Additional file 1: Figure S1. of Endophilin A2 promotes HER2 internalization and sensitivity to trastuzumab-based therapy in HER2-positive breast cancers

Endo II expression and association with risk of metastasis and chemotherapy response in HER2+ breast cancers. Figure S2. Endo II silencing in SK-BR-3 cells. Figure S3. Endo II overexpression leads to reduced surface HER2. Figure S4. Endo II promotes EGF-induced Akt activation in SK-BR-3 cells. Figure S5. Endo II promotes HER2 internalization in HCC1954 cells. Figure S6. Endo II promotes SK-BR-3 cell motility. Figure S7. Endo II silencing has no overt effects on HER2+ tumor growth. Figure S8. Endo II promotes HER2 internalization and downregulation following trastuzumab treatment of HCC1954 cells. Figure S9. Endo II promotes cytoxicity upon treatment with trastuzumab, but not with lapatinib, in HER2+ cancer cells. Figure S10. Endo II overexpression results in increased sensitivity to T-DM1. (DOCX 1965 kb

Hypoxia–ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia–ischemia. This relatively brief period of hypoxia–ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13±0.25 counts/µm2), and declined significantly during hypoxia–ischemia (0.93±0.15 counts/µm2: P0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia–ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90±0.20 counts/µm2), and ependymal cells (2.20±0.28 counts/µm2), and hypoxia–ischemia produced a large fall in ependymal glutamate (0.97±0.08 counts/µm2: P>0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia

The average relative quantities of mtDNA observed in association with female age at the cleavage stage.

<p>The mtDNA values (2-^{Delta Delta Ct}) were obtained during real-time PCR analysis. All examined blastomeres were characterised as being chromosomally normal.</p><p>The average relative quantities of mtDNA observed in association with female age at the cleavage stage.</p

The average relative quantities of mtDNA observed in association to female age and blastocyst chromosome status.

<p>The mtDNA values (2-^{Delta Delta Ct}) were obtained during real-time PCR analysis.</p><p>The average relative quantities of mtDNA observed in association to female age and blastocyst chromosome status.</p

mtDNA quantities and clinical outcomes of 23 TE samples assessed via real-time PCR and NGS.

<p>*The threshold for considering a sample to have elevated mtDNA levels, incompatible with implantation, was 0.003 for real-time PCR and 0.07 for NGS.</p><p>mtDNA quantities and clinical outcomes of 23 TE samples assessed via real-time PCR and NGS.</p

The relationship between mtDNA quantity, female age and embryo chromosome constitution.

<p><b>a)</b> Data obtained during quantitative real-time PCR analysis of TE samples removed from 302 blastocysts demonstrated a statistically significant increase (P = 0.003) in the level of mtDNA in relation to advancing female age. This phenomenon was evident for both euploid and aneuploid blastocysts. <b>b)</b> Real-time PCR analysis of 39 blastomeres showed that cleavage stage embryos from reproductively younger women contained significantly (P = 0.01) higher mtDNA levels, compared to those generated by reproductively older women. <b>c)</b> Real-time PCR analysis of TE samples also demonstrated that aneuploid blastocysts (n = 99) contained significantly (P = 0.025) larger quantities of mtDNA at all ages, compared to those that were euploid (n = 203). Statistical analysis of mtDNA values took place with the use of unpaired two-tailed t-tests.</p

The mtDNA content of chromosomally normal blastocysts in relation to clinical outcome.

<p>On average, chromosomally normal blastocysts capable of establishing a clinical pregnancy contained significantly (P = 0.007) lower levels of mtDNA compared to chromosomally normal blastocysts that failed to do so.</p

mtDNA quantification via NGS analysis of chromosomally normal and abnormal blastocysts.

<p>NGS analysis of TE samples biopsied from 38 embryos showed a statistically significant increase (P = 0.006) in the mtDNA levels occurring in the presence of chromosome errors.</p

Blastocyst mtDNA quantity threshold in relation to clinical outcome.

<p><b>a)</b> The mtDNA quantity viability threshold for euploid blastocysts, established via retrospective analysis of TE biopsies from transferred embryos with known outcomes. All blastocysts producing viable pregnancies contained mtDNA quantities below the 0.003 value (red line) whereas mtDNA quantities above this value were associated with failure to achieve an ongoing clinical pregnancy. <b>b)</b> Results of the prospective blinded study. The mtDNA threshold used was the same as that established in the retrospective study (4a). Validity was confirmed, since all blastocysts producing viable pregnancies contained mtDNA quantities below the cut-off (red line) and no blastocysts with mtDNA quantities above this value achieved an ongoing clinical pregnancy. <b>c)</b> NGS analysis of the mtDNA level in 23 euploid TE samples. The corresponding embryos were transferred during SET cycles, and clinical outcomes were known for 21 of them. As with the real-time PCR experiments, mtDNA levels were lower in the seven implanting embryos (note- the y-axis scale is different for NGS analyses and consequently cut-off values differ).</p

Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes

KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel