Biology subjects

Early, J.

Publications and source records attributed to Early, J..

4 recordsLinked to original sources

bioRxiv2026

Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and crystal structures of TvPPi-PFK are reported. TvPPi-PFK is the first reported eukaryotic PPi-PFK structure. TvPPi-PFK retains the overall PPi-PFK topology observed in bacterial PPi-PFK including conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. In addition to the catalytic PPi-PFK binding sites, TvPPi-PFK has two additional ligand binding sites. The first binds AMP usurped during protein production and helps stabilize the TvPPi-PFK tetramer. A second ligand binding site was observed in proximity to the AMP-binding site and accommodates sugar phosphates soaked into preformed crystals. This sugar phosphates binding site is distinct from the TvPPi-PFK active site that binds fructose-6-phosphate. Future mutagenesis and activity studies are planned to determine the relevance of both sites. SynopsisThe production, crystallization, and crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis (TvPPi-PFK) are reported. TvPPi-PFK has a prototypical PPi-PFK active site as well as unexpected AMP and sugar-phosphate binding sites at the dimer interface.

biochemistry↗

bioRxiv2026

Perinatal Exposure to Organophosphate Flame Retardants Induces Sex- and Hormone-Dependent Alterations in Anxiety, Memory, Neurotransmitter Content, and Hippocampal Gene Expression

Developmental exposure to organophosphate flame retardants (OPFRs) is a public health concern due to their endocrine-disrupting potential. We examined perinatal exposure to tris(1,3-dichloro-2-propyl) phosphate, triphenyl phosphate, and tricresyl phosphate in mice. Adult male and female offspring were assessed for memory and anxiety-like behavior. Dopamine and norepinephrine were quantified in the hippocampus and prefrontal cortex (PFC), and bulk RNA sequencing was conducted for the hippocampus. OPFR-treated females in high ovarian hormone states spent less time in the open field test (OFT) center, the Y-maze unknown arm, and with the displaced object in spatial object recognition (SOR) indicating increased anxiety-like behavior and impaired spatial memory. These females also illustrated improved memory on the short-term Barnes maze, and a trending improvement in the novel object recognition test. Females in low ovarian hormone states, demonstrated a trend in center OFT exploration. OPFR-treated males displayed disruption in memory in the SOR and the short- and long-term Barnes maze. Perinatal OPFR reduced hippocampal dopamine in males and altered prefrontal dopamine in females in a hormone-dependent manner. OPFR-treated females in high ovarian hormones states demonstrated a trending decrease in PFC norepinephrine. Perinatal OPFR treatment caused differential gene expression in 121 individual genes and alteration to functional modules related to RNA processing, cellular metabolism, and extracellular organization. Hormone status also affected gene OPFR-induced altered expression, with similarity between males and high ovarian hormone state females. Our findings suggest that perinatal OPFR exposure causes widespread, sex specific, and hormone dependent disruptions in behavior, neurochemistry, and gene expression in adulthood. HighlightsO_LIAnxiety-like behavior in OPFR-treated females varied with ovarian hormone status C_LIO_LIHigh ovarian hormone OPFR females showed task-dependent changes in memory C_LIO_LIMales displayed impaired spatial memory following perinatal OPFR treatment C_LIO_LIPerinatal OPFR modifies hippocampal and prefrontal dopamine and norepinephrine C_LIO_LIOPFR treatment altered individual gene and functional gene module expression C_LI

pharmacology and toxicology↗

bioRxiv2022

Dynamic Compartmentalisation of Intracellular Sodium in collecting duct cells

In Principal cells (PC) of the cortical collecting duct (CCD), the highly regulated and coordinated reabsorption of sodium occurs through the epithelial sodium channel (ENaC) at the apical membrane and Na/K ATPase at the basolateral membrane. However, it is not known how sodium ions (Na+) are transported across the cell. We investigated intracellular transport in mCCDcl1 cells using a fluorescent sodium dye, CoroNa Green AM. Dye uptake was stimulated by aldosterone, blocked by amiloride (an ENaC inhibiter), and basolateral transport was prevented by ouabain (an Na/K ATPase blocker) thus validating the dyes apparently faithful replication of sodium transport. Cells exhibited a consistent pattern of sodium-containing vesicles, of various sizes, surrounded by cytoskeleton and lipid membrane. While the smallest vesicles ([~]0.5m) co-stained with lysotracker, larger vesicles (up to 6.4m) did not co-stain with either lysosomal- or mitochondrial-specific dyes and appeared to have internal structure, suggesting that they were multivesicular bodies. Time-lapse imaging showed a subset of these multivesicular bodies release or take up sodium dye in a controlled manner. Our novel data suggest that intracellular sodium compartmentalisation is highly regulated and offer new insights into intracellular sodium dynamics in the collecting duct, revealing potential new targets for control of sodium homeostasis. New and NoteworthyThe article shows for the first time, to our knowledge, intracellular sodium transport mechanism in mCCDcl1 cells in the form of dynamic vesicular bodies. These structures offer new targets for the regulation of sodium homeostasis and transport in the kidney collecting duct, with wider implications for blood pressure regulation.

cell biology↗

bioRxiv2019

A rapid method to determine the bactericidal activity of compounds against non-replicating Mycobacterium tuberculosis at low pH

There is an urgent need for new anti-tubercular agents which can lead to a shortened treatment time by targeting persistent or non-replicating bacilli. In order to assess compound activity against non-replicating Mycobacterium tuberculosis, we developed a method to detect the bactericidal activity of novel compounds within 7 days. Our method uses incubation at low pH in order to induce a non-replicating state. We used a strain of M. tuberculosis expressing luciferase; we first confirmed the linear relationship between luminescence and viable bacteria (determined by colony forming units) under our assay conditions. We optimized the assay parameters in 96-well plates in order to achieve a reproducible assay. Our final assay used M. tuberculosis in phosphate-citrate buffer, pH 4.5 exposed to compounds for 7 days; viable bacteria were determined by luminescence. We recorded the minimum bactericidal concentration at pH 4.5 (MBC4.5) representing >2 logs of kill. We confirmed the utility of the assay with control compounds. The ionophores monensin, niclosamide, and carbonyl cyanide 3-chlorophenylhydrazone and the anit-tubercular drugs pretomanid and rifampicin were active, while several other drugs such as isoniazid, ethambutol, and linezolid were not.

microbiology↗