Pinus taeda (native host) and Populus trichocarpa (non-native host). Gene expression of interacting fungal and bacterial rhizosphere communities was compared among three plant-pairs: Populus growing with Populus, Populus with Pinus, and Pinus with Pinus. Our results demonstrate that heterospecific plant partners affect the assembly of root microbiomes, including the changes in the structure of host specific community. Comparative metatranscriptomics reveals that several species of ectomycorrhizal fungi (EMF) and saprotrophic fungi exhibit different patterns of functional and regulatory gene expression with these two plant hosts. Heterospecific plants affect the transcriptional expression pattern of EMF host-specialists  on both plant species, mainly including the genes involved in the transportation of amino acids, carbohydrates, and inorganic ions. Alteration of root microbiome by neighboring plants may help regulate basic plant physiological processes via modulation of molecular functions in the root microbiome.Within the forest community, competition and facilitation between adjacent-growing conspecific and heterospecific plants are mediated by interactions involving common mycorrhizal networks. The ability of plants to alter their neighbor’s microbiome is well documented, but the molecular biology of plant-fungal interactions during competition and facilitation has not been previously examined. We used a common soil-plant bioassay experiment to study molecular plant-microbial interactions among rhizosphere communities associated with  Temperate and boreal forest communities are often composed of a mix of plant species. These forest plants may release up to 40% of their photo-assimilated carbon into the soil, where it is dynamically transferred, along with soil nutrients, among neighboring plants through common mycorrhizal networks (CMNs) or “wood wide web”  and rhizPinus trees utilize ectomycorrhizal CMNs to shunt nutrients from overstory plant hosts to the nutrient-demanders  are the most dominant fungal symbionts of tree species . For exaosystems . In addiosystems . Previouosystems , which iosystems . Relativosystems .Populus and Pinus species in heterospecific and conspecific combinations to assess the influence of host’s microbiome on that of its plant neighbors. We hypothesized that heterospecific neighbor plants would alter each other’s root microbiome structure  and gene expression, impacting the biomass of plant partners. In this study, we define root microbiome (root bacteria and root fungi) as the microbes living in or around the roots. We characterized gene expression of the whole root microbial community using metatranscriptomics to reveal molecular mechanisms governing the selection of fungal microorganisms and plant growth promotion. Within the same soil, different species of plants have been reported to harbor different communities of microorganisms  vs. non-native (Populus trichocarpa) hosts, a plant bioassay experiment was performed with fresh soils collected from a P. taeda forest site  in October 2014. Around 30 Kg soils  were collected with a spade shovel. At the time of soil collection, the average temperature in these forest soils was 20°C. Collected soils were mixed with sterile sand [soil:sand = 3:7 (w/w)] and plants were grown with a partner in three combinations: (1) Populus × Populus, (2) Populus × Pinus, and (3) Pinus × Pinus  and 12 months (n = 4). At these times, plant dry weight biomass and mycorrhization levels were examined .Metatranscriptomic data was generated for samples taken at the 4-month time period .The means that were presented with bars  and in tSRP156890). Bioinformatic pipelines for data assembly, quality filtering, annotation and comparison are described in Supporting Information Text A2. Briefly, ∼440 millions of qualified reads with a quality score >20  were used for downstream analysis .DNA and RNA of root samples were co-extracted and purified according to analysis , Step S1analysis , and NCBanalysis . The alide novo approaches to sort out RNA-seq reads belonging to key functional genes of fungi . We then pooled and assembled de novo reads into contigs to serve as references (P. trichocarpa roots” (FunGene_P. tri) and “Fungal genes originally expressed in P. taeda root” (FunGene_P. taeda). Here, the terms “FunGene_P. tri” and “FunGene_P. taeda” references represent the assembled reads that were originally collected from P. trichocarpa and P. taeda root, respectively. Since most of the pine root tips were heavily colonized by EMF . Comparative transcriptomics were furthered applied for the normalized mapped-reads with the DESeq package (FunGene_P. tri and FunGene_P. taeda were assigned to the function (KOG) of closely relative fungal taxa using BlastX. The annotated protein sequences from 24 fungal genomes (Supporting Information Text A2_4) were used as references for mapping transcripts. These references were selected based upon their phylogenetic association with dominant fungal taxa detected in our dataset , Leptosphaeria, Fusarium, Pleurotus, and Glonium], the dominant soil/root microbiomes that were presented in the Populus sites  . The representative ecological group of each assigned gene was further designated based on the predicted ecological function of the assigned reference. The R-scripts used for this study can be accessed through: https://github.com/NFREC-Liao-Lab/Neighbor_plant_microbiome_RNA.To determine the genes belonging to root fungi that were differentially expressed across the three combinations of plant partners, we applied a combination of genome-based mapping and of fungi . Firstlyferences  to identferences –7. Usingd by EMF , we expe package . Here, taset see  [e.g., Rdophora) . The seqdophora) . Here, tPinus and Populus plants grown together in pairwise combinations in a common soil to study neighbor effects on plant growth, root community and gene expression  with non-host plant species, however, might still be able to communicate with non-hosts through molecular interactions  were 24% (stdev. 3.2%), 27% (stdev. 1.9%), and 47%, respectively  were 5% (stdev 0.4%), 59% (stdev 1.6%), and 37%, respectively. Excluding ribosomal RNA reads (see next section), 37% (from Pinus root samples) and 47% of the reads (from Populus root samples) did not map to fungal genomes or plant transcriptomic database. Some of these reads may belong to plant and fungi, however, were not mapped due to lower abundance of these genes or genome variation within a species. Some of the unmapped reads may belong to the fungi which have not been molecular characterized. These sequences were excluded from the analysis. For example, relatively few full genome sequences are publicly available to serve as the databases for AMF and EMF species, indicating that some of the excluded reads might belong to the unknown taxa of mycorrhizal fungi. The attempts to identify the expression pattern of mycorrhizal fungi and other root microbes can be limited by the limitation of current available genome databases.We employed metatranscriptomics to examine root tips of eatments . For P. ectively . In contPinus and Populus together resulted in an increase of fungal-to-bacterial rRNA ratio to 2:1 in both plant species  . Fungal rRNAs belonging to putative saprotrophs (18%), pathogens (16%) and endophytic fungi (4%) were also consistently observed in Populus roots (Populus roots include Claroideoglomus (AMF), Diversispora (AMF), Hebeloma (EMF), Hyaloscypha (EMF), Peziza (putative-EMF) and Tuber (EMF). Interestingly, Populus roots were dominated by a similar group of fungal taxa regardless of the presence of heterospecific neighboring plants. Previous studies where P. trichocarpa cuttings were grown individually also found that Claroideoglomus, Diversispora, Glomus, Rhizophagus, Tuber, Hebeloma, and Hyaloscypha were dominated in Populus roots independent of whether cuttings were grown in the soils collected from Pinus sites  . Populusus roots . The mosus sites  or Populus sites , indicatPinus roots. Two host-specific taxa Suillus and Rhizopogon were dominant on Pinus roots, and represented over 80% of fungal rRNA D1/D2 reads, implying that compared to other EMF taxa, Suillus and Rhizopogon may better adapt to young