Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells.

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide's ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.

Enhancement of Collagen-I Levels in Human Gingival Fibroblasts by Small Molecule Activation of HIF-1α.

Collagen is the most abundant protein in various mammalian tissues and has an essential role in various cellular processes. Collagen is necessary for food-related biotechnological applications such as cultivated meat, medical engineering, and cosmetics. High-yield expression of natural collagen from mammalian cells is challenging and not cost-effective. Thus, external collagen is obtained primarily from animal tissues. Under cellular hypoxia, overactivation of the transcription factor hypoxia-inducible factor (HIF) was shown to correlate with enhanced accumulation of collagen. Herein, we showed that the small molecule ML228, a known molecular activator of HIF, enhances the accumulation of collagen type-I in human fibroblast cells. We report an increase in collagen levels by 2.33 ± 0.33 when fibroblasts were incubated with 5 µM of ML228. Our experimental results demonstrated, for the first time, that external modulation of the hypoxia biological pathway can boost collagen levels in mammalian cells. Our findings pave the way for enhancing natural collagen production in mammals by altering cellular signaling pathways.

Funneling modulatory peptide design with generative models: Discovery and characterization of disruptors of calcineurin protein-protein interactions.

Design of peptide binders is an attractive strategy for targeting "undruggable" protein-protein interfaces. Current design protocols rely on the extraction of an initial sequence from one known protein interactor of the target protein, followed by in-silico or in-vitro mutagenesis-based optimization of its binding affinity. Wet lab protocols can explore only a minor portion of the vast sequence space and cannot efficiently screen for other desirable properties such as high specificity and low toxicity, while in-silico design requires intensive computational resources and often relies on simplified binding models. Yet, for a multivalent protein target, dozens to hundreds of natural protein partners already exist in the cellular environment. Here, we describe a peptide design protocol that harnesses this diversity via a machine learning generative model. After identifying putative natural binding fragments by literature and homology search, a compositional Restricted Boltzmann Machine is trained and sampled to yield hundreds of diverse candidate peptides. The latter are further filtered via flexible molecular docking and an in-vitro microchip-based binding assay. We validate and test our protocol on calcineurin, a calcium-dependent protein phosphatase involved in various cellular pathways in health and disease. In a single screening round, we identified multiple 16-length peptides with up to six mutations from their closest natural sequence that successfully interfere with the binding of calcineurin to its substrates. In summary, integrating protein interaction and sequence databases, generative modeling, molecular docking and interaction assays enables the discovery of novel protein-protein interaction modulators.

Hyperbaric oxygen therapy improves neurocognitive functions and symptoms of post-COVID condition: randomized controlled trial.

Post-COVID-19 condition refers to a range of persisting physical, neurocognitive, and neuropsychological symptoms after SARS-CoV-2 infection. The mechanism can be related to brain tissue pathology caused by virus invasion or indirectly by neuroinflammation and hypercoagulability. This randomized, sham-control, double blind trial evaluated the effect of hyperbaric oxygen therapy (HBOT or HBO2 therapy) on post-COVID-19 patients with ongoing symptoms for at least 3 months after confirmed infection. Seventy-three patients were randomized to receive daily 40 session of HBOT (n = 37) or sham (n = 36). Follow-up assessments were performed at baseline and 1-3 weeks after the last treatment session. Following HBOT, there was a significant group-by-time interaction in global cognitive function, attention and executive function (d = 0.495, p = 0.038; d = 0.477, p = 0.04 and d = 0.463, p = 0.05 respectively). Significant improvement was also demonstrated in the energy domain (d = 0.522, p = 0.029), sleep (d = - 0.48, p = 0.042), psychiatric symptoms (d = 0.636, p = 0.008), and pain interference (d = 0.737, p = 0.001). Clinical outcomes were associated with significant improvement in brain MRI perfusion and microstructural changes in the supramarginal gyrus, left supplementary motor area, right insula, left frontal precentral gyrus, right middle frontal gyrus, and superior corona radiate. These results indicate that HBOT can induce neuroplasticity and improve cognitive, psychiatric, fatigue, sleep and pain symptoms of patients suffering from post-COVID-19 condition. HBOT's beneficial effect may be attributed to increased brain perfusion and neuroplasticity in regions associated with cognitive and emotional roles.

