Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency.

Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a "failure to thrive" phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology.

PS48 can replace bovine serum albumin in pig embryo culture medium, and improve in vitro embryo development by phosphorylating AKT.

The application of embryo-related technology is dependent on in vitro culture systems. Unfortunately, most culture media are suboptimal and result in developmentally compromised embryos. Since embryo development is partially dependent upon Warburg Effect-like metabolism, our goal was to test the response of embryos treated with compounds that are known to stimulate or enhance this Effect. One such compound is 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48). When added during oocyte maturation, the quality of the resultant embryos was compromised, whereas when added to the culture medium after fertilization, PS48 improved both the percentage of embryos that reach the blastocyst stage and the number of nuclei in those blastocysts. Embryonic PS48 treatment resulted in more phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) in blastocyst-stage embryos as compared to the controls. Further, PS48 could replace bovine serum albumin in embryo culture medium, as demonstrated by high-quality embryos that were developmentally competent. The action of PS48 appears to be via stimulation of phosphoinositide-3 kinase and phosphorylation of AKT, which is consistent with stimulation of the Warburg Effect.

Gray matter volume in women with the BRCA mutation with and without ovarian removal: evidence for increased risk of late-life Alzheimer's disease or dementia.

OBJECTIVE: Ovarian removal prior to spontaneous/natural menopause (SM) is associated with increased risk of late life dementias including Alzheimer's disease. This increased risk may be related to the sudden and early loss of endogenous estradiol. Women with breast cancer gene mutations (BRCAm) are counseled to undergo oophorectomy prior to SM to significantly reduce their risk of developing breast, ovarian, and cervical cancers. There is limited evidence of the neurological effects of ovarian removal prior to the age of SM showing women without the BRCAm had cortical thinning in medial temporal lobe structures. A second study in women with BRCAm and bilateral salpingo-oophorectomy (BSO) noted changes in cognition. METHODS: The present, cross-sectional study examined whole-brain differences in gray matter (GM) volume using high-resolution, quantitative magnetic resonance imaging in women with BRCAm and intact ovaries (BRCA-preBSO [study cohort with BRCA mutation prior to oophorectomy]; n = 9) and after surgery with (BSO + estradiol-based therapy [ERT]; n = 10) and without (BSO; n = 10) postsurgical estradiol hormone therapy compared with age-matched women (age-matched controls; n = 10) with their ovaries. RESULTS: The BRCA-preBSO and BSO groups showed significantly lower GM volume in the left medial temporal and frontal lobe structures. BSO + ERT exhibited few areas of lower GM volume compared with age-matched controls. Novel to this study, we also observed that all three BRCAm groups exhibited significantly higher GM volume compared with age-matched controls, suggesting continued plasticity. CONCLUSIONS: The present study provides evidence, through lower GM volume, to support both the possibility that the BRCAm, alone, and early life BSO may play a role in increasing the risk for late-life dementia. At least for BRCAm with BSO, postsurgical ERT seems to ameliorate GM losses.

Piglets produced from cloned blastocysts cultured in vitro with GM-CSF.

In general, pig embryos established by somatic cell nuclear transfer (SCNT) are transferred at the one-cell stage because of suboptimal embryo culture conditions. Improvements in embryo culture can increase the practical application of late embryo transfer. The goal of this study was to evaluate embryos cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro, and to track the in vivo developmental competency of SCNT-derived blastocysts from these GM-CSF embryos. The receptor for GM-CSF was up-regulated in in vitro-produced embryos when compared to in vivo-produced cohorts, but the level decreased when GM-CSF was present. In vitro fertilized (IVF) embryos, supplemented with GM-CSF (2 or 10 ng/ml), showed a higher frequency of development to the blastocyst stage compared to controls. The total cell numbers of the blastocysts also increased with supplementation of GM-CSF. Molecular analysis demonstrates that IVF-derived blastocysts cultured with GM-CSF exhibit less apoptotic activity. Similarly, an increase in development to the blastocyst stage and an increase in the average total-cell number in the blastocysts were observed when SCNT-derived embryos were cultured with either concentration of GM-CSF (2 or 10 ng/ml). When SCNT-derived embryos, cultured with 10 ng/ml GM-CSF, were transferred into six surrogates at Day 6, five of the surrogates became pregnant and delivered healthy piglets. Our findings suggest that supplementation of GM-CSF can provide better culture conditions for IVF- and SCNT-derived embryos, and pig SCNT-derived embryos cultured with GM-CSF in vitro can successfully produce piglets when transferred into surrogates at the blastocyst stage. Thus, it may be practical to begin performing SCNT-derived embryo transfer at the blastocyst stage.