seedlings. It also implies their ability to facilitate seedling establishment in pine forests (Sporisorium) and saprotrophic (Cladosporium) fungi were also detected in ectomycorrhizal roots of Pinus. Pairwise comparisons between P. trichocarpa (P. trichocarpa × P. trichocarpa) vs. P. taeda (P. taeda × P. taeda) suggested that Populus harbored a higher relative abundance of six AMF taxa , four EMF taxa , and 12 other fungal taxa with endophytic, pathogenic or/and saprotrophic activities , Pseudomonadales (14%), Burkholderiales (9%), Rhizobiales (8%), and Sphingomonadales (5%). Acidobacteria, previously reported to be reduced in the Populus root endosphere , Comamonadaceae , and Bradyrhizobiaceae  (P. trichocarpa (P. trichocarpa × P. trichocarpa) vs. P. taeda (P. taeda × P. taeda), and only two families (Comamonadaceae and Bradyrhizobiaceae) showed higher relative abundance in P. taeda (P. taeda × P. taeda)  . These tes, 38%) . Of the . taeda) . Taken tP. trichocarpa × P. trichocarpa; P. trichocarpa × P. taeda; P. taeda × P. taeda. Distinct patterns of fungal and bacterial community were detected on P. trichocarpa and P. taeda root tips when these plant species were grown together (P < 0.05). The results verify that plant species can select for specific root microbiomes (in terms of maintaining high relative abundances of specific microbiome), even with heterospecific plants growing nearby. However, the community structures of certain fungal and bacterial taxa can be significantly affected by the species of neighboring plants  or decreased (15 taxa) when P. trichocarpa was grown with P. taeda compared to when P. trichocarpa was grown with a conspecific neighbor  while growing with P. trichocarpa, a few P. trichocarpa-associated EMF , AMF  and endophytes  and other five taxa increased their relative abundance of rRNA on these Pinus roots  compared to Pinus roots. Boletales (particularly Suillus and Rhizopogon) were consistently abundant in Pinus, which dominate the Pinus rhizosphere as uncovered by RNA-seq data  were abundant in RNA-seq data of Pinus but not in cDNA amplicon sequencing data. RNA-seq reads assigned to Suillus were examined by aligning to Suillus reference sequences (D1/D2 regions obtained from fruiting bodies). Results confirm that merged short reads could be aligned to Suillus reference sequences, mostly along the conserved area between D1 and D2 regions  and “Fungal genes originally expressed in P. taeda root” (FunGene_P. taeda) were compared. Comparative transcriptomics was further analyzed for the normalized mapped-reads with the DESeq package (FunGene_P. tri and FunGene_P. taeda) were detected on P. trichocarpa and P. taeda roots    than P. trichocarpa roots  . The 1,15 genes) . Intereschocarpa , and P. pa roots . Howeverpa roots  and totapa roots  were greFunGene_P. tri and FunGene_P. taeda were assigned to the function (KOG) of closely relative fungal taxa using BlastX  were stimulated by means of enrichment of expressed genes, when the plant was grown with heterospecific neighbors compared to when it was grown with conspecifics , were also detected when plants were grown with their heterospecific partners  in and/or on the roots, including Pinus-associated EMF. The results imply that certain EMF  may disproportionately contribute to the function of the CMNs. However, changes in gene expression of these Pinus-associated EMF on non-host roots may also reflect a response to different biotic and environmental cues or a shift in the relative abundance of this fungal taxa. For example, we cannot exclude the possibilities that the colonization events and the neighboring effects on fungal gene expression can also be mediated directly by the diffusible metabolites between plant species  relationships with non-EMF host plants . For example, the hyphae of Suillus and Rhizopogon may interact with the rhizodeposits released from P. trichocarpa to mine nitrogen or/and alter P. trichocarpa associated microbiome without colonizing the roots of P. trichocarpa. Future studies are needed to understand whether rhizodeposits from P. trichocarpa structure the molecular function of Pinus EMF specialists.To determine the genes belonging to dominant root fungi that were differentially expressed across the three combination of plant partners, the g BlastX . Most ofl traits , 7. For wn alone . Consequizopogon . The molpecifics . On the pecifics . In addipartners , howeverpartners . Taking g effect , 7A, les species . Compares levels . The SuiSuillus and Rhizopogon were both enriched for genes encoding WD-40 repeats  and cytochrome P450  when interacting with heterospecific hosts compared to conspecifics, but only when these fungal taxa colonized Pinus but not Populus  were enriched across different groups of fungi when grown with heterospecific plants compared to conspecifics . However Populus . Previouognition . Thus, tSuillus and Rhizopogon also expressed higher proportions of transporter genes (0.6 to 0.9% of significantly expressed genes) during interaction with heterospecific plants compared to conspecifics (Suillus and Rhizopogon were significantly increased in P. trichocarpa (P. trichocarpa × P. taeda) compared to P. trichocarpa (P. trichocarpa × P. trichocarpa) roots  for transporters (7%), amino acid transporters (4%), transcriptional co-activators (3%) and chitinases (2%) were enriched, when saprotrophs and endophytes interacted with Pinus compared to all other plant host combinations (FunGene_P. tri) encoding for transporters (5%) were expressed when plant pathogens interacted with Pinus compared to their conspecific host (Populus). Together, these results suggest that activation of transporter genes from multiple fungal taxa with diverse ecological functions is needed to regulate the nutritional flux between organisms. Similarly, changes in the plant community composition may alter the molecular mechanisms of fungal transportation systems and may directly affect source-sink dynamics of any nutritional flux. Overall, these results show that although many differentially expressed genes of fungi for fungal development may be functionally redundant, fungal genes involved in host compatibility and nutrient reallocation are functionally diverse and are differentially regulated depending on plant species.pecifics , 4. For a) roots . Of 401 inations . In addiP. trichocarpa and P. taeda growing in a common soil can still establish distinct community structure and functional capacity of their respective root microbiomes; (c) Plant species promotes rhizobiome diversity and the ratio fungal-to-bacterial rRNA reads; (d) Compared to the compatible fungal-plant interaction, fungal specialists  can express different gene sets to interact with neighboring incompatible (heterospecific) plant hosts. (e) Heterospecific plant species may enrich the fungal genes for transporter systems across different fungal taxa with diverse ecological functions, compared to conspecifics, which may in turn help to determine changes in nutritional flux between organisms and in soil ecosystems. Together, these results provide an important step to scaling from individual plants to community and ecosystem levels.Plant growth is influenced by diverse and complex biotic and abiotic drivers. Using metatranscriptomics, we demonstrate that interactions between different plant host species shape their root microbial community structure and molecular function. We observed that a shift in adjacent plant partners had a direct and asymmetric effect on plant growth and rhizosphere community structure (including at the transcriptome level). Key findings in our study include: (a) individual plants can alter the biomass of their heterospecific plant neighbors, presumably through the action of their CMNs, plant-plant competition or the interactive effect of both drivers; (b) https://www.ncbi.nlm.nih.gov/, SRP156890.