Raymond M. Anchan

Disruption of local retinoid-mediated gene expression accompanies abnormal development in the mammalian olfactory pathway

We have evaluated the role of retinoid signaling in the early development of the olfactory epithelium and olfactory bulb. When retinoid‐mediated gene expression is blocked briefly in mouse embryos at midgestation with citral (a general alcohol dehydrogenase antagonist that is thought to interfere with retinoid synthesis), the spectrum of morphogenetic abnormalities includes disruption of olfactory pathway development. It is difficult, however, to assess the specificity of this pharmacological manipulation, insofar as it also compromises several other aspects of central nervous system development. In homozygous Pax6 mutant mice (small eye: Pax6Sey‐Neu), there is a more discrete lesion to the olfactory pathway: The epithelium and bulb cannot be recognized at any time during development, whereas other forebrain subdivisions can still be recognized. This loss of the entire primary olfactory pathway is accompanied by a failure of retinoid‐mediated gene expression limited to the frontonasal region and forebrain. Retinoid receptors are expressed in the forebrain of Pax6Sey‐Neu/Pax6Sey‐Neu embryos, and the mutant forebrain remains responsive to exogenous retinoic acid. However, in Pax6Sey‐Neu/Pax6Sey‐Neu embryos, retinoic acid (RA) is not produced by the frontonasal mesenchyme, which normally provides local retinoid signals to the placode and forebrain. Together, these results suggest that local retinoid signaling is essential for the normal development of the mammalian olfactory pathway. J. Comp. Neurol. 379:171–184, 1997.

Vitrification and levitation of a liquid droplet on liquid nitrogen

The vitrification of a liquid occurs when ice crystal formation is prevented in the cryogenic environment through ultrarapid cooling. In general, vitrification entails a large temperature difference between the liquid and its surrounding medium. In our droplet vitrification experiments, we observed that such vitrification events are accompanied by a Leidenfrost phenomenon, which impedes the heat transfer to cool the liquid, when the liquid droplet comes into direct contact with liquid nitrogen. This is distinct from the more generally observed Leidenfrost phenomenon that occurs when a liquid droplet is self-vaporized on a hot plate. In the case of rapid cooling, the phase transition from liquid to vitrified solid (i.e., vitrification) and the levitation of droplets on liquid nitrogen (i.e., Leidenfrost phenomenon) take place simultaneously. Here, we investigate these two simultaneous physical events by using a theoretical model containing three dimensionless parameters (i.e., Stefan, Biot, and Fourier numbers). We explain theoretically and observe experimentally a threshold droplet radius during the vitrification of a cryoprotectant droplet in the presence of the Leidenfrost effect.

RETINOID SIGNALING DISTINGUISHES A SUBPOPULATION OF OLFACTORY RECEPTOR NEURONS IN THE DEVELOPING AND ADULT MOUSE

We asked whether retinoic acid (RA) influences olfactory receptor neurons (ORNs) in the developing and mature mouse olfactory epithelium (oe). The distribution of retinoid receptors and binding proteins in the oe changes between embryonic days 11.5 and 13.5, the period when ORNs first differentiate and send axons into the nascent olfactory nerve. Coincident with this change, RA, which is produced in the frontonasal mesenchyme at these ages, begins to activate gene expression in a bilaterally symmetric subset of ORNs in the dorsolateral oe, as judged by the expression of an RA‐responsive transgene. Axons from these RA‐activated ORNs are segregated in the olfactory nerve as it extends through the frontonasal mesenchyme toward the forebrain. In vitro, RA potentiates ORN neurite growth on laminin, which, in the embryo, is found in a stripe of frontonasal mesenchyme directly associated with the olfactory nerve. RA does not modify growth on fibronectin, type IV collagen, or L1, which olfactory axons encounter in different regions of the territory between the olfactory epithelium and the brain. The pattern of RA‐mediated transcriptional activation and axon segregation persists in early postnatal mice, and RA signaling can be recognized in a subset of adult ORNs in the dorsolateral oe. Thus, RA‐mediated gene expression distinguishes a subpopulation of ORNs in a distinct region of the oe during the early development of the olfactory pathway, and may influence differentiation and axonal projections of ORNs in this region throughout life. J. Comp. Neurol. 394:445–461, 1998.