Sex and Gender Science: The World Writes on the Body.

Sex and Gender Science seeks to better acknowledge that the body cannot be removed from the world it inhabits. We believe that to best answer any neuroscience question, the biological and the social need to be addressed through both objective means to learn, "how it is like" and subjective means to learn, "what it is like." We call bringing the biological and social together, "Situated Neuroscience" and the mixing of approaches to do so, Very Mixed Methods. Taken together, they constitute an approach to Sex and Gender Science. In this chapter, we describe neural phenomena for which considering sex and gender together produces a fuller knowledge base: sleep, pain, memory, and concussion. For these brain phenomena examples, studying only quantitative measures does not reveal the full impact of these lived experiences on the brain but studying only the qualitative would not reveal how the brain responds. We discuss how Sex and Gender Science allows us to begin to bring together biology and its social context and acknowledge where context can contribute to resolving ignorance to offer more expansive, complementary, and interrelating pictures of an intricate neuro-landscape.

Sex and menstrual cycle influence human spatial navigation strategies and performance.

Which facets of human spatial navigation do sex and menstrual cycle influence? To answer this question, a cross-sectional online study of reproductive age women and men was conducted in which participants were asked to demonstrate and self-report their spatial navigation skills and strategies. Participants self-reported their sex and current menstrual phase [early follicular (EF), late follicular/periovulatory (PO), and mid/late luteal (ML)], and completed a series of questionnaires and tasks measuring self-reported navigation strategy use, topographical memory, cognitive map formation, face recognition, and path integration. We found that sex influenced self-reported use of cognitive map- and scene-based strategies, face recognition, and path integration. Menstrual phase moderated the influence of sex: compared to men, women had better face recognition and worse path integration, but only during the PO phase; PO women were also better at path integration in the presence of a landmark compared to EF + ML women and men. These findings provide evidence that human spatial navigation varies with the menstrual cycle and suggest that sensitivity of the entorhinal cortex and longitudinal axis of the hippocampus to differential hormonal effects may account for this variation.

High-resolution ultrasound in the evaluation of musculoskeletal infections.

Soft tissue and osseous musculoskeletal infections are common but can be difficult to diagnose clinically. Signs, symptoms, and physical examination findings may be nonspecific, and laboratory values can be inconclusive. The extent of disease may also be underestimated on physical examination. Soft tissue infections most commonly occur secondary to direct inoculation from broken skin and less frequently due to the seeding of the soft tissues from hematogenous spread, while osseous infections are more commonly due to hematogenous seeding. Infections may also be iatrogenic, following surgery or other procedural interventions. High-resolution ultrasound is an extremely useful imaging modality in the evaluation of musculoskeletal soft tissue and joint infections, and can occasionally be used to evaluate osseous infections as well. Ultrasound can aid in the early diagnosis of musculoskeletal infections, allowing for prompt treatment, decreased risk of complications, and treatment optimization. Ultrasound is sensitive and specific in evaluating soft tissue edema and hyperemia; soft tissue abscesses; joint, bursal and tendon sheath effusions/synovitis; and subperiosteal abscesses. This article describes the typical high-resolution grayscale as well as color and power Doppler ultrasound imaging findings of soft tissue infections including cellulitis, fasciitis, necrotizing deep soft tissue infection, pyomyositis, soft tissue abscess, infectious bursitis, and infectious tenosynovitis. Ultrasound findings of septic arthritis as well as osteomyelitis, such as subperiosteal spread of infection (subperiosteal abscess). are also reviewed. In addition, the use of ultrasound to guide fluid and tissue sampling is discussed.

Women's Brain Health: Midlife Ovarian Removal Affects Associative Memory.

Women with early bilateral salpingo-oophorectomy (BSO; removal of ovaries and fallopian tubes) have greater Alzheimer's disease (AD) risk than women in spontaneous/natural menopause (SM), but early biomarkers of this risk are not well-characterized. Considering associative memory deficits may presage preclinical AD, we wondered if one of the earliest changes might be in associative memory and whether younger women with BSO had changes similar to those observed in SM. Women with BSO (with and without 17ß-estradiol replacement therapy (ERT)), their age-matched premenopausal controls (AMC), and older women in SM completed a functional magnetic resonance imaging face-name associative memory task shown to predict early AD. Brain activation during encoding was compared between groups: AMC (n=25), BSO no ERT (BSO; n=15), BSO+ERT (n=16), and SM without hormone therapy (n=16). Region-of-interest analyses revealed AMC did not contribute to functional group differences. BSO+ERT had higher hippocampal activation than BSO and SM. This hippocampal activation correlated positively with urinary metabolite levels of 17ß-estradiol. Multivariate partial least squares analyses showed BSO+ERT had a different network-level activation pattern than BSO and SM. Thus, despite being approximately 10 years younger, women with BSO without ERT had similar brain function to those with SM, suggesting early 17ß-estradiol loss may lead to an altered functional brain phenotype which could influence late-life AD risk, making face-name encoding a potential biomarker for midlife women with increased AD risk. Despite similarities in activation, BSO and SM groups showed opposite within-hippocampus connectivity, suggesting menopause type is an important consideration when assessing brain function.