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: H-LL, GB, KH, JL, CS, GT, FM, and RV: design and perform the experiments. H-LL, SW, and K-HC: data assembly and analysis. AK, KB, and IG: genome references publication (Joint Genome Institute). H-LL, GB, and RV: manuscript writing. All authors contributed to manuscript editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The incidence and mortality of pancreatic cancer (PC) has gradually increased. The aim of this study was to identify survival-related DNA methylation (DNAm)-driven genes and establish a nomogram to predict outcomes in patients with PC.The gene expression, DNA methylation database, and PC clinical samples were downloaded from TCGA. DNAm-driven genes were identified by integrating analyses of gene expression and DNA methylation data. Survival-related DNAm-driven genes were screened via univariate, least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression analyses to develop a risk score model for prognosis. Based on analyses of clinical parameters and risk score, a nomogram was built and validated. The independent cohort from GEO database were used for external validation.P < 0.1). In the multivariate analysis, stage, chemotherapy, and risk score were significantly correlated to overall survival (P < 0.05). The nomogram was constructed with the three variables  for predicting the 1-year, 2-year, and 3-year survival rates of PC patients. Nomogram performance was assessed by receiver operating characteristic (ROC) curves and calibration curves. 1-year, 2-year and 3-year AUC of nomogram model was 0.899, 0.765 and 0.776, respectively.A total of 16 differentially expressed methylation-driven genes were identified. Based on LASSO Cox regression and multivariate Cox regression analysis, six genes  were chosen to develop the risk score model. In the Kaplan–Meier analysis, age, T stage, N stage, AJCC stage, radiation therapy history, tumor size, surgery type performed, pathological type, chemotherapy history, and risk score were potential prognostic factors in PC  with a relationship to the outcomes of PC patients. The nomogram including stage, chemotherapy, and risk score could be used to predict survival in PC patients.The online version contains supplementary material available at 10.1186/s12864-021-08097-w. Pancreatic cancer (PC), a malignant tumor with uniformly poor outcomes, is the sixth leading cause of cancer-related mortalities in China . PC remaWith the development of molecular biology technologies, increasing attention has been paid to efficient gene prediction. An increasing number of studies have attempted to identify prognostic genes at the mRNA level and have constructed different gene prognostic models to improve prognosis , 12. ForGene expression levels can be influenced by epigenetic dysregulation . DNA metIn this study, we screened effective PC-related DNA methylation-driven genes by merging methylation and mRNA expression profiles from TCGA (The Cancer Genome Atlas) database. We constructed a model based on DNA methylation-driven genes to predict outcomes in PC.https://portal.gdc.cancer.gov/). The mRNA expression of TCGA and GTEx (Genotype-Tissue Expression) was downloaded from the UCSC Xena website (https://xenabrowser.net/datapages/). There were 195 samples with DNA methylation data , 182 samples with mRNA expression data , and 185 cases with clinical data from TCGA. Additionally, 167 data points for normal mRNA expression were obtained from GTEx. The study flowchart is shown in Fig. We downloaded clinical survival data and DNA methylation of PC from TCGA dataset  and GTEx  with the Limma package in R. Then, we identified the DEGs  from the merged data  with the Limma package in R. Cutoff criteria were | Log2FC | > 2, and false discovery rate (FDR) < 0.05.P < 0.05, and Cor < − 0.3 were set as the cutoff criteria.We performed a comprehensive analysis to acquire three data matrices . Then, we used the MethylMix package in R to screen the DNAm-driven genes. First, MethylMix was used to compare the methylation state of tumor tissues with that of normal tissues. Correlation analyses were performed between the gene expression data of DEGs and DNAm data to distinguish the DNA methylation events, which may affect gene expression. Second, a mixture model of gene methylation state was built. Third, the differential DNA methylation state between tumor and normal tissues was calculated via Wilcoxon rank sum test. http://cpdb.molgen.mpg.de/). P < 0.05 was the cutoff criterion.Gene ontology (GO) analysis was performed using the clusterProfiler package in R. GO analysis contained cellular component, biological process, and molecular function. We applied the GOplot package in R to visualize the data. Pathway analysis of the methylation-driven genes was conducted with ConsensusPathDB . We combined GSE21501 and GSE57495 datasets into one data set for external validation.The potential predictive value of the risk score model was validated in the GSE21501, GSE57495, GSE78229 and GSE62452 cohort. They were downloaded from the Gene Expression Omnibus (GEO)  were estimated using the Kaplan–Meier method. The variable with a P value < 0.1 was selected for further analysis. In the Cox proportional hazards regression model, we used backward stepwise selection with AIC (Akaike information criterion) to identify the final prognostic variable. The variable with a statistical significance level of 0.05 was added to the nomogram model. The nomogram was used to predict the 1-year, 2-year, and 3-year OS rates with the rms package in R.The performance of the model was estimated using the C statistic and calibration. The C statistic was used to evaluate the discriminating ability of the model and is equal to the area under the receiver operating characteristic (ROC) curve. Calibration estimated the accuracy of the model and was visualized by calibration plot. The nomogram was validated by the bootstrap method with 1,000 resamples. We also applied the ROC curve to measure the accuracy of the nomogram.http://www.r-project.org/), and X-tile version 3.6.1  was used to find the optimal cut-off value for stratifying patients[P < 0.05 was considered statistically significant. All methods in our manuscript were performed in accordance with the relevant guidelines and regulations.All statistical analyses were performed using R  were used for further study. Among these DEGs, 743 were upregulated and 846 were downregulated  were screened. An adjusted s Table S. The mets Table S A. AmongP < 0.001)  such as cell adhesion molecule binding, integrin binding, and serine−type endopeptidase activity Fig.  A. Pathw01) Fig.  B.Fig. P < 0.05). Then, based on 1,000 repetitions of LASSO regression analyses, the 11 genes with a non-zero coefficient were selected as seed genes using 10-fold cross-validation  analysis to explore the connection between the gene expression of sixteen DNAm-driven genes and OS Table S. Eleven ion Fig.  A, B. FiP = 2.429e−07)  Fig.  C. The s07) Fig.  D. 1-yea07) Fig.  E.Fig. P = 0.002, P = 0.022, and P =0.024, respectively) in the K-M survival analysis  were used to explore the prognostic performance of the six-gene risk score model. Risk scores of patients in the validation cohort were computed with same method as previously described. The patients were also classified into low-risk and high-risk groups by X-tile. The survival status distribution of patients in different risk groups was visualized via scatter plot , T stage , N stage , AJCC TNM stage , radiation therapy history , tumor size (p = 0.005), surgery type performed , pathological type , chemotherapy history , and risk score (p < 0.001) were regarded as potential predictive variables. Finally, in the multivariate Cox regression, we used backward stepwise elimination and AIC to screen independent prognostic factors for the final nomogram model: stage, chemotherapy, and risk score (P < 0.05)  Fig.  A. We de05) Fig. B. The C-05) Fig.  C. The 105) Fig. D.Fig. 7In recent years, new PC cases and cancer-related deaths have been gradually increasing , 24. RadPrior studies had confirmed that tumorigenesis is correlated with aberrant methylation status, which can change expression levels of oncogenes and tumor suppressor genes , 31. NumIn our study, we identified abnormal gene methylation by comparing normal and PC samples using MethylMix. In this analysis, 16 DNAm-driven genes were screened. To explore the function of DNAm-driven genes, we performed GO analysis and pathway analysis. DNAm-driven gene function was enriched in molecular function (MF) including cell adhesion molecule binding, integrin binding, and serine−type endopeptidase activity. Function analysis and pathway analysis showed that the function of these genes could regulate tumor cell migration and metastasis.Six genes  correlated with survival were screened by LASSO Cox regression and multivariate Cox regression. A risk model was constructed using gene expression levels and multivariate Cox regression coefficients. The survival analysis of the risk score showed that the patients with a high-risk score had worse survival status. The AUC of the risk model in the ROC curves wasgreater than 0.720. The performance of the risk model was demonstrated by external validation. To our knowledge, this is the first report of the six-gene risk model, which may be a new PC prognostic biomarker.To further explore the prognostic value of the risk model and potential clinical variables, we developed a nomogram to calculate a score for each patient and predict survival rate. The C-index of the nomogram validated by 1,000 bootstrap resamples was 0.768. The calibration curves and ROC curves showed that the predictive ability of the nomogram was excellent.The six genes  had high expression and hypomethylation in PC. FERMT1, named fermitin family member 1, can reduce the phosphorylation level of β-catenin and lead to the activation of the Wnt/β-catenin pathway. These changes lead to EMT  and have a relationship with tumor aggressiveness and invasiveness . Sandra Our study had some limitations. First, this was a retrospective study, and the development and validation of the nomogram model was based on the TCGA dataset. Therefore, further external validation using other independent databases is necessary. Second, the sample size of PC in the TCGA database was relatively small, and only half of the patients had complete clinical information. Therefore, we also need to utilize our own data for validation.Based on the TCGA database, we screened six methylation-driven genes  associated with prognosis in PC patients. A risk score model comprising the six methylation-driven genes was established and validated to predict overall survival. The risk score was combined with different clinical factors to construct a good predictive nomogram for PC patients. Our results support the viewpoint that DNAm controlled genes are associated with prognosis. In clinical practice, use of the six DNAm-driven genes for prognosis would be a cost-effective and accurate predictive method in PC.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


Mycobacterium tuberculosis (M.tb) is the main thrust area in today’s scenario. In this direction global efforts were made to use aptamers, the chemical antibodies as tool for TB diagnostics and therapeutics. This review describes the various aptamers introduced for targeting M.tb and highlights the need for development of novel aptamers to selectively target virulent proteins of M.tb for vaccine and anti-TB drugs. The objective of this review is to highlight the diagnostic and therapeutic application of aptamers used for tuberculosis. The discovery of aptamers, SELEX technology, different types of SELEX development processes, DNA and RNA aptamers reported for diseases and pathogenic agents as well have also been described in detail. But the emphasis of this review is on the development of aptamers which can block the function of virulent mycobacterial components for developing newer TB vaccine candidates and/or drug targets. Aptamers designed to target M.tb cell wall proteins, virulent factors, secretory proteins, or combination could orchestrate advanced diagnosis and therapeutic measures for tuberculosis.Tuberculosis (TB) has been plaguing human civilization for centuries, and currently around one-third of the global population is affected with TB. Development of novel intervention tools for early diagnosis and therapeutics against  Mycobacterium tuberculosis (M.tb) is a leading cause of death from a single infectious agent worldwide. World health organization (WHO) estimates that 10 million people died of TB in 2018. In this scenario, development of novel approaches for early diagnosis and therapeutics against M.tb is main thrust area. Since conventional control measures have been partially successful in keeping TB epidemic under check, identification of biomolecules which can block the function of virulent mycobacterial components is crucial for developing newer TB vaccine candidates and/or drugs. In this context, aptamers, also known as ‘chemical antibodies’, that specifically recognize M.tb or inhibit the function of its virulent proteins are being developed (M.tb.Tuberculosis (TB) caused by eveloped . The apteveloped . Aptamereveloped  and are eveloped . In thisin vitro synthesis of aptamers is known as “Systematic Evolution of Ligands by Exponential enrichment (SELEX)” ((i) generation of random library of 1014−1016 single stranded oligonucleotides, (ii) incubation of oligonucleotides with its target, (iii) separation of bound oligonucleotides from unbound ones, (iv) selection of specific oligonucleotides, amplification by PCR (DNA aptamers) or RT−PCR (RNA aptamer), and (v) finally characterization of aptamer by sequencing (in vitro transcription is performed to generate the desired RNA aptamers. All the steps are repeated till the desired oligonucleotide (or aptamer) with high binding affinity is obtained. Once the desired clones are obtained, they are further optimized to maximize the function. They are truncated or reduced in size to achieve minimal aptamer length with maximum binding affinity for the target. The preferred optimal length for aptamers is 15−45 nucleotides with molecular weight of ∼5−15 kDa. Aptamers bind to their targets with pico to micromolar binding affinity. In recent times, various types of SELEX processes have been developed for specific purposes ” . Aptamerquencing Figure 1purposes . “Slow opurposes . Naturalpurposes . Suitablpurposes . After ipurposes .Trypanosama cruzi is a blood-borne parasite and known to cause Chagas disease. RNA aptamer generated using whole cell SELEX method was able to detect T. cruzi trypomastigotes in the blood  convert a blue-dye Resazurin to Resorufin (pink). Magnetic bead-coated aptamers were developed to capture and separate PLDH, PfGDH from serum. The conversion from blue to pink color was measured using either spectrophotometer or by qualitative visual interpretation. This is a low cost, portable, user-friendly diagnostic test , 27-nucleotide aptamer (LA27) was developed to detect LPS from Salmonella typhimurium -Luc tumor xenograft  which is administered locally to treat age-related macular degeneration (AMD) by targeting vascular endothelial growth factor. Macugen received FDA approval in December 2004 for the treatment of AMD . RB006 aenograft . Cytotoxenograft .+ T-cells, macrophages, and dendritic cells. An incapacitated immune system fails to protect humans and leads to development of acquired immunodeficiency syndrome (AIDS). Gp120 is a glycoprotein of HIV virus that binds to CD4+ T-cell receptor and invades the host. A novel dual inhibitory function-based anti-gp120 aptamer-siRNA chimera (A-1 and B-68) could bind to gp120, thus preventing the viral entry in CHO cell line , food-borne infection, and hospital-acquired infection. Enterotoxin B (SEB) from S. aureus activates T cells to generate cytokine storm. Aptamer A11 was developed to inhibit SEB activity, preventing cytokine storm  that has protease activity. LF cleaves and activates mitogen-activated protein kinase kinase (MAPKK). ssDNA aptamer (ML12) prevents the protease activity of LF reducing its toxicity. It is being considered as a potential drug target against LF (It is known that human immunodeficiency virus (HIV) attacks CD4ell line . A modifell line . Ebola vin vitro . Staphylne storm . Anthraxainst LF .in vitro and has shown activity against human tumor xenografts in vivo  Figure 3M.tb continued to kill millions of humans every year. Limitations of rapid and accurate diagnosis of tuberculosis, emergence of drug-resistance and HIV-TB co-infections are complicating TB treatment. For TB diagnosis, smear microscopy is a less sensitive test. Culturing M.tb is the most accurate method to identify the pathogenic species, but it is time