Gestational diabetes is associated with depressed adiponectin levels.

Objective: Adiponectin is a 29-kd adipocyte-secreted protein that has been linked to insulin resistance in obesity and diabetes. The aim of the present study was to evaluate adiponectin levels in the insulinresistant state of diabetes in gestation. Methods: Term, gravid subjects with diabetes (n = 31; age, 30.0±0.9 years; weight, 98.8±4.6 kg) and healthy, term, gravid subjects (n = 27; age, 26.1±1.1 years; weight, 91.2±3.78 kg) were examined. The diabetes group consisted of 11 class A1, 11 class A2, and nine class B subjects. Plasma insulin, glucose, adiponectin, and leptin were measured on samples obtained immediately before Cesarean or vaginal delivery. Data were presented as means ± SE, and signficance is set at P ≤ .05. Results: We observed decreased adiponectin levels in class A2 (4.93 ± 0.58,μg/mL; P = .013) and class B diabetics (3.33 ± 0.56,μg/mL; P = .001) as compared to controls (8.17 ± 0.82,μg/mL), while a nonsignficant decrease was also observed in class A 1 (6.58 ± 1. 13,μg/mL; P = .213). When grouping all gravid subjects, we observed that non-Caucasian subjects (n = 42) (5.51 ± 0.51,μg/mL; P - .003) had lower adiponectin levels than Caucasian subjects (n = 16) (8.88 ± 1. 11 ug/mL). Within tIe non-Caucasian group, wefound significantly lower adiponectin levels in diabetic gravid subjects (class A2: 4.24 ± 0.75,μg/mL; P = .044; and class B: 3.33 ± 0.56 μg/mL; P = .005) compared tvith nondiabetic gravid subjects (7.05 ± 0.80 μg/mL). Conclusion: Class A2 and B gestational diabetes are associated with suppressed levels of adiponectin, similar to thatfound in other insulin-resistant states (type II diabetes and obesity).

Amniocytes can serve a dual function as a source of iPS cells and feeder layers

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5–7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4+ or c-Kit+ amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

Exhaustion of racing sperm in nature-mimicking microfluidic channels during sorting.

Fertilization is central to the survival and propagation of a species, however, the precise mechanisms that regulate the sperms journey to the egg are not well understood. In nature, the sperm has to swim through the cervical mucus, akin to a microfluidic channel. Inspired by this, a simple, cost-effective microfluidic channel is designed on the same scale. The experimental results are supported by a computational model incorporating the exhaustion time of sperm.

Preserving human cells for regenerative, reproductive, and transfusion medicine

Cell cryopreservation maintains cellular life at sub‐zero temperatures by slowing down biochemical processes. Various cell types are routinely cryopreserved in modern reproductive, regenerative, and transfusion medicine. Current cell cryopreservation methods involve freezing (slow/rapid) or vitrifying cells in the presence of a cryoprotective agent (CPA). Although these methods are clinically utilized, cryo‐injury due to ice crystals, osmotic shock, and CPA toxicity cause loss of cell viability and function. Recent approaches using minimum volume vitrification provide alternatives to the conventional cryopreservation methods. Minimum volume vitrification provides ultra‐high cooling and rewarming rates that enable preserving cells without ice crystal formation. Herein, we review recent advances in cell cryopreservation technology and provide examples of techniques that are utilized in oocyte, stem cell, and red blood cell cryopreservation.

MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

Cardiac left ventricular outflow tract (LVOT) defects represent a common but heterogeneous subset of congenital heart disease for which gene identification has been difficult. We describe a 46,XY,t(1;5)(p36.11;q31.2)dn translocation carrier with pervasive developmental delay who also exhibited LVOT defects, including bicuspid aortic valve (BAV), coarctation of the aorta (CoA) and patent ductus arteriosus (PDA). The 1p breakpoint disrupts the 5′ UTR of AHDC1, which encodes AT-hook DNA-binding motif containing-1 protein, and AHDC1-truncating mutations have recently been described in a syndrome that includes developmental delay, but not congenital heart disease [Xia, F., Bainbridge, M.N., Tan, T.Y., Wangler, M.F., Scheuerle, A.E., Zackai, E.H., Harr, M.H., Sutton, V.R., Nalam, R.L., Zhu, W. et al. (2014) De Novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea. Am. J. Hum. Genet., 94, 784–789]. On the other hand, the 5q translocation breakpoint disrupts the 3′ UTR of MATR3, which encodes the nuclear matrix protein Matrin 3, and mouse Matr3 is strongly expressed in neural crest, developing heart and great vessels, whereas Ahdc1 is not. To further establish MATR3 3′ UTR disruption as the cause of the probands LVOT defects, we prepared a mouse Matr3Gt-ex13 gene trap allele that disrupted the 3′ portion of the gene. Matr3Gt-ex13 homozygotes are early embryo lethal, but Matr3Gt-ex13 heterozygotes exhibit incompletely penetrant BAV, CoA and PDA phenotypes similar to those in the human proband, as well as ventricular septal defect (VSD) and double-outlet right ventricle (DORV). Both the human MATR3 translocation breakpoint and the mouse Matr3Gt-ex13 gene trap insertion disturb the polyadenylation of MATR3 transcripts and alter Matrin 3 protein expression, quantitatively or qualitatively. Thus, subtle perturbations in Matrin 3 expression appear to cause similar LVOT defects in human and mouse.

Contained tissue extraction using power morcellation: prospective evaluation of leakage parameters

BACKGROUND Safe tissue removal is a challenge for minimally invasive procedures such as myomectomy, supracervical hysterectomy, or total hysterectomy of a large uterine specimen. There is concern regarding disruption or dissemination of tissue during this process, which may be of particular significance in cases of undetected malignancy. Contained tissue extraction techniques have been developed in an effort to mitigate morcellation-related risks. OBJECTIVE The objective of the study was to quantify perioperative outcomes of contained tissue extraction using power morcellation, specifically evaluating parameters of tissue or fluid leakage from within the containment system. STUDY DESIGN This was a study including a multicenter prospective cohort of adult women who underwent minimally invasive hysterectomy or myomectomy using a contained power morcellation technique. Blue dye was applied to the tissue specimen prior to removal to help identify cases of fluid or tissue leakage from within the containment system. RESULTS A total of 76 patients successfully underwent the contained power morcellation protocol. Mean time for the contained morcellation procedure was 30.2 minutes (±22.4). The mean hysterectomy specimen weight was 480.1 g (±359.1), and mean myomectomy specimen weight was 239.1 g (±229.7). The vast majority of patients (73.7%) were discharged home the same day of surgery. Final pathological diagnosis was benign in all cases. Spillage of dye or tissue was noted in 7 cases (9.2%), although containment bags were intact in each of these instances. CONCLUSION Findings are consistent with prior work demonstrating the feasibility of contained tissue extraction; however, further refinement of this technique is warranted.

Sperm processing for advanced reproductive technologies: Where are we today?

Assisted reproductive technologies (ARTs) utilize sperm sorting methods to select viable sperm from the semen samples. Conventional sperm sorting techniques in current use are density gradient centrifugation, direct swim-up, and conventional swim-up. These methods use multiple centrifugation steps, which have been shown to generate reactive oxygen species (ROS) that decrease DNA integrity and damage sperm. Newer technologies, such as microfluidics, electrophoresis, motile sperm organelle morphology examination (MSOME), and birefringence eliminate the centrifugation steps and can improve the selection of sperm with higher DNA integrity, normal morphology, and motility as well as improved artificial insemination outcomes. In this review, we discuss some recent research in centrifugation and non-centrifugation based techniques and their effect on sperm quality and ART outcomes.