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Fulltext Functional characterization of quorum sensing LuxR-type transcriptional regulator, EasR in Enterobacter asburiae strain L1

Lau YY, How KY, Yin WF, Chan KG

PeerJ, 2020;8:e10068.
PMID: 33150063 DOI: 10.7717/peerj.10068

Over the past decades, Enterobacter spp. have been identified as challenging and important pathogens. The emergence of multidrug-resistant Enterobacteria especially those that produce Klebsiella pneumoniae carbapenemase has been a very worrying health crisis. Although efforts have been made to unravel the complex mechanisms that contribute to the pathogenicity of different Enterobacter spp., there is very little information associated with AHL-type QS mechanism in Enterobacter spp. Signaling via N-acyl homoserine lactone (AHL) is the most common quorum sensing (QS) mechanism utilized by Proteobacteria. A typical AHL-based QS system involves two key players: a luxI gene homolog to synthesize AHLs and a luxR gene homolog, an AHL-dependent transcriptional regulator. These signaling molecules enable inter-species and intra-species interaction in response to external stimuli according to population density. In our recent study, we reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. Whole genome sequencing and in silico analysis revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easI/R, in strain L1. In a QS system, a LuxR transcriptional protein detects and responds to the concentration of a specific AHL controlling gene expression. In E. asburiae strain L1, EasR protein binds to its cognate AHLs, N-butanoyl homoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL), modulating the expression of targeted genes. In this current work, we have cloned the 693 bp luxR homolog of strain L1 for further characterization. The functionality and specificity of EasR protein in response to different AHL signaling molecules to activate gene transcription were tested and validated with β-galactosidase assays. Higher β-galactosidase activities were detected for cells harboring EasR, indicating EasR is a functional transcriptional regulator. This is the first report documenting the cloning and characterization of transcriptional regulator, luxR homolog of E. asburiae.
Fulltext Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerantPseudomonassp. AMS8

Garba L, Mohamad Yussoff MA, Abd Halim KB, Ishak SNH, Mohamad Ali MS, Oslan SN, et al.

PeerJ, 2018;6:e4347.
PMID: 29576935 DOI: 10.7717/peerj.4347

Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerantPseudomonassp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed inEscherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerantPseudomonassp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, -6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with thein vivoactivity of the Δ9-fatty acid desaturase on the membrane phospholipids.
Fulltext Infectious hematopoietic necrosis virus: advances in diagnosis and vaccine development

Yong CY, Ong HK, Tang HC, Yeap SK, Omar AR, Ho KL, et al.

PeerJ, 2019;7:e7151.
PMID: 31341728 DOI: 10.7717/peerj.7151

The aquaculture of salmonid fishes is a multi-billion dollar industry with production over 3 million tons annually. However, infectious hematopoietic necrosis virus (IHNV), which infects and kills salmon and trout, significantly reduces the revenue of the salmon farming industry. Currently, there is no effective treatment for IHNV infected fishes; therefore, early detection and depopulation of the infected fishes remain the most common practices to contain the spread of IHNV. Apart from hygiene practices in aquaculture and isolation of infected fishes, loss of fishes due to IHNV infection can also be significantly reduced through vaccination programs. In the current review, some of the diagnostic methods for IHNV, spanning from clinical diagnosis to cell culture, serological and molecular methods are discussed in detail. In addition, some of the most significant candidate vaccines for IHNV are also extensively discussed, particularly the DNA vaccines.
Fulltext The Plasmodium knowlesi Pk41 surface protein diversity, natural selection, sub population and geographical clustering: a 6-cysteine protein family member