consuming and not suitable for point-of-care (POC) diagnosis. In PCR-based diagnosis, infection is confirmed by the presence of mycobacterial DNA or RNA sequence; however, the thermal cyclers are not readily available in resource-limited settings. On the other hand, rapid and less expensive POC, such as serological assays, is banned for testing the TB. WHO endorsed Gene-Xpert MTB/RIF, a specific, fast technique and revolutionary TB detection method based on PCR-DNA amplification, but it has certain limitations (M.tb requires skilled manpower and sophisticated instrument setup. Tuberculin skin test (TST) and Interferon-γ-Release-Assay (IGRA) are immunological tests based on the principle of antigen–antibody reaction. Interferon-γ is a pro-inflammatory cytokine, released during tuberculosis infection. IFN-γ is measured by ELISA from TB patients’ blood. These tests fail to differentiate between active TB, latent TB, or relapsed TB. Despite these advancements, the diagnosis of M.tb infection still requires a rapid, accurate, and efficient diagnostic platform. It has been estimated that 0.5 million people fell ill with drug resistant TB, and only one in three TB patients had access to treatment  and extensively drug resistant (XDR) M.tb arise due to over and improper use of antibiotics during the treatment of TB   can detect CFP-10 and ESAT-6 proteins respectively in serum samples from active pulmonary tuberculosis patients, extrapulmonary TB patients, and healthy individuals  . A fluorcomplex) . Entry onfection . NK2 aptith M.tb . The 6-kividuals . Mannoseogenesis . ManLam ogenesis . An apta vaccine . Acetoac of M.tb . Aptamerce model . BM2 aptce model . Thioaptce model . CD44 isrophages . Conjugaice lung .M.tb cell wall proteins, virulent factors, secretory proteins, or combination of above factors that could be a substitute of conventional diagnosis approaches. Aptamers generated against whole bacterium are regarded suitable for diagnostic purposes as they can recognize different epitopes present on M.tb and detect them in human fluid i.e. blood, serum, bronchoalveolar lavage  therapy for tuberculosis management, a combination of antibiotics is given for a long period of time. M.tb develops drug resistance, and thus the currently available drugs generally fail to clear the infection. Most antibiotics are generated against enzymes important for cell wall formation, biofilm synthesis, or DNA replication, and protein synthesis pathway, or they target they metabolic pathway of M.tb , Government of India (BT/PR20669/MED/29/1072/2016), Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India (DST/SERB/CRG/2019/000239), TATA Innovation Fellowship, DBT (BT/HRD/35/01/03/2018), Council of Scientific and Industrial Research (CSIR), Govt. of India (27(0364)/20/EMR-II), and a core grant from the Centre for DNA Fingerprinting and Diagnostics by the Department of Biotechnology (DBT) to SM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anopheles mosquitoes are responsible for malaria transmission, and while there has been a reduction in malaria-related deaths worldwide, growing insecticide resistance is a cause for concern. Aedes mosquitoes are known vectors of viral infections, including dengue, yellow fever, chikungunya, and Zika. Aminoacyl-tRNA synthetases (aaRSs) are key players in protein synthesis and are potent anti-infective drug targets. The structure–function activity relationship of aaRSs in mosquitoes  remains unexplored.Mosquito-borne diseases have a devastating impact on human civilization. A few species of Anopheles culicifacies, Anopheles stephensi, Anopheles gambiae, Anopheles minimus, and Aedes aegypti). The VectorBase database (https://vectorbase.org/vectorbase/app) and web-based tools were utilized to predict the subcellular localizations , physicochemical characteristics (ProtParam), and domain arrangements  of the aaRSs. Structural models for prolyl (PRS)-, and phenylalanyl (FRS)-tRNA synthetases—were generated using the I-TASSER and Phyre protein modeling servers.We employed computational techniques to identify aaRSs from five different mosquito species (An. gambiae), 37 (An. culicifacies), 37 (An. stephensi), 37 (An. minimus), and 35 (Ae. aegypti) different aaRSs were characterized within their respective mosquito genomes. Sequence identity amongst the aaRSs from the four Anopheles spp. was > 80% and in Ae. aegypti was > 50%.Among the vector species, a total of 37  and bicyclic azetidine (BRD1369)] binding sites. This suggests the potential for repurposing of these inhibitors against the studied Anopheles spp. and Ae. aegypti. Structural analysis of two important aminoacyl-tRNA synthetases [prolyl (PRS) and phenylanalyl (FRS)] of The online version contains supplementary material available at 10.1186/s13071-021-05106-5. Aedes aegypti transmits viruses , whereas Anopheles spp. transmit the malaria-causing parasites of Plasmodium spp. . A. APlasmoPRS PDB: YDQ, HsPRPRS PDB: YDQ, HsPRy pocket , 36. FurPRS PDB: YDQ, HsPRAnopheles spp.  and Ae. aegypti. We show variation in the number of aaRSs present in the four Anopheline species compared to Ae. aegypti, as has been seen for several other species including Plasmodium, H. sapiens, and Babesia [Gln and Asn-tRNAAsn synthesis that coexist in parallel with the classical synthesis pathways [Owing to increased insecticide resistance in several of the mosquito species, there is an immediate need to develop novel strategies to control mosquito populations. The availability of the genomes from several mosquito species has opened new avenues for screening novel insecticides. In the current study, we catalogued aaRSs from four different  Babesia , 37. In  Babesia . Howeverpathways . In addipathways .Anopheles spp. and Ae. aegypti.Among all aaRSs analyzed here, FRS is found to be the most interesting, as this is the only protein found in our analysis that consists of two chains, one α and one β, and similar to other species it likely exists as a heterodimer in solution . It is wAnopheles spp. and Ae. aegypti. The aaRSs are essential for protein synthesis, and their inhibition is detrimental to the organism. AaRSs are a gene family that is considered a high-value drug target against parasites . Moderate sequence identity (40–80%) within the core synthetase domain of aaRSs from H. sapiens and mosquito species in our analysis suggests that targeting aaRSs in mosquito species can be effectively translated in designing safe (nontoxic) inhibitors against mosquitoes. Furthermore, the partial structural and sequence similarity between Plasmodium and mosquito aaRS binding sites offers a window for drug repurposing. Indeed, targeting any particular aaRS that is conserved between the mosquito (Anopheles) and the parasite (Plasmodium) presents a very unique opportunity. This comprehensive study of aaRSs from Anopheles and Aedes mosquitoes will be beneficial for new insecticide development and thus vector control via targeting aaRSs as potent insecticidal targets.In conclusion, our analysis provides in-depth data on genome-wide identification and annotation of their potential localization along with domain arrangements of aaRSs from four different Additional file 1: Table S1. Annotation of aminoacyl-tRNA synthetases (aaRSs) in five different mosquito genomes. Domain annotations were made using Pfam and InterPro. Physicochemical parameters of domains, for instance isoelectric points (pI), were calculated using ProtoParam. Table S2. Class I and II type aaRS average size (core synthetase domain) and isoelectric point. Table S3. Sequence-level comparison of core synthetase domain between human and An. gambiae and An. culicifacies aaRSs. Figure S1. Phylogenetic trees were constructed for three representative aminoacyl tRNA synthetases . a For lysyl and prolyl aminoacyl tRNA synthetases, a phylogenetic tree was generated using the maximum likelihood method (MLM). b In the case of FRS, both alpha and beta subunits were considered for phylogenetic analysis. Sc: Saccharomyces cerevisiae; Pf: P. falciparum; Hs: H. sapiens; Ec: Escherichia coli; Bv: Babesia bovis; Ae: Ae. aegypti; Ag: An. gambiae; Ac: An. culicifacies; As: An. stephensi; Am: An. minimus; Mtb: Mycobacterium tuberculosis; Pfu: Pyrococcus furiosus.


