PE1

Extracellular recording were performed from mushroom body-extrinsic neurons while the animal was exposed to differential conditioning to two odors, the forward-paired conditioned stimulus (CS+; the odor that will be or has been paired with sucrose reward) and the unpaired CS- (the odor that will be or has been specifically unpaired with sucrose reward). A single neuron, the pedunculus-extrinsic neuron number 1 (PE1), was identified on the basis of its firing pattern, and other neurons were grouped together as non-PE1 neurons. PE1 reduces its response to CS+ and does not change its response to CS- after learning. Most non-PE1 neurons do not change their responses during learning, but some decrease, and one neuron increases its response to CS+. PE1 receives inhibitory synaptic inputs, and neuroanatomical studies indicate closely attached GABA-immune reactive profiles originating at least partially from neurons of the protocerebral-calycal tract (PCT). Thus, either the associative reduction of odor responses originates within the PE1 via a long-term depression (LTD)-like mechanism, or PE1 receives stronger inhibition for the learned odor from the PCT neurons or from Kenyon cells. In any event, as the decreased firing of PE1 correlates with the increased probability of behavioral responses, our data suggest that the mushroom bodies exert general inhibition over sensory-motor connections, which relaxes selectively for learned stimuli.

The distinctive PE and PPE families of proteins in Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacteria, have been associated primarily with antigenicity, immune-modulation and virulence. Earlier, using structure-based sequence annotation, we identified a 225 amino acid conserved PE-PPE domain (Pfam: PF08237) commonly present in some PE and PPE proteins which was observed to comprise α/β-serine hydrolase fold. The prediction was supported by experimental validations of PE16 that was shown to exhibit esterase activity. In this study, we undertook the characterization of the probable operonic ORFs Rv0151c (pe1) and Rv0152c (pe2). Here we demonstrated that pe1 and pe2 are operonic in organization and are co-transcribed. Both PE1 and PE2 proteins possess esterase activity and hydrolyze short to medium chain p-nitrophenyl esters with more specific activity for p-nitrophenyl caproate (C6) with the optimal catalytic conditions of 37-38 °C and pH 7.0-8.0. The thermal denaturation temperature of PE1 and PE2 proteins were found to be 50 °C. The esterase activity of full length PE1, PE2 and their PE-PPE (α/β-serine hydrolase) domains are similar indicating that the function of PE-PPE domain is independent of the rest of the protein. The esterase activity of these proteins was validated by mutagenesis of the active site Ser; using PE1 Ser246Ala and PE2 Ser163Ala mutants. With these experiments, we conclusively show that the co-transcribed pe1 and pe2 genes code for enzymes belonging to the esterase family of proteins.

Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions and deletions in cells and animals without requiring double-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3' ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (>5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5 and twinPE) for a given application. Finally, we provide detailed instructions on how to perform PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2-4 weeks.