Functional Genes and Transcripts Indicate the Existent and Active Microbial Mercury-Methylating Community in Mangrove Intertidal Sediments of an Urbanized Bay.

Mercury (Hg) methylation in mangrove sediments can result in the accumulation of neurotoxic methylmercury (MeHg). Identification of Hg methyltransferase gene hgcA provides the means to directly characterize the microbial Hg-methylating consortia in environments. Hitherto, the microbial Hg-methylating community in mangrove sediments was scarcely investigated. An effort to assess the diversity and abundance of hgcA genes and transcripts and link them to Hg and MeHg contents was made in the mangrove intertidal sediments along the urbanized Shenzhen Bay, China. The hgcA genes and transcripts associated with Thermodesulfobacteria [mainly Geobacteraceae, Syntrophorhabdaceae, Desulfobacterales, and Desulfarculales (these four lineages were previously classified into the Deltaproteobacteria taxon)], as well as Euryarchaeota (mainly Methanomicrobia and Theionarchaea) dominated the hgcA-harboring communities, while Chloroflexota, Nitrospirota, Planctomycetota, and Lentisphaerota-like hgcA sequences accounted for a small proportion. The hgcA genes appeared in greater abundance and diversity than their transcript counterparts in each sampling site. Correlation analysis demonstrated that the MeHg content rather than Hg content significantly correlated with the structure of the existent/active hgcA-harboring community and the abundance of hgcA genes/transcripts. These findings provide better insights into the microbial Hg methylation drivers in mangrove sediments, which could be helpful for understanding the MeHg biotransformation therein.

Interleukin-1 increases SERPINE1 expression in human granulosa-lutein cell via P50/P52 signaling pathways.

It has been reported that immune factors are associated with the occurrence of polycystic ovary syndrome (PCOS). Interleukin-1 (IL-1) is a member of the interleukin family that widely participates in the regulation of the inflammatory response in the immune system. In addition, it has been reported that aberrant IL-1 accumulation in serum is associated with the occurrence of PCOS. However, little is known about how IL-1 participates in the pathogenesis of PCOS. In the present study, we demonstrated that the immune microenvironment was altered in follicular fluid from PCOS patients and that the expression levels of two IL-1 cytokines, IL-1α and IL-1ß were increased. Transcriptome analysis revealed that IL-1α and IL-1ß treatment induced primary human granulosa-lutein (hGL) cell inflammatory response and increased the expression of serpin family E member 1 (SERPINE1). Mechanistically, we demonstrated that IL-1α and IL-1ß upregulated SERPINE1 expression through IL-1R1-mediated activation of downstream P50 and P52 signaling pathways in human granulosa cells. Our study highlighted the role of immune state changes in the occurrence of PCOS and provided new insight into the treatment of patients with IL-1-induced ovarian function disorders.

The impact and inflammatory characteristics of SARS-CoV-2 infection during ovarian stimulation on the outcomes of assisted reproductive treatment.

Introduction: Despite the global prevalence of coronavirus disease 2019 (COVID-19), limited research has been conducted on the effects of SARS-CoV-2 infection on human reproduction. The aims of this study were to investigate the impact of SARS-CoV-2 infection during controlled ovarian stimulation (COS) on the outcomes of assisted reproductive treatment (ART) and the cytokine status of patients. Methods: This retrospective cohort study included 202 couples who received ART treatment, 101 couples infected with SARS-CoV-2 during COS and 101 matched uninfected couples. The parameters of ovarian stimulation and pregnancy outcomes were compared between the two groups. The All-Human Inflammation Array Q3 kit was utilized to measure cytokine levels in both blood and follicular fluid. Results: No difference was found in the number of good-quality embryos (3.3 ± 3.1 vs. 3.0 ± 2.2, P = 0.553) between the infected and uninfected groups. Among couples who received fresh embryo transfers, no difference was observed in clinical pregnancy rate (53.3% vs. 51.5%, P = 0.907). The rates of fertilization, implantation, miscarriage, ectopic pregnancy and live birth were also comparable between the two groups. After adjustments were made for confounders, regression models indicated that the quality of embryos (B = 0.16, P = 0.605) and clinical pregnancy rate (P = 0.206) remained unaffected by SARS-CoV-2 infection. The serum levels of MCP-1, TIMP-1, I-309, TNF-RI and TNF-RII were increased, while that of eotaxin-2 was decreased in COVID-19 patients. No significant difference was found in the levels of cytokines in follicular fluid between the two groups. Conclusion: Asymptomatic or mild COVID-19 during COS had no adverse effects on ART outcomes. Although mild inflammation was present in the serum, it was not detected in the follicular fluid of these patients. The subsequent immune response needs further investigation.

