Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2-treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. https://www.selleckchem.com/products/plx5622.html In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).Severe acute pancreatitis (AP) is a life-threatening disease with up to 30% mortality. Therefore, prevention of AP aggravation and promotion of pancreatic regeneration are critical during the course and treatment of AP. Hypertriglyceridemia (HTG) is an established aggravating factor for AP that hinders pancreatic regeneration; however, its exact mechanism remains unclear. Using miRNA sequencing and further verification, we found that miRNA-153 (miR-153) was upregulated in the pancreas of HTG animal models and in the plasma of patients with HTG-AP. Increased miR-153 aggravated HTG-AP and delayed pancreatic repair via targeting TRAF3. Furthermore, miR-153 was transcriptionally suppressed by sterol regulatory element-binding transcription factor 1c (SREBP1c), which was suppressed by lipoprotein lipase malfunction-induced HTG. Overexpressing SREBP1c suppressed miR-153 expression, alleviated the severity of AP, and facilitated tissue regeneration in vivo. Finally, therapeutic administration of insulin also protected against HTG-AP via upregulating SREBP1c. Collectively, our results not only provide evidence that HTG leads to the development of more severe AP and hinders pancreatic regeneration via inducing persistent dysregulation of SREBP1c/miR-153 signaling, but also demonstrate that SREBP1c activators, including insulin, might be used to treat HTG-AP in patients.Pneumocystis is an important opportunistic fungus that causes pneumonia in children and immunocompromised individuals. Recent genomic data show that divergence of major surface glycoproteins may confer speciation and host range selectivity. On the other hand, immune clearance between mice and humans is well correlated. Thus, we hypothesized that humanize mice may provide information about human immune responses involved in controlling Pneumocystis infection. CD34-engrafted huNOG-EXL mice controlled fungal burdens to a greater extent than nonengrafted mice. Moreover, engrafted mice generated fungal-specific IgM. Fungal control was associated with a transcriptional signature that was enriched for genes associated with nonopsonic recognition of trophs (CD209) and asci (CLEC7A). These same genes were downregulated in CD4-deficient mice as well as twins with bare lymphocyte syndrome with Pneumocystis pneumonia.BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease that causes severe mucocutaneous fragility due to mutations in COL7A1 (encoding type VII collagen [C7]). In this phase I/IIa trial, we evaluated the safety and possible clinical efficacy of intravenous infusion of allogeneic human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in patients with RDEB.METHODSFour adult and two pediatric patients with RDEB were treated with 3 intravenous injections of hUCB-MSCs (1 × 106 to 3 × 106 cells/kg) every 2 weeks and followed up for 8-24 months after treatment. The primary endpoint was safety. Secondary endpoints related to efficacy included clinical parameters, such as disease severity score, wound assessment, itch and pain score, and quality of life. C7 expression levels and inflammatory infiltrates in the skin, as well as serum levels of inflammatory markers and neuropeptides, were also assessed.RESULTSIntravenous hUCB-MSC infusions were well tolerated, without serious adverse events. Improvements in the Birmingham Epidermolysis Bullosa Severity Score, body surface area involvement, blister counts, pain, pruritus, and quality of life were observed with maximal effects at 56-112 days after treatment. hUCB-MSC administration induced M2 macrophage polarization and reduced mast cell infiltration in RDEB skin. Serum levels of substance P were decreased after therapy. Increased C7 expression was observed at the dermoepidermal junction in 1 of 6 patients at day 56.CONCLUSIONTo the best of our knowledge, this is the first clinical trial of systemic administration of allogeneic hUCB-MSCs in patients with RDEB, demonstrating safety and transient clinical benefits.TRIAL REGISTRATIONClinicalTrials.gov NCT04520022.FUNDINGThis work was supported by Daewoong Pharmaceutical Co. Ltd. and Kangstem Biotech Co. Ltd.