Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters

Improved blastocyst development of single cow OPU-derived presumptive zygotes by group culture with agarose-embedded helper embryos

<p>Abstract</p> <p>Background</p> <p>The <it>in vitro </it>culture of presumed zygotes derived from single cow ovum pick-up (OPU) is important for the production of quality blastocysts maintaining pedigree. The aim of the present study was to evaluate the agar chip-embedded helper embryo coculture system for single cow OPU-derived zygotes by assessing embryo quality.</p> <p>Methods</p> <p>Cumulus oocyte complexes (COCs) were collected from Hanwoo cows with high genetic merit twice a week using the ultra-sound guided OPU technique and from slaughterhouse ovaries. The Hanwoo cow COCs and slaughterhouse ovaries were matured <it>in vitro</it>, fertilized <it>in vitro </it>with thawed Hanwoo sperm and cultured for 24 h. The presumed zygotes were subsequently placed in three different culture systems: (1) control OPU (controlOPU) with single cow OPU-derived presumed zygotes (2~8); (2) agar chip-embedded slaughterhouse helper embryo coculture (agarOPU) with ten presumed zygotes including all presumed zygotes from a cow (2~8) and the rest from agar chip-embedded slaughterhouse presumed zygotes (8~2); and (3) slaughterhouse <it>in vitro </it>embryo production (sIVP) with ten slaughterhouse ovary-derived presumed zygotes, each in 50 μL droplets. Day 8 blastocysts were assayed for apoptosis and gene expression using real time PCR.</p> <p>Results</p> <p>The coculture system promoted higher blastocyst development in OPU zygotes compared to control OPU zygotes cultured alone (35.2 vs. 13.9%; P < 0.01). Genes predicted to be involved in implantation failure and/or embryo resorption were down-regulated (P < 0.05) in control OPU zygotes (<it>CD9</it>, 0.4-fold; <it>AKRAB</it>1, 0.3-fold) and in cocultured zygotes (<it>CD9</it>, 0.3-fold; <it>AKRAB</it>1, 0.3-fold) compared to sIVP blastocysts (1.0-fold). Moreover, genes involved in implantation and/or normal calf delivery were up-regulated (P < 0.05 to P < 0.01) in control OPU zygotes (<it>PGSH</it>2, 5.0-fold; <it>TXN</it>, 4.3-fold; <it>PLAU</it>, 1.7-fold) and cocultured zygotes (<it>PGSH</it>2, 14.5-fold; <it>TXN</it>, 3.2-fold; <it>PLAU</it>, 6.8-fold) compared to sIVP (1.0-fold) blastocysts. However, the expression of <it>PLAC8, TGF-β1, ODC1</it>, <it>ATP5A1 </it>and <it>CASP3 </it>did not differ between the three culture groups.</p> <p>Conclusions</p> <p>Results show that the agar chip-embedded helper embryo coculture system enhances developmental competence and embryo quality in cultures of limited numbers of high pedigree single cow OPU presumed zygotes.</p

Effect of income level on stroke incidence and the mediated effect of simultaneous diagnosis of metabolic syndrome diseases; a nationwide cohort study in South Korea

Background : This study aimed to determine whether a simultaneous diagnosis of main components of metabolic syndrome (MetS) (hypertension, diabetes mellitus, and dyslipidemia) plays a mediator between income level and stroke. Methods : We used the National Health Insurance Service National Sample Cohort database from 2006 to 2015. The mediator variables were the number of main MetS components diagnosed simultaneously (two or more/three or more). We used a weighting approach method of causal mediation analysis to apply counterfactual frameworks to the Cox proportional hazards regression model. Results : A total of 213,526 people were included with 1,690,665.3 person-years of followed up. Compared with the high-income group, the risk of being diagnosed with two or more components of MetS significantly increased in all other income groups [middle-income OR 1.05 (95% CI 1.02–1.08); low-income OR 1.09 (95% CI 1.05–1.12); Medical Aid beneficiaries OR 1.39 (95% CI 1.32–1.47)]. A lower level of income was significantly associated with a higher risk of stroke compared with the high-income group [middle-income HR 1.15 (95% CI 1.07–1.25); low-income HR 1.19 (95% CI 1.10–1.29); Medical Aid beneficiaries HR 1.63 (95% CI 1.48–1.80)]. In the Medical Aid beneficiaries, simultaneous diagnosis of the main metabolic components acted as a significant mediator between income levels and stroke incidence, with 26.6% mediated when diagnosed with two or more diseases and 21.1% when diagnosed with all three. Conclusions : Co-diagnosis of MetS components played a significant mediator role between income level and stroke incidence