Effects of Hyperbaric Oxygen Therapy on Mitochondrial Respiration and Physical Performance in Middle-Aged Athletes: A Blinded, Randomized Controlled Trial.

INTRODUCTION: Hyperbaric oxygen therapy (HBOT) has been used to increase endurance performance but has yet to be evaluated in placebo-controlled clinical trials. The current study aimed to evaluate the effect of an intermittent HBOT protocol on maximal physical performance and mitochondrial function in middle-aged master athletes. METHODS: A double-blind, randomized, placebo-controlled study on 37 healthy middle-aged (40-50) master athletes was performed between 2018 and 2020. The subjects were exposed to 40 repeated sessions of either HBOT [two absolute atmospheres (ATA), breathing 100% oxygen for 1 h] or SHAM (1.02ATA, breathing air for 1 h). RESULTS: Out of 37 athletes, 16 HBOT and 15 SHAM allocated athletes were included in the final analysis. Following HBOT, there was a significant increase in the maximal oxygen consumption (VO2Max) (p = 0.010, effect size(es) = 0.989) and in the oxygen consumption measured at the anaerobic threshold (VO2AT)(es = 0.837) compared to the SHAM group. Following HBOT, there were significant increases in both maximal oxygen phosphorylation capacity (es = 1.085, p = 0.04), maximal uncoupled capacity (es = 0.956, p = 0.02) and mitochondrial mass marker MTG (p = 0.0002) compared to the SHAM sessions. CONCLUSION: HBOT enhances physical performance in healthy middle-age master athletes, including VO2max, power and VO2AT. The mechanisms may be related to significant improvements in mitochondrial respiration and increased mitochondrial mass. Trial Registration ClinicalTrials.gov Identifier: https://clinicaltrials.gov/ct2/show/NCT03524989 (May 15, 2018).

Human mesenchymal stromal cells ameliorate complement induced inflammatory cascade and improve renal functions in a rat model of ischemia-reperfusion induced acute kidney injury.

INTRODUCTION: The primary rational for using mesenchymal stromal cells (MSCs) to rejuvenate damaged tissue is mostly based on their capacity to trans-differentiate and repair injured organs. However, previous studies have demonstrated that MSCs are beneficial even at very early stages, before differentiation and proliferation can be expected. The aim of the current study was to investigate the multifaceted immunological effects of systemically administrating MSCs in the setting of acute kidney injury (AKI) induced by ischemic-reperfusion (I/R). METHODS: A rat model of I/R induced AKI was used. The rats underwent a unilateral nephrectomy with simultaneously clamping the contralateral kidney for 60 minutes. Four treatment groups received intravenously, increasing doses of human MSCs and after 48 hours, the rats were sacrificed. Blood was taken to evaluate renal functions and to measure systemic inflammatory markers. Kidneys were taken for histopathologic examinations and evaluations of intra-renal complement activation and inflammatory mediators. RESULTS: Renal functions improved in U shaped dose dependent manner. Mean serum creatinine levels were 4.5, 2.9, 2.6, 1.7 and 4.1 mg/dL in I/R + placebo, I/R + 150x103 cells, I/R + 250x103 cells, I/R + 500x103 cells and I/R + 1,000x103 cells respectfully (p-values<0.05). Urea demonstrated consistent results with the same U shape improvement manner. The extensive activation of the complement system was ameliorated in the MSCs treatment groups. In addition, MSCs significantly decreased intra-renal levels of IL-1ß and TNF-α. It should be noted that the highest doses of MSCs induced renal hypoxia, marked by the Hypoxy-probe staining. CONCLUSIONS: The early beneficial effect of MSCs in the setting of AKI may be attributed to their immunomodulatory effects. Safe treatment with MSCs can block the deleterious activation of the complement cascade and alleviate the hazardous inflammatory mediator-related cascade.