ALKBH5 controls the meiosis-coupled mRNA clearance in oocytes by removing the N 6-methyladenosine methylation.

N6-methyladenosine (m6A) maintains maternal RNA stability in oocytes. One regulator of m6A, ALKBH5, reverses m6A deposition and is essential in RNA metabolism. However, the specific role of ALKBH5 in oocyte maturation remains elusive. Here, we show that Alkbh5 depletion causes a wide range of defects in oocyte meiosis and results in female infertility. Temporal profiling of the maternal transcriptomes revealed striking RNA accumulation in Alkbh5-/- oocytes during meiotic maturation. Analysis of m6A dynamics demonstrated that ALKBH5-mediated m6A demethylation ensures the timely degradation of maternal RNAs, which is severely disrupted following Alkbh5-/- depletion. A distinct subset of transcripts with persistent m6A peaks are recognized by the m6A reader IGF2BP2 and thus remain stabilized, resulting in impaired RNA clearance. Additionally, reducing IGF2BP2 in Alkbh5-depleted oocytes partially rescued these defects. Overall, this work identifies ALKBH5 as a key determinant of oocyte quality and unveil the facilitating role of ALKBH5-mediated m6A removal in maternal RNA decay.

Interleukin-1 increases cyclooxygenase-2 expression and prostaglandin E2 production in human granulosa-lutein cell via nuclear factor kappa B/P65 and extracellular signal-regulated kinase 1/2 signaling pathways.

A multitude of cytokines have been reported to participate in the folliculogenesis process in female. Interleukin-1 (IL-1), belonging to interleukin family, is originally identified as an important immune factor involved in inflammation response. Besides the immunity system, IL-1 is also expressed in reproductive system. However, the role of IL-1 in regulating ovarian follicle function remains to be elucidated. In the current study, using the primary human granulosa-lutein (hGL) and immortalized human granulosa-like tumor cell line (KGN) models, we demonstrated that both IL-1α and IL-1ß increased prostaglandin E2 (PGE2) production via upregulating its cyclooxygenase (COX) enzyme COX-2 expression in human granulosa cells. Mechanistically, IL-1α and IL-1ß treatment activated nuclear factor kappa B (NF-κB) signaling pathway. Using the specific siRNA to knock down endogenous gene expression, we found that the inhibition of p65 expression abolished IL-1α and IL-1ß-induced upregulation of COX-2 expression whereas knockdown of p50 and p52 had no effect. Moreover, our results also showed that IL-1α and IL-1ß promoted the nuclear translocation of p65. ChIP assay demonstrated the transcriptional regulation of p65 on COX-2 expression. Additionally, we also found that IL-1α and IL-1ß could activate the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The inhibition of ERK1/2 signaling pathway activation reversed IL-1α and IL-1ß-induced upregulation of COX-2 expression. Our findings shed light on the cellular and molecular mechanisms by which IL-1 modulates the COX-2 expression through NF-κB/P65 and ERK1/2 signaling pathways in human granulosa cells.

Phenotypes of peripheral CD4+ T helper cell subsets in pregnant women with HBeAg-negative chronic asymptomatic HBV carriers.