Ahmed MA, Chu KB, Quan FS

PeerJ, 2018;6:e6141.
PMID: 30581686 DOI: 10.7717/peerj.6141

Introduction: The zoonotic malaria parasite Plasmodium knowlesi has currently become the most dominant form of infection in humans in Malaysia and is an emerging infectious disease in most Southeast Asian countries. The P41 is a merozoite surface protein belonging to the 6-cysteine family and is a well-characterized vaccine candidate in P. vivax and P. falciparum; however, no study has been done in the orthologous gene of P. knowlesi. This study investigates the level of polymorphism, haplotypes and natural selection of pk41 genes in clinical isolates from Malaysia.
Method: Thirty-five full-length pk41 sequences from clinical isolates of Malaysia along with four laboratory lines (along with H-strain) were downloaded from public databases. For comparative analysis between species, orthologous P41 genes from P. falciparum, P. vivax, P. coatneyi and P. cynomolgi were also downloaded. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. Phylogenetic relationships between Pk41 genes were determined using MEGA 5.0 software.
Results: Analysis of 39 full-length pk41 sequences along with the H-strain identified 36 SNPs (20 non-synonymous and 16 synonymous substitutions) resulting in 31 haplotypes. Nucleotide diversity across the full-length gene was low and was similar to its ortholog in P. vivax; pv41. Domain-wise amino acid analysis of the two s48/45 domains indicated low level of polymorphisms for both the domains, and the glutamic acid rich region had extensive size variations. In the central domain, upstream to the glutamate rich region, a unique two to six (K-E)n repeat region was identified within the clinical isolates. Overall, the pk41 genes were indicative of negative/purifying selection due to functional constraints. Domain-wise analysis of the s48/45 domains also indicated purifying selection. However, analysis of Tajima's D across the genes identified non-synonymous SNPs in the s48/45 domain II with high positive values indicating possible epitope binding regions. All the 6-cysteine residues within the s48/45 domains were conserved within the clinical isolates indicating functional conservation of these regions. Phylogenetic analysis of full-length pk41 genes indicated geographical clustering and identified three subpopulations of P. knowlesi; one originating in the laboratory lines and two originating from Sarawak, Malaysian Borneo.
Conclusion: This is the first study to report on the polymorphism and natural selection of pk41 genes from clinical isolates of Malaysia. The results reveal that there is low level of polymorphism in both s48/45 domains, indicating that this antigen could be a potential vaccine target. However, genetic and molecular immunology studies involving higher number of samples from various parts of Malaysia would be necessary to validate this antigen's candidacy as a vaccine target for P. knowlesi.
Fulltext Borneo coral reefs subject to high sediment loads show evidence of resilience to various environmental stressors

Browne N, Braoun C, McIlwain J, Nagarajan R, Zinke J

PeerJ, 2019;7:e7382.
PMID: 31428541 DOI: 10.7717/peerj.7382

For reefs in South East Asia the synergistic effects of rapid land development, insufficient environmental policies and a lack of enforcement has led to poor water quality and compromised coral health from increased sediment and pollution. Those inshore turbid coral reefs, subject to significant sediment inputs, may also inherit some resilience to the effects of thermal stress and coral bleaching. We studied the inshore turbid reefs near Miri, in northwest Borneo through a comprehensive assessment of coral cover and health in addition to quantifying sediment-related parameters. Although Miri's Reefs had comparatively low coral species diversity, dominated by massive and encrusting forms of Diploastrea, Porites, Montipora, Favites, Dipsastrea and Pachyseris, they were characterized by a healthy cover ranging from 22 to 39%. We found a strong inshore to offshore gradient in hard coral cover, diversity and community composition as a direct result of spatial differences in sediment at distances <10 km. As well as distance to shore, we included other environmental variables like reef depth and sediment trap accumulation and particle size that explained 62.5% of variation in benthic composition among sites. Miri's reefs showed little evidence of coral disease and relatively low prevalence of compromised health signs including bleaching (6.7%), bioerosion (6.6%), pigmentation response (2.2%), scars (1.1%) and excessive mucus production (0.5%). Tagged colonies of Diploastrea and Pachyseris suffering partial bleaching in 2016 had fully (90-100%) recovered the following year. There were, however, seasonal differences in bioerosion rates, which increased five-fold after the 2017 wet season. Differences in measures of coral physiology, like that of symbiont density and chlorophyll a for Montipora, Pachyseris and Acropora, were not detected among sites. We conclude that Miri's reefs may be in a temporally stable state given minimal recently dead coral and a limited decline in coral cover over the last two decades. This study provides further evidence that turbid coral reefs exposed to seasonally elevated sediment loads can exhibit relatively high coral cover and be resilient to disease and elevated sea surface temperatures.
Fulltext Genome sequencing and analysis of Salmonella enterica subsp. enterica serovar Stanley UPM 517: Insights on its virulence-associated elements and their potentials as vaccine candidates

