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Abstract Mitochondrial DNAs (mtDNAs) appear in almost all eukaryotic species and are useful molecular markers for phylogenetic studies and species identification. Kinetoplast DNAs (kDNAs) are structurally complex circular mtDNA networks in kinetoplastids, divided into maxicircles and minicircles. Despite several kDNAs of many Leishmania species being examined, the kDNAs of the new species, Leishmania orientalis (formerly named Leishmania siamensis) strain PCM2, have not been explored. This study aimed to investigate the maxicircle and minicircle DNAs of L. orientalis strain PCM2 using hybrid genome sequencing technologies and bioinformatic analyses. The kDNA sequences were isolated and assembled using the SPAdes hybrid assembler from the Illumina short-read and PacBio long-read data. Circular contigs of the maxicircle and minicircle DNAs were reconstructed and confirmed by BLASTn and rKOMICs programs. The kDNA genome was annotated by BLASTn before the genome comparison and phylogenetic analysis by progressiveMauve, MAFFT, and MEGA programs. The maxicircle of L. orientalis strain PCM2 (18,215 bp) showed 99.92% similarity and gene arrangement to Leishmania enriettii strain LEM3045 maxicircle with variation in the 12s rRNA gene and divergent region. Phylogenetics of the whole sequence, coding regions, divergent regions, and 12s rRNA gene also confirmed this relationship and subgenera separation. The identified 105 classes of minicircles (402–1177 bp) were clustered monophyletically and related to the Leishmania donovani minicircles. The kinetoplast maxicircle and minicircle DNAs of L. orientalis strain PCM2 contained a unique conserved region potentially useful for specific diagnosis of L. orientalis and further exploration of this parasite population genetics in Thailand and related regions.

Background Mounting evidence has linked cancer metabolic reprogramming with altered redox homeostasis. The pentose phosphate pathway (PPP) is one of the key metabolism-related pathways that has been enhanced to promote cancer growth. The glucose 6-phosphate dehydrogenase (G6PD) of this pathway generates reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is essential for controlling cellular redox homeostasis. Objective This research aimed to investigate the growth-promoting effects of G6PD in non-small cell lung cancer (NSCLC). Methods Clinical characteristics and G6PD expression levels in lung tissues of 64 patients diagnosed with lung cancer at the King Chulalongkorn Memorial Hospital (Bangkok, Thailand) during 2009-2014 were analyzed. G6PD activity in NSCLC cell lines, including NCI-H1975 and NCI-H292, was experimentally inhibited using DHEA and siG6PD to study cancer cell proliferation and migration. Results The positive expression of G6PD in NSCLC tissues was detected by immunohistochemical staining and was found to be associated with squamous cells. G6PD expression levels and activity also coincided with the proliferation rate of NSCLC cell lines. Suppression of G6PD-induced apoptosis in NSCLC cell lines by increasing Bax/Bcl-2 ratio expression. The addition of D-(-)-ribose, which is an end-product of the PPP, increased the survival of G6PD-deficient NSCLC cell lines. Conclusion Collectively, these findings demonstrated that G6PD might play an important role in the carcinogenesis of NSCLC. Inhibition of G6PD might provide a therapeutic strategy for the treatment of NSCLC.

Abstract Background The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated. Methods This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis. Results We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species. Conclusions This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite. Graphical abstract

Background: Leishmania orientalis (formerly named Leishmania siamensis) has been neglected for years in Thailand. The genomic study of L. orientalis has gained much attention recently after the release of the first high-quality reference genome of the isolate LSCM4. The integrative approach of multiple sequencing platforms for whole-genome sequencing has proven effective at the expense of considerably expensive costs. This study presents a preliminary bioinformatic workflow including the use of multi-step de novo assembly coupled with the reference-based assembly method to produce high-quality genomic drafts from the short-read Illumina sequence data of L. orientalis isolate PCM2. Results: The integrating multi-step de novo assembly by MEGAHIT and SPAdes with the reference-based method using the L. enriettii genome and salvaging the unmapped reads resulted in the 30.27 Mb genomic draft of L. orientalis isolate PCM2 with 3367 contigs and 8887 predicted genes. The results from the integrated approach showed the best integrity, coverage, and contig alignment when compared to the genome of L. orientalis isolate LSCM4 collected from the northern province of Thailand. Similar patterns of gene ratios and frequency were observed from the GO biological process annotation. Fifty GO terms were assigned to the assembled genomes, and 23 of these (accounting for 61.6% of the annotated genes) showed higher gene counts and ratios when results from our workflow were compared to those of the LSCM4 isolate. Conclusions: These results indicated that our proposed bioinformatic workflow produced an acceptable-quality genome of L. orientalis strain PCM2 for functional genomic analysis, maximising the usage of the short-read data. This workflow would give extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation.

(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis) and Leishmania martiniquensis, has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.

Abstract Background Pasteurella multocida is a Gram-negative bacterium that causes economically significant infections of a broad range of animal species. Pneumonic and septicaemic pasteurellosis caused by this bacterium remain important problems in pigs, cattle, and water buffaloes in Thailand. The aim of this study was to characterise the virulence-associated gene profiles and to develop an OmpA molecular typing scheme for classifying 191 bovine and porcine isolates of P. multocida collected between 1989 and 2012 in Thailand using polymerase chain reactions (PCRs), nucleotide sequencing, and sequence and structural bioinformatics analyses. Results PCR screening successfully characterised the profiles of 25 virulence-associated genes in all isolates. The gene profiles separated these isolates into bovine and porcine clusters based on eight genes (hgbB, hsf1, tadD, nanH, pfhA, plpE, pmHAS, and tbpA). Phylogenetic analyses of the nucleotide and protein sequences corresponding to the ompA gene, which encodes a major outer membrane surface protein, showed two major bovine and porcine clusters. Structural prediction and analysis of the dN/dS ratio revealed four hypervariable extracellular loops of the OmpA transmembrane domains. These four loops were used to develop an OmpA typing scheme. This scheme classified 186 isolates into five major loop sequence types (LST8, LST12, LST15, LST18, and LST19), consistent with the phylogenetic results. The loop regions of the bovine isolates were predicted to be more antigenic than those of the porcine isolates. Thus, molecular evolution of the OmpA proteins could be used to classify P. multocida isolates into different capsular types, host types, and, possibly, pathogenicity levels. Conclusions Together with the virulence-associated gene profiles, the typing reported in this work provides a better understanding of P. multocida virulence. Effective monitoring and potential strain-specific subunit vaccines could be developed based on these loop oligopeptides.