Background: Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide, and mother-to-child transmission is the key mode of HBV infection. CD4+ T helper (Th) cells play a critical role in the immune microenvironment of specific maternal tolerance to the foetus during pregnancy. However, the roles of Th cell subsets in pregnant women (PW) with chronic asymptomatic HBV carriers (ASCs) remain completely unclear. Here, we aimed to characterize CD4+ T-cell immunity in PW with hepatitis Be antigen (HBeAg)-negative chronic ASCs. Methods: Human peripheral blood mononuclear cells (PBMCs) from PW without HBV infection or with chronic ASCs and healthy controls (HC) were isolated, and CD4+ Th cell subsets were detected by flow cytometry in addition to serum cytokines. Serological HBV markers, liver function and hormone levels of these individuals were also tested. Results: The frequencies of circulating T follicular helper (Tfh) type 2 (Tfh2) cells were significantly evaluated, but Tfh1 cell frequencies were notably decreased in PW compared to HC. Moreover, the frequencies of Th22 cells were only notably increased in PW with chronic ASCs in comparison with PW. Additionally, increased levels of serum IL-4 were positively correlated with Tfh2 cell frequencies in healthy PW. Interestingly, serum P4 levels were positively associated with the frequencies of circulating Tfh2 or Th2 cells but were negatively related to the frequencies of circulating Tfh17 or Th17 cells in healthy PW. Although there were some changes in the other CD4+ Th cell frequencies and cytokine levels or other references, significant differences were not found among HC, healthy PW, PW with HBeAg-negative chronic ASCs. Conclusion: CD4+ Th cell subsets played a critical role in the immune microenvironment of PW, and these findings provided potential evidence for why PW with chronic ASCs did not receive antenatal antiviral prophylaxis.

Risk of Higher Blood Pressure in 3 to 6 Years Old Singleton Born From OHSS Patients Undergone With Fresh IVF/ICSI.

Background: A large registry-based study found the increasing disorders of cardiovascular and metabolism in IVF children but underlying mechanism is still unknown. Few studies have investigated any association between OHSS and cardiovascular or metabolic function in subsequent children. Objective: To evaluate the effect of ovarian hyperstimulation syndrome (OHSS) on blood pressure of singletons after in vitro fertilization (IVF) with or without intracytoplasmic sperm injection (ICSI). Study Design: The singlet-center corhort study included 1780 singletons born with IVF/ICSI and 83 spontaneously conceived children from 2003 to 2014. Follow-up has lasted more than 10 years, and is still ongoing. This study analyzed data from follow-up surveys at 3 to 6 years of age. Participants Setting and Methods: We recruited 83 children (Group E) spontaneously conceived (SC) as control group and 1780 children born with IVF/ICSI including 126 children born to OHSS-fresh embryo transfer (ET) women (Group A), 1069 children born to non OHSS-ET women (Group B), 98 children conceived by women who developed into moderate or severe OHSS after oocyte retrieval and selected the frozen-thawed embryo transfer (FET) (Group C), 487 children conceived with non OHSS-FET (Group D). We evaluated cardiometabolic function, assessed BP in mmHg, heart rate, anthropometrics, and metabolic index including glucose, serum lipid (triglyceride, total cholesterol, low density lipoprotein, high density lipoprotein), thyroid function, of those children. The BP and heart rate were measured twice on the same day. We applied several multiple regression analyses to investigate the effect of OHSS in the early pregnancy. Main Findings: By the single factor analysis, the SBP and DBP in the SC group (SBP: 99.84 ± 8.9; DBP: 55.27 ± 8.8) were significantly lower than OHSS-ET group's, while the blood pressure was similar between the SC group and other three ART groups. Children had higher BP in the OHSS-ET group (SBP: 101.93 ± 8.17; DBP: 58.75 ± 8.48) than in the non OHSS-ET (SBP: 99.49 ± 8.91; DBP: 56.55 ± 8.02) or OHSS-FET group (SBP: 99.38 ± 8.17; DBP: 55.72 ± 7.94). After using multiple regression analysis to adjust current, early life, parental and ART characteristics, the differences in the SBP and DBP (B (95% confidence interval)) between OHSS-ET and non OHSS-ET remained significant (SBP: 3.193 (0.549 to 2.301); DBP: 3.440 (0.611 to 2.333)). And the BP showed no significant difference complementarily when compared non OHSS-FET group with non OHSS-ET group. In addition, the anthropometrics, fast glucose, serum lipid, and thyroid index did not differ among the ART groups. Principal Conclusions: OHSS might play an independent key role on offspring's BP even cardiovascular function. Electing frozen-thawed embryo transfer for high risk of OHSS population may reduce the risk of the high BP trend. Wider Implications of the Findings: It is a large sample study to investigate the effect of OHSS on offspring's health. These findings provide a clinic evidence of the impact of early environment (embryo even oocyte stage) on the offspring's cardiovascular health. Our study emphasis the importance of the accuracy of IVF clinic strategy and preventing the OHSS after fresh embryo transfer.

