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Diabetes Complicat. Hemopoiesis in human fetal and embryonic liver. Universities offer Computer Science degrees which give you a strong basis of knowledge if you wish to become a programmer . Retrieved January 10, Importantly, we observed a substantial long-term postnatal survival advantage Betta the in utero treated animals versus untreated controls, highlighting the potential for clinical translation of our work.
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Additional data and movies are available in the supplementary material. Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality.
Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders.
Every year, an estimated 8 million children are born worldwide with severe genetic disorders or birth defects. Recent advances in non—invasive genetic testing allow for diagnosis of genetic disorders such as thalassemia early in gestation 2 , providing a window during which genetic correction could be pursued prior to birth.
In utero gene therapy thus far has focused on stem-cell transplantation and viral-mediated gene delivery [reviewed in ref. Considerable advances in gene therapy approaches have occurred, but they still face challenges associated with the use of viruses and with the risk of ectopic integration into deleterious sites in the genome, issues of particular concern for a developing fetus. As an alternative, our group has recently shown that gene correction can be coordinated efficiently and safely in postnatal animals via the intravenous or inhalational administration of polymeric, biodegradable nanoparticles NPs loaded with triplex-forming peptide nucleic acids PNAs and single-stranded donor DNAs 5 — 7.
This process is, in part, dependent on the nucleotide excision repair and homology dependent repair pathways 10 , 13 [reviewed in ref. We find that NPs can be delivered to multiple fetal mouse tissues intravenously, with the most pronounced accumulation in the fetal liver, the site of fetal hematopoiesis.
In contrast, intra-amniotic NP delivery results in preferential NP accumulation in the fetal lung and gut at gestational ages later than 15 days. We find that both delivery approaches are minimally invasive and do not hinder fetal development, long-term survival, or reproductive potential.
Importantly, we observed a substantial long-term postnatal survival advantage for the in utero treated animals versus untreated controls, highlighting the potential for clinical translation of our work. Fetal surgeons and maternal fetal medicine physicians can safely access the amniotic cavity for amniocentesis and cannulate umbilical vessels for fetal blood transfusions under ultrasound guidance as early as 18 weeks of gestation in humans 20 , We hypothesized that similar techniques could be used to introduce NPs safely in utero.
Fluorescent NPs were administered to fetal B6 mice either intravenously via the vitelline vein, as a proxy for human umbilical vein transfusion, or directly into the amniotic cavity at gestational ages later than E15 Fig.
Administration of NPs to fetal mice results in particle retention within the fetuses with no detectable particle accumulation in the maternal mouse Fig. As a positive control, fluorescent NPs were directly administered to the maternal circulation of a mouse pregnant with fetuses at E Substantial accumulation of NPs in the fetal liver is expected during development because the extraembryonic vitelline veins anastomose to form the portal circulation.
During physiologic mammalian fetal development, the fetus breaths amniotic fluid into and out of the developing lungs, providing the necessary forces to direct lung development and growth Developing fetuses also swallow amniotic fluid, which aids the formation of the gastrointestinal tract Thus, introduction of NPs into the amniotic fluid at gestational ages after the onset of fetal breathing and swallowing could result in their direct delivery to the respiratory and gastrointestinal tracts, respectively.
However, IA injection at E NP accumulation in the lung and gut was also observed after IA injections at E These NPs showed a spherical shape with sizes and zeta potentials Supplementary Fig. We chose to focus first on wild-type mice to assess the impact of the NPs on growth and survival in the absence of disease. We found no significant differences in the survival of pups to weaning between those treated in utero with NPs, either intravenously or intra-amniotically, compared to pups that received sham surgery, but no in utero NP treatment, which we refer to as untreated mice Fig.
For the mice that lived to weaning, we observed no significant differences in the long-term survival between untreated mice and those that received NP treatment in utero Fig.
