Understanding mechanisms of JAK1 inhibition on synovial fibroblasts using combinatorial approaches of bulk and single cell RNAseq analyses.

OBJECTIVES: The aim of these studies was to characterise the molecular effects of a tool JAK1 inhibitor on cultured primary fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) through both total and individual cell analysis. METHODS: RA-FLS cultures from 6 (Bulk RNA-seq) or 4 (ScRNA-seq) donors were pre-treated with various concentrations (100 nM and 1µM) of ABT-317 with/without exposure to 25% SEB-conditioned PBMC medium to mimic the RA inflammatory milieu. Cells were subjected to both bulk RNA-seq (36 libraries) and single cell RNA-seq (scRNA-seq; 24 libraries) to identify biological processes impacted by CM and ABT-317 treatments. RESULTS: In our bulk RNA-seq analysis, a total of 2,605 differentially expressed genes (DEGs) were identified between CM-stimulation and unstimulated groups, while 1,122 DEGs were found between ABT-317 1µM and DMSO in CM-stimulated groups using thresholds of log2 (fold change) ≥ |0.58| and FDR ≤ 10%. Both bulk and single cell mRNA analysis of RA-FLS treated with a combination of CM and ABT-317 demonstrated the expected changes in inflammatory pathways such as interferon and IL-6 signalling. However, other non-inflammation associated pathways were also altered by ABT-317. In addition, the single cell analysis highlighted that FLS segregate into distinctive clusters upon combination CM and ABT-317 treatment, suggesting JAK inhibition can drive RA-FLS into multiple heterogenous cell populations. Interestingly, one of the unique RA-FLS clusters that emerged from the CM and ABT-317 treatment showed matrix metalloproteinase-3 (MMP3)high expression as well as several gene signatures that are not found in any other ABT-317 derived clusters. CONCLUSIONS: JAK inhibition with ABT-317 is effective at globally inhibiting CM-induced pro- and non-inflammatory pathways in FLS cultures, but also results in several distinct fibroblast populations with unique gene-associated pathways. This study advances the molecular understanding of JAK1 inhibitor effects on fibroblasts that may contribute to clinical efficacy.

Fusion Protein of RBP and Albumin Domain III Reduces Lung Fibrosis by Inactivating Lung Stellate Cells.

Activated stellate cells play a role in fibrosis development in the liver, pancreas, and kidneys. The fusion protein R-III, which consists of retinol-binding protein and albumin domain III, has been demonstrated to attenuate liver and renal fibrosis by suppressing stellate cell activation. In this study, we investigated the efficacy of R-III against bleomycin-induced lung fibrosis in mice. R-III reduced lung fibrosis and primarily localized in autofluorescent cells in the lung tissue. Furthermore, we isolated lung stellate cells (LSCs) from rat lungs using the isolation protocol employed for hepatic stellate cells (HSCs). LSCs shared many characteristics with HSCs, including the presence of vitamin A-containing lipid droplets and the expression of alpha-smooth muscle actin and collagen type I, markers for activated HSCs/myofibroblasts. LSCs spontaneously transdifferentiated into myofibroblasts in in vitro culture, which was inhibited by R-III. These findings suggest that R-III may reduce lung fibrosis by inactivating LSCs and could be a promising treatment for extrahepatic fibrosis.

Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill.

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae has made it a pre-eminent genetic model organism. However, since efficient protein isolation depends upon optimal disruption of cells, the use of yeast for biochemical analysis of cellular proteins is hampered by its cell wall which is expensive to digest enzymatically (using lyticase or zymolyase), and difficult to disrupt mechanically (using a traditional bead beater, a French press or a coffee grinder) without causing heating of samples, which in turn causes protein denaturation and degradation. Although manual grinding of yeast cells under liquid nitrogen (LN2) using a mortar and pestle avoids overheating of samples, it is labor intensive and subject to variability in cell lysis between operators. For many years, we have been successfully preparing high quality yeast extracts using cryogrinding of cells in an automated freezer mill. The temperature of -196 °C achieved with the use of LN2 protects the biological material from degradation by proteases and nucleases, allowing the retrieval of intact proteins, nucleic acids and other macromolecules. Here we describe this technique in detail for budding yeast cells which involves first freezing a suspension of cells in a lysis buffer through its dropwise addition into LN2 to generate frozen droplets of cells known as "popcorn". This popcorn is then pulverized under LN2 in a freezer mill to generate a frozen "powdered" extract which is thawed slowly and clarified by centrifugation to remove insoluble debris. The resulting extracts are ready for downstream applications, such as protein or nucleic acid purification, proteomic analyses, or co-immunoprecipitation studies. This technique is widely applicable for cell extract preparation from a variety of microorganisms, plant and animal tissues, marine specimens including corals, as well as isolating DNA/RNA from forensic and permafrost fossil specimens.

Treatment of keloids with a single dose of low-energy superficial X-ray radiation to prevent recurrence after surgical excision: An in vitro and in vivo study.

BACKGROUND: Although keloids have been empirically treated with steroids and radiation, evidence-based radiation parameters for keloid therapy are lacking. OBJECTIVE: To determine evidence-based radiation parameters for blocking keloid fibroblast proliferation in vitro and apply them to patients. METHODS: The effects of various radiation parameters and steroids on cell proliferation, cell death, and collagen production in keloid explants and fibroblasts were evaluated with standard assays. Effective radiation parameters were then tested on patients. RESULTS: No differences were observed between the effects of 50 and 320 kV radiation or between single and fractionated radiation doses on keloid fibroblasts. A 3 Gy, 50 kV dose inhibited keloid fibroblast proliferation in culture, whereas 9 Gy completely blocked their outgrowth from explants by inducing multiple cell death pathways and reducing collagen levels. Thirteen of 14 keloids treated with a single 8 Gy, 50 kV dose of radiation did not recur, although 4 patients with 6 keloids were lost to follow-up. LIMITATIONS: Seventy-five percent of patients received steroids for pruritus, whereas approximately 25% of patients were lost to follow-up. CONCLUSIONS: A single 8 Gy dose of superficial 50 kV radiation delivered an average of 34 days after keloid excision maybe sufficient to minimize recurrence, including in individuals resistant to steroids. Higher radiation energies, doses, or fractions may be unnecessary for keloid therapy.

Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.