Dynamics of early post-operative plasma ddcfDNA levels in kidney transplantation: a single-center pilot study.

Donor-derived cell-free DNA (ddcfDNA) is reported to be a promising noninvasive biomarker for acute rejection in organ transplant. However, studies on monitoring ddcfDNA dynamics during the early periods after organ transplantation are scarce. Our study assessed the dynamic variation in ddcfDNA in early period with various types and status of kidney transplantation. Target region capture sequencing used identifies ddcfDNA level in 21 kidney transplant recipients. Median ddcfDNA level was 20.69% at the initial time post-transplant, and decreased to 5.22% on the first day and stayed at the stable level after the second day. The ddcfDNA level in DCD (deceased donors) group (44.99%) was significantly higher than that in LDRT (living donor) group (10.24%) at initial time, P < 0.01. DdcfDNA level in DGF (delayed graft function) recipients was lower (23.96%) than that in non-DGF (47.74%) at the initial time, P = 0.89 (19.34% in DGF and 4.46% in non-DGF on the first day, P = 0.17). DdcfDNA level at initial time significantly correlated with serum creatinine (r2  = 0.219, P = 0.032) and warm ischemia time (r2  = 0.204, P = 0.040). Plasma ddcfDNA level decreased rapidly follow an L-shaped curve post-transplant, and level in DGF declined slower than non-DGF. The rebound of ddcfDNA level may indicate the occurrence of acute rejection.

Crustacean intersexuality is feminization without demasculinization: implications for environmental toxicology.

The dysfunction associated with intersexuality in vertebrates and molluscs is often a serious threat to ecosystems. Although poorly understood, crustacean intersexuality is associated with contamination and includes forms linked to increased sex-ratio distorting parasites at polluted sites. Despite the importance of crustaceans for monitoring vulnerable aquatic habitats, little is known about the molecular basis of this abnormal sexual differentiation and any associated sexual dysfunction. To increase the relevance of crustaceans to environmental toxicologists, we comprehensively analyzed gene expression in amphipods presenting parasite- and nonparasite-associated intersexuality. Our findings reveal existing vertebrate biomarkers of feminization should not be applied to crustaceans, as orthologous genes are not induced in feminized amphipods. Furthermore, in contrast to vertebrates, where feminization and intersexuality is often associated with deleterious demasculinization, we find males maintain masculinity even when unambiguously feminized. This reveals a considerable regulatory separation of the gene pathways responsible for male and female characteristics and demonstrates that evidence of feminization (even if detected with appropriate biomarkers) is not a proxy for demasculinization in crustaceans. This study has also produced a comprehensive spectrum of potential molecular biomarkers that, when combined with our new molecular understanding, will greatly facilitate the use of crustaceans to monitor aquatic habitats.

Analysis of cell-free fetal DNA in 16,843 pregnant women from a single center in China using targeted sequencing approach.

INTRODUCTION: Non-invasive prenatal testing (NIPT) using cell-free fetal DNA (cffDNA) has been widely accepted for detecting common fetal chromosome aneuploidies, but few large-scale studies have reported the kinetics of cffDNA concentration during gestation. This study examines cffDNA kinetics spanning gestational periods. METHODS: In this retrospective cohort study, cffDNA concentration from maternal plasma of 16,843 pregnant women between 4 and 39 weeks of pregnancy were determined by SNP-based targeted deep sequencing. RESULTS: Maternal plasma cffDNA could be detected as early as the fourth gestational week. After detection, cffDNA concentration begun to increase to the 39th week showing three conspicuous inflection points characterized by growth and stabilization phases. The rapid increase in cffDNA (∼1.19% per week) from the 4th to 9th week represents the first growth stage. The first plateau phase spanned from the 10th to 19th week (∼0.03% increase per week). cffDNA begun to rise dramatically (∼0.85% per week) from the 19th to 29th week, stabilizing at week 30 and onwards with only 0.27% increase per week representing the second plateau period. The proportion of cases with cffDNA ≥4% increased rapidly before the 10th gestational week with no significant increase from the 10th week onwards. About 92.00% of all the maternal plasma had a cffDNA greater than 4% from 10 weeks. DISCUSSION: We indicate that cffDNA had 3 inflection points at the 10th, 19th and 30th week of gestation, an observation not yet reported. Moreover, we show that cffDNA concentration has met the NIPT requirements after 9 weeks gestational age.