Disturbed sleep is associated with reduced verbal episodic memory and entorhinal cortex volume in younger middle-aged women with risk-reducing early ovarian removal.

Introduction: Women with early ovarian removal (<48 years) have an elevated risk for both late-life Alzheimer's disease (AD) and insomnia, a modifiable risk factor. In early midlife, they also show reduced verbal episodic memory and hippocampal volume. Whether these reductions correlate with a sleep phenotype consistent with insomnia risk remains unexplored. Methods: We recruited thirty-one younger middleaged women with risk-reducing early bilateral salpingo-oophorectomy (BSO), fifteen of whom were taking estradiol-based hormone replacement therapy (BSO+ERT) and sixteen who were not (BSO). Fourteen age-matched premenopausal (AMC) and seventeen spontaneously peri-postmenopausal (SM) women who were ~10y older and not taking ERT were also enrolled. Overnight polysomnography recordings were collected at participants' home across multiple nights (M=2.38 SEM=0.19), along with subjective sleep quality and hot flash ratings. In addition to group comparisons on sleep measures, associations with verbal episodic memory and medial temporal lobe volume were assessed. Results: Increased sleep latency and decreased sleep efficiency were observed on polysomnography recordings of those not taking ERT, consistent with insomnia symptoms. This phenotype was also observed in the older women in SM, implicating ovarian hormone loss. Further, sleep latency was associated with more forgetting on the paragraph recall task, previously shown to be altered in women with early BSO. Both increased sleep latency and reduced sleep efficiency were associated with smaller anterolateral entorhinal cortex volume. Discussion: Together, these findings confirm an association between ovarian hormone loss and insomnia symptoms, and importantly, identify an younger onset age in women with early ovarian removal, which may contribute to poorer cognitive and brain outcomes in these women.

Glycolysis in preimplantation development is partially controlled by the Warburg Effect.

Glucose metabolism in preimplantation embryos has traditionally been viewed from a somatic cell viewpoint. Here, we show that gene expression in early embryos is similar to rapidly dividing cancer cells. In vitro-produced pig blastocysts were subjected to deep-sequencing, and were found to express two gene variants that have been ascribed importance to cancer cell metabolism (HK2 and the M2 variant of PKM2). Development was monitored and gene expression was quantified in additional embryos cultured in low or high O(2) (5% CO(2), 5% O(2), 90% N(2) vs. 5% CO(2) in air). Development to the blastocyst stage in the two atmospheres was similar, except low O(2) resulted in more total and inner cell mass nuclei than high O(2). Of the 15 candidate genes selected that are involved in glucose metabolism, only TALDO1 and PDK1 were increased in the low O(2) environment. One paradigm that has been used to explain glycolysis under low oxygen tension is the Warburg Effect (WE). The WE predicts that expression of both HK2 and PKM2 M2 results in a slowing of glucose metabolism through the TCA cycle, thereby forcing the products of glycolysis to be metabolized through the pentose phosphate pathway and to lactic acid. This charging of the system is apparently so important to the early embryo that redundant mechanisms are present, that is, a fetal form of PKM2 and high levels of PDK1. Here, we set the framework for using the WE to describe glucose metabolism and energy production during preimplantation development.

Scene memory and hippocampal volume in middle-aged women with early hormone loss.

The present study explored whether early midlife bilateral salpingo-oophorectomy (BSO), a female-specific risk factor for dementia, is associated with reduced medial temporal lobe structure and function. Younger middle-aged women with the BRCA1/2 mutation and a BSO prior to spontaneous menopause (SM) were recruited. We determined the performance of women with BSO not taking estradiol-based hormone therapy (n = 18) on a task measuring object and scene recognition and quantified medial temporal lobe subregion volumes using manually segmented high-resolution T2-weighted MRI scans. Comparisons were made to those with BSO taking estradiol-based hormone therapy (n = 20), age-matched premenopausal controls (n = 28), and older women in SM not taking hormone therapy matched for duration of hormone deprivation (n = 17). Reduced hippocampal integrity specific to the BSO group not taking hormone therapy was observed, reflected by significantly smaller dentate gyrus/CA2/CA3 volumes and lower scene recognition memory performance. These findings show that hippocampal subfield volume may be useful for identifying early midlife changes in women at elevated risk for dementia.