After pups were born and matured, we also observed no significant differences between untreated mice and those that received in utero NP treatment with respect to growth patterns or body weights Fig. There were no significant increases in levels of any of the proinflammatory cytokines measured in the NP treated groups compared to untreated fetuses Fig.
For reference, administration of a small dose of lipopolysaccharide LPS as a positive control elicits a response in which inflammatory cytokines are elevated over fold 6. Safety of in utero nanoparticle NP delivery. As in the human hematopoietic system 32 , murine hematopoietic stem cells HSCs , the target cells for gene correction in thalassemia, first emerge in the para-aortic splanchnopleure after the 7 th day of gestation, E7.
HSCs that develop in these tissues do not differentiate, but go on to seed the fetal liver on E Within the fetal liver, HSCs undergo a massive expansion before further seeding the spleen, thymus, and finally the bone marrow at the beginning of E The rapid cycling and expansion of HSCs in the fetal liver contrasts with adult bone marrow HSCs, which are largely quiescent Because our prior work identified increased DNA repair and gene editing in activated stem cells 5 , 6 , we hypothesized that the ability to target this rapidly dividing and expanding stem cell population in the fetal liver might represent an important therapeutic opportunity for gene editing.
Given our observation that NPs injected in the vitelline vein on day E The resulting pups were genotyped prior to weaning. The blood hemoglobin concentrations of treated heterozygous mice were measured at six and ten weeks of age. The sustained elevation in postnatal hemoglobin was accompanied by a clear improvement in red blood cell RBC morphology on peripheral blood smear Fig. The observed reduction in splenomegaly correlated with improved splenic architecture in treated mice, with prominently defined red and white pulp, similar to that normally seen in wild-type Fig.
Decreased immunostaining for CD44 stains erythroid precursors and staining decreases during terminal erythroid differentiation, but is not specific 39 , 40 , CD71 specifically stains early erythroblasts 39 , 41 , E-cadherin immature erythroid precursors 42 and CD61 megakaryocytes in treated compared to untreated mice additionally suggests a reduction in extramedullary hematopoiesis Supplementary Figs.
Taken together, the reduction of splenomegaly and improvement in splenic histology pattern in treated mice further indicates alleviation of anemia. In addition to elevated hemoglobin levels, improved RBC morphology, and reduction of splenomegaly, we also found significantly reduced reticulocyte counts in the peripheral blood of treated mice Fig.
Importantly, the mice treated with NPs in utero also showed a significant postnatal survival advantage compared to untreated controls. To quantify the extent of gene editing achieved in the mice that leads to these observed postnatal improvements in anemia, we performed deep sequencing analysis on genomic DNA extracted from total bone marrow of postnatal mice at a 15 week time-point counting from the day of the in utero NP treatment. Genomic DNA from total bone marrow cells 15 weeks post NP treatment was subject to deep sequencing analysis at these loci of partial homology as well as the target locus.
The size of the region sequenced around each site, the total number of amplicons sequenced and the number of amplicons with modified sequences are listed. In these cells, we observed 8. Deep sequencing analysis of E The total number of amplicons sequenced and the number of amplicons with modified sequences are listed.
Genetic correction during pregnancy could provide treatment or cure of genetic diseases and allow normal fetal development, a possible advantage compared to treatments given after birth. By delivering gene-editing therapies in utero, it is possible to gain access to dividing stem and progenitor cell populations, which can result in propagation of the corrected gene in all progeny cells.
We observed increased hemoglobin concentrations into the wild-type range, improved red cell morphology, reduced reticulocyte counts, and decreased extramedullary hematopoiesis. In addition, we found that in utero gene editing conferred a significant survival advantage on the treated mice compared to untreated controls.
We also found that intra-amniotic administration results in nanoparticle distribution to the fetal lung and gut at gestational ages later than E The distribution of particles at later time-points merits further study and may reveal that the tissue distribution shifts over time, which could open the possibility of editing other developing tissues of interest, such as the fetal brain, which might require particles with longer release profiles.