Preimplantation Genetic Testing Prevented Intergenerational Transmission of X-Linked Alport Syndrome.

BACKGROUND: Alport syndrome (AS) is a hereditary renal basement membrane disease that can lead to end-stage renal disease in young adults. It can be diagnosed by genetic analysis, being mostly caused by mutations in COL4A3, COL-4A4, and COL4A5. To date, there is no radical cure for this disease. OBJECTIVES: The aim of this study was to avoid the transmission of AS within an affected family by selecting healthy embryos for uterine transfer. The embryos were identified by preimplantation genetic testing for monogenic disorders (PGT-M). METHODS: We used next-generation sequencing (NGS) to identify mutations in the proband and his parents. The results of NGS were confirmed by Sanger sequencing. Targeted NGS combined with targeted single-nucleotide polymorphism haplotyping was used for the in vitro identification of COL4A5 mutations in human embryos to prevent their intergenerational transmission. RESULTS: The c.349_359delGGACCTCAAGG and c.360_361insTGC mutations in COL4A5 were identified in a family affected by X-linked AS. Whole-genome sequencing by NGS with targeted haplotyping was performed on biopsied trophectoderm cells. A healthy baby was born after transfer of a single freeze-thawed blastocyst. CONCLUSIONS: The use of targeted NGS for identifying diagnostic markers combined with targeted haplotyping is an easy and efficient PGT-M method for preventing intergenerational transmission of AS.

The BMP2 Signaling Axis Promotes Invasive Differentiation of Human Trophoblasts.

Embryo implantation and trophoblast invasion are principal limiting factors of pregnancy establishment. Aberrant embryo development or improper trophoblast differentiation and invasion may lead to various unfavorable pregnancy-related outcomes, including early pregnancy loss (EPL). Our clinical data show that the serum BMP2 levels were significantly increased during the first trimester of pregnancy and that the serum and BMP2 expression levels were lower in women with EPL than in women with normal early pregnancies. Moreover, we observed that BMP2 was expressed in oocytes and trophoblast cells of cleaved embryos and blastocysts prior to implantation in both humans and mice. Exogenous BMP2 promoted embryonic development by enhancing blastocyst formation and hatching in mice. LncRNA NR026833.1 was upregulated by BMP2 and promoted SNAIL expression by competitively binding to miR-502-5p. SNAIL induced MMP2 expression and promoted cell invasion in primary extravillous trophoblast cells. BMP2 promotes the invasive differentiation of mouse trophoblast stem cells by downregulating the expression of TS cell marker and upregulating the expression of trophoblast giant cell marker and labyrinthine/spongiotrophoblast marker. Our findings provide significant insights into the regulatory roles of BMP2 in the development of the placenta, which may give us a framework to explore new therapeutic strategies to pregnancy-related complications.

Influence of cleavage-stage embryo quality on the in-vitro fertilization outcome after single embryo transfer in fresh cycles.

OBJECTIVE: Embryo quality is crucial for determining the outcome of embryo implantation. This study aimed to assess the impact of embryo quality on the outcome of in vitro fertilization/single-embryo transfer (IVF-SET). MATERIALS AND METHODS: This retrospective study included 2531 fresh IVF-SET cycles, including 277 poor-quality and 2254 top-quality embryos. The clinical pregnancy rate, miscarriage rate, live birth, implantation rate, pregnancy outcome and complication were analyzed and compared. Risk factors associated with miscarriage rate and pregnancy complication were identified using logistics regression analysis. RESULTS: Top-quality embryos resulted in higher clinical pregnancy rate (30.5% vs. 12.6%, P < 0.001) and live birth rate (23.9% vs. 9.7%, P < 0.001) compared with poor-quality embryos. Logistics regression analysis revealed that embryo quality was not correlated with miscarriage rate (95% CI 0.33-1.89) and pregnancy complications (95% CI 0.12-7.84). Maternal age and body mass index was a risk factor for miscarriage rate (95% CI 1.05-1.22) and pregnancy complication (95% CI 1.01-1.29), respectively. CONCLUSION: Clinical miscarriage rate and pregnancy complication were embryo quality independent. Maternal age was the risk factor for miscarriage rate. Embryo quality did not affect miscarriage once a clinical pregnancy is achieved.

