Rescue of Mutant CFTR Trafficking Defect by the Investigational Compound MCG1516A

Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects.

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

The Prevalence of Mesangial Electron-Dense Deposits in PLA2R-Positive Membranous Nephropathy

<b><i>Introduction:</i></b> Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults and can be primary or secondary. The antigenic target of antibodies in 70% of primary cases is phospholipase A2 receptor (PLA2R). The presence or absence of mesangial electron-dense deposits has been used to distinguish between primary and secondary MN. Mesangial deposits suggest MN due to lupus, infection, or other causes, though they are reported to occur in approximately 10% of primary MN. Staining for PLA2R is now frequently used for confirming a diagnosis of primary MN. If mesangial deposits predict a secondary cause, they should be more frequent in PLA2R-negative biopsies. <b><i>Methods:</i></b> A review of institutional kidney biopsies between March 2017 and June 2020 identified all cases of MN. Cases with a diagnosis of lupus or near “full-house” staining by immunofluorescence microscopy (IF) were excluded. Light microscopy, IF, and electron microscopy (EM) were performed. PLA2R staining was performed by IF. EM for all cases was reviewed and electron-dense deposit location, distribution, and size were determined. <b><i>Results:</i></b> Ninety-three cases of MN were identified, of which 86 had both PLA2R staining and EM performed. Of these, 51 cases (59%) were positive for PLA2R and 35 (41%) were negative. Mesangial electron-dense deposits were present in 22 (25.6%) of the 86 cases, including 27.5% (14/51) of PLA2R-positive cases and 22.8% (8/35) of PLA2R-negative cases. No difference was seen in size or distribution of deposits, or other features considered suggestive of secondary MN. <b><i>Conclusion:</i></b> PLA2R-negative cases were not more likely to have mesangial deposits than PLA2R-positive cases. Mesangial deposits should not be used as an indicator of secondary MN.

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90–100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.

Generating Large Numbers of Human Megakaryocytes from Pluripotent Stem Cells

The process of hemostasis and blood coagulation relies heavily on a sufficient supply of platelets (PLTs, also known as thrombocytes) within a person's bloodstream. Platelet transfusion is an effective treatment for thrombocytopenia-related diseases, yet paucity of supply and limited shelf-life (5 - 7 days) remain challenging. PLTs are generated by the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) into megakaryocytes (MKs), a rare subset of large polyploid bone marrow cells. Methods to produce MKs in vitro by inducing mesodermal specification and hematopoietic differentiation of human pluripotent stem cells (hPSCs) could provide a reliable and safe supply of PLTs for transfusion and would also be amenable to gene editing for generation of HLA-null universal PLTs. Culture methods to generate hPSC-derived MKs and PLTs have been described, yet published protocols lack standardization, are PSC line dependent and/or promote differentiation of other lineages, resulting in low MK cell yields and purity. The use of feeder cells and/or viral vectors also limits the clinical and scale-up applicability. Here, we describe an efficient feeder cell-free and serum-free culture system that promotes the selective differentiation of hPSCs from multiple ES and iPS lines into polyploid MKs with high purity and yields and ability to generate platelets. The 17-day protocol includes two stages: a 12-day stage to differentiate hPSCs into megakaryocytic-biased HSPCs through endothelial-to-hematopoietic transition (H-phase), and a 5-day stage to further differentiate HSPCs into mature MKs (MK-phase). at the start of the H-phase, hPSC aggregates were plated in mTeSR TM media on Matrigel ®-coated plates at 16 aggregates (100 - 200 µm in diameter, ~100 cells per aggregate) per cm 2 to allow attachment overnight (Day -1). The cells were then cultured in mesoderm-induction medium for 3 days (Day 0 - 3), and subsequently in hematopoietic specification medium for 9 days (Day 3 - 12). During this phase, PSC-derived HSPCs emerged from an adherent layer of endothelial cells and were released into suspension. On day 12 these nonadherent cells were harvested and seeded at 1 - 3.5 × 10 5 cells/mL in MK maturation medium containing thrombopoietin (TPO) and other hematopoietic growth factors and cultured for 5 additional days (MK-phase, Day 12 - 17). At the end of H-phase (day 12) and MK-phase (day 17) the cells were counted and assessed for HSPC markers (CD34/CD45), the erythroid marker glycophorin A (GlyA), MK markers (CD41a/CD42b), DNA ploidy profile and PLT production by flow cytometry and immunofluorescence microscopy. Two embryonic stem (ES) cell lines (H1 and H9), and two induced pluripotent stem (iPS) cell lines (WLS-1C and STiPS-R038) were used in this study. At the end of H-phase (Day 12), on average 48% (range: 34 - 72%) of cells released into suspension co-expressed CD41a and CD42b markers, with an average yield of 93 CD41a +CD42b + cells per seeded hPSC (range: 30 - 200, n = 4 for H9/1C, n = 3 for H1/R038). The cells also expressed CD34 (average of 78% CD34 + cells) and GlyA (average of 71% GlyA + cells), indicating that the H-phase may support differentiation of PSCs to megakaryocyte-erythroid progenitors. At the end of MK-phase (Day 17), on average 82% of the cells expressed CD41a (range: 70 - 99%), 62% of the cells co-expressed CD41a and CD42b (range: 40 - 85%), and an average of 253 CD41a +CD42b + cells were generated per seeded hPSC (range: 70 - 700 MKs, n = 11 for H1/H9/R038, n = 7 for 1C). Of note, less than 5% of cells expressed GlyA, showing that the culture system is specific for megakaryocytic differentiation. The DNA ploidy profile of the CD41a +CD42b + cells generated on Day 17 showed that on average 26% and 9% of cells had 4N and 8N+ DNA ploidy, respectively (n = 11). Multinucleated MKs could also be readily observed by immunofluorescence microscopy. These PSC-derived MKs produced an average of 3.5 PLTs (range: 1 - 10 PLTs, n = 11) based on viable CD41a +CD45 -GlyA - PLT-like particles with a similar size and CD41 expression as control PLTs prepared from fresh blood. In conclusion, we have developed a simple two-step, yet robust serum- and feeder-free culture system for generating high numbers of hPSC-MKs that are large, polyploid, and capable of shedding PLTs. This culture method provides a platform to study thrombopoiesis and is amenable to scale-up method development. Disclosures No relevant conflicts of interest to declare.

