The genomic basis of sporadic and recurrent pregnancy loss: a comprehensive in-depth analysis of 24,900 miscarriages.

RESEARCH QUESTION: What is the genetic cause of sporadic and recurrent pregnancy loss and does the frequency and nature of chromosomal abnormalities play a role? Types and frequency of all identifiable chromosomal abnormalities were determined to inform our understanding, medical management and recurrence risk for patients experiencing pregnancy loss. DESIGN: Genome-wide single-nucleotide polymorphism-based chromosomal microarray (SNP-CMA) were used to evaluate 24,900 products of conception samples from various forms of pregnancy losses. RESULTS: Sporadic miscarriage (64.7%) or recurrent pregnancy loss (RPL) (22%) were the most common referrals. Clinically significant abnormalities were observed in 55.8% (13,910) of samples, variants of uncertain significance in 1.8%, and normal results in 42.4%. In addition to autosomal trisomies (in 36% of samples), polyploidy and large segmental imbalances were identified in 7.8% and 2.8% of samples, respectively. Analysis of sequential samples from 1103 patients who had experienced RPL provided important insight into possible predispositions to RPL. CONCLUSIONS: This expansive chromosomal microarray analyses of pregnancy loss samples illuminates our understanding of the full spectrum, relative frequencies and the role of genomic abnormalities in pregnancy loss. The empiric observations described here provide useful insight for clinicians and highlight the importance of high-resolution genomic testing for comprehensive evaluation and risk assessment of individuals experiencing pregnancy loss.

Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia.

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

Bayesian modeling predicts age and sex are not required for accurate stature estimation from femoral length.

Despite the recognized flaws in applying traditional stature estimation equations such as those of Trotter and Gleser (1952) [5] to a contemporary population, there are currently no available alternatives for stature estimation in Australia that address these limitations. Post mortem computed tomography (PMCT) DICOM scans of the left and right femora were acquired from 76 Australian deceased individuals aged 17-76 years for metric analysis. Femoral bicondylar length, femoral epicondylar breadth and anterior-posterior (AP) diameter, medial-lateral (ML) diameter, circumference and cortical area at the femoral midshaft were measured on three-dimensional (3D) models to build statistical models for estimating stature. In addition, Australian individuals aged 16-63 years (n=111) were measured in standing and supine positions to aid in the adjustment of supine stature of deceased individuals utilized in this study to standing stature. The results of this preliminary evaluation strongly indicate that the optimal model for estimating stature includes bicondylar femoral length and epicondylar breadth, that the effect of sex as an independent variable is very low, and there is limited practical benefit in including age in the estimation of stature. Our study indicates that the Australian population sampled represents a small yet significant shift in stature from the original Trotter and Gleser sample. Additionally, in the case of fragmentary remains, it was found that epicondylar breadth and AP diameter had the highest probability of accurate stature estimation in the absence of bicondylar femoral length. As stature forms a significant component of a biological profile and therefore aids in the personal identification of human remains, it is important that forensic anthropologists utilize the most accurate methodologies available. Stature estimation of Australian individuals is therefore achieved with higher accuracy through utilizing the femoral equations proposed in this study.

Chimerism for 20q11.2 microdeletion of GDF5 explains discordant phenotypes in monochorionic-diamniotic twins.

Microdeletions of 20q11.2 are rare but have been associated with characteristic clinical findings. A 1.6 Mb minimal critical region has been identified that includes three OMIM genes: GDF5, EPB41L1, and SAMHD. Here we describe a male monozygotic, monochorionic-diamniotic twin pair with discordant phenotypes, one with multiple findings that overlap with those reported in 20q11.2 deletions, and the other unaffected. Microarray analysis revealed mosaicism for a 363 Kb deletion encompassing GDF5 in the peripheral blood of both twins, which was confirmed by FISH. Subsequent FISH on buccal cells identified the deletion only in the affected twin. The blood FISH findings were interpreted as representing chimerism resulting from anastomosis and the blood exchange between the twins in utero. The implications of this finding are discussed, as is the contribution of GDF5 to the associated clinical findings of 20q11.2 deletions.

Standardized anthropological measurement of postcranial bones using three-dimensional models in CAD software.

