Presentation_1_Contralateral seventh cervical nerve transfer for central spastic arm paralysis: a systematic review and meta-analysis.pdf

ObjectivesThe specific benefits of a contralateral cervical 7 nerve transplant in people with spastic paralysis of the upper extremity caused by cerebral nerve injury are unclear. To evaluate the efficacy and safety of contralateral C7 nerve transfer for central spastic paralysis of the upper extremity, we conducted a comprehensive literature search and meta-analysis.Materials and methodsPRISMA guidelines were used to search the databases for papers comparing the efficacy of contralateral cervical 7 nerve transfer vs. rehabilitation treatment from January 2010 to August 2022. The finishing indications were expressed using SMD ± mean. A meta-analysis was used to assess the recovery of motor function in the paralyzed upper extremity.ResultsThe meta-analysis included three publications. One of the publications offers information about RCTs and non-RCTs. A total of 384 paralyzed patients were included, including 192 who underwent CC7 transfer and 192 who received rehabilitation. Results from all patients were combined and revealed that patients who had CC7 transfer may have regained greater motor function in the Fugl-Meyer score (SMD 3.52, 95% CI = 3.19–3.84, p ConclusionOur findings suggested that a contralateral C7 nerve transfer, which has no additional adverse effects on the healthy upper limb, is a preferable method to restore motor function.</p

Presentation_2_Contralateral seventh cervical nerve transfer for central spastic arm paralysis: a systematic review and meta-analysis.pdf

ObjectivesThe specific benefits of a contralateral cervical 7 nerve transplant in people with spastic paralysis of the upper extremity caused by cerebral nerve injury are unclear. To evaluate the efficacy and safety of contralateral C7 nerve transfer for central spastic paralysis of the upper extremity, we conducted a comprehensive literature search and meta-analysis.Materials and methodsPRISMA guidelines were used to search the databases for papers comparing the efficacy of contralateral cervical 7 nerve transfer vs. rehabilitation treatment from January 2010 to August 2022. The finishing indications were expressed using SMD ± mean. A meta-analysis was used to assess the recovery of motor function in the paralyzed upper extremity.ResultsThe meta-analysis included three publications. One of the publications offers information about RCTs and non-RCTs. A total of 384 paralyzed patients were included, including 192 who underwent CC7 transfer and 192 who received rehabilitation. Results from all patients were combined and revealed that patients who had CC7 transfer may have regained greater motor function in the Fugl-Meyer score (SMD 3.52, 95% CI = 3.19–3.84, p ConclusionOur findings suggested that a contralateral C7 nerve transfer, which has no additional adverse effects on the healthy upper limb, is a preferable method to restore motor function.</p

Cx46G143R enhances gap junctional plaque formation.

<p><i>A)</i> Lysates of HeLa cells expressing exogenous wild-type Cx46 or Cx46G143R were prepared, and the Triton X-100-soluble and insoluble fractions were separated by centrifugation and analyzed by western blotting using anti-Cx46 antibody. <i>B)</i> The ratio of Triton X-100-insoluble fraction to total Cx46 protein was quantified using densitometry. Cx46G143R verses wild-type Cx46, ^**, <i>P</i><0.01. n = 4.</p

The G143R mutation increases the interaction between intracellular loop and Cx46.

<p>Crude membrane extract of cells expressing Cx46 were prepared and equal amount of Cx46 protein, as determined by immunoblotting with anti-Cx46 antibody (upper left panel), were incubated with glutathione beads conjugated with GST, GST-Cx46 loop or GST-Cx46G143R loop fusion proteins or beads alone. The eluted fractions of Cx46 from beads were immunoblotted with anti-Cx46 antibody, then stripped and reblotted with anti-GST antibody (lower left panels). The band intensity was quantified and the ratio of eluted (bound) Cx46 from wild-type and mutant Cx46 loop domains was calculated (right panel). Cx46G143R verses wild-type Cx46, ^**, <i>P</i><0.01. n = 3.</p

Cx46G143R increases hemichannel activity.

<p><i>A)</i> Time lapse recording of Etd⁺ uptake in HeLa cells transfected with Cx46 or Cx46G143R. Cells were washed with recording media with (left panel) or without (right panel) 1.8 mM of CaCl₂ 3 times and then incubated with 25 µM Etd⁺ for 15 min. During this time frame, fluorescence images were taken every 30 sec. LaCl₃, a known connexin hemichannel blocker, was added after 10 min (arrow). <i>B)</i> Etd⁺ dye uptake or <i>C)</i> Alexa350 dye uptake was performed in HeLa cells stably transfected with Cx46 or Cx46G143R. Cells were washed with recording media with (left panel) or without (right panel) 1.8 mM of CaCl₂ 3 times and then incubated with 50 µM mM Etd⁺ for 5 min or Alexa350 for 10 min. The intensity of Etd⁺ or Alexa350 fluorescence was measured and quantified. Untransfected HeLa cells were used as control. In absence or presence of extracellular Ca²⁺, Cx46G143R versus wild-type Cx46, ^*, <i>P</i><0.05; ^**, <i>P</i><0.01; ^***, <i>P</i><0.001. n = 5.</p

Cx46G143R, like wild-type Cx46, is localized at junctional plaques, but inhibits dye coupling in a dominant negative manner.