Ashari KS, Roslan NS, Omar AR, Bejo MH, Ideris A, Mat Isa N

PeerJ, 2019;7:e6948.
PMID: 31293824 DOI: 10.7717/peerj.6948

Salmonella enterica subsp. enterica serovar Stanley (S. Stanley) is a pathogen that contaminates food, and is related to Salmonella outbreaks in a variety of hosts such as humans and farm animals through products like dairy items and vegetables. Despite the fact that several vaccines of Salmonella strains had been constructed, none of them were developed according to serovar Stanley up to this day. This study presents results of genome sequencing and analysis on our S. Stanley UPM 517 strain taken from fecal swabs of 21-day-old healthy commercial chickens in Perak, Malaysia and used Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) as a reference to be compared with. First, sequencing and assembling of the Salmonella Stanley UPM 517 genome into a contiguous form were done. The work was then continued with scaffolding and gap filling. Annotation and alignment of the draft genome was performed with S. Typhimurium LT2. The other elements of virulence estimated in this study included Salmonella pathogenicity islands, resistance genes, prophages, virulence factors, plasmid regions, restriction-modification sites and the CRISPR-Cas system. The S. Stanley UPM 517 draft genome had a length of 4,736,817 bp with 4,730 coding sequence and 58 RNAs. It was discovered via genomic analysis on this strain that there were antimicrobial resistance properties toward a wide variety of antibiotics. Tcf and ste, the two fimbrial virulence clusters related with human and broiler intestinal colonizations which were not found in S. Typhimurium LT2, were atypically discovered in the S. Stanley UPM 517 genome. These clusters are involved in the intestinal colonization of human and broilers, respectively. There were seven Salmonella pathogenicity islands (SPIs) within the draft genome, which contained the virulence factors associated with Salmonella infection (except SPI-14). Five intact prophage regions, mostly comprising of the protein encoding Gifsy-1, Fels-1, RE-2010 and SEN34 prophages, were also encoded in the draft genome. Also identified were Type I-III restriction-modification sites and the CRISPR-Cas system of the Type I-E subtype. As this strain exhibited resistance toward numerous antibiotics, we distinguished several genes that had the potential for removal in the construction of a possible vaccine candidate to restrain and lessen the pervasiveness of salmonellosis and to function as an alternative to antibiotics.
Fulltext First record of Phyllorhiza sp. (Cnidaria: Scyphozoa) in a Chinese coastal aquaculture pond

Dong Z, Morandini AC, Schiariti A, Wang L, Sun T

PeerJ, 2019;7:e6191.
PMID: 30643699 DOI: 10.7717/peerj.6191

Background: It has been suggested that aquaculture ponds on the Chinese coast could act as breeding grounds for scyphozoans. Here, we present the first record of the scyphomedusa Phyllorhiza sp. in an aquaculture pond on the coast of the southern Yellow Sea, based on a combination of morphological characteristics and mitochondrial 16S DNA sequence data.
Methods: A field survey was performed on June 29, 2017 in a pond used for culturing the shrimp Penaeus japonicus, located in the southern Yellow Sea, China. Jellyfish specimens were collected for morphological and genetic analysis. The morphological characters of the jellyfish specimens were compared to taxonomic literature. Additionally, phylogenetic analysis of the mitochondrial 16S fragments of these specimens were also conducted.
Results: These specimens had the following morphological characters: hemispherical umbrella without scapulets; J-shaped oral arms; a single larger terminal club on each arm; bluish colored with a slightly expanded white tip; and mouthlets present only in the lower half to one-third of each arm. These morphological features of the medusae indicated that the specimens found in the shrimp culture ponds belong to the genus Phyllorhiza Agassiz, 1862, but did not match with the description of any of the known species of the genus Phyllorhiza. Phylogenetic analyses of the mtDNA 16S regions revealed that these specimens, together with Phyllorhiza sp. from Malaysian coastal waters, belong to a sister group of Phyllorhiza punctata. Juveniles and ephyrae of Phyllorhiza sp. were observed in the aquaculture pond. The mean density of Phyllorhiza sp. medusa in the surface water within the pond was estimated to be 0.05 individuals/m2.
Discussion: Based on our observations of the gross morphology and molecular data, we state that the specimens collected in the aquaculture pond can be identified as Phyllorhiza sp. This is the first record of Phyllorhiza sp. in Chinese seas. Large scale dispersal through ballast water or the expansion of jellyfish aquarium exhibitions are possible pathways of invasion, but this needs to be confirmed in further studies.
Fulltext Targeted inactivation of Salmonella Agona metabolic genes by group II introns and in vivo assessment of pathogenicity and anti-tumour activity in mouse model