Unusual male size vs sperm count relationships in a coastal marine amphipod indicate reproductive impairment by unknown toxicants.

Sperm quantity/quality are significant reproductive endpoints with clear links to population level dynamics. Amphipods are important model organisms in environmental toxicology. Despite this, field monitoring of male fertility in invertebrates has rarely been used in monitoring programs. The aim of this study was to compare sperm quality/quantity in an amphipod collected at six UK locations with differing water quality. Due to low sperm counts and an observed lack of relationship between sperm count and weight in amphipods collected from a nationally protected conservation area (Langstone Harbour, England), we also compared datasets from this site over a decade to determine the temporal significance of this finding. One collection to evaluate a female reproductive endpoint was also performed at this site. Interestingly, this harbour consistently presented some of the lowest sperm counts comparable to highly industrial sites and low eggs number from females. Amphipods collected from all the sites, except from Langstone Harbour, presented strong positive correlations between sperm count and weight. Given Langstone Harbour has several international and national protected statutes primarily for marine life and birds, our results indicate that E. marinus, one important food component for wading birds, might be impacted by unknown reproductive stressors. These unknown stressors maybe related to agricultural runoff, leachate from historical landfills and effluent from storm water overflows. This study highlights the importance of exploring new reproductive endpoints such as sperm quantity/quality in marine monitoring programs.

A Noninvasive and Donor-independent Method Simultaneously Monitors Rejection and Infection in Patients With Organ Transplant.

BACKGROUND: Rejection and infection are 2 major complications affecting the health and survival of patients receiving an allograft organ transplantation. We describe a diagnostic assay that simultaneously monitors for rejection and infection in recipients of kidney transplant by sequencing of cell-free DNA (cfDNA) in plasma. METHODS: By using cfDNA in plasma, we established a noninvasive method that simultaneously monitors rejection and infection in patients with a history of organ transplant. A total of 6200 single-nucleotide polymorphisms were captured by liquid hybridization and sequenced by next-generation sequencing. The donor-derived cfDNA (ddcfDNA) level was calculated based on maximum likelihood estimation, without relying on the donor's genotype. We also analyzed the nonhuman cfDNA to test for infections in the patients' plasma. RESULTS: Artificial ddcfDNA levels quantified by a donor-dependent and donor-independent algorithm were significantly correlated, with the multivariate coefficient of determination, or R2 value, of 0.999. This technique was applied on 30 patients (32 samples) after kidney transplantation, and a significant difference was observed on the ddcfdNA levels between nonrejection and rejection. Furthermore, 1 BK virus infection and 1 cytomegalovirus infection were revealed by this method, and the enrichment efficiency of the viral sequences was 114 and 489 times, respectively, which are consistent with clinical results. CONCLUSION: This method can be used to simultaneously monitor for acute rejection as well as a broad spectrum of infections for patients of allograft organ transplant because it provides comprehensive information for clinicians to optimize immunosuppression therapy.

Infertility in a marine crustacean: have we been ignoring pollution impacts on male invertebrates?