Background: Mounting evidence has linked cancer metabolic reprogramming with altered redox homeostasis. The pentose phosphate pathway (PPP) is one of the key metabolism-related pathways that has been enhanced to promote cancer growth. The glucose 6-phosphate dehydrogenase (G6PD) of this pathway generates reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is essential for controlling cellular redox homeostasis., Objective: This research aimed to investigate the growth-promoting effects of G6PD in non-small cell lung cancer (NSCLC)., Methods: Clinical characteristics and G6PD expression levels in lung tissues of 64 patients diagnosed with lung cancer at the King Chulalongkorn Memorial Hospital (Bangkok, Thailand) during 2009-2014 were analyzed. G6PD activity in NSCLC cell lines, including NCI-H1975 and NCI-H292, was experimentally inhibited using DHEA and siG6PD to study cancer cell proliferation and migration., Results: The positive expression of G6PD in NSCLC tissues was detected by immunohistochemical staining and was found to be associated with squamous cells. G6PD expression levels and activity also coincided with the proliferation rate of NSCLC cell lines. Suppression of G6PD-induced apoptosis in NSCLC cell lines by increasing Bax/Bcl-2 ratio expression. The addition of D-(-)-ribose, which is an end-product of the PPP, increased the survival of G6PD-deficient NSCLC cell lines., Conclusion: Collectively, these findings demonstrated that G6PD might play an important role in the carcinogenesis of NSCLC. Inhibition of G6PD might provide a therapeutic strategy for the treatment of NSCLC., Competing Interests: The authors declare there are no competing interests., (©2023 Chanda et al.)

Background: Leishmania orientalis (formerly named Leishmania siamensis ) has been neglected for years in Thailand. The genomic study of L. orientalis has gained much attention recently after the release of the first high-quality reference genome of the isolate LSCM4. The integrative approach of multiple sequencing platforms for whole-genome sequencing has proven effective at the expense of considerably expensive costs. This study presents a preliminary bioinformatic workflow including the use of multi-step de novo assembly coupled with the reference-based assembly method to produce high-quality genomic drafts from the short-read Illumina sequence data of L. orientalis isolate PCM2., Results: The integrating multi-step de novo assembly by MEGAHIT and SPAdes with the reference-based method using the L. enriettii genome and salvaging the unmapped reads resulted in the 30.27 Mb genomic draft of L. orientalis isolate PCM2 with 3367 contigs and 8887 predicted genes. The results from the integrated approach showed the best integrity, coverage, and contig alignment when compared to the genome of L. orientalis isolate LSCM4 collected from the northern province of Thailand. Similar patterns of gene ratios and frequency were observed from the GO biological process annotation. Fifty GO terms were assigned to the assembled genomes, and 23 of these (accounting for 61.6% of the annotated genes) showed higher gene counts and ratios when results from our workflow were compared to those of the LSCM4 isolate., Conclusions: These results indicated that our proposed bioinformatic workflow produced an acceptable-quality genome of L. orientalis strain PCM2 for functional genomic analysis, maximising the usage of the short-read data. This workflow would give extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation.

(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis ) and Leishmania martiniquensis , has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions.

Background: Pasteurella multocida is a Gram-negative bacterium that causes economically significant infections of a broad range of animal species. Pneumonic and septicaemic pasteurellosis caused by this bacterium remain important problems in pigs, cattle, and water buffaloes in Thailand. The aim of this study was to characterise the virulence-associated gene profiles and to develop an OmpA molecular typing scheme for classifying 191 bovine and porcine isolates of P. multocida collected between 1989 and 2012 in Thailand using polymerase chain reactions (PCRs), nucleotide sequencing, and sequence and structural bioinformatics analyses., Results: PCR screening successfully characterised the profiles of 25 virulence-associated genes in all isolates. The gene profiles separated these isolates into bovine and porcine clusters based on eight genes (hgbB, hsf1, tadD, nanH, pfhA, plpE, pmHAS, and tbpA). Phylogenetic analyses of the nucleotide and protein sequences corresponding to the ompA gene, which encodes a major outer membrane surface protein, showed two major bovine and porcine clusters. Structural prediction and analysis of the dN/dS ratio revealed four hypervariable extracellular loops of the OmpA transmembrane domains. These four loops were used to develop an OmpA typing scheme. This scheme classified 186 isolates into five major loop sequence types (LST8, LST12, LST15, LST18, and LST19), consistent with the phylogenetic results. The loop regions of the bovine isolates were predicted to be more antigenic than those of the porcine isolates. Thus, molecular evolution of the OmpA proteins could be used to classify P. multocida isolates into different capsular types, host types, and, possibly, pathogenicity levels., Conclusions: Together with the virulence-associated gene profiles, the typing reported in this work provides a better understanding of P. multocida virulence. Effective monitoring and potential strain-specific subunit vaccines could be developed based on these loop oligopeptides.