Video_1_Cues to Opening Mechanisms From in Silico Electric Field Excitation of Cx26 Hemichannel and in Vitro Mutagenesis Studies in HeLa Transfectans.AVI

<p>Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family.</p

Image_1_Cues to Opening Mechanisms From in Silico Electric Field Excitation of Cx26 Hemichannel and in Vitro Mutagenesis Studies in HeLa Transfectans.PNG

<p>Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family.</p

Comparison of (A) HCMV-specific and (B) total T-cells/µl in group 4 non-protected patients <b><i>vs</i></b><b> groups 1+2+3 protected patients.</b>

<p>While both total and HCMV-specific CD4⁺ T-cells are significantly higher in protected patients, no difference is observed between protected and non-protected patients for both total and specific CD8⁺ T-cells.</p

Virological and immunological monitoring of the four groups of solid-organ transplant recipients with or without HCMV infection reactivation.

<p>NA, not applicable.</p>1<p>Follow-up.</p>2<p>Patients of this group showed HCMV specific CD8⁺ only, until about 3 months after transplantation, then developed HCMV-specific CD4⁺ T-cells.</p>3<p>In some cases, clinicians preferred initiating antiviral therapy after reaching 100,000 (instead of 300,000) DNA copies/ml blood due to presence of end-organ disease.</p><p>Virological and immunological monitoring of the four groups of solid-organ transplant recipients with or without HCMV infection reactivation.</p

Kinetics of absolute numbers/µl blood of total and HCMV-specific CD4⁺ and CD8⁺ T-cells in four SOTR patients (each representative of one of the four patient groups).

<p>Patient A (group 1): no HCMV infection (no viral DNA) is detected and HCMV-specific CD4⁺ and CD8⁺ T-cells are consistently above the cut-off (black dotted line corresponding to 0.4 T-cells/µl blood); Patient B (group 2): self-resolving infection in the presence of low viral load and specific CD4⁺ and CD8⁺ T-cells consistently above the cut-off; Patient C (group 3): self-resolving infection in the presence of a high viral load peak and a number of HCMV-specific CD8⁺ T-cells above the cut-off, but in the absence of specific CD4⁺ T-cells or in the presence of CD4⁺ T-cells at a level close to the cut-off for the first two-three months after transplantation; Patient D (group 4): uncontrolled infection in the presence of high viral load above the cut-off (requiring antiviral treatment) and absence of specific CD4⁺ T-cells until 12 months after transplantation. The dashed line indicates the cut-off of viral load to start preemptive therapy. KTR, kidney transplant recipient; HTR, heart transplant recipient; VGCV, valganciclovir.</p

Polyfunctional hierarchy of HCMV-specific CD4⁺ T-cells in groups 1 and 2 SOTR.

<p>Predominance of polyfunctional vs monofunctional CD4⁺ T-cells in healthy controls and in protected patients of groups 1+2.</p

Kinetics of median levels of polyfunctional HCMV-specific CD8⁺ T-cells in groups 1–4 SOTR.

<p>No consistent statistically significant difference was observed between the four patient groups.</p

Kinetics of median levels of (A) total and (B) HCMV-specific CD4⁺ T-cells/µl blood in SOTR.

<p>A significantly higher number of HCMV-specific CD4⁺ T-cells was found in groups 1 and 2 <i>vs</i> groups 3 and 4 at 30 and 60 days after transplantation, and in groups 1 and 2 <i>vs</i> group 4 at 90 days after transplantation. *, P<0.05; **, P<0.01; ***, P<0.001.</p

Kinetics of median levels of (A) total and (B) HCMV-specific CD8⁺ T-cells in SOTR.

<p>No significant difference was found in the number/µl blood of HCMV-specific CD8⁺ T-cells among the different groups at any time post-transplant.</p

Kinetics of median levels of (A) Vδ2⁻ γδ T-cells and (B) Vδ2⁻/Vδ2⁺ γδ T-cell ratio.

<p>No significant difference was observed among groups in the number of Vδ2⁻ γδ T-cells. The Vδ2<b>⁻</b>/Vδ2⁺ γδ T-cell ratio was significantly higher in group 4 than in groups 1 and 3 at days 60 and 360. Stars above columns indicate significant differences among transplanted patient groups: *, P<0.05.</p