Aggregated chromosomes/chromatin transfer: a novel approach for mitochondrial replacement with minimal mitochondrial carryover: the implications of mouse experiments for human aggregated chromosome transfer.

Nuclear transfer techniques, including spindle chromosome complex (SC) transfer and pronuclear transfer, have been employed to mitigate mitochondrial diseases. Nevertheless, the challenge of mitochondrial DNA (mtDNA) carryover remains unresolved. Previously, we introduced a method for aggregated chromosome (AC) transfer in human subjects, offering a potential solution. However, the subsequent rates of embryonic development have remained unexplored owing to legal limitations in Japan, and animal studies have been hindered by a lack of AC formation in other species. Building upon our success in generating ACs within mouse oocytes via utilization of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (IBMX), this study has established a mouse model for AC transfer. Subsequently, a comparative analysis of embryo development rates and mtDNA carryover between AC transfer and SC transfer was conducted. Additionally, the mitochondrial distribution around SC and AC structures was investigated, revealing that in oocytes at the metaphase II stage, the mitochondria exhibited a relatively concentrated arrangement around the spindle apparatus, while the distribution of mitochondria in AC-formed oocytes appeared to be independent of the AC position. The AC transfer approach produced a marked augmentation in rates of fertilization, embryo cleavage, and blastocyst formation, especially as compared to scenarios without AC transfer in IBMX-treated AC-formed oocytes. No significant disparities in fertilization and embryo development rates were observed between AC and SC transfers. However, relative real-time PCR analyses revealed that the mtDNA carryover for AC transfers was one-tenth and therefore significantly lower than that of SC transfers. This study successfully accomplished nuclear transfers with ACs in mouse oocytes, offering an insight into the potential of AC transfers as a solution to heteroplasmy-related challenges. These findings are promising in terms of future investigation with human oocytes, thus advancing AC transfer as an innovative approach in the field of human nuclear transfer methodology.

Cadmium(II) complexes of (arylazo)imidazoles: synthesis, structure, photochromism, and density functional theory calculation.

Reaction between CdX2 and 1-alkyl-2-(phenylazo)imidazole (RaaiR') has isolated complexes of composition Cd(RaaiR')2X2 in MeOH or MeCN. Crystallization of Cd(RaaiR')2I2 from N,N-dimethylformamide (DMF) has separated [Cd(RaaiR')I2.DMF], while Cd(RaaiR')2X2 (X = Cl and Br) remains unchanged in its composition upon crystallization under identical conditions. The structure has been established by spectral (UV-vis and 1H NMR) data and confirmation in the latter case by a single-crystal X-ray diffraction study of [Cd(TaiMe)I2.DMF] [where TaiMe = 1-methyl-2-(p-tolylazo)imidazole]. UV-light irradiation in a MeCN solution of Cd(RaaiR')2I2 and [Cd(RaaiR')I2.DMF] shows trans-to-cis isomerization of coordinated azoimidazole. The reverse transformation, cis-to-trans, is very slow with visible light irradiation. Quantum yields (phit-->c) of trans-to-cis isomerization are calculated, and the free ligand shows higher phi values than their cadmium(II) iodo complexes. The cis-to-trans isomerization is a thermally induced process. The activation energy (Ea) of cis-to-trans isomerization is calculated by a controlled-temperature experiment. The effects of the anions (Cl-, Br-, I-, and ClO4-) and the number of coordinated azoimidazoles (RaaiR') [Cd(RaaiR') or Cd(RaaiR')2] on the rate and quantum yields of photochromism are established in this work. A slow rate of photoisomerization of [Cd(RaaiR')4](ClO4)2 compared to Cd(RaaiR')I2 or Cd(RaaiR')2X2 may be associated with the increased mass and rotor volume of the complexes. The rate of isomerization is also dependent on the nature of X and follows the sequence Cd(RaaiR')2Cl2 < Cd(RaaiR')2Br2 < Cd(RaaiR')2I2. It may be related to the size and electronegativity of halide, which reduces the effective molar association in the order of I < Br < Cl and hence the rate. Gaussian 03 calculations of representative complexes and free ligands are used to explain the difference in the rates and quantum yields of photoisomerization.

Structural studies and photochromism of mercury(II)-iodo complexes of (arylazo)imidazoles.

Neat reaction between HgI2 and 1-methyl-2-(phenylazo)imidazole (Pai-Me) under microwave irradiation has isolated a novel compound whose structure shows intercalated HgI2 in the layers of Pai-Me. They exist independently in interpenetrated arrays. In a solution phase study, the same reaction has synthesized an iodo-bridged azoimidazole-Hg(II) complex, [Hg(RaaiR')(mu-I)(I)]2 (RaaiR' = 1-alkyl-2-(arylazo)imidazole). The structures have been characterized by X-ray diffraction studies. Chloro-bridged Hg(II) complexes of azoimidazoles, [Hg(RaaiR')(mu-Cl)(Cl)]2, are also known. These complexes upon irradiation with UV light show trans-to-cis isomerization. The reverse transformation, cis-to-trans isomerization, is very slow with visible light irradiation. Quantum yields (phi t-->c) of trans-to-cis isomerization are calculated, and the free ligand shows higher phi than their Hg(II) complexes. The cis-to-trans isomerization is a thermally induced process. The activation energy (Ea) of cis-to-trans isomerization is calculated by controlled temperature reaction. The Ea's of free ligands are much higher than that of halo-bridged Hg(II)-azoimidazole complexes. Chloro-bridged Hg(II) complexes show lower Ea's than those of iodo-bridged complexes. DFT calculation has been adopted to rationalize the experimental results.