This study introduces a standardized protocol for conducting linear measurements of postcranial skeletal elements using three-dimensional (3D) models constructed from post-mortem computed tomography (PMCT) scans. Using femoral DICOM datasets, reference planes were generated and plane-to-plane measurements were conducted on 3D surface rendered models. Bicondylar length, epicondylar breadth, anterior-posterior (AP) diameter, medial-lateral (ML) diameter and cortical area at the midshaft were measured by four observers to test the measurement error variance and observer agreement of the protocol (n=6). Intra-observer error resulted in a mean relative technical error of measurement (%TEM) of 0.11 and an intraclass correlation coefficient (ICC) of 0.999 (CI=0.998-1.000); inter-observer error resulted in a mean %TEM of 0.54 and ICC of 0.996 (CI=0.979-1.000) for bicondylar length. Epicondylar breadth, AP diameter, ML diameter and cortical area also yielded minimal error. Precision testing demonstrated that the approach is highly repeatable and is recommended for implementation in anthropological investigation and research. This study exploits the benefits of virtual anthropology, introducing an innovative, standardized alternative to dry bone osteometric measurements.

Inducible targeting of cDCs and their subsets in vivo.

Conventional dendritic cells (cDCs) are essential immune cells linking the innate and adaptive immune system. cDC depletion in mice is an important method to study the function of these cells in vivo. Here we report an inducible in vivo system for cDC depletion in which excision of a loxP flanked Stop signal enables expression of the human diphtheria toxin receptor (DTR) under the control of Zbtb46 (zDC(lSlDTR)). cDCs can be specifically depleted by combining zDC(lSlDTR) mice with a Csf1r(Cre) driver line. In addition, we show that zDC(Cre) mice can be used to produce cDC specific conditional knockout mice (Irf8, Irf4, Notch2) which lack specific subsets of cDCs.

Absence of MHC class II on cDCs results in microbial-dependent intestinal inflammation.

Conventional dendritic cells (cDCs) play an essential role in host immunity by initiating adaptive T cell responses and by serving as innate immune sensors. Although both innate and adaptive functions of cDCs are well documented, their relative importance in maintaining immune homeostasis is poorly understood. To examine the significance of cDC-initiated adaptive immunity in maintaining homeostasis, independent of their innate activities, we generated a cDC-specific Cre mouse and crossed it to a floxed MHC class II (MHCII) mouse. Absence of MHCII on cDCs resulted in chronic intestinal inflammation that was alleviated by antibiotic treatment and entirely averted under germ-free conditions. Uncoupling innate and adaptive functions of cDCs revealed that innate immune functions of cDCs are insufficient to maintain homeostasis and antigen presentation by cDCs is essential for a mutualistic relationship between the host and intestinal bacteria.

Aire controls gene expression in the thymic epithelium with ordered stochasticity.

The transcription factor Aire controls immunological tolerance by inducing the ectopic thymic expression of many tissue-specific genes, acting broadly by removing stops on the transcriptional machinery. To better understand Aire's specificity, we performed single-cell RNA-seq and DNA-methylation analysis of Aire-sufficient and Aire-deficient medullary epithelial cells (mTECs). Each of Aire's target genes was induced in only a minority of mTECs, independently of DNA-methylation patterns, as small inter-chromosomal gene clusters activated in concert in a proportion of mTECs. These microclusters differed between individual mice. Thus, our results suggest an organization of the DNA or of the epigenome that results from stochastic determinism but is 'bookmarked' and stable through mTEC divisions, which ensures more effective presentation of self antigens and favors diversity of self-tolerance between individuals.

Classical Flt3L-dependent dendritic cells control immunity to protein vaccine.

DCs are critical for initiating immunity. The current paradigm in vaccine biology is that DCs migrating from peripheral tissue and classical lymphoid-resident DCs (cDCs) cooperate in the draining LNs to initiate priming and proliferation of T cells. Here, we observe subcutaneous immunity is Fms-like tyrosine kinase 3 ligand (Flt3L) dependent. Flt3L is rapidly secreted after immunization; Flt3 deletion reduces T cell responses by 50%. Flt3L enhances global T cell and humoral immunity as well as both the numbers and antigen capture capacity of migratory DCs (migDCs) and LN-resident cDCs. Surprisingly, however, we find immunity is controlled by cDCs and actively tempered in vivo by migDCs. Deletion of Langerin(+) DC or blockade of DC migration improves immunity. Consistent with an immune-regulatory role, transcriptomic analyses reveals different skin migDC subsets in both mouse and human cluster together, and share immune-suppressing gene expression and regulatory pathways. These data reveal that protective immunity to protein vaccines is controlled by Flt3L-dependent, LN-resident cDCs.

Morphometric modelling of ageing in the human pubic symphysis: sexual dimorphism in an Australian population.

