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Background: Primary amoebic meningoencephalitis (PAM) is an acute and fulminant CNS infection caused by Naegleria fowleri. Recreational activities and ritual ablution with contaminated warm fresh water are the main reason of PAM. Pakistan ranked the second most affected country, where most of the PAM incidences were reported from Karachi, Pakistan. Methods: In May, 2019, a 28-yr-old suspected PAM patient came to the Imam Zain-Ul-Abdin Hospital, Karachi. Biochemical and cytological investigations of patient`s CSF were carried out at Karachi Diagnostic Center and Molecular Biology Lab. Sequencing of Naegleria sp. specific (ITS) primer-based amplicons was performed from both patient`s CSF and water samples followed by multiple sequence alignment and phylogenetic studies. Results: Biochemical and cytological investigations of patient`s CSF showed 5 mg/dl glucose, 240 mg/dl total protein and 2260/mm3 TLC suggesting acute meningoencephalitis. PCR-based analyses of patient`s CSF and his residential tap water samples using Naegleria sp. specific (ITS) and N. fowleri specific primers revealed the presence of N. fowleri DNA. Nucleotide sequences of ITS primer-based amplicons from both patient`s CSF and water samples were submitted in GenBank under the accession numbers MT726981.1 and MT726226.1, respectively. According to phylogenetic analysis, N. fowleri isolate from Pakistan has shown the least node age of seven. Conclusion: Here, for the very first time in Pakistan, N. fowleri genotype has been identified as type-2. Phylogenetic analysis showed that N. fowleri isolate from Pakistan is among the latest descendants, i.e., evolved later in life.

Neisseria meningitidis, a gram negative bacterium, is the leading cause of bacterial meningitis and severe sepsis. Neisseria meningitidis genome contains 2,160 predicted coding regions including 1,000 hypothetical genes. Re-annotation of N. meningitidis hypothetical proteins identified nine putative peptidases. Among them, the NMB1620 protein was annotated as LD-carboxypeptidase involved in peptidoglycan recycling. Structural bioinformatics studies of NMB1620 protein using homology modeling and ligand docking were carried out. Structural comparison of substrate binding site of LD-carboxypeptidase was performed based on binding of tetrapeptide substrate 'L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine'. Inspection of different subsite-forming residues showed changeability in the S1 subsite across different bacterial species. This variability was predicted to provide a structural basis to S1-subsite for accommodating different amino acid residues at P1 position of the tetrapeptide substrate 'L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine'.

We have previously reported that squalene overproducing yeast self-downregulate the expression of the ethanol pathway (non-essential pathway) to divert the metabolic flux to the squalene pathway. In this study, the effect of co-production of squalene and ethanol on other non-essential pathways (fusel alcohol pathway, FA) of Saccharomyces cerevisiae was evaluated. However, before that, 13 constitutive promoters, like IRA1p, PET9p, RHO1p, CMD1p, ATP16p, USA3p, RER2p, COQ1p, RIM1p, GRS1p, MAK5p, and BRN1p, were engineered using transcription factor bindings sites from strong promoters HHF2p (−300 to −669 bp) and TEF1p (−300 to −579 bp), and employed to co-overexpress squalene and ethanol pathways in S. cerevisiae. The FSE strain overexpressing the key genes of the squalene pathway accumulated 56.20 mg/L squalene, a 16.43-fold higher than wild type strain (WS). The biogenesis of lipid droplets was stimulated by overexpressing DGA1 and produced 106 mg/L squalene in the FSE strain. AFT1p and CTR1p repressible promoters were also characterized and employed to downregulate the expression of ERG1, which also enhanced the production of squalene in FSE strain up to 42.85- (148.67 mg/L) and 73.49-fold (255.11 mg/L) respectively. The FSE strain was further engineered by overexpressing the key genes of the ethanol pathway and produced 40.2 mg/mL ethanol in the FSE1 strain, 3.23-fold higher than the WS strain. The FSE1 strain also self-downregulated the expression of the FA pathway up to 73.9%, perhaps by downregulating the expression of GCN4 by 2.24-fold. We demonstrate the successful tuning of the strength of yeast promoters and highest coproduction of squalene and ethanol in yeast, and present GCN4 as a novel metabolic regulator that can be manipulated to divert the metabolic flux from the non-essential pathway to engineered pathways.