Changes in preoperative and postoperative lipoprotein-associated phospholipase A2 activity in patients with malignant obstructive jaundice.

BACKGROUND: Malignant obstructive jaundice (MOJ) leads to hyperbilirubinemia and systemic pathophysiological changes. The main clinical treatments for MOJ include radical pancreatoduodenectomy, palliative surgical treatment, and minimally invasive treatment, which can relieve biliary obstruction, drain bile, reduce jaundice, and improve liver function. In rat models, hepatic exposure to endotoxin resulted in rapid increases in biliary and plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) levels, and our previous study revealed that Lp-PLA2 activity was strongly associated with liver damage. The present study aimed to clarify the serum Lp-PLA2 activity changes and evaluate the associations between Lp-PLA2 activity and laboratory parameters in MOJ patients preoperatively and postoperatively. METHODS: Twenty-one patients with MOJ were enrolled in this prospective study. Lp-PLA2 activity and other laboratory parameters were analyzed using a Hitachi 7600 automatic biochemical analyzer. Spearman correlation coefficients, percent differences, and dynamic difference plots were used to evaluate the changes in preoperative and postoperative Lp-PLA2 activity and the associations of Lp-PLA2 activity with other laboratory parameters. RESULTS: The postoperative Lp-PLA2 activity at 1 day [646 (range, 175-1,025) U/L], 1 week [419 (range, 144-949) U/L], and day of hospital discharge [347 (range, 165-698) U/L] differed significantly from the preoperative baseline activity (636 (range, 172-1,664) U/L; P<0.05 for all). Lp-PLA2 activity was correlated with total bilirubin (TB) at specific time points (P<0.05 for all). The percent differences and dynamic difference graphs revealed that Lp-PLA2 activity, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and TB gradually decreased after biliary obstruction was relieved by surgical treatment. CONCLUSIONS: Lp-PLA2 activity in MOJ patients was associated with biliary obstruction and liver damage. Serum Lp-PLA2 can be used as a novel indicator for biliary obstruction severity and treatment monitoring.

Association between ratio of γ-glutamyl transpeptidase to high-density lipoprotein cholesterol and prevalence of nonalcoholic fatty liver disease and metabolic syndrome: a cross-sectional study.

BACKGROUND: Metabolic risk factors including obesity, insulin resistance, dyslipidemia, metabolic syndrome (MS), and diabetes are associated with nonalcoholic fatty liver disease (NAFLD). γ-Glutamyl transpeptidase (GGT) and high-density lipoprotein cholesterol (HDL-C) are associated with insulin resistance, dyslipidemia, oxidative stress, and obesity. We investigated the associations between GGT/HDL-C ratio and prevalence of NAFLD in a Chinese population. METHODS: The study included 1,813 NAFLD (526 females, 1,287 males) and 4,513 non-NAFLD (3,077 females, 1,436 males) participants. The diagnosis of NAFLD was based on ultrasonography. RESULTS: Participants with NAFLD had higher GGT/HDL-C ratio, BMI, WC, TG, TC, and HOMA-IR, but lower HDL-C than participants without NAFLD. GGT/HDL-C ratio was significantly associated with prevalence of NAFLD. Specifically, for each 1 unit increase in GGT/HDL-C ratio, the prevalence of NAFLD will increase by 0.3%. As GGT/HDL-C ratio quartiles increased, prevalence of NAFLD/MS in Q4 (highest GGT/HDL-C ratio quartile) was 6.362/3.968 times higher than that in Q1 (lowest GGT/HDL-C ratio quartile). The AUC [0.799 (0.788-0.810)] for GGT/HDL-C ratio was significantly higher than those for GGT and HDL-C alone. CONCLUSIONS: The present results suggest that GGT/HDL-C ratio can be used as a predictive factor for prevalence of NAFLD after adjustment for confounding variables.

Emergent complication of assisted reproductive technology: Clinical analysis of 17 pregnant women with adnexal torsion.

