The characterization of therapeutic phage genomes plays a crucial role in the success rate of phage therapies. There are three checkpoints that need to be examined for the selection of phage candidates, namely, the presence of temperate markers, antimicrobial resistance (AMR) genes, and virulence genes. However, currently, no single-step tools are available for this purpose. Hence, we have developed a tool capable of checking all three conditions required for the selection of suitable therapeutic phage candidates. This tool consists of an ensemble of machine-learning-based predictors for determining the presence of temperate markers (integrase, Cro/CI repressor, immunity repressor, DNA partitioning protein A, and antirepressor) along with the integration of the ABRicate tool to determine the presence of antibiotic resistance genes and virulence genes. Using the biological features of the temperate markers, we were able to predict the presence of the temperate markers with high MCC scores (>0.70), corresponding to the lifestyle of the phages with an accuracy of 96.5%. Additionally, the screening of 183 lytic phage genomes revealed that six phages were found to contain AMR or virulence genes, showing that not all lytic phages are suitable to be used for therapy. The suite of predictors, PhageLeads, along with the integrated ABRicate tool, can be accessed online for in silico selection of suitable therapeutic phage candidates from single genome or metagenomic contigs.

Parkinson’s disease (PD) is a neurodegenerative disorder defined by progressive deterioration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Dental pulp stem cells (DPSCs) have been proposed to replace the degenerated dopaminergic neurons due to its inherent neurogenic and regenerative potential. However, the effective delivery and homing of DPSCs within the lesioned brain has been one of the many obstacles faced in cell-based therapy of neurodegenerative disorders. We hypothesized that DPSCs, delivered intranasally, could circumvent these challenges. In the present study, we investigated the therapeutic efficacy of intranasally administered DPSCs in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Human deciduous DPSCs were cultured, pre-labelled with PKH 26, and intranasally delivered into PD mice following MPTP treatment. Behavioural analyses were performed to measure olfactory function and sensorimotor coordination, while tyrosine hydroxylase (TH) immunofluorescence was used to evaluate MPTP neurotoxicity in SNpc neurons. Upon intranasal delivery, degenerated TH-positive neurons were ameliorated, while deterioration in behavioural performances was significantly enhanced. Thus, the intranasal approach enriched cell delivery to the brain, optimizing its therapeutic potential through its efficacious delivery and protection against dopaminergic neuron degeneration.

Complete genomes of xenobiotic-degrading microorganisms provide valuable resources for researchers to understand molecular mechanisms involved in bioremediation. Despite the well-known ability of Sphingomonas paucimobilis to degrade persistent xenobiotic compounds, a complete genome sequencing is lacking for this organism. In line with this, we report the first complete genome sequence of Sphingomonas paucimobilis (strain AIMST S2), an organophosphate and hydrocarbon-degrading bacterium isolated from oil-polluted soil at Kedah, Malaysia. The genome was derived from a hybrid assembly of short and long reads generated by Illumina HiSeq and MinION, respectively. The assembly resulted in a single contig of 4,005,505 bases which consisted of 3,612 CDS and 56 tRNAs. An array of genes involved in xenobiotic degradation and plant-growth promoters were identified, suggesting its’ potential role as an effective microorganism in bioremediation and agriculture. Having reported the first complete genome of the species, this study will serve as a stepping stone for comparative genome analysis of Sphingomonas strains and other xenobiotic-degrading microorganisms as well as gene expression studies in organophosphate biodegradation.

Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform. TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique. Amresco’s Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.