Despite the prominent use of the pubic symphysis for age estimation in forensic anthropology, little has been documented regarding the quantitative morphological and micro-architectural changes of this surface. Specifically, utilising post-mortem computed tomography data from a large, contemporary Australian adult population, this study aimed to evaluate sexual dimorphism in the morphology and bone composition of the symphyseal surface; and temporal characterisation of the pubic symphysis in individuals of advancing age. The sample consisted of multi-slice computed tomography (MSCT) scans of the pubic symphysis (slice thickness: 0.5mm, overlap: 0.1mm) of 200 individuals of Caucasian ancestry aged 15-70 years, obtained in 2011. Surface rendering reconstruction of the symphyseal surface was conducted in OsiriX(®) (v.4.1) and quantitative analyses in Rapidform XOS™ and Osteomeasure™. Morphometric variables including inter-pubic distance, surface area, circumference, maximum height and width of the symphyseal surface and micro-architectural assessment of cortical and trabecular bone compositions were quantified using novel automated engineering software capabilities. The major results of this study are correlated with the macroscopic ossification and degeneration pattern of the symphyseal surface, demonstrating significant age-related changes in the morphometric and bone tissue variables between 15 and 70 years. Regardless of sex, the overall dimensions of the symphyseal surface increased with age, coupled with a decrease in bone mass in the trabecular and cortical bone compartments. Significant differences between the ventral, dorsal and medial cortical surfaces were observed, which may be correlated to bone formation activity dependent on muscle activity and ligamentous attachments. Our study demonstrates significant sexual dimorphism at this site, with males exhibiting greater surface dimensions than females. These baseline results provide a detailed insight into the changes in the structure of the pubic symphysis with ageing and sexually dimorphic features associated with the cortical and trabecular bone profiles.

Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium.

Dendritic cells (DCs), monocytes, and macrophages are closely related phagocytes that share many phenotypic features and, in some cases, a common developmental origin. Although the requirement for DCs in initiating adaptive immune responses is well appreciated, the role of monocytes and macrophages remains largely undefined, in part because of the lack of genetic tools enabling their specific depletion. Here, we describe a two-gene approach that requires overlapping expression of LysM and Csf1r to define and deplete monocytes and macrophages. The role of monocytes and macrophages in immunity to pathogens was tested by their selective depletion during infection with Citrobacter rodentium. Although neither cell type was required to initiate immunity, monocytes and macrophages contributed to the adaptive immune response by secreting IL-12, which induced Th1 polarization and IFN-γ secretion. Thus, whereas DCs are indispensable for priming naive CD4(+) T cells, monocytes and macrophages participate in intestinal immunity by producing mediators that direct T cell polarization.

Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens.

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4(+) and NKp46(+) innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b(+) cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103(+) cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b(+) cDCs in the response to pathogens in vivo.

Inflammatory Flt3l is essential to mobilize dendritic cells and for T cell responses during Plasmodium infection.

Innate sensing mechanisms trigger a variety of humoral and cellular events that are essential to adaptive immune responses. Here we describe an innate sensing pathway triggered by Plasmodium infection that regulates dendritic cell homeostasis and adaptive immunity through Flt3 ligand (Flt3l) release. Plasmodium-induced Flt3l release in mice requires Toll-like receptor (TLR) activation and type I interferon (IFN) production. We found that type I IFN supports the upregulation of xanthine dehydrogenase, which metabolizes the xanthine accumulating in infected erythrocytes to uric acid. Uric acid crystals trigger mast cells to release soluble Flt3l from a pre-synthesized membrane-associated precursor. During infection, Flt3l preferentially stimulates expansion of the CD8-α(+) dendritic cell subset or its BDCA3(+) human dendritic cell equivalent and has a substantial impact on the magnitude of T cell activation, mostly in the CD8(+) compartment. Our findings highlight a new mechanism that regulates dendritic cell homeostasis and T cell responses to infection.

Evaluation of the Suchey-Brooks method of age estimation in an Australian subpopulation using computed tomography of the pubic symphyseal surface.

Despite the prominent use of the Suchey-Brooks (S-B) method of age estimation in forensic anthropological practice, it is subject to intrinsic limitations, with reports of differential interpopulation error rates between geographical locations. This study assessed the accuracy of the S-B method to a contemporary adult population in Queensland, Australia and provides robust age parameters calibrated for our population. Three-dimensional surface reconstructions were generated from computed tomography scans of the pubic symphysis of male and female Caucasian individuals aged 15-70 years (n = 195) in Amira and Rapidform. Error was analyzed on the basis of bias, inaccuracy and percentage correct classification for left and right symphyseal surfaces. Application of transition analysis and Chi-square statistics demonstrated 63.9 and 69.7% correct age classification associated with the left symphyseal surface of Australian males and females, respectively, using the S-B method. Using Bayesian statistics, probability density distributions for each S-B phase were calculated, providing refined age parameters for our population. Mean inaccuracies of 6.77 (±2.76) and 8.28 (±4.41) years were reported for the left surfaces of males and females, respectively; with positive biases for younger individuals (<55 years) and negative biases in older individuals. Significant sexual dimorphism in the application of the S-B method was observed; and asymmetry in phase classification of the pubic symphysis was a frequent phenomenon. These results recommend that the S-B method should be applied with caution in medico-legal death investigations of Queensland skeletal remains and warrant further investigation of reliable age estimation techniques.