Gwee CP, Khoo CH, Yeap SK, Tan GC, Cheah YK

PeerJ, 2019;7:e5989.
PMID: 30671294 DOI: 10.7717/peerj.5989

The fight against cancer has been a never-ending battle. Limitations of conventional therapies include lack of selectivity, poor penetration and highly toxic to the host. Using genetically modified bacteria as a tumour therapy agent has gained the interest of scientist from the past few decades. Low virulence and highly tolerability of Salmonella spp. in animals and humans make it as the most studied pathogen with regards to anti-tumour therapy. The present study aims to construct a genetically modified S. Agona auxotroph as an anti-tumour agent. LeuB and ArgD metabolic genes in ΔSopBΔSopD double knockout S. Agona were successfully knocked out using a Targetron gene knockout system. The knockout was confirmed by colony PCR and the strains were characterized in vitro and in vivo. The knockout of metabolic genes causes significant growth defect in M9 minimal media. Quadruple knockout ΔSopBΔSopDΔLeuBΔArgD (BDLA) exhibited lowest virulence among all of the strains in all parameters including bacterial load, immunity profile and histopathology studies. In vivo anti-tumour study on colorectal tumour bearing-BALB/c mice revealed that all strains of S. Agona were able to suppress the growth of the large solid tumour as compared with negative control and ΔLeuBΔArgD (LA) and BDLA auxotroph showed better efficacy. Interestingly, higher level of tumour growth suppression was noticed in large tumour. However, multiple administration of bacteria dosage did not increase the tumour suppression efficacy. In this study, the virulence of BDLA knockout strain was slightly reduced and tumour growth suppression efficacy was successfully enhanced, which provide a valuable starting point for the development of S. Agona as anti-tumour agent.
Fulltext Histological changes of female reproductive organs subjected to different jumping exercise intensities and honey supplementation in rats

Mosavat M, Mohamed M, Ooi FK, Mirsanjari M, Mat Zin AA, Che Romli A

PeerJ, 2019;7:e7646.
PMID: 31565571 DOI: 10.7717/peerj.7646

Background: We assessed histopathological changes of ovaries and uterus in female rats subjected to different jumping exercise intensities combined with honey supplementation at one g/kg body weight/day.
Methods: A total of 72 rats were divided into six groups, 12 rats in each: control (C), 20 and 80 jumps (20E, 80E), honey (H), and 20 and 80 jump with honey (20EH, 80EH).
Results: The endometrium was significantly thicker in the rats in H, 20EH and 80EH groups compared to C, 20E, and 80E. The myometrium thickness was significantly lower in 80E and significantly higher in 80EH compared to C, respectively. There was significantly higher myometrium thickness in 20EH and 80EH compared to 20E and 80E and H. The number of glands of the uterus in 20E and 80E was significantly lower than C. However, there was a significantly higher number of glands in H, 20EH, and 80EH compared to 20E and 80E. The numbers of uterus vessels were significantly lower in 80E compared to 20E. However, the numbers of vessels were significantly higher in H, 20EH, and 80EH compared to 80E. The number of ovarian haemorregia was significantly lower in 20E, 80E, H, 20EH, and 80EH compared to C. The number of corpora lutea was significantly lower in 80EH, H, 80E, and 20E compared to C. However, the number of corpora lutea was significantly higher in 20EH compared to J20 and H.
Conclusion: This study suggested that jumping exercises in particularly high-intensity exercise may induce histopathological changes in uterus and ovary in rats, and honey supplementation may ameliorate these effects.
Fulltext Cellular accumulation and cytotoxic effects of zinc oxide nanoparticles in microalga Haematococcus pluvialis

