Fig 2: Analysis of bacterial membrane vesicles extracted from murine stool. Stool samples were collected 24 hpi from C57BL/6 mice which were either infected with mock inoculum or MNV. a) Stool derived vesicles were probed with anti-CD9 antibody. Bands from CD9 tetraspanin are observed in pooled exosomes from depletion steps (Lane 1) as well as from exosomes harvested from RAW 264.7 cells in vitro (Lanes 8–10). Bands were not observed in bEV extracts (Lanes 2–7). b) Exosome depleted vesicles derived from murine stool were also probed with anti-HSP40 antibody. While RAW 264.7 cell lysates (Lane 1) and stool derived exosomes (Lane 4) were positive for HSP70, bacterial lysates (Lanes 2–3) and stool derived bEVs (Lanes 5–8) were negative for this protein. c) Stool derived vesicles were probed for anti-LPS antibody. Bands for LPS appeared in the gram-negative bacterial lysate (Lane 1), in vitro generated OMVs (Lane 7) and in the exosome-depleted, stool derived bEVs (Lanes 9–12). LPS was not detected in any other sample

Fig 3: Schematic summary. After a 6 h of a 30 min heat shock at 37°C, several immune genes are upregulated in the mosquitoes. These include genes that have been shown responsible for limiting a Plasmodium infection such as ppo, duox, frep3 in the whole mosquito, and antimicrobial peptides in the midgut. hsp70 is also upregulated along with dnmt2, a gene responsible for the methylation of DNA and RNA in mosquitoes. This might indicate an increment in methylcytosines in the nucleic acids, which is also supported by the down regulation of the enzyme that removes these epigenetic marks, tet2 (not shown). The PO activity also increases in the hemolymph of the Plasmodium-challenged and heat-shocked mosquitoes, although the total amount of NO produced did not changed (not shown). This overall response caused the mosquitoes to become less infected with the parasite. Only statistically significant results are portrayed.

Fig 4: Proposed mechanism for HSF1-mediated clearance of Tau. In normal conditions HSF1 is present as an inactive monomer in cytosol. HSF1 in inactive state forms complex with Hsp70. Triggering of Tau-induced stress or treatment of cells with limonoids leads to dissociation of Hsp70 from HSF1. HSF1 monomer is now subjected to phosphorylation and is transported into nucleus. In nucleus, HSF1 trimerizes and interacts with heat shock element (HSE) present in the upstream of Hsp70 promoter. This leads to increase in expression of Hsp70 and thus enhancing the cytosolic levels of Hsp70.

Fig 5: HSF1 localization to nuclear compartment. (A) HSF1-Hsp70/40/90 complex are present as inactive form in the cytosol. During stress condition, HSF1 is signalled to dissociate from Hsps and allocated to nucleus for active transcription of Hsp70 in order to manage the cellular protein burden. The altered balance between nuclear vs. cytosolic HSF1 is the key determinant of controlling effective proteostasis in AD. (B) Tau aggregates induced the Hsp70 level in HEK293T cells while epoxyazadiradione has induced the HSF1 with reduced Hsp70 level, which indicates the feed-back control of cellular proteostasis signalling. (C) The quantification showed increase in nuclear as well as cytosolic levels of HSF1 in epoxyazadiradione treatment which is also maintained in Tau stressed cells. Quantification was carried out by Zen 2.3 software (https://www.zeiss.com/microscopy/int/products/light-microscopes/axio-observer-for-biology.html).