Leisure sedentary time is differentially associated with hypertension, diabetes mellitus, and hyperlipidemia depending on occupation

Abstract Background Sedentary behavior is considered an independent cause of cardio-metabolic diseases, regardless of physical activity level and obesity. Few studies have reported the association between leisure sedentary time and cardio-vascular diseases in terms of occupation. Methods We performed a cross-sectional study using data from the Korean Community Health Survey (KCHS) for 240,086 participants assessed in 2011 and 2013. Occupation was categorized into four groups: farmer or fisherman, laborer, and soldier (Group I); service worker, salesperson, technician, mechanic, production worker, and engineer (Group II); manager, expert, specialist, and clerk (Group III); and unemployed (Group IV). Leisure sedentary time was divided into five groups: 0 h, 1 h, 2 h, 3 h, and 4+ h. The association between leisure sedentary time on weekdays and hypertension/diabetes mellitus/hyperlipidemia for different occupations was analyzed using simple and multiple logistic regression analyses with complex sampling. Results In Groups I, II and III, no length of sedentary time was associated with hypertension, and only 3 h or 4+ h of sedentary time was associated with diabetes mellitus and hyperlipidemia. Group IV showed a significant association with hypertension and diabetes mellitus for the 2 h, 3 h, and 4+ h sedentary times. Conclusions The unemployed are more susceptible than other occupation groups to cardio-metabolic diseases when leisure time is sedentary

Glacial history and depositional environments in little Storfjorden and Hambergbukta of Arctic Svalbard since the younger dryas

Geophysical and lithological data provide crucial information for the understanding of glacial history in Arctic Svalbard. In this study, we reconstructed the glacier-induced depositional environments of Little Storfjorden and its tributary, Hambergbukta, over the last 13 ka to better understand the glacial history of southeastern Svalbard. The combined uses of swath-bathymetry, high-resolution seismic stratigraphy, and multiple-proxy measurements of sediment cores allowed us to define five steps of glacier-induced depositional environments: 1) deposition of massive, semi-consolidated gravelly sandy mud (Facies 1) during re-advance or still-stand of the marine-based glaciers/ice streams in Little Storfjorden during Younger Dryas (13–12 ka); 2) deposition of massive mud to gravelly sandy mud (Facies 2A and B) during glacial retreat until the earliest Holocene (12–10.1 ka); 3) sediment winnowing by enhanced bottom currents during the early to middle Holocene (10.1–3.7 ka); 4) deposition of bioturbated sandy mud (Facies 3) with high productivity under seasonal sea ice conditions during the late Holocene (3.7–0.7 ka); and 5) deposition of (slightly) bioturbated sandy to gravelly mud (Facies 4) affected by glacier surges since Little Ice Age (LIA) (Facies 4). In addition to seismic stratigraphy, depositional patterns of IRD in Little Storfjorden indicate that the glacier surges in Hambergbukta occurred only after ∼0.7 ka. This suggests that the terminal moraine complex (TMC) represents the maximum extent of the LIA surges, which argues against the recent inference for the TMC formation during pre-LIA. This study shows the importance of multiple parameters to better understand the current behavior of tidewater glaciers in the Svalbard fjords in response to rapid climate change

Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba

Observation of oxide precipitates in InN nanostructures

We observed the formation of oxide precipitates (bcc-In(2)O(3)) in InN nanostructures formed during metal-organic chemical vapor deposition (MOCVD) and/or subsequent postgrowth procedures in H(2) ambient. It was found that InN is extremely unstable in H(2) ambient and the activation energy of N(2) desorption of InN is measured to be similar to 0.28 eV, which is one order of magnitude smaller than that of reported value of InN in vacuum. Instability of InN nanostructures under H(2) ambient together with residual oxidant in the reactor facilitates the formation of indium oxide precipitates in the nanostructure matrix during MOCVD or the oxidation of residual indium at the surface, resulting in indium oxide dots.open3

Spontaneous Uterine Rupture in the First Trimester: A Case Report

Uterine rupture is one of the most feared obstetric complications affecting the pregnant woman and fetus. Most of the cases have various risk factors and mainly occur during the second or third trimester. However, spontaneous uterine rupture during the first trimester is extremely rare. We experienced a case of spontaneous uterine rupture in a 36-yr-old multiparous woman without definite risk factors. The initial impression was a hemoperitoneum of an unknown origin with normal early pregnancy. Intensive surgical method would be needed for accurate diagnosis and immediate management in bad situation by hemoperitoneum even though a patient was early pregnancy