Djearamane S, Lim YM, Wong LS, Lee PF

PeerJ, 2019;7:e7582.
PMID: 31579572 DOI: 10.7717/peerj.7582

Background: Zinc oxide nanoparticles (ZnO NPs) are widely used in household and cosmetic products which imply an increased releasing of these particles into the environment, especially aquatic ecosystems, resulting in the need of assessing the potential toxic effects of ZnO NPS on the aquatic organisms, particularly on microalgae which form the base for food chain of aquatic biota. The present study has investigated the dose- and time-dependent cellular accumulation and the corresponding cytotoxic effects of increasing concentrations of ZnO NPs from 10-200 μg/mL on microalga Haematococcus pluvialis at an interval of 24 h for 96 h.
Methods: The scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX) was used to qualitatively detect the cellular accumulation of ZnO NPs in algal cells, while inductively coupled plasma optical emission spectrometry (ICP OES) was performed to quantify the cell associated-zinc in algal cells. The percentage of cell death, reduction in algal biomass, and loss in photosynthetic pigments were measured to investigate the cytotoxic effects of ZnO NPs on H. pluvialis. Extracellular and intracellular changes in algal cells resulted from the treatment of ZnO NPs were demonstrated through optical, scanning, and transmission electron microscopic studies.
Results: SEM-EDX spectrum evidenced the accumulation of ZnO NPs in algal biomass and ICP OES results reported a significant (p < 0.05) dose- and time-dependent accumulation of zinc in algal cells from 24 h for all the tested concentrations of ZnO NPs (10-200 μg/mL). Further, the study showed a significant (p < 0.05) dose- and time-dependent growth inhibition of H. pluvialis from 72 h at 10-200 μg/mL of ZnO NPs. The morphological examinations revealed substantial surface and intracellular damages in algal cells due to the treatment of ZnO NPs.
Discussion: The present study reported the significant cellular accumulation of ZnO NPs in algal cells and the corresponding cytotoxic effects of ZnO NPs on H. pluvialis through the considerable reduction in algal cell viability, biomass, and photosynthetic pigments together with surface and intracellular damages.
Fulltext Computational analysis of functional SNPs in Alzheimer's disease-associated endocytosis genes

Tey HJ, Ng CH

PeerJ, 2019;7:e7667.
PMID: 31592138 DOI: 10.7717/peerj.7667

Background: From genome wide association studies on Alzheimer's disease (AD), it has been shown that many single nucleotide polymorphisms (SNPs) of genes of different pathways affect the disease risk. One of the pathways is endocytosis, and variants in these genes may affect their functions in amyloid precursor protein (APP) trafficking, amyloid-beta (Aβ) production as well as its clearance in the brain. This study uses computational methods to predict the effect of novel SNPs, including untranslated region (UTR) variants, splice site variants, synonymous SNPs (sSNPs) and non-synonymous SNPs (nsSNPs) in three endocytosis genes associated with AD, namely PICALM, SYNJ1 and SH3KBP1.
Materials and Methods: All the variants' information was retrieved from the Ensembl genome database, and then different variation prediction analyses were performed. UTRScan was used to predict UTR variants while MaxEntScan was used to predict splice site variants. Meta-analysis by PredictSNP2 was used to predict sSNPs. Parallel prediction analyses by five different software packages including SIFT, PolyPhen-2, Mutation Assessor, I-Mutant2.0 and SNPs&GO were used to predict the effects of nsSNPs. The level of evolutionary conservation of deleterious nsSNPs was further analyzed using ConSurf server. Mutant protein structures of deleterious nsSNPs were modelled and refined using SPARKS-X and ModRefiner for structural comparison.
Results: A total of 56 deleterious variants were identified in this study, including 12 UTR variants, 18 splice site variants, eight sSNPs and 18 nsSNPs. Among these 56 deleterious variants, seven variants were also identified in the Alzheimer's Disease Sequencing Project (ADSP), Alzheimer's Disease Neuroimaging Initiative (ADNI) and Mount Sinai Brain Bank (MSBB) studies.
Discussion: The 56 deleterious variants were predicted to affect the regulation of gene expression, or have functional impacts on these three endocytosis genes and their gene products. The deleterious variants in these genes are expected to affect their cellular function in endocytosis and may be implicated in the pathogenesis of AD as well. The biological consequences of these deleterious variants and their potential impacts on the disease risks could be further validated experimentally and may be useful for gene-disease association study.
Fulltext Expression of leptin and leptin receptors in colorectal cancer-an immunohistochemical study

Al-Shibli SM, Harun N, Ashour AE, Mohd Kasmuri MHB, Mizan S

PeerJ, 2019;7:e7624.
PMID: 31592340 DOI: 10.7717/peerj.7624

Obesity is demonstrated to be a risk factor in the development of cancers of various organs, such as colon, prostate, pancreas and so on. Leptine (LEP) is the most renowned of the adipokines. As a hormone, it mediates its effect through leptin receptor (LEPR), which is widely expressed in various tissues including colon mucosa. In this study, we have investigated the degree of expression of LEP and LEPR in colorectal cancer (CRC). We collected 44 surgically resected colon cancer tissues along with normal adjacent colon tissue (NACT) from a sample of CRC patients from the Malaysian population and looked for leptin and leptin receptors using immunohistochemistry (IHC). All the samples showed low presence of both LEP and LEPR in NACT, while both LEP and LEPR were present at high intensity in the cancerous tissues with 100% and 97.7% prevalence, respectively. Both were sparsed in the cytoplasm and were concentrated beneath the cell membrane. However, we did not find any significant correlation between their expression and pathological parameters like grade, tumor size, and lymph node involvement. Our study further emphasizes the possible causal role of LEP and LEPR with CRC, and also the prospect of using LEPR as a possible therapeutic target.
Fulltext Evaluation of female Aedes aegypti proteome via LC-ESI-MS/MS using two protein extraction methods

