Ubx Regulates Differential Enlargement and Diversification of Insect Hind Legs

Differential enlargement of hind (T3) legs represents one of the hallmarks of insect evolution. However, the actual mechanism(s) responsible are yet to be determined. To address this issue, we have now studied the molecular basis of T3 leg enlargement in Oncopeltus fasciatus (milkweed bug) and Acheta domesticus (house cricket). In Oncopeltus, the T3 tibia displays a moderate increase in size, whereas in Acheta, the T3 femur, tibia, and tarsus are all greatly enlarged. Here, we show that the hox gene Ultrabithorax (Ubx) is expressed in the enlarged segments of hind legs. Furthermore, we demonstrate that depletion of Ubx during embryogenesis has a primary effect in T3 legs and causes shortening of leg segments that are enlarged in a wild type. This result shows that Ubx is regulating the differential growth and enlargement of T3 legs in both Oncopeltus and Acheta. The emerging view suggests that Ubx was co-opted for a novel role in regulating leg growth and that the transcriptional modification of its expression may be a universal mechanism for the evolutionary diversification of insect hind legs

Plasmodium falciparum erythrocytic stage parasites require the putative autophagy protein PfAtg7 for normal growth.

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite's erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth

Western blotting confirms expression of PfAtg7 fusion proteins.

<p>(A) PfATG7-HA transgenic parasites confirm protein expression. Western blot of two independent clones detects PfAtg7-HA at ∼150 kDa and ∼65 kDa. Lane 1: PfAtg7-HA clone C1; Lane 2: PfAtg7-HA clone F2; Lane 3: UCH-HA positive control (see Materials and Methods). (B) PfAtg7-RFA transgenic parasites confirm expression of the two PfAtg7 species. Western blot detection of PfATG7-RFA fusion protein incubated in the presence of TMP reveals PfATG7 at ∼200 kDa and ∼100 kDa. RFA tag is 47 kDa.</p

PfAtg7 has an unusual primary structure.

<p>(A) PfAtg7 contains long insertions within the C-terminal and N-terminal domains. A schematic of PfATG7 and ScATG7 domains illustrates the presence of insertions in PfAtg7 as compared to the well-described C-terminal and N-terminal domains of ScAtg7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067047#pone.0067047-Noda1" target="_blank">[42]</a>. (B) Alignment of ScAtg7 and PfAtg7 C-terminal regions reveals conservation of key motifs. Alignment between yeast (Sc) and <i>P. falciparum</i> (Pf) ATG7 C-terminal domain illustrate similarity, with conservation of the ATP binding domain (black bar), the catalytic cysteine (box), amino acids required for hydrogen bonding (black arrowheads), and salt bridges (open arrowheads) between ATG7 and ATG8 (for complete alignment see Fig. S1C in File S1).</p

PfAtg7 is essential for normal parasite growth.

<p>Short-term growth experiment reveals slow growth phenotype upon TMP removal. PfAtg7-RFA clone A1 (A) B4 (B) and parental PM1 (C) were washed free of TMP and parasitemia was monitored by flow cytometry every 24 h for 3 days. Parasitemia in the absence of TMP (white bars/circles) for clones A1 and B4 was significantly reduced compared to growth in the presence of TMP (black bars/circles; **p<0.001, student’s T test). Parental line (PM1) shows significant growth difference at 48 h (*p<0.05, student’s T test) but no significant difference in the presence and absence of TMP at the 24 h or 72 h timepoints. (D) PfATG7 attenuation results in a sustained slow growth phenotype. Two independent PfATG7-RFA transgenic clones, A1 and B4 (not shown), were monitored every 24 h by staining with acridine orange and enumeration of parasitemia by flow cytometry for up to 8 days. Dilutions were performed on parasite cultures every three days to maintain the parasites at optimal parasitemia and avoid parasite death. Overall dilutions were factored in, resulting in “cumulative growth.” (E) PM1 parental parasites exhibit no significant change in growth over the 8 day period.</p

PfAtgs 3, 4, 7, 8 are expressed in<i>P. falciparum erythrocytic stage parasites.</i>

<p>Genomic DNA (gDNA) and RNA was extracted from asynchronous erythrocytic stage parasites, the latter used for cDNA production. Primers flanking introns were chosen, except for PfATG8, which has no predicted introns. No RT control shows lack of gDNA contamination of RNA preparation.</p

The emergence of SARS-CoV-2 lineages and associated saliva antibody responses among asymptomatic individuals in a large university community.

SARS-CoV-2 (CoV2) infected, asymptomatic individuals are an important contributor to COVID transmission. CoV2-specific immunoglobulin (Ig)-as generated by the immune system following infection or vaccination-has helped limit CoV2 transmission from asymptomatic individuals to susceptible populations (e.g. elderly). Here, we describe the relationships between COVID incidence and CoV2 lineage, viral load, saliva Ig levels (CoV2-specific IgM, IgA and IgG), and ACE2 binding inhibition capacity in asymptomatic individuals between January 2021 and May 2022. These data were generated as part of a large university COVID monitoring program in Ohio, United States of America, and demonstrate that COVID incidence among asymptomatic individuals occurred in waves which mirrored those in surrounding regions, with saliva CoV2 viral loads becoming progressively higher in our community until vaccine mandates were established. Among the unvaccinated, infection with each CoV2 lineage (pre-Omicron) resulted in saliva Spike-specific IgM, IgA, and IgG responses, the latter increasing significantly post-infection and being more pronounced than N-specific IgG responses. Vaccination resulted in significantly higher Spike-specific IgG levels compared to unvaccinated infected individuals, and uninfected vaccinees' saliva was more capable of inhibiting Spike function. Vaccinees with breakthrough Delta infections had Spike-specific IgG levels comparable to those of uninfected vaccinees; however, their ability to inhibit Spike binding was diminished. These data are consistent with COVID vaccines having achieved hoped-for effects in our community, including the generation of mucosal antibodies that inhibit Spike and lower community viral loads, and suggest breakthrough Delta infections were not due to an absence of vaccine-elicited Ig, but instead limited Spike binding activity in the face of high community viral loads