In this regard, altering the polymer composition, size, or surface modifications could also be investigated as means of changing the distribution or nucleic acid release profile of nanoparticles, as we have shown in other studies 44 — We elected to deliver these molecules using PLGA, a biodegradable and biocompatible polymer that is known to be safe in humans.
In prior work, clinically relevant levels of gene editing were achieved in adult mice after multiple doses of nanoparticles were administered in combination with stem cell factor SCF. During fetal development, however, it is well known that SCF is highly expressed at sites of hematopoiesis, including the yolk sac, fetal liver, and bone marrow 48 — High levels of SCF within the fetal liver may create an environment that is particularly amenable to gene editing using our approach.
These improvements suggest there may be an advantage to gene editing in the fetus because it is possible to access a rapidly cycling population of HSCs within in the fetal liver.
The fact that gene editing at these frequencies could yield a discernable phenotypic improvement is consistent with transplantation studies in thalassemic mice and humans, in which low numbers of engrafted donor cells are sufficient to correct anemia 53 — This finding is attributed to a positive in vivo selection of genetically corrected erythroblasts Similarly, others have observed a selective advantage of corrected hematopoietic progenitor cells in patients with severe combined immunodeficiency SCID -X1 disease who received viral-mediated gene therapy 59 — The relatively short gestation of mice limited us to delivering just one treatment of nanoparticles.
Due to the low toxicity of one dose, we speculate that multiple treatments should be possible in humans or mammals with longer gestational periods, which may result in higher gene editing frequencies. For instance, during human fetal development the liver is the main site of hematopoiesis and HSC cycling from about 6 to 22 weeks of gestation 62 , which could allow for weeks of access to the fetal liver HSCs via umbilical vessel cannulation. Treatment at later time points, when the HSCs primarily reside within the fetal BM, could also be therapeutic since the HSCs are still rapidly cycling [reviewed in 63 ].
Additionally, PNA-modifications that enhance target binding such as guanidine-G-clamp PNA monomers 64 , could be used to further improve gene-editing efficiencies. In utero gene editing may hold great promise for treating or curing numerous inherited human genetic diseases. Success in future investigations could provide a compelling rationale for clinical application.
The sample sizes of experiments were selected on the basis of previous experience. Data collection was stopped at a priori defined points for all experiments. For all in utero treatment experiments, animals were randomly assigned to IV, IA, or control treatment groups in a blinded manner.
All sample measurements were blinded. The number of replicates for each experiment is stated in each figure legend. At each step, the respective product was purified by column chromatography PNA oligomers were synthesized on solid support using Boc chemistry The oligomers were synthesized on MBHA 4-methylbenzhydrylamine resin according to standard procedures of Boc chemistry. A kaiser test was performed at each step to measure complete coupling and double coupling was performed if it was required.
The single-stranded donor DNA oligomer was prepared by standard DNA synthesis except for the inclusion of three phosphorothioate internucleoside linkages at each end to protect against nuclease degradation Midland Certified Reagent Company; Midland, TX. PLGA ester-terminated, 0. C6 or DiD was added to the polymer solution at a 0. To form the second emulsion, the first emulsion was added slowly dropwise to 1. Litters from this mouse model were genotyped prior to weaning.
Only heterozygous mice were included in this study. The gravid uterus was exposed through a midline laparotomy incision. Intravascular injections were performed at E Intra-amniotic injections were performed at E Fetuses were delivered via cesarean section and washed in PBS.
Ex vivo fetal fluorescence stereomicrope imaging was performed on a Leica M80 stereomicroscope Wetzlar, Germany. Untreated pregnant mice were anesthetized and the gravid uterus was exposed as described above.
The fetuses were counted, the uterus was returned to the abdomen, and the midline incision was closed. The number of untreated, intravenously and intra-amniotically treated pups surviving was counted at the time of weaning, 21 days.