Shettima A, Ishak IH, Abdul Rais SH, Abu Hasan H, Othman N

PeerJ, 2021;9:e10863.
PMID: 33717682 DOI: 10.7717/peerj.10863

Background: Proteomic analyses have broadened the horizons of vector control measures by identifying proteins associated with different biological and physiological processes and give further insight into the mosquitoes' biology, mechanism of insecticide resistance and pathogens-mosquitoes interaction. Female Ae. aegypti ingests human blood to acquire the requisite nutrients to make eggs. During blood ingestion, female mosquitoes transmit different pathogens. Therefore, this study aimed to determine the best protein extraction method for mass spectrometry analysis which will allow a better proteome profiling for female mosquitoes.
Methods: In this present study, two protein extractions methods were performed to analyze female Ae. aegyti proteome, via TCA acetone precipitation extraction method and a commercial protein extraction reagent CytoBusterTM. Then, protein identification was performed by LC-ESI-MS/MS and followed by functional protein annotation analysis.
Results: The CytoBusterTM reagent gave the highest protein yield with a mean of 475.90 µg compared to TCA acetone precipitation extraction showed 283.15 µg mean of protein. LC-ESI-MS/MS identified 1,290 and 890 proteins from the CytoBusterTM reagent and TCA acetone precipitation, respectively. When comparing the protein class categories in both methods, there were three additional categories for proteins identified using CytoBusterTM reagent. The proteins were related to scaffold/adaptor protein (PC00226), protein binding activity modulator (PC00095) and intercellular signal molecule (PC00207). In conclusion, the CytoBusterTM protein extraction reagent showed a better performance for the extraction of proteins in term of the protein yield, proteome coverage and extraction speed.
Fulltext Historical and current distribution ranges and loss of mega-herbivores and carnivores of Asia

Mahmood T, Vu TT, Campos-Arceiz A, Akrim F, Andleeb S, Farooq M, et al.

PeerJ, 2021;9:e10738.
PMID: 33628635 DOI: 10.7717/peerj.10738

Ecosystem functioning is dependent a lot on large mammals, which are, however, vulnerable and facing extinction risks due to human impacts mainly. Megafauna of Asia has been declining for a long, not only in numbers but also in their distribution ranges. In the current study, we collected information on past and current occurrence and distribution records of Asia's megafauna species. We reconstructed the historical distribution ranges of the six herbivores and four carnivores for comparison with their present ranges, to quantify spatially explicit levels of mega-defaunation. Results revealed that historically the selected megafauna species were more widely distributed than at current. Severe range contraction was observed for the Asiatic lion, three rhino species, Asian elephant, tigers, and tapirs. Defaunation maps generated have revealed the vanishing of megafauna from parts of the East, Southeast, and Southwest Asia, even some protected Areas losing up to eight out of ten megafaunal species. These defaunation maps can help develop future conservation policies, to save the remaining distribution ranges of large mammals.
Fulltext Metabolite profiling of endophytic Streptomyces spp. and its antiplasmodial potential

Ahmad SJ, Mohamad Zin N, Mazlan NW, Baharum SN, Baba MS, Lau YL

PeerJ, 2021;9:e10816.
PMID: 33777509 DOI: 10.7717/peerj.10816

Background: Antiplasmodial drug discovery is significant especially from natural sources such as plant bacteria. This research aimed to determine antiplasmodial metabolites of Streptomyces spp. against Plasmodium falciparum 3D7 by using a metabolomics approach.
Methods: Streptomyces strains' growth curves, namely SUK 12 and SUK 48, were measured and P. falciparum 3D7 IC50 values were calculated. Metabolomics analysis was conducted on both strains' mid-exponential and stationary phase extracts.
Results: The most successful antiplasmodial activity of SUK 12 and SUK 48 extracts shown to be at the stationary phase with IC50 values of 0.8168 ng/mL and 0.1963 ng/mL, respectively. In contrast, the IC50 value of chloroquine diphosphate (CQ) for antiplasmodial activity was 0.2812 ng/mL. The univariate analysis revealed that 854 metabolites and 14, 44 and three metabolites showed significant differences in terms of strain, fermentation phase, and their interactions. Orthogonal partial least square-discriminant analysis and S-loading plot putatively identified pavettine, aurantioclavine, and 4-butyldiphenylmethane as significant outliers from the stationary phase of SUK 48. For potential isolation, metabolomics approach may be used as a preliminary approach to rapidly track and identify the presence of antimalarial metabolites before any isolation and purification can be done.
Fulltext Gluconobacter dominates the gut microbiome of the Asian palm civet Paradoxurus hermaphroditus that produces kopi luwak