First person – Anna D'Amico

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Anna D'Amico is first author on ‘ PKC-ε regulates vesicle delivery and focal exocytosis for efficient IgG-mediated phagocytosis’, published in JCS. Anna is a PhD student in the lab of Dr Michelle R. Lennartz at Albany Medical College, Albany, NY, USA, where she is using immunofluorescence microscopy to investigate the mechanism underlying the role of PKC-ε in membrane trafficking.

SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia

In addition to respiratory complications produced by SARS‐CoV‐2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS‐CoV‐2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF-α, IL-6, IL-1β and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-IκBα, as well as enhanced DNA binding and transcriptional activity of NF-κB. These effects of the protein were blocked in the presence of BAY11-7082 (1 µM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/ml), an action that was reduced in the presence of SKF 86,002 (1 µM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 µM) and transfection with TLR4 small interfering RNA resulted in significant reduction in TNF-α and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-κB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

Environmental Stress Determines The Colonization and Impact of An Endophytic Fungus On Invasive Knotweed

There is increasing evidence that microbes play a key role in some plant invasions. A diverse and widespread but little understood group of plant-associated microbes are the fungal root endophytes of the order Sebacinales. They are associated with exotic populations of invasive knotweed (Reynoutria ssp.) in Europe, but their effects on the invaders are unknown. We used the recently isolated Sebacinales root endophyte Serendipita herbamans to experimentally inoculate invasive knotweed and study root colonisation and effects on knotweed growth under different environmental conditions. We verified the inoculation success and fungal colonisation through immunofluorescence microscopy and qPCR. We found that S. herbamans strongly colonized invasive knotweed in low-nutrient and shade environments, but much less under drought or benign conditions. At low nutrients, the endophyte had a positive effect on plant growth, whereas the opposite was true under shaded conditions. Our study demonstrates that the root endophyte S. herbamans has the potential to colonize invasive knotweed fine roots and impact its growth, and it could thus also play a role in natural populations. Our results also show that effects of fungal endophytes on plants can be strongly environment-dependent, and may only be visible under stressful environmental conditions.

Hydrogen-Rich Water Improves Cognitive Ability and Induces Antioxidative, Antiapoptotic, and Anti-Inflammatory Effects in an Acute Ischemia-Reperfusion Injury Mouse Model

Background. Cerebral ischemia and its reperfusion injury facilitate serious neurodegenerative diseases such as dementia due to cell death; however, there is currently no treatment for it. Reactive oxygen species is one of the many factors that induce and worsen the development of such diseases, and it can be targeted by hydrogen treatment. This study examined the effect of molecular hydrogen in cerebral ischemia-reperfusion injury, which is emerging as a novel therapeutic agent for various diseases. Methods. Ischemia-reperfusion injury was generated through bilateral common carotid artery occlusion in C57BL/6 mice. The test group received hydrogen-rich water orally during the test period. To confirm model establishment and the effect of hydrogen treatment, behavioural tests, biochemical assays, immunofluorescence microscopy, and cytokine assays were conducted. Results. Open field and novel object recognition tests revealed that the hydrogen-treated group had improved cognitive function and anxiety levels compared to the nontreated group, while hematoxylin and eosin stain showed abundant pyknotic cells in a model mouse brain, and this was attenuated in the hydrogen-treated mouse brain. Total antioxidant capacity and thiobarbituric acid reactive substance assays revealed that hydrogen treatment induced antioxidative effects in the mouse brain. Immunofluorescence microscopy revealed attenuated apoptosis in the striatum, cerebral cortex, and hippocampus of hydrogen-treated mice. Western blotting showed that hydrogen treatment reduced Bax and TNFα levels. Finally, cytokine assays showed that IL-2 and IL-10 levels significantly differed between the hydrogen-treated and nontreated groups. Conclusion. Hydrogen treatment could potentially be a future therapeutic strategy for ischemia and its derived neurodegenerative diseases by improving cognitive abilities and inducing antioxidative and antiapoptotic effects. Hydrogen treatment also decreased Bax and TNFα levels and induced an anti-inflammatory response via regulation of IL-2 and IL-10. These results will serve as a milestone for future studies intended to reveal the mechanism of action of molecular hydrogen in neurodegenerative diseases.