<p><i>A)</i> HeLa cells were stably-transfected with wild-type Cx46 or Cx46G143R. Crude membrane extracts of parental HeLa cells (control) and stably transfected cells expressing wild-type Cx46 or Cx46G143R mutant were immunoblotted with anti-Cx46, β-actin or GAPDH antibody. <i>B)</i> Cx46 and Cx46G143R-transfected HeLa cells were fixed and labeled by anti-Cx46 antibody and followed by labeling with rhodamine-conjugated anti-rabbit secondary IgG and countered stained with DAPI. Wild-type Cx46 and Cx46G143R are localized at gap junction plaques. <i>C)</i> HeLa cells were untransfected (control) or transfected individually with Cx46, Cx46G143R or co-transfected with a 1∶1 of the plasmids. Forty-eight hr after transfection, cells were microinjected with Alexa Fluor 350 dye (20 mM). The cells were fixed 5 min after microinjection and the degree by dye transfer was quantified. Frequency of dye transfer was measured as the% of injected cells showing dye transfer to at least one neighbor. Untransfected HeLa cells were used as a negative control. Cx46G143R or Cx46G143R plus Cx46 versus Cx46, ^***, <i>P</i><0.001. n = 3 (∼15 cells/each experiment). The ratios of Alexa Fluor 350 fluorescent intensity in each recipient cell compared to the injected (donor) cell were calculated to show the efficiency of transfer by gap junctions, Cx46G143R versus wild-type Cx46,^**, <i>P</i><0.01.</p

Cx46G143R decreases cell surface expression.

<p>HeLa cells stably transfected with Cx46, Cx46G143R, or untransfected controls were labeled with Sulfo-NHS-LC-Biotin. Equal amounts of total protein in cell lysates were mixed with neutravidin-conjugated avidin beads. The total lysate (left panel) and isolated biotinylated (right panel) proteins were analyzed by western blotting using anti-Cx46 antibody. β-actin and GAPDH were used as biotinylation controls. The bands of Cx46 were quantified using densitometry (NIH Image J software). The normalized ratios of biotinylated and total wild-type Cx46 or Cx46G143R were calculated (bottom panel). Cx46G143R versus Cx46, *, <i>P</i><0.05. n = 4.</p

Cx46G143R decreases cell viability and promotes cell death.

<p><i>A)</i> HeLa cells transfected with wild-type Cx46, Cx46G143R or untransfected control were incubated with WST-1 reagent and the absorbance at 450 nm was measured, which indicates the number of viable cells. The absorbance of the solution only containing culture medium and WST-1 reagent was measured as the background control. The absorbance of Cx46G143R versus that of Cx46, ^***, <i>P</i><0.001. n = 5. <i>B)</i> HeLa cells stably transfected with wild-type Cx46, Cx46G143R or non-transfected cells were treated with (right panel) or without (left panel) 0.5 mM H₂O₂. Twelve hr after treatment, cells at 90% confluency were labeled with annexin V and analyzed by flow cytometry to measure the percentage of annexin V positive cells. Cx46G143R versus Cx46, ^*, <i>P</i><0.05, ^**, <i>P</i><0.01. n = 5. <i>C)</i> Sparse cultured cells with minimal cell contacts were treated with (right panel), or without (left panel), 0.5 mM H₂O₂. Flow cytometry was used to measure the percentage of annexin V (upper panels) and PI (lower panels) positive cells. Cx46G143R versus Cx46, ^*, <i>P</i><0.05, ^**, <i>P</i><0.01. n = 3.</p

<i>L</i>. <i>japonicus scarn</i> mutant alleles.

<p><i>L</i>. <i>japonicus scarn</i> mutant alleles.</p

Complementation of <i>scarn-1</i> nodulation phenotype by hairy root transformation.

<p>^a Ratio indicates numbers of successfully transformed plants that formed nodules versus all plants of the indicated line that were successfully transformed with the indicated construct.</p><p>^b Mean nodule number per successfully transformed plant.</p><p>Complementation of <i>scarn-1</i> nodulation phenotype by hairy root transformation.</p