BACKGROUND: The purpose of this study was to evaluate the clinical symptoms, surgical management, and outcomes of pregnant women with adnexal torsion due to assisted reproductive technology. METHODS: It was a retrospective study that include 17 pregnant women with adnexal torsion, in which the maternal age, type of fertilization, gestational age, clinical symptoms, ultrasonic findings, side affected by the disease, surgical method, and pregnancy outcomes were evaluated. RESULTS: A total of 17 patients with adnexal torsion were included in this study, of which 8 patients conceived by in vitro fertilization-embryo transfer (IVF-ET), 1 by artificial insemination (AIH), and the other 8 conceived naturally after ovulation induction. About 14 were reported to have occurred in the first trimester of pregnancy, 1 case in the second trimester, and the other 2 in the third trimester. Clinical symptoms were abdominal pain with or without nausea and vomiting. 14 cases occurred in the right adnexa and the other 3 in the left. 5 of the patients underwent laparoscopy, and the other 12 underwent laparotomy. 8 cases were of full- term delivery, 6 twins gave birth prematurely, and 3 patients had inevitable abortion. CONCLUSIONS: Adnexal torsion is an acute onset of lower abdominal pain in women, which seldom occurs during pregnancy. However, because of the wide application of assisted reproductive technology (ART), its incidence has increased. Early diagnosis and treatment can lead to better results.

Analysis of functional gene transcripts suggests active CO2 assimilation and CO oxidation by diverse bacteria in marine sponges.

Bacteria are the dominant symbionts in sponges and are regarded as important contributors to ocean nutrient cycling; however, their roles in carbon utilization in sponge holobionts are seldom identified. Here, the in situ active bacteria and their CO2 assimilation and CO oxidation functions in sponges Theonella swinhoei, Plakortis simplex and Phakellia fusca were evaluated using the analysis of functional gene transcripts. Phylogenetically diverse bacteria belonging to 16 phyla were detected by 16S rRNA analysis. Particularly, some of the active bacteria appeared to be sponge-specific or even sponge species-specific. Transcribed autotrophic CO2 assimilation genes rbcL and rbcM, anaplerotic CO2 assimilation gene accC and aerobic CO oxidation gene coxL were uncovered and assigned to a wide variety of bacterial lineages. Some of these carbon metabolism genes showed specificity to sponge species or different transcriptional activity among the sponge species. This study uncovered the phylogenetic diversity of transcriptionally active bacteria especially with CO2 assimilation or CO oxidation functions, providing insights into the ecological functions of the sponge-symbiotic bacteria regarding carbon metabolism.

Reference Intervals of Apolipoprotein E in Healthy Chinese Han Adults.

BACKGROUND: Reference intervals (RIs) of Apo E levels are an important parameter for the clinical evaluation of patient health, and the RIs of serum Apo E could be variable in different population. We plan to establish RIs of apolipoprotein E (Apo E) according to the CLSI EP28-A3 guideline in healthy Chinese Han adults. METHODS: Serum Apo E values of 1,206 healthy adults (from 19 to 87 years old) were measured by immunoturbidimetry. The relationship between Apo E and age was analyzed by using Spearman's correlation. The differences between the gender and age groups were compared using Mann-Whitney U test/Kruskal-Wallis H test. We calculated recommended nonparametric Q2.5 and Q97.5 percentile intervals and the 90% confidence intervals (CI) of lower and upper limits to define the age- and gender- related RIs. RESULTS: The level of Apo E was higher in females than males. Apo E was significantly associated with aging in adult females (r = 0.108, p < 0.05), but not in males (p = 0.518). The RIs of Apo E for females were 0.0268 - 0.0619, 0.0247 - 0.0603, and 0.0269 - 0.0658 g/L for 18 - 29, 30 - 59, and ≥ 60 years old, respectively, that for males was 0.0242 - 0.0579 g/L. CONCLUSIONS: Our results established the age- and gender-specific RIs of serum Apo E in healthy Chinese Han adults in our laboratory.

Effects of Freeze-Thaw Times on Screening Coagulation Tests and Factors VIII and IX Activities in Citrate-Anticoagulated Plasma at -20°C and -80°C.