Polymerase γ (POLG) is a protein that plays a pivotal role in the replication of the mitochondrial genome. POLG-related disorders constitute a sequence of overlying phenotypes that can present from early infancy to late adulthood. Parkinsonism is the most common movement disorder associated with POLG mutation. We also summarize all reported cases of POLG-related Parkinsonism, along with a literature review.We present the case of an 80-year-old male presented with complaints of episodic confusion, tremors, and restlessness. He has been on risperidone for psychosis. A normal DaT scan ruled out Parkinson’s disease, and molecular analysis for POLG was positive (E1143G). He was diagnosed with drug-induced Parkinsonism and tardive akathisia with an incidental POLG mutation.A literature search revealed 55 cases of “POLG-related Parkinsonism” that met our criteria. These mutations can clinically affect multiple organ systems. Parkinsonism is the most common movement disorder reported in association with POLG mutations. We conducted a molecular analysis in our patient due to his Parkinsonism and recurrent episodes of encephalopathy. E1143G mutation found in our case was coincidental and reported a non-pathogenic or benign variant in literature.E1143G is a non-pathogenic variant and multiple studies have shown that its co-occurrence with other POLG mutations can aggravate disease occurrence and severity. Literature findings and the experience from our own case indicate that the pathogenicity of E1143G is debatable, and future studies involving this particular variant may help understand its pathological consequences. A group of proteins replicates the mitochondrial deoxyribonucleic acid (mtDNA), a major one being DNA polymerase γ (POLG). POLG-related disorders constitute a continuum of overlying phenotypes that can present from early infancy to late adulthood. The most common movement disorder found in association with POLG mutation is Parkinsonism. These patients usually show clinical symptoms at a young age and are commonly associated with progressive external ophthalmoplegia (PEO). We present an 80-year-old male with drug-induced Parkinsonism and tardive akathisia with an incidental POLG mutation. We performed a literature search to summarize all reported cases of POLG-related Parkinsonism.An 80-year-old Caucasian male with a past medical history of diabetes mellitus, chronic kidney disease stage III, obstructive sleep apnea, psoriatic arthritis on methotrexate, and depression on mirtazapine; presented with complaints of episodic confusion of 5 years duration, restlessness, and tremors of 2 years duration.The episodic confusion was associated with apparent auditory/visual hallucination and delusions and typically occurred at night. The inciting event was a recognized infection during most episodes. He was started on risperidone to be used intermittently during these attacks to control the psychotic symptoms. According to his wife, he has taken risperidone only a few times after beginning the medication.The restlessness comprised repeated crossing and uncrossing legs, sitting and standing, and moving his arms around. It began 3 years after initiating antipsychotic medications. Around the same time, he also developed tremors in his hands bilaterally (right >left). It has remained stable, is present predominantly at rest, and does not affect his day-to-day activities. He denied any loss of sense of smell, change in facial expression, voice change, drooling, constipation, and orthostatic symptoms. He also reported no similar complaints among his family members.On examination, he had symmetric, akinetic, rigid Parkinsonism, and prominent akathisia. His total Movement Disorders Society Unified Parkinson’s Disease Rating Scale score was 46/104. Sensory examination showed impaired vibration sense in bilateral lower limbs. We advised him to avoid risperidone, as this could explain his Parkinsonism. When there was no noticeable improvement, we started him on a trial of Sinemet. A DaT scan was normal, ruling out Parkinson’s disease. We prompted genetic testing due to the occurrence of marked encephalopathy from minor infections and Parkinsonism from low and intermittent doses of risperidone. Molecular analysis was positive for a heterozygous POLG mutation- c.3428 A >G, p. Glu1143Gly (E1143G).At 85 years of age, follow-up demonstrated a good response to Sinemet. The patient denied any new delirium episodes and mentions his tremors and akathisia is still present but improving. He was diagnosed with drug-induced Parkinsonism and tardive akathisia, with an incidental POLG mutation and peripheral neuropathy, most likely due to poor diabetic control.https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV000021312.5), it could be a contributing factor to our patient’s clinical presentation.Although E1143G mutation is a non-pathogenic POLG variant  were males, 25 (44.6%) were females, and 1 patient had no gender specified.The age of onset of Parkinsonism ranged between 18 and 78 years- 4 patients (8.5%) had the onset before 20 years, 10 patients (21.3%) had between 21 and 40 years, 21 patients (44.7%) had between 41 and 60 years, and 12 patients (25.5%) had the onset after 60 years of age. PEO was a common manifestation in most cases, except 13 patients (23.2%). Myopathy was present among 28 patients (50%) and neuropathy among 30 patients (53.6%).Among the 25 patients who underwent DaT scan, 16 patients (64%) were positive and 43 patients (76.8%) showed good response to treatment with dopaminergic medications.The mitochondria are known as the cell’s powerhouse, generating adenosine triphosphate. Through the electron transport chain, this allows the proper functioning of cells, including the neurons. Multiple proteins that make up the system are derived from the mitochondrial genome, consisting of circular mtDNA.POLG is a major DNA-binding protein involved in mDNA replication. POLG-related disorders can present with clinically overlapping symptoms in affected individuals. However, they are characterized into six clinical forms-Alpers-Huttenlocher syndrome- severe progressive encephalopathy, intractable epilepsy, and liver failure.Myocerebrohepatopathy spectrum- developmental delay, lactic acidosis, myopathy, and hepatic impairment.Myoclonic epilepsy myopathy sensory ataxia- a spectrum of epilepsy, myopathy, ataxia, and disorders previously described as a spinocerebellar ataxia with epilepsy.Ataxia neuropathy spectrum- sensory ataxia, neuropathy, dysarthria, ophthalmoplegia, and mitochondrial recessive ataxia syndrome.Autosomal recessive PEO- progressive extraocular muscle weakness causing ptosis and ophthalmoparesis without any systemic involvement.Autosomal dominant PEO- generalized myopathy, sensorineural hearing loss, axonal neuropathy, depression, Parkinsonism, hypogonadism, and cataracts.Clinical signs and symptoms associated with POLG mutations include hypotonia, developmental delay, myoclonus, focal motor seizures, generalized seizures, status epilepticus, migraine, stroke-like episodes, Parkinsonism, chorea, sensory neuronopathy, ganglionopathy, axonal sensorimotor neuropathy, cerebellar ataxia, depression, psychosis, dementia, sensorineural hearing loss, PEO/ptosis, cataracts, retinopathy, liver failure, gastrointestinal dysmotility, proximal myopathy, exercise intolerance, diabetes mellitus, primary ovarian failure, ovarian dysgenesis, primary testicular failure, and cardiomyopathy .rd–4th decade [et al. described 11 patients with POLG-related encephalopathy. The patients had no signs of Parkinsonism; however, DaT scans indicated severe nigrostriatal neuronal loss [Parkinsonism is the most common movement disorder associated with POLG mutations. The exact mechanism by which mitochondrial dysfunction leads to Parkinsonism is still not well understood. The existing hypothesis indicates that an alteration in the mtDNA can result in impaired protein synthesis resulting in mitochondrial dysfunction in the dopaminergic neurons . Parkinsh decade . Although decade . Tzoulisnal loss . Degeneret al. studied seven families, and for the 1st time, described a significant link between Parkinsonism and POLG mutations [et al. also conducted a post-mortem examination on two patients with POLG mutation. This revealed loss of pigmented dopaminergic neurons in the substantia nigra, though Lewy bodies were not seen [et al. performed an autopsy on a patient with multiple heterozygous POLG mutations. Microscopic examination of the brain showed a severe neuronal loss in the substantia nigra and Lewy bodies’ presence in the remaining nigral neurons [In 2004, Luoma utations . The extutations . Luoma enot seen . Betts-H neurons .https://www.ncbi.nlm.nih.gov/snp/rs2307441#frequency%20tab). The mutation is rendered benign due to its occurrence outside the identified pathogenic cluster [http://www.snps3d.org/), another program predicted it to be damaging protein structure and function (http://genetics.bwh.harvard.edu/pph/). The commonly encountered pathogenic POLG variants include A467T, W748S, G848S, and T251I-P587L [et al. reported five patients with E1143G mutation, of which two individuals had compound mutations- A467T/E1143G and S433C/E1143G [et al. investigated the biochemical consequences of POLG proteins with E1143G polymorphism. They described that the occurrence of W748S in cis with E1143 mutation could modulate the former’s phenotypic effects [E1143G mutation is a non-pathogenic variant found at varying frequencies in different population subgroups . The variant probably remained silent till the dopamine blocking effects of risperidone were in effect. It could also be contributing to his episodic encephalopathy and amplifying his drug-induced Parkinsonism.The mutation identified in our patient, a benign heterozygous POLG variant (E1143G), was incidental. The pathogenicity of E1143G is debatable, and future studies involving this particular variant may help understand the pathological consequences when it occurs as single or compound mutations.The authors declare that they have no competing interests.