Role of Ovarian Hormones in the Modulation of Sleep in Females Across the Adult Lifespan.

Ovarian hormones, including 17ß-estradiol, are implicated in numerous physiological processes, including sleep. Beginning at puberty, girls report more sleep complaints than boys, which is maintained throughout the reproductive life stage. Sleep problems are exacerbated during the menopausal transition, evidenced by greater risk for sleep disorders. There is emerging evidence that menopause-associated hormone loss contributes to this elevated risk, but age is also an important factor. The extent to which menopause-associated sleep disturbance persists into postmenopause above and beyond the effects of age remains unknown. Untreated sleep disturbances have important implications for cognitive health, as they are emerging as risk factors for dementia. Given that sleep loss impairs memory, an important knowledge gap concerns the role played by menopause-associated hormone loss in exacerbating sleep disturbance and, ultimately, cognitive function in aging women. In this review, we take a translational approach to illustrate the contribution of ovarian hormones in maintaining the sleep-wake cycle in younger and middle-aged females, with evidence implicating 17ß-estradiol in supporting the memory-promoting effects of sleep. Sleep physiology is briefly reviewed before turning to behavioral and neural evidence from young females linking 17ß-estradiol to sleep-wake cycle maintenance. Implications of menopause-associated 17ß-estradiol loss is also reviewed before discussing how ovarian hormones may support the memory-promoting effects of sleep, and why menopause may exacerbate pathological aging via effects on sleep. While still in its infancy, this research area offers a new sex-based perspective on aging research, with a focus on a modifiable risk factor for pathological aging.

Sex difference or hormonal difference in mental rotation? The influence of ovarian milieu.

Sex differences in visuospatial cognition have long been reported, with men being advantaged on the Mental Rotations Test (MRT). The data, however, are variable, and sensitive to design parameters. When men and women are compared directly, with women in different hormonal milieus combined, there seem to be sex differences. When women alone are studied, taking into account different ovarian steroid concentrations and treatments, MRT performance varies with these changes. Indeed, several reports describe better performance among women with reduced estrogens. To better understand whether the sex difference in MRT persists once hormonal status is considered, we recruited reproductive age adults designated male and female at birth (MAB, FAB), and administered the Vandenberg-Kuse (V/K) MRT-comparing performance among MAB (n = 169) and FAB (n = 219). For FAB combined, we found a sex difference with MAB performing better than FAB. However, when FAB were analyzed by current menstrual cycle phase (Early Follicular (EF), Periovulatory (PO), Midluteal (ML)) or by hormone therapy (transmasculine testosterone administration (TM+), oral contraceptive (OC) ingestion prior to (OC+) or after cognitive testing (OC-)), low-estradiol groups (EF, OC-, TM+) performed as strongly as MAB, and had better MRT than cycling FAB in high-estradiol menstrual cycle phases (PO, ML). On a verbal memory control task, neither a sex difference nor a low estrogen advantage was detected, although performance varied with hormonal milieu. Our findings support a dynamic model of spatial performance and suggest that both MAB and FAB perform strongly on MRT, contingent on hormonal status.

Cognitive markers of dementia risk in middle-aged women with bilateral salpingo-oophorectomy prior to menopause.

Oophorectomy prior to menopause is associated with late-life dementia. Memory decline may start within 6 months after oophorectomy in middle-aged women, suggested by lower verbal and working memory performance. Unknown is whether such changes persist beyond 6 months, and whether they are reversed by estradiol. Short-term benefits of estradiol on verbal memory following oophorectomy were observed in one study, but longer term effects remain unknown. In the present study, middle-aged BRCA1/2 mutation carriers with early oophorectomy at least 1 year prior to study onset were tested on verbal and working memory with results stratified by (1) current estradiol use (n = 22) or (2) no history of estradiol use (n = 24), and compared to age-matched premenopausal controls (n = 25). Both memory abilities were adversely affected by oophorectomy, but only working memory was maintained by estradiol. Estrogen metabolite levels correlated with working memory, suggesting a role for estradiol in preserving this ability. Memory decline appears to persist after early oophorectomy, particularly for women who do not take estradiol.

Mutational disruption of the ABCC2 gene in fall armyworm, Spodoptera frugiperda, confers resistance to the Cry1Fa and Cry1A.105 insecticidal proteins.