Invertebrate infertility has been under-explored as a potential ecological issue or biomarker of stress within ecotoxicology. To date, the majority of studies focussing on contaminant induced infertility have centred on vertebrate groups. This study aimed to address the question whether industrial pollution has the ability to influence the sperm counts and testicular morphology of male amphipod, Echinogammarus marinus (Leach). In addition, the sperm counts of normal and intersex specimens were compared to assess the potential impact of a crustacean with a disrupted endocrine system. Specimens of E. marinus were collected at one industrially impacted (Inverkeithing) and two reference (Thurso and Loch Fleet) sites along the north and eastern coasts of Scotland. Significantly higher sperm counts ( approximately 20%) were observed from normal males collected from reference sites compared to the industrially impacted site. Higher proportions (30%) of intersex specimens were observed at the industrially impacted site compared to 17 and 6% male intersexuality observed at Thurso and Loch Fleet, respectively. Intersex male specimens from Thurso had lower mean sperm counts ( approximately 15%) than normal male specimens, however, this result was not significant (P=0.089). No significant differences in sperm counts were observed between normal and intersex males at Inverkeithing. Our results indicate that industrial pollution does have the potential to affect the sperm counts of male crustaceans. Whether the quality of sperm in Crustacea from contaminated sites is also compromised or whether this is an endocrine mediated effect is yet to be confirmed. To date, many of the studies of endocrine disruption in crustaceans have, surprisingly, focussed on female fecundity parameters, growth and moulting despite many of the vertebrate studies initially focussing on the male gender. Whether this could be an ecological issue needs to be addressed through further field and laboratory based studies. It is recommended that, strategically, considerable information regarding developing methodologies could be gained from scientists working in the crustacean aquaculture industries and crustacean taxonomists.

Vitellogenin is not an appropriate biomarker of feminisation in a crustacean.

The expression of the yolk protein vitellogenin (Vtg) has been used as a biomarker of feminisation in multiple fish species throughout the world. Since the late 1990s, researchers have attempted to develop similar biomarkers to address whether reproductive endocrine disruption also occurs in the males of invertebrate groups such as the Crustacea. To date, the vast majority of studies investigating Vtg induction in male Crustacea have resulted in negative or inconclusive results, leading researchers to question the utility of Vtg expression as a biomarker in this taxon. This study measured the expression of Vtg genes in two intersex phenotypes (termed internal and external) found in the male amphipod, Echinogammarus marinus, and compared them with those of normal males and females. Males presenting the external intersex phenotype are infected with known feminising parasites and display a variety of feminised traits including oviduct structures on their testes and external female brood plates (oostegites). The internal intersex male phenotype, that displays a pronounced oviduct structure on the testes without the external intersex characteristics, is not parasite infected and it is thought to be a result of environmental contamination. Given their morphology, these phenotypes might be considered highly 'feminised' or 'de-masculinised' and can be utilised to test the suitability of feminisation biomarkers. The E. marinus transcriptome was searched for genes resembling Vtg and two sequences were revealed, that we subsequently refer to as Vtg1 and Vtg2. Results from a high-throughput transcriptomic sequencing screen of gonadal cDNA libraries suggested that very low expression (in this manuscript gene transcription is taken to represent gene expression, although it is acknowledged that in addition to transcription, translation, transcript processing, mRNA stability and protein stability can regulate gene expression) of Vtg1 and Vtg2 in normal males (ESTs=1 and 0 for Vtg1 and Vtg2, respectively), internal intersex males (ESTs=0 for both Vtg sequences) and external intersex males (ESTs=5 and 0 for Vtg1 and Vtg2, respectively). In contrast, the sequencing suggested notable levels of expression of both Vtg genes in females (ESTs=1133 and 84 for Vtg1 and Vtg2, respectively). Subsequent qPCR analysis validates these expression levels, with the signal for Vtg1 and Vtg2 transcripts in all male phenotypes being indistinguishable from that caused by contamination of trace levels of genomic DNA or the low-level amplification non-target sequences. These findings suggest that Vtg expression is not notably induced in highly feminised amphipods and is therefore not an appropriate biomarker of feminisation/de-masculination in crustaceans. We discuss our findings in the context of previous attempts to measure Vtg in male crustaceans and suggest a requirement for more appropriate taxon-specific biomarkers to monitor feminisation in these groups.

Paramyxean-microsporidian co-infection in amphipods: is the consensus that Microsporidia can feminise their hosts presumptive?

