Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1؉ LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. IMPORTANCEEbola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1 ؉ LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1 ؉ LE/Lys, as a therapeutic target for SARS and EBOV. Filoviruses are large filamentous viruses that cause deadly hemorrhagic fevers (1-3). Recently, much has been learned about how these viruses enter cells to initiate replication (for reviews, see references 4-7). After engaging host cell surface proteins, including C-type lectins and T-cell immunoglobulin and mucin domain proteins and Tyro3/Axl/Mer family members, Ebola virus (EBOV) particles are internalized by macropinocytosis and traffic through endosomes. En route, the receptor binding subunit of the EBOV g...

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Media coverage on coronavirus disease 2019 (COVID-19) has been extensive, yet large gaps remain in our understanding of the role of social media platforms during worldwide health crisis. The purpose of this study was to assess the most viewed YouTube videos on COVID-19 for medical content. We coded video characteristics, source, and medical content of the 113 most-widely viewed videos about COVID-19. Seventynine (69.9%) videos were classified as useful, and 10 (8.8%) videos were classified as misleading. Independent users were more likely to post misleading videos than useful videos (60.0% vs 21.5%, P = 0.009). News agencies were more likely to post useful videos than misleading videos (72.2% vs 40.0%, P = 0.039). Useful videos were more likely to present any information on prevalence or incidence (79.7% vs 20.0%, P < 0.001), as well as information on outcomes or prognosis (84.8% vs 30.0%, P < 0.001) compared to misleading videos. The World Health Organization contributed one useful video (1.3%), while no videos from the Center for Disease Control were included. Although YouTube generally is a useful source of medical information on the COVID-19 pandemic, increased efforts to disseminate accurate information from reputable sources is desired to help mitigate disease spread and decrease unnecessary panic in the general population. ARTICLE HISTORY

bEbolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two-pore Ca 2؉ channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging, we obtained evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain both NPC1 and TPC2.A s evidenced by the recent crisis in West Africa, ebolavirus (EBOV) can cause widespread disease and death in human populations. Entry of EBOV into cells, which is mediated by its sole glycoprotein (GP), is a target for therapeutic intervention (1, 2). EBOV entry is unusual in that it requires proteolytic priming of GP in endosomes followed by engagement of Niemann-Pick C1 (NPC1), a thirteen-pass membrane protein that aids cholesterol transport from endolysosomes (LE/Lys). Since the realization that NPC1 functions as an EBOV receptor (3-6), models have invoked entry through NPC1 ϩ LE/Lys (7-9). However, a recent study proposed a very different model: passage through NPC1 ϩ LE/Lys followed by trafficking to and entry into endosomes that lack NPC1 but contain two-pore Ca 2ϩ channel 2 (TPC2), a recently emerged EBOV entry factor (10, 11). The recent studies for (9) and against (10) entry in NPC1 ϩ LE/Lys were based on static microscopic assessment of colocalization of virus-like particles (VLPs) with endosomal markers. We found (9) that VLPs bearing EBOV GP enter the cytoplasm shortly after colocalization with NPC1 ϩ LE/Lys, assessed at fixed timed intervals in parallel samples analyzing VLP colocalization with NPC1 and VLP entry, which was based on delivery of VP40-␤-lactamase into the cytoplasm. A limitation of the static colocalization analyses is that the observer The image is from an early frame of a movie in which MLV pseudovirions expressing EBOV 21-kDa GP were added to a BSC-1 cell expressing NPC1-GFP. An overlay of the red (to detect Gag-mKO) and and far-red (to detect the lipid dye, DiD) fluorescent images is presented, with the far-red (DiD) fluorescence pseudocolored blue. Scale bar, 2 m. (c) Expected behavior of Gag-mKO and DiD (from a double-labeled MLV pseudovirus) upon delivery, hemifusion, and fusion in a GFP-labeled endosome.

Spermiation is the final phase of spermatogenesis leading to release of mature spermatids into the lumen of the seminiferous tubules. Morphologically, it involves a series of events, namely removal of excess spermatid cytoplasm, removal of ectoplasmic specialization, formation of tubulobulbar complex, and final disengagement of the spermatid from the Sertoli cell. Previous studies in our laboratory have shown that administration of 17beta-estradiol at a dose of 100 microg/kg body weight for 10 d resulted in failure of spermiation. This was accompanied by a suppression of FSH and intratesticular testosterone with a concomitant rise in intratesticular 17beta-estradiol. The present study was undertaken to determine the cause of failure and subsequently the molecular events in spermiation. Electron microscopic and confocal studies revealed an absence of tubulobulbar complex in step 19 spermatids after estradiol treatment, highlighting the significance of these structures in spermiation. It was further observed that treatment affected the Sertoli cell cytoskeleton and Arp2/3 complex that is critical for de novo polymerization of actin during tubulobulbar complex formation. In conclusion, the present study reports the role of 17beta-estradiol in inhibiting the formation of tubulobulbar complex, which could be one of the mechanism by which environmental estrogens influence male fertility.

Summary Background Early, sorting endosomes are a major crossroad of membrane traffic, at the intersection of the endocytic and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations is mediated by a number of distinct coat protein complexes, including AP-1, AP-3 and GGAs. Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal membrane, but the mechanisms of coat recruitment and assembly at this site remain poorly understood. Results Here we report that the endosomal Rab protein Rab4 orchestrates a GTPase cascade which results in the sequential recruitment of the Arf-like protein Arl1, the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2, and the class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1/2 activity with Brefeldin A results in the loss of AP-1, AP-3 and GGA-3, but not Arl1, from endosomal membranes and the formation of elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal membranes, inhibits the formation of tubular subdomains and blocks recruitment of BIG1/2, Arfs and adaptor complexes to the endosome. Conclusion Together these findings indicate that Arl1 links Rab4-dependent formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that constitute the endosomal sorting machinery.

Background:We previously showed that BRAG2, a known activator of Arf6, regulates cell surface levels of ␤1 integrin. Results: BRAG2 can also activate Arf4 and Arf5, but it is Arf5 that regulates integrin endocytosis. Conclusion: BRAG2 interacts with clathrin and activates Arf5 to enhance integrin internalization. Significance: This is the first evidence of a role for Arf5 at the plasma membrane in the regulation of endocytosis.