Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore. Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins. 1-3 Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices. 4,5 They are activated by BH3-only proteins binding to the α2-α5 surface groove, 6-12 or for Bax, to the α1/α6 'rear pocket'. 13 Binding triggers dissociation of the latch domain (α6-α8) from the core domain (α2-α5), together with exposure of N-terminal epitopes and the BH3 domain. 6,7,14-16 The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers. 6,7,14,17,18 In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer. 19 In Bax, the α5 and α6 helices may insert into the MOM, 20 although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface. 7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation. 26,27 However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the...
By disrupting microRNA (miRNA) biogenesis, we previously showed that this pathway is critical for the differentiation and function of T cells. While various cloning studies have shown that many miRNAs are expressed during T cell development, and in a dynamic manner, it was unclear how comprehensive these earlier analyses were. We therefore decided to profile miRNA expression by means of Next Generation Sequencing. Furthermore, we profiled miRNA expression starting from the hematopoietic stem cell. This analysis revealed that miRNA expression during T cell development is extremely dynamic, with 645 miRNAs sequenced, and the expression of some varying by as much as 3 orders of magnitude. Furthermore, changes in precursor processing led to altered mature miRNA sequences. We also analyzed the structures of the primary miRNA transcripts expressed in T cells, and found that many were extremely long. The longest was pri-mir-29b-1/29a at ~168kb. All the long pri-miRNAs also displayed extensive splicing. Our findings indicate that miRNA expression during T cell development is both a highly dynamic and a highly regulated process.
routine protocol and were block randomized by age at the time of blastocyst formation by the embryology team utilizing serially numbered opaque envelopes. The treatment group had trophectoderm biopsy, PGS, and fresh or frozen euploid embryo transfer. The control group underwent fresh or frozen unscreened blastocyst transfer. The primary outcome was time to live birth. Secondary outcomes included clinical loss and ongoing aneuploid pregnancies. Life table analyses and Kaplan-Meier survival curves were employed for the primary outcome and Chi-square analysis for secondary outcomes. RESULTS: There were 181 patients enrolled and 128 underwent randomization with designated outcomes of live birth, completed study protocol, drop out of care, and ongoing treatment. The mean age was 37.4 (AE3.3) in the control group and 37.1 (AE2.9) in the PGS group; mean AMH was 0.59 (AE.28) ng/mL and 0.68 (AE0.41); and mean AFC was 8.2 (AE2.4) and 8.0 (AE2.4) respectively with no statistically significant differences between groups. When time to live birth was analyzed, there were 69 total deliveries, 37 in the control and 32 in the PGS groups. Randomization to PGS resulted in significantly less time to live birth from a mean of 301 days in the control group to 209 days in the PGS group (p¼0.02). There were 18 clinical loses, 5 in the PGS group and 13 in the control group (p¼0.079). This included two instances of termination of pregnancy for a trisomy 18 and a trisomy 13 pregnancy in the control group. CONCLUSIONS: PGS significantly decreased time to live birth by an average of three months in patients with diminished ovarian reserve. Further, PGS appears to have a decreased risk for ongoing aneuploid gestations as there were two terminations required for trisomic gestations in the control group. Offering this embryonic screening paradigm to these patients serves as a way to decrease time to live birth and may decrease the risk of clinical miscarriage and abnormal ongoing gestations.
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