Watanabe H, Ng CH, Limviphuvadh V, Suzuki S, Yamada T

PeerJ, 2020;8:e9579.
PMID: 32821539 DOI: 10.7717/peerj.9579

Coffee beans derived from feces of the civet cat are used to brew coffee known as kopi luwak (the Indonesian words for coffee and palm civet, respectively), which is one of the most expensive coffees in the world owing to its limited supply and strong market demand. Recent metabolomics studies have revealed that kopi luwak metabolites differ from metabolites found in other coffee beans. To produce kopi luwak, coffee beans are first eaten by civet cats. It has been proposed that fermentation inside the civet cat digestive tract may contribute to the distinctively smooth flavor of kopi luwak, but the biological basis has not been determined. Therefore, we characterized the microbiome of civet cat feces using 16S rRNA gene sequences to determine the bacterial taxa that may influence fermentation processes related to kopi luwak. Moreover, we compared this fecal microbiome with that of 14 other animals, revealing that Gluconobacter is a genus that is, uniquely found in feces of the civet cat. We also found that Gluconobacter species have a large number of cell motility genes, which may encode flagellar proteins allowing colonization of the civet gut. In addition, genes encoding enzymes involved in the metabolism of hydrogen sulfide and sulfur-containing amino acids were over-represented in Gluconobacter. These genes may contribute to the fermentation of coffee beans in the digestive tract of civet cats.
Fulltext Explicating anti-amyloidogenic role of curcumin and piperine via amyloid beta (Aβ) explicit pathway: recovery and reversal paradigm effects

Abdul Manap AS, Madhavan P, Vijayabalan S, Chia A, Fukui K

PeerJ, 2020;8:e10003.
PMID: 33062432 DOI: 10.7717/peerj.10003

Previously, we reported the synergistic effects of curcumin and piperine in cell cultures as potential anti-cholinesterase and anti-amyloidogenic agents. Due to limited findings on the enrolment of these compounds on epigenetic events in AD, we aimed at elucidating the expression profiles of Aβ42-induced SH-SY5Y cells using microarray profiling. In this study, an optimized concentration of 35 µM of curcumin and piperine in combination was used to treat Aβ42 fibril and high-throughput microarray profiling was performed on the extracted RNA. This was then compared to curcumin and piperine used singularly at 49.11 µM and 25 µM, respectively. Our results demonstrated that in the curcumin treated group, from the top 10 upregulated and top 10 downregulated significantly differentially expressed genes (p
Fulltext Morphological and molecular systematic review of Marphysa Quatrefages, 1865 (Annelida: Eunicidae) species from South Africa

Kara J, Molina-Acevedo IC, Zanol J, Simon C, Idris I

PeerJ, 2020;8:e10076.
PMID: 33150064 DOI: 10.7717/peerj.10076

A vast polychaete fauna is hidden behind complexes of cryptic and pseudo-cryptic species, which has greatly hindered our understanding of species diversity in several regions worldwide. Among the eunicids, Marphysa sanguinea Montagu, 1813 is a typical example, recorded in three oceans and with various species considered its junior synonyms. In South Africa, specimens previously misidentified as M. sanguinea are now known as Marphysa elityeni Lewis & Karageorgopoulos, 2008. Of the six Marphysa Quatrefages, 1865a species recorded from the same region, three have their distributions restricted to South Africa while the others are considered to have worldwide distributions. Here, we evaluated the taxonomic status of the indigenous M. elityeni and investigated the presence of the widespread species Marphysa macintoshi Crossland, 1903 and Marphysa depressa Schmarda, 1861 in South Africa using morphological and molecular data. Our results reveal that M. elityeni is a junior synonym of Marphysa haemasoma, a species previously described from South Africa which is herein reinstated as a valid species. Both M. macintoshi and M. depressa are not present in South Africa and their status as being distributed worldwide deserves further investigation. Marphysa durbanensis Day, 1934 and the new species described here, M. sherlockae n. sp., had been misidentified as M. macintoshi and M. depressa respectively. Thus, the number of Marphysa species with distributions restricted to South Africa increased from three to five. This study reiterates the importance of implementing an integrated taxonomic framework to unravel local biodiversity.
Fulltext Current and future trends in socio-economic, demographic and governance factors affecting global primate conservation

