of dams, especially those on river mainstreams, are likely to be greater, more predictable and more immediately pressing for fishes than the consequences of global warming. Limits upon dam construction should therefore be a priority action for conserving fish biodiversity in the Indo-Burma hotspot. This would minimize synergistic impacts attributable to dams plus global warming, and help ensure the continued provision of ecosystem services represented by the Lower Mekong fishery.

A differential proteomic approach to assess the effects of chemotherapeutics and production management strategy on giant tiger shrimp Penaeus monodon.

The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg(-1)) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were under-expressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.

Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon hypophthalmus, exposed to trichlorfon.

Trichlorfon (TRC) is the most common organophosphorous insecticide used in aquaculture practices in Southeast Asian countries. Indiscriminate use of TRC can either damage or alter the enzymatic and hormonal activities in the living organisms. In this present study, therefore, toxicogenomic analyses using real time PCR was used to characterize expression levels of various genes in Pangasianodon hypophthalmus after exposure to three concentrations, the 96 h 1/100LC(50) (0.01 mg/L), the 96 h 110LC(50) (0.1 mg/L) and the 96 h 12LC(50) (0.5 mg/L) of TRC for 6 h, 24 h, 96 h, 7 days, 14 days, 28 days and 56 days respectively. The expression kinetics of stress and other cellular toxicity representative genes such as heat shock protein70 (HSP70), growth hormone, acetylcholinesterase (AChE), trypsinogen, cytochrome P4501B (CYP1B) and cytochrome oxidase subunit 1 (COI) were investigated in liver and gills. TRC at a level of 0.1 mg/L and 0.5 mg/L induced a time and dose-dependent increase in the expression of the HSP70, COI and CYPIB while the transcript level of AChE, growth hormone and trypsinogen were significantly down-regulated. These results could permit to develop a "molecular biomarker system" which can be applied as a first-tier method of identifying contaminant exposure before effects at population level occur.