The current consensus is that Microsporidia belong to a select group of parasites capable of causing both intersexuality and complete sex reversal in their hosts. Indeed, species such as Nosema granulosis and Dictyocoela duebenum, which infect amphipod crustaceans, are regularly referred to as 'feminising microsporidians'. This categorisation is based on a combination of findings: that these species are vertically transmitted and occur at a high prevalence of infection in intersex and female amphipods, that infected amphipod populations are female-biased, and that infected females have significantly female-biased broods with no concurrent increase in mortality. In this study, we report on a population of the amphipod Echinogammarus marinus presenting both female bias and high levels of intersexuality, which are infected with D. deubenum. In keeping with its feminising classification, infection is prevalent in animals presenting female and intersex phenotypes. However, a further screen revealed the presence of a previously unknown paramyxean parasite related to organisms of the genus Marteilia, a group known to cause catastrophic sexual dysfunction in bivalves. We found that the paramyxean parasite was also vertically transmitted, with infections being more prevalent in females and intersex animals. Critically, every animal infected with D. deubenum was also co-infected with the paramyxean, with few animals presenting an independent paramyxean infection. In contrast, co-infection of E. marinus with a paramyxean and the non-feminising microsporidian Dictyocoela berillonum rarely occurred. These observations raise a new hypothesis, namely, that D. duebenum and other feminising microsporidians are not actually capable of host feminisation but instead 'hitch-hike' together with a feminising paramyxean parasite.

Low dose TBT exposure decreases amphipod immunocompetence and reproductive fitness.

The antifouling agent tributyltin (TBT) is a highly toxic pollutant present in many aquatic ecosystems. Despite of regulations on the usage of TBT, it remains in high concentrations in sediments both in harbors and in off-shore sites. The toxicity of TBT in mollusks is well documented. However, adverse effects in other aquatic organisms, such as crustaceans, are less well known. This study is an effort to assess the effects of environmentally realistic concentrations of TBT on an ecologically important species in Swedish fresh and brackish water ecosystems, the benthic amphipod Monoporeia affinis. Field collected animals were exposed during gonad maturation to TBT (70 and 170 ng/g sediment d wt) for five weeks in static microcosms with natural sediment. Exposure concentrations were chosen to reflect effects at concentrations found in Swedish coastal sediment, but below expected effects on survival. TBT exposure resulted in a statistically significant adverse effect on oocyte viability and a doubling of the prevalence of microsporidian parasites in females, from 17% in the control to 34% in the 170 ng TBT/g sediment d wt exposure. No effects on survival were observed. Borderline significant effects were observed on male sexual maturation in the 70 ng TBT/g d wt exposure and on ecdysteroid levels in the 170 ng/g sediment d wt exposure. Both reproduction and parasite infection effects are of ecological importance since they have the potential to affect population viability in the field. This study gives further evidence to the connection between low dose contaminant exposure and increases in microsporidian parasite infection.

Perfluorooctane sulfonate accumulation and parasite infestation in a field population of the amphipod Monoporeia affinis after microcosm exposure.

Perfluorooctane sulfonate (PFOS) is the focus of intense toxicity research due to its persistence and widespread occurrence in biota. Studies on benthic invertebrates have shown them to be subjects of high PFOS exposure. However, effects on benthic invertebrates exposed to PFOS in the field are still far from elucidated. To fill a knowledge gap on concentrations and effects in benthic invertebrates, a microcosm study on the benthic amphipod Monoporeia affinis was performed. Field collected M. affinis were analysed for PFOS and showed average background concentrations 39 and 58 ng/g (wet weight) in two different samplings. The field collected animals were exposed to three concentrations of PFOS (50, 200 and 5000 microg PFOS/L water) for 3 weeks during gonad development. Body burdens of PFOS were determined after experiment termination. Results showed negative effects on survival and reproduction effects such as decreased sexual maturation and decreased oocyte viability caused by PFOS exposure. Additionally, a follow-up experiment demonstrated a significant increase in the infection incidence by a microsporidian muscle parasite in animals exposed to PFOS at tissue concentrations in the range of concentrations found in field collected benthic amphipods. This is the first study to demonstrate increased microsporidian infection with pollutant exposure and it suggests that ecologically relevant PFOS concentrations could be sufficient to elicit these effects.