Concomitant antibiotic and mercury resistance among gastrointestinal microflora of feral brook trout, Salvelinus fontinalis.

Twenty-nine bacterial isolates representing eight genera from the gastrointestinal tracts of feral brook trout Salvelinus fontinalis (Mitchell) demonstrated multiple maximal antibiotic resistances and concomitant broad-spectrum mercury (Hg) resistance. Equivalent viable plate counts on tryptic soy agar supplemented with either 0 or 25 µM HgCl(2) verified the ubiquity of mercury resistance in this microbial environment. Mercury levels in lake water samples measured 1.5 ng L(-1); mercury concentrations in fish filets ranged from 81.8 to 1,080 ng g(-1) and correlated with fish length. The presence of similar antibiotic and Hg resistance patterns in multiple genera of gastrointestinal microflora supports a growing body of research that multiple selective genes can be transferred horizontally in the presence of an unrelated individual selective pressure. We present data that bioaccumulation of non-point source Hg pollution could be a selective pressure to accumulate both antibiotic and Hg resistant bacteria.

Zinc finger transcription factor zDC is a negative regulator required to prevent activation of classical dendritic cells in the steady state.

Classical dendritic cells (cDCs) process and present antigens to T cells. Under steady-state conditions, antigen presentation by cDCs induces tolerance. In contrast, during infection or inflammation, cDCs become activated, express higher levels of cell surface MHC molecules, and induce strong adaptive immune responses. We recently identified a cDC-restricted zinc finger transcription factor, zDC (also known as Zbtb46 or Btbd4), that is not expressed by other immune cell populations, including plasmacytoid DCs, monocytes, or macrophages. We define the zDC consensus DNA binding motif and the genes regulated by zDC using chromatin immunoprecipitation and deep sequencing. By deleting zDC from the mouse genome, we show that zDC is primarily a negative regulator of cDC gene expression. zDC deficiency alters the cDC subset composition in the spleen in favor of CD8(+) DCs, up-regulates activation pathways in steady-state cDCs, including elevated MHC II expression, and enhances cDC production of vascular endothelial growth factor leading to increased vascularization of skin-draining lymph nodes. Consistent with these observations, zDC protein expression is rapidly down-regulated after TLR stimulation. Thus, zDC is a TLR-responsive, cDC-specific transcriptional repressor that is in part responsible for preventing cDC maturation in the steady state.

Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization.

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage.

Classical dendritic cells (cDCs), monocytes, and plasmacytoid DCs (pDCs) arise from a common bone marrow precursor (macrophage and DC progenitors [MDPs]) and express many of the same surface markers, including CD11c. We describe a previously uncharacterized zinc finger transcription factor, zDC (Zbtb46, Btbd4), which is specifically expressed by cDCs and committed cDC precursors but not by monocytes, pDCs, or other immune cell populations. We inserted diphtheria toxin (DT) receptor (DTR) cDNA into the 3' UTR of the zDC locus to serve as an indicator of zDC expression and as a means to specifically deplete cDCs. Mice bearing this knockin express DTR in cDCs but not other immune cell populations, and DT injection into zDC-DTR bone marrow chimeras results in cDC depletion. In contrast to previously characterized CD11c-DTR mice, non-cDCs, including pDCs, monocytes, macrophages, and NK cells, were spared after DT injection in zDC-DTR mice. We compared immune responses to Toxoplasma gondii and MO4 melanoma in DT-treated zDC- and CD11c-DTR mice and found that immunity was only partially impaired in zDC-DTR mice. Our results indicate that CD11c-expressing non-cDCs make significant contributions to initiating immunity to parasites and tumors.

Biofuel cells: enhanced enzymatic bioelectrocatalysis.

Enzymatic biofuel cells represent an emerging technology that can create electrical energy from biologically renewable catalysts and fuels. A wide variety of redox enzymes have been employed to create unique biofuel cells that can be used in applications such as implantable power sources, energy sources for small electronic devices, self-powered sensors, and bioelectrocatalytic logic gates. This review addresses the fundamental concepts necessary to understand the operating principles of biofuel cells, as well as recent advances in mediated electron transfer- and direct electron transfer-based biofuel cells, which have been developed to create bioelectrical devices that can produce significant power and remain stable for long periods.