The use of Bt proteins in crops has revolutionized insect pest management by offering effective season-long control. However, field-evolved resistance to Bt proteins threatens their utility and durability. A recent example is field-evolved resistance to Cry1Fa and Cry1A.105 in fall armyworm (Spodoptera frugiperda). This resistance has been detected in Puerto Rico, mainland USA, and Brazil. A S. frugiperda population with suspected resistance to Cry1Fa was sampled from a maize field in Puerto Rico and used to develop a resistant lab colony. The colony demonstrated resistance to Cry1Fa and partial cross-resistance to Cry1A.105 in diet bioassays. Using genetic crosses and proteomics, we show that this resistance is due to loss-of-function mutations in the ABCC2 gene. We characterize two novel mutant alleles from Puerto Rico. We also find that these alleles are absent in a broad screen of partially resistant Brazilian populations. These findings confirm that ABCC2 is a receptor for Cry1Fa and Cry1A.105 in S. frugiperda, and lay the groundwork for genetically enabled resistance management in this species, with the caution that there may be several distinct ABCC2 resistances alleles in nature.

Partial loss of interleukin 2 receptor gamma function in pigs provides mechanistic insights for the study of human immunodeficiency syndrome.

In this study, we described the phenotype of monoallelic interleukin 2 receptor gamma knockout (mIL2RG+/Δ69-368 KO) pigs. Approximately 80% of mIL2RG+/Δ69-368 KO pigs (8/10) were athymic, whereas 20% (2/10) presented a rudimentary thymus. The body weight of IL2RG+/Δ69-368KO pigs developed normally. Immunological analysis showed that mIL2RG+/Δ69-368 KO pigs possessed CD25+CD44- or CD25-CD44+ cells, whereas single (CD4 or CD8) or double (CD4/8) positive cells were lacking in mIL2RG+/Δ69-368 KO pigs. CD3+ cells in the thymus of mIL2RG+/Δ69-368 KO pigs contained mainly CD44+ cells and/or CD25+ cells, which included FOXP3+ cells. These observations demonstrated that T cells from mIL2RG+/Δ69-368 KO pigs were able to develop to the DN3 stage, but failed to transition toward the DN4 stage. Whole-transcriptome analysis of thymus and spleen, and subsequent pathway analysis revealed that a subset of genes differentially expressed following the loss of IL2RG might be responsible for both impaired T-cell receptor and cytokine-mediated signalling. However, comparative analysis of two mIL2RG+/Δ69-368 KO pigs revealed little variability in the down- and up-regulated gene sets. In conclusion, mIL2RG+/Δ69-368 KO pigs presented a T-B+NK- SCID phenotype, suggesting that pigs can be used as a valuable and suitable biomedical model for human SCID research.

Dickkopf-related protein 1 inhibits the WNT signaling pathway and improves pig oocyte maturation.

The ability to mature oocytes in vitro provides a tool for creating embryos by parthenogenesis, fertilization, and cloning. Unfortunately the quality of oocytes matured in vitro falls behind that of in vivo matured oocytes. To address this difference, transcriptional profiling by deep sequencing was conducted on pig oocytes that were either matured in vitro or in vivo. Alignment of over 18 million reads identified 1,316 transcripts that were differentially represented. One pathway that was overrepresented in the oocytes matured in vitro was for Wingless-type MMTV integration site (WNT) signaling. In an attempt to inhibit the WNT pathway, Dickkopf-related protein 1 was added to the in vitro maturation medium. Addition of Dickkopf-related protein 1 improved the percentage of oocytes that matured to the metaphase II stage, increased the number of nuclei in the resulting blastocyst stage embryos, and reduced the amount of disheveled segment polarity protein 1 protein in oocytes. It is concluded that transcriptional profiling is a powerful method for detecting differences between in vitro and in vivo matured oocytes, and that the WNT signaling pathway is important for proper oocyte maturation.

Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs.

After the knock-out (KO) of α1,3 galactosyltransfease (Gal-T), the Hanganutziu-Deicher antigen became a major antigen of the "non-Gal antigen" that is implicated in subsequent xenograft rejection. For deletion of non-Gal antigen, we successfully produced zinc finger nuclease (ZFN)-mediated monoallelic/biallelic male and female CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO miniature pigs: the efficiency of the gene targeting (41.7%) was higher when donor DNA was used with the ZFN than those of ZFN alone (9.1%). Monoallelic KO pigs had no integration of exogenous DNA into their genome, indicating that this technique would provide a new avenue to reduce the risk of antibiotics resistance when organs from genetically modified pigs are transplanted into patients. Until now, both monoallelic and biallelic CMAH KO pigs are healthy and show no sign of abnormality and off-target mutations. Therefore, these CMAH null pigs on the Gal-T KO background could serve as an important model for the xenotransplantation.