Estrada A, Garber PA, Chaudhary A

PeerJ, 2020;8:e9816.
PMID: 32884865 DOI: 10.7717/peerj.9816

Currently, ~65% of extant primate species (ca 512 species) distributed in 91 countries in the Neotropics, mainland Africa, Madagascar, South Asia and Southeast Asia are threatened with extinction and 75% have declining populations as a result of deforestation and habitat loss resulting from increasing global market demands, and land conversion for industrial agriculture, cattle production and natural resource extraction. Other pressures that negatively impact primates are unsustainable bushmeat hunting, the illegal trade of primates as pets and as body parts, expanding road networks in previously isolated areas, zoonotic disease transmission and climate change. Here we examine current and future trends in several socio-economic factors directly or indirectly affecting primates to further our understanding of the interdependent relationship between human well-being, sustainable development, and primate population persistence. We found that between 2001 and 2018 ca 191 Mha of tropical forest (30% canopy cover) were lost as a result of human activities in the five primate range regions. Forty-six percent of this loss was in the Neotropics (Mexico, Central and South America), 30% in Southeast Asia, 21% in mainland Africa, 2% in Madagascar and 1% in South Asia. Countries with the greatest losses (ca 57% of total tree cover loss) were Brazil, Indonesia, DRC, China, and Malaysia. Together these countries harbor almost 50% of all extant primate species. In 2018, the world human population was estimated at ca 8bn people, ca 60% of which were found in primate range countries. Projections to 2050 and to 2100 indicate continued rapid growth of the human populations in these five primate range regions, with Africa surpassing all the other regions and totaling ca 4bn people by the year 2100. Socioeconomic indicators show that, compared to developed nations, most primate range countries are characterized by high levels of poverty and income inequality, low human development, low food security, high levels of corruption and weak governance. Models of Shared Socioeconomic Pathway scenarios (SSPs) projected to 2050 and 2100 showed that whereas practices of increasing inequality (SSP4) or unconstrained growth in economic output and energy use (SSP5) are projected to have dire consequences for human well-being and primate survivorship, practices of sustainability-focused growth and equality (SSP1) are expected to have a positive effect on maintaining biodiversity, protecting environments, and improving the human condition. These results stress that improving the well-being, health, and security of the current and future human populations in primate range countries are of paramount importance if we are to move forward with effective policies to protect the world's primate species and promote biodiversity conservation.
Fulltext Improved genome of Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium genomospecies 4

Gan HM, Lee MVL, Savka MA

PeerJ, 2019;7:e6366.
PMID: 30775173 DOI: 10.7717/peerj.6366

The reported Agrobacterium radiobacter DSM 30174T genome is highly fragmented, hindering robust comparative genomics and genome-based taxonomic analysis. We re-sequenced the Agrobacterium radiobacter type strain, generating a dramatically improved genome with high contiguity. In addition, we sequenced the genome of Agrobacterium tumefaciens B6T, enabling for the first time, a proper comparative genomics of these contentious Agrobacterium species. We provide concrete evidence that the previously reported Agrobacterium radiobacter type strain genome (Accession Number: ASXY01) is contaminated which explains its abnormally large genome size and fragmented assembly. We propose that Agrobacterium tumefaciens be reclassified as Agrobacterium radiobacter subsp. tumefaciens and that Agrobacterium radiobacter retains it species status with the proposed name of Agrobacterium radiobacter subsp. radiobacter. This proposal is based, first on the high pairwise genome-scale average nucleotide identity supporting the amalgamation of both Agrobacterium radiobacter and Agrobacterium tumefaciens into a single species. Second, maximum likelihood tree construction based on the concatenated alignment of shared genes (core genes) among related strains indicates that Agrobacterium radiobacter NCPPB3001 is sufficiently divergent from Agrobacterium tumefaciens to propose two independent sub-clades. Third, Agrobacterium tumefaciens demonstrates the genomic potential to synthesize the L configuration of fucose in its lipid polysaccharide, fostering its ability to colonize plant cells more effectively than Agrobacterium radiobacter.

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