BACKGROUND: Accurate determination of screening coagulation tests and factors VIII and IX activities (FVIII:C and FIX:C) in fresh plasma is very important for diagnosing abnormalities in the intrinsic or extrinsic coagulation pathways and factor deficiencies. If thawed samples cannot be detected for all required items at the same time, or need to be re-tested or re-stored, the thawed samples need to be re-frozen. We planned to perform in-house validation studies on freeze-thawed samples for screening coagulation tests, FVIII:C and FIX:C. METHODS: Mean percent changes, numbers of samples with > 10% changes, and difference plots were evaluated to determine clinically relevant differences between results for fresh and freeze-thawed samples. The statistical significance of differences between repeated-measure multiple groups and baseline values were evaluated by repeated-measures analysis of variance. RESULTS: The acceptable freeze-thaw cycles for activated partial thromboplastin time, fibrinogen, prothrombin time/international normalized ratio, thrombin time, and FIX:C were three times at -20°C/-80°C, while the acceptable freeze-thaw cycles for FVIII:C were three times at -80°C and once at -20°C. CONCLUSIONS: The freeze-thaw results on stabilities were affected by time and temperature, with lower temperature and fewer times associated with more stable activity.

Functional Transcripts Indicate Phylogenetically Diverse Active Ammonia-Scavenging Microbiota in Sympatric Sponges.

Symbiotic ammonia scavengers contribute to effective removal of ammonia in sponges. However, the phylogenetic diversity and in situ activity of ammonia-scavenging microbiota between different sponge species are poorly addressed. Here, transcribed ammonia monooxygenase genes (amoA), hydrazine synthase genes (hzsA), and glutamine synthetase genes (glnA) were analyzed to reveal the active ammonia-scavenging microbiota in the sympatric sponges Theonella swinhoei, Plakortis simplex, and Phakellia fusca, and seawater. Archaeal amoA and bacterial glnA transcripts rather than bacterial amoA, hzsA, and archaeal glnA transcripts were detected in the investigated sponges and seawater. The transcribed amoA genes were ascribed to two Thaumarchaeota ecotypes, while the transcribed glnA genes were interspersed among the lineages of Cyanobacteria, Tectomicrobia, Poribacteria, Alpha-, Beta-, Gamma-, and Epsilonproteobacteria. In addition, transcribed abundances of archaeal amoA and bacterial glnA genes in these sponges have been quantified, showing significant variation among the investigated sponges and seawater. The transcriptome-based qualitative and quantitative analyses clarified the different phylogenetic diversity and transcription expression of functional genes related to microbially mediated ammonia scavenging in different sympatric sponges, contributing to the understanding of in situ active ecological functions of sponge microbial symbionts in holobiont nitrogen cycling.

Effects of pre-analytical storage time, temperature, and freeze-thaw times on coagulation factors activities in citrate-anticoagulated plasma.

BACKGROUND: Coagulation factor assays are very important for diagnosing, treating, and monitoring inherited and acquired factor deficiencies. Appropriate pre-analytical storage conditions of citrate-anticoagulated plasma are essential for detection of coagulation factor activity. We aimed to investigate the effects of storage temperature and time on coagulation factor (F) II, FV, FVII, FX, FXI, and FXII activity up to 24 h and the effects of freeze-thaw times at -80 °C on factor activity. METHODS: Twenty-two blood samples were analyzed after storage for 0 (baseline), 2, 4, 6, 8, 12, and 24 h at 25 and 4 °C. Mean percent changes, numbers of samples with >10% changes, percent change trend plots, and difference plots were evaluated to determine clinically relevant differences. RESULTS: The acceptable storage times for FII coagulation activity (FII:C), FV:C, FVII:C, FX:C, FXI:C, and FXII:C were 24, 8, 8, 24, 12, and 12 h at 4 °C and 24, 4, 8, 8, 12, and 12 h at 25 °C, respectively. The acceptable freeze-thaw times for FII:C, FV:C, FVII:C, FX:C, FXI:C, and FXII:C were 2, 2, 3, 3, 2, and 1, respectively. CONCLUSIONS: When factor activity cannot be determined within these acceptable timeframes, we recommend that plasma samples should be frozen and thawed at appropriate times for analysis.