In muscle and other mechanically active tissue, cell membranes are constantly injured and their repair depends on the injury induced increase in cytosolic calcium. Here we show that injury-triggered Ca2+ increase results in assembly of ESCRTIII and accessory proteins at the site of repair. This process is initiated by the calcium binding protein - Apoptosis Linked Gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2 interacting protein X (ALIX), ESCRT III, and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX, or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca2+-dependent accumulation of ESCRTIII-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRTIII-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.
Objectives: Convalescent plasma (CP) as a passive source of neutralizing antibodies and immunomodulators is a century-old therapeutic option used for the management of viral diseases. We investigated its effectiveness for the treatment of COVID-19.
Design: Open-label, parallel-arm, phase II, multicentre, randomized controlled trial.
Setting: Thirty-nine public and private hospitals across India.
Participants: Hospitalized, moderately ill confirmed COVID-19 patients (PaO2/FiO2: 200-300 or respiratory rate > 24/min and SpO2 ≤ 93% on room air).
Intervention: Participants were randomized to either control (best standard of care (BSC)) or intervention (CP + BSC) arm. Two doses of 200 mL CP was transfused 24 hours apart in the intervention arm.
Main Outcome Measure: Composite of progression to severe disease (PaO2/FiO2<100) or all-cause mortality at 28 days post-enrolment.
Results: Between 22 nd April to 14 th July 2020, 464 participants were enrolled; 235 and 229 in intervention and control arm, respectively. Composite primary outcome was achieved in 44 (18.7%) participants in the intervention arm and 41 (17.9%) in the control arm [aOR: 1.09; 95%
CI: 0.67, 1.77]. Mortality was documented in 34 (13.6%) and 31 (14.6%) participants in intervention and control arm, respectively [aOR) 1.06 95% CI: -0.61 to 1.83].
Interpretation: CP was not associated with reduction in mortality or progression to severe COVID-19. This trial has high generalizability and approximates real-life setting of CP therapy in settings with limited laboratory capacity. A priori measurement of neutralizing antibody titres
in donors and participants may further clarify the role of CP in management of COVID-19.
Background: Cellular processes involved in healing injured skeletal muscle fibers are poorly understood. Results: Using an improved quantitative membrane proteomics approach for cells and tissues, we have identified accumulation of mitochondria at the site of sarcolemma injury as a key requirement for myofiber healing. Conclusion: Mitochondria are the earliest responders to myofiber injury. Significance: This work identifies a novel function of mitochondria in muscle injury.
A major role in the regulation of eukaryotic protein-coding genes is played by the gene-specific transcriptional regulators, which recruit the RNA polymerase II holoenzyme to the specific promoter. Several components of the mediator complex within the holoenzyme also have been shown to affect activation of different subsets of genes. Only recently has it been suggested that besides the largest subunit of RNA polymerase II, smaller subunits like Rpb3 and Rpb5 may have regulatory roles in expression of specific sets of genes. We report here, the role of Rpb4, a non-essential subunit of core RNA polymerase II, in activation of a subset of genes in Saccharomyces cerevisiae. We have shown below that whereas constitutive transcription is largely unaffected, activation from various promoters tested is severely compromised in the absence of RPB4. This activation defect can be rescued by the overexpression of cognate activators. We have localized the region of Rpb4 involved in activation to the C-terminal 24 amino acids. We have also shown here that transcriptional activation by artificial recruitment of the TATA-binding protein (TBP) to the promoter is also defective in the absence of RPB4. Surprisingly, the overexpression of RPB7 (the interacting partner of Rpb4) does not rescue the activation defect of all the promoters tested, although it rescues the activation defect of the heat shock element-containing promoter and the temperature sensitivity associated with RPB4 deletion. Overall, our results indicate that Rpb4 and Rpb7 play independent roles in transcriptional regulation of genes.
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