The relationship between pregnancy outcome and smooth endoplasmic reticulum clusters in MII human oocytes.

BACKGROUND: During ICSI, we occasionally observe pronucleus sized translucent vacuoles. We investigated why these vacuoles occur and determined the effect on pregnancy outcome. METHODS: Translucent vacuole-positive oocytes and the corresponding cohort were examined by transmission electron microscopy (TEM) and histochemical staining with DiI and ER-Tracker. Stimulation methods, hormonal levels, patients' condition and grade of transferred embryos were compared between vacuole-positive and vacuole-negative cycles. RESULTS: By TEM, we confirmed that the vacuoles were tubular-type smooth endoplasmic reticulum clusters (sERCs). Numerous small sERCs were also observed in the oocytes from the same cohort. Veeck's grades of transferred embryos were higher in sERC-positive cycles and fertilization rate was similar to those of sERC-negative cycles. However, in sERC-positive cycles, significantly lower pregnancy and higher biochemical pregnancy rates were shown. Serum estradiol levels on the day of hCG administration were significantly higher in sERC-positive cycles. CONCLUSIONS: The presence of sERCs is associated with lower chances of successful pregnancy, even in sERC-negative oocytes from the same cohort that are transferred along with the sERC-positive oocytes. High estradiol levels could be one of the causes of sERC formation.

The use of standardized patients within a procedural competency model to teach death disclosure.

OBJECTIVE: To design, implement, and evaluate a multi-dimensional, interdisciplinary, educational training module that enables residents to deliver an effective and empathic death disclosure in the emergency setting. The Accreditation Council for Graduate Medical Education (ACGME) "Toolbox of Assessment Methods" to assess competency was adopted as the foundation of this project. METHODS: Sixteen emergency medicine residents, eight postgraduate year 1 (PGY-1) and eight PGY-2, underwent a one-day training and evaluation exercise. The exercise consisted of: 1) a large-group didactic session, 2) a small-group didactic session, and 3) two standardized patient (SP) examinations. Changes in comfort levels, training helpfulness, and competency were measured. Inter-rater agreement between evaluators was examined. RESULTS: Trainees reported improvement in comfort levels and high levels of satisfaction regarding the helpfulness of the training. Good interrater agreement was obtained regarding resident competency to perform a death disclosure between the faculty and SP evaluators [kappa 0.61; 95% confidence interval (95% CI) = 0.33 to 0.88]. However, overall agreement among raters was poor (kappa 0.16; standard error = 0.26). This poor agreement reflected a lack of agreement between resident and SP evaluators (kappa 0.08; 95% CI = 0.16 to 0.33) and resident and faculty evaluators (kappa -0.02; 95% CI = 0.30 to 0.26). CONCLUSIONS: This project used the ACGME "Toolbox of Assessment Methods" to evaluate the competency of emergency medicine trainees to perform an effective and empathic death disclosure. The finding of inconsistent competency assessments by resident self-evaluators compared with those assessments made by faculty and standardized patients have important implications in future curricular design.

Skin Necrosis Associated With Heparin-Induced Thrombocytopenia and Thrombosis.

Skin necrosis is a rare complication of heparin therapy. Strong evidence suggests an immune-mediated mechanism in which heparin-antibody complexes bind to platelets, resulting in platelet aggregation, thromboembolism, and ischemic necrosis. Heparin-induced thrombocytopenia (HIT) may also occur in response to immune-mediated platelet aggregation. The presence, of heparin-dependent antibodies can be confirmed by platelet aggregometry, (14)C-serotonin release assay (SRA), or enzyme-linked immunosorbent assay (ELISA). Clinical suspicion, early detection and immediate cessation of heparin therapy are important in preventing the potentially severe complications of heparin-induced platelet aggregation. Potential therapeutic approaches include plasmapheresis and alternative forms of anticoagulation such as warfarin, aspirin, dipyridamole, or other novel investigational agents.

Neurologically intact survivor of prolonged ventricular fibrillation: a case for intermediate dose epinephrine and postresuscitation infusion.

The authors point out that our understanding of the optimal dose of epinephrine used in the resuscitation of patients in cardiac arrest continues to evolve. Doses greater than the standard 1 mg of epinephrine every 5 minutes have been studied and shown to increase the rate of return of spontaneous circulation. However, reports of neurologically intact survivors of prolonged cardiac arrest are rare. The authors report a neurologically intact survivor of prolonged ventricular fibrillation with severe mixed acidosis who responded to intermediate doses of epinephrine and epinephrine infusion, where standard amounts had failed. Further research should be directed into the relationship between postresuscitation epinephrine infusions and neurologic outcome.

The use of 3H serotonin release from mast cells of the mouse as an assay for mediator liberation.

The release of 3H 5-HT from murine mast cells is shown to be a simple reproducible method for studying the activation of such cells by various agents. 3H-serotonin was taken up by peritoneal cell suspensions in vitro and was released by antigen, Concanavalin A, Forssman antiserum, anti-mouse immunoglobulin, or a polypeptide antibiotic, Cinnamycin.