Background: We assessed the relationship between preoperative prognostic nutritional index (PNI) and short- and long-term outcomes among gastric cancer patients because the clinical significance of PNI in these patients remains controversial.Materials and Methods: We retrospectively reviewed the medical records of 434 consecutive patients who underwent curative laparoscopic gastrectomy for gastric cancer.n = 118), those with postoperative complications experienced significantly poorer OS than those without complications. Among the low PNI group with pTNM stage I and II disease, those with postoperative complications experienced significantly worse OS than those without complications. However, among the high PNI group and patients with stage II and III disease in the low PNI group, OS was similar with respect to postoperative complications.Results: Patients with postoperative complications had a significantly poorer overall survival (OS) than those without. On multivariate analyses, postoperative complications were independently associated with PNI value and operative procedure type. In the low PNI group (Conclusions: The present study confirmed that long-term prognosis was unaffected by postoperative complications in well-nourished gastric cancer patients. In addition, preoperative nutritional status and postoperative complications, may be crucial in determining the prognosis of gastric cancer, especially in early-stage cancer. Cancer-associated malnutrition is a common and significant clinical issue because it is often unrecognized and undertreated. Gastric cancer patients are often malnourished at the time of diagnosis, which is related to impaired digestion, malabsorption, and bleeding and/or protein loss from the primary lesion , 2. ReceThe prognostic nutrition index (PNI), which predicts the risk of postoperative complications, is calculated using serum albumin level and total number of lymphocytes . Serum aBased on a PNI cut-off of 44.2, 118 (27.2%) and 316 (72.8%), patients were classified into the low and high PNI groups, respectively. Significant differences were observed in age, body mass index (BMI), tumor diameter, tumor stage, pathological TNM (pTNM) stage, surgical procedure, intraoperative blood loss, and C-reactive protein (CRP) level .Among the 434 patients, 128 (29.4%) experienced postoperative complications . Signifip < 0.001), CEA (p = 0.003), and PNI (p < 0.001) were found to be independent predictive factors for OS , CRP level, PNI, postoperative complications, and postoperative adjuvant chemotherapy were significant relevant factors. In multivariate analyses, pTNM stage (s for OS .p < 0.001)  .p = 0.031) and surgical procedure (p = 0.011) were independent risk factors   .p = 0.007)  .p = 0.350)  .p = 0.029)  .However, among patients with pTNM stages II and III, the OS of those with and without postoperative complications did not differ significantly  and 4C.p = 0.028) (p = 0.029) (p = 0.667)  . However= 0.028) . Similar= 0.029) . However= 0.667) .Meanwhile, among pTNM stage III patients, there was no significant difference in OS between those with and without postoperative complications when stratified according to PNI value  and 7B.Recent studies have shown that postoperative complications such as anastomotic leakage and postoperative infections adversely affect the prognosis of gastric cancer patients , 17–20. In the stage-stratified analysis, pTNM stage I patients who developed postoperative complications had a significantly inferior prognosis than those without complications, whereas there was no difference in prognosis with respect to postoperative complications in stage II and III patients. In addition, among stage I and II gastric cancer patients, those with low PNI who developed postoperative complications had a significantly inferior OS compared to those without postoperative complications, whereas there was no difference in high PNI patients. And among pTNM stage III patients, there was no significant difference in OS between those with and without complications, regardless of PNI value. These findings suggest that preoperative nutritional status, similar to postoperative complications, is crucial in the prognosis of gastric cancer, especially in relatively early-stage cancer, whereas the association of degree of cancer stage outweighs that of postoperative complications with OS in advanced-stage cancer , 28.In clinical practice, TNM staging alone cannot be used to predict clinical outcomes, since it only classifies patients according to postoperative pathological outcomes and does not include nutritional or inflammatory status , 30. ThuWhile the current study has some advantages over previous reports, it has some potential limitations and caution should be exercised when interpreting the results. First, this was a single-institutional study with a relatively small number of patients and a relatively short follow-up period for evaluation of long-term prognosis. The follow-up period was sufficient to assess the outcome of postoperative complications, but further research is needed to investigate the impact of PNI on long-term prognosis. Second, immunonutritional parameters and systemic levels of inflammatory cytokines were not assessed. Third, the subgroup-derived evidence did not have sufficient statistical power to validate various conclusions because of the small number of patients. Fourth, the analysis did not include factors that could affect inflammation and nutritional markers, such as medication and comorbidity. Fifth, OS was evaluated in this study as it is considered the gold standard endpoint in cancer prognosis studies. However, disease-specific survival and recurrence-free survival data analysis would also provide to be useful. They were not analyzed.In conclusion, this study suggested that PNI is useful in identifying gastric cancer patients who would benefit from preoperative nutritional management and have an improved prognosis. However, it is unclear whether PNI serve as a nutritional parameter for selecting candidates for nutritional management. Future prospective studies are needed to determine whether aggressive preoperative nutritional management can increase preoperative PNI and improve short-term outcomes and long-term prognosis.We retrospectively investigated the medical records of 434 gastric cancer patients who undergoing curative laparoscopic gastrectomy between 2010 and 2018. The gastrectomy and lymphadenectomy were determined by the Japanese Gastric Cancer Treatment Guidelines (version 4) . The ethPostoperative complications of grade II or higher according to the Clavien-Dindo (CD) classification were retrospectively determined from the patients’ records . PostopePNI and BMI, were calculated using laboratory data obtained within 1 week preoperatively. PNI was proposed by Onodera et al. .The receiver operating characteristics (ROC) curve and area under curve (AUC) analyses were performed to determine the optimal cut-off value of PNI for OS. Based on this analysis, cut-off value of PNI was set at 44.2  .t-test. Differences between categorical variables were analyzed using the chi-square test. The OS was analyzed using the Kaplan-Meier method and the Log-rank test. In addition, a univariate analysis was performed to identify significantly relevant variables, and variables with a univariate p-value <0.05 were included in the subsequent multivariate analysis. The Cox proportional hazards model used HR and a 95% confidence interval (95% CI). Statistical analysis was performed using JMP software , and p < 0.05 was judged to be a statistically significant difference.Continuous variables are represented as median and range. Differences between groups were assessed using Student’s


Algal species still show unrevealed and unexplored potentiality for the identification of new compounds. Photosynthetic organisms represent a valuable resource to exploit and sustain the urgent need of sustainable and green technologies. Particularly, unconventional organisms from extreme environments could hide properties to be employed in a wide range of biotechnology applications, due to their peculiar alleles, proteins, and molecules. In this review we report a detailed dissection about the latest and advanced applications of protein derived from algae. Furthermore, the innovative use of modified algae as bio-reactors to generate proteins or bioactive compounds was discussed. The latest progress about pharmaceutical applications, including the possibility to obtain drugs to counteract virus (as SARS-CoV-2) were also examined. The last paragraph will survey recent cases of the utilization of extremophiles as bio-factories for specific protein and molecule production. Plants and algae possess still untouched possibilities; thus, these organisms are emerging as formidable sustainable tools with a number of advantages over the conventional biotechnologies [In recent years, public opinion, research community, and commercial stakeholders paid great attention to green and sustainable biotechnologies; these are not anymore considered a luxury, but a necessity in all countries. Actually, the current world population is estimated in 7.8 billion people, and an increase is expected of about 2 billions in the next 30 years , lysophosphatidic acid acyl- transferase (LPAAT), and acyl-CoA:diacylglycerol acyltransferase (DGAT). These enzymes regulate fatty acid and TAG biosynthetic pathways in plants and microalgae [P. tricornutum resulting in a 2.9- and 2.3-fold change increased lipid content, respectively [Neochloris oleoabundans where the plastidial NeoLPAAT1 and endoplasmic reticulum-located NeoDGAT2 were co-overexpressed. This strategy increased the TAG content of about 2-fold compared to wild type; furthermore, engineered strain reporting no effects on algal biomass [T. pseudonana and P. tricornutum, respectively. The suppression of TAG lipase in these two diatoms significantly increased the lipid content. Interestingly both did not show effects on growth [P. tricornutum strain [PtTES1, a hotdog-fold thioesterase involved in acyl-CoA hydrolysis. This modification lead to a 1.7-fold change increase of TAG content [P. tricornutum and T. pseudonana. These preliminary results opened new perspectives to obtain improved microalgae strains using this technique [Thus, a number of strategies have been used to hyperaccumulate fatty acids and/or triacylglycerol (TAG) content . Phaeodacroalgae ,23,24. Tectively ,20. Inte biomass . Signifi biomass  and Bark biomass , developn growth ,26,27. Tm strain  reportin content . Successechnique ,30,31. Arabidopsis thalian(AtLEC1) represents a key transcription factor involved in plants lipid metabolism [AtLEC1 improves fatty acids and total lipids content in Chlorella ellipsoidea both under mixotrophic and autotrophic culture conditions. These differences between wild type and the engineered algal strain were related to an enhanced regulation of key enzymes namely ACCase, GPDH, PDAT1, and DGAT1 [A. thaliana in microalgae showed the heterologous overexpression of 1-deoxy-D-xylulose 5-phosphate synthase from A. thaliana in Nannochloropsis oceanica. This strain reported an improved CO2 fixation, thus increasing biomass, lipids, and carbohydrates productions upon different nutritional and stressed conditions [Nannochloropsis represented a further model used as platform for biofuel production from microalgae [Nannochloropsis, particularly focused on the increase on lipid and fatty acids content. Different approaches, namely overexpression [Nannochloropsis. For example, Jeon et al. [NsME1) in Nannochloropsis salina. This enzyme is an important NADPH supplier, playing the central role in the C4-like cycle of microalgae. The overexpressed strain showed an increased fatty acid and lipids contents compared with wild type, up to 53% and 38%, respectively [An increase in lipids content has been obtained by modifying different microalgal pathways. LEAFY COTYLEDON1 from tabolism . Liu et tabolism  demonstrnd DGAT1 . Anothernditions . Particucroalgae ,35,36,37pression  were sucP. tricornutum induced by PtG6PDH overexpression stimulated an increase fatty acid production upon control and high CO2 level conditions [PtG6PDH and NoG6PDH (from Nannochloropsis oceanica) in Chlorella pyrenoidosa showed an increase in lipid content of about 1.79 and 3.09-fold-change, respectively.Recently, several papers reported the role of glucose-6-phosphate dehydrogenase (G6PDH) in lipogenesis regulation ,39. ThisCyclotella cryptica, Nitzschia laevis, Navicula incerta, Navicula pelliculosa, and others [P. tricornutum represents a model organism for diatoms, mostly studied for biomass, lipid, and EPA production [P. tricornutum overexpressing malonyl CoA-acyl carrier protein transacylase (PtMCAT) and fatty acid desaturase 5b (PtFAD5b) significantly improved the content of EPA [P.tricornutum strain expressing Δ-5 elongase from Ostreococcus tauri, and a glucose transporter from the moss Physcomitrella patens. This strain accumulated ω-3 long-chain polyunsaturated fatty acid and showed an unconventional ability to grow in the dark [Aspergillus niger and Escherichia coli to overcome this constraint. P. tricornutum strains expressing the phytases from these two species showed an enhanced content of EPA and DHA [Among lipids, long-chain polyunsaturated fatty acid  represent a major fraction of microalgae biomass such as d others ,43,44. Pd others ,43,44. Ht of EPA . Similart of EPA , obtainethe dark . Recentlthe dark . Pudney the dark , propose and DHA . Porphyridium sp. [Porphyridium sp. engineered strains, mainly obtained by Agrobacterium-mediated and biolistic transformation methods [Porphyridium sp. increasing the production of phycoerythrin. The authors manipulated the chlorophyll synthase gene (CHS1), obtaining an improved strain able to produce an increased content of phycoerythrin from 44% to 63% depending by the light sources [Successful manipulations were also obtained using red microalgae as well as dium sp. ,50. Thesdium sp. . There a methods . InteresHaematococcus pluvialis in response to adverse conditions namely excess of light, salinity, and nutrient starvation [H. pluvialis using physical and chemical mutagens such as UV radiation, ethyl methanesulphonate (EMS), diethyl sulphate (DES) and other. In some case desirable improved strains were obtained showing high growth rates at the vegetative stage and high astaxanthin accumulation rates at the encystment stage [H. pluvialis. For example, Waissman-Levy et al. [H. pluvialis by insertion of the hexose uptake protein (HUP1) gene from the green microalga Parachlorella kesslerii. The engineered strain was able to grow upon heterotrophy conditions in glucose-supplemented media [Analogous to phycoerythrin, astaxanthin is a desirable product for human consumption. This is mainly produced by the green microalga arvation . A numbent stage . In recey et al. , manipuled media .Apart from the utilization of microalgae for the production of biomass, biofuels , and recombinant proteins, the exploitation of these organisms as makers of proteins and complex molecules of pharmacological interest is a major theme of current research. The flexible metabolism of photosynthetic cells represents an intriguing tool for the synthesis of those compounds requiring complex biosynthesis not always possible in a single cell compartment as in bacteria.Microalgae represent an important biotechnology resource for the production of recombinant proteins for pharmacological applications. Successful results were reported by the genetic manipulation of microalgae for the production of antigens for vaccines, antibodies, immunotoxins, hormones, and antimicrobial agents ,85,86,87Recombinant protein production was attained by engineering both nuclear and chloroplast microalgal genomes . ChloropChlamydomonas reinhardtii has been widely used for this type of applications.Recently, the utilization of different engineering strategies, many biopharmaceutical compounds have been effectively produced in microalgae, and Aeromonas hydrophila bacteria [A list of engineered strains is shown in bacteria , and vibbacteria . Ch. reinhardtii strain able to produce the antimicrobial peptide Mytichitin-A. This peptide—not toxic for human cells—caused the inhibition of bacteria growth at the concentration of 60–80 ug/mL [H. pluvialis by biolistic manipulation of chloroplast of this microalga [In addition, recombinant proteins for therapy against human diseases and pathogens have been produced in microalgae. Dong et al.  obtained80 ug/mL . An inteicroalga .Ch. reinhardtii by Agrobacterium nuclear transformation [Human interferon α (huIFN α) is widely used to treat cancer and some viral infections including hepatitis C; huIFN α was engineered and expressed in ormation . The recormation .Ch. reinhardtii and Dunaliella salina producing about 0.94% and 0.59% of iL-2 respectively [Human interleukin-2 (iL-2), used in different cancers therapies, was successfully expressed in ectively . The staectively .Chlorella vulgaris and Ch. reinhardtii [Schizochytrium sp.  [In recent years, microalgae were tested as suitable platforms for drugs production, even to counteract virus and other pathogens. Particularly, the receptor binding domain (RBD) of SARS-CoV-2, able to bind the angiotensin-converting 2 receptor (ACE2) to entry into human host cells was produced in nhardtii ,84. Thushulales) ,86,87.Agrobacterium-mediated transformation in Schizochytrium sp. [Zika virus (ZIKV) infection cause severe symptoms including fever, rash, conjunctivitis, muscle and joint pains. ZK microalgae-made vaccine was tested by oral administration in mice showing immunogenic potential and inducing both systemic (IgG) and mucosal (IgA) humoral responses. Promising antigens against ZIKV were obtained by rium sp. .Schizochytrium sp. Furthermore, algae-based recombinant vaccine showed high stability to heat treatment (up to 60 °C), therefore, this was proposed as a possible vaccine for the treatment of AD [RAGE (receptor of advanced glycation end-products) is a receptor related to development and progression of Alzheimer’s disease (AD), its expression was increased in the brain in AD patients, inducing the release of proinflammatory mediators; thus, RAGE is considered an interesting target for AD therapy . RAGE funt of AD .C. reinhardtii chloroplast obtaining a CelTOS antigen from Plasmodium falciparum developing a sensitive ELISA test to identify malaria infection.CelTOS  is crucial protein, which mediated the malaria virus infection. Shamriz et al.  manipulaSchizochytrium sp. multi-epitope protein (BCB) against breast cancer (BC). They selected different B cell epitopes developed from tumor associated antigens (TAAs) recognized by the immune system. Biomass from transgenic algae expressing BCB were administered to mice, recording specific antibodies production [One of the great challenges is the development of an algae-based vaccines against cancer. Hernandez-Ramirez et al.  expresseoduction . P. tricornutum, reporting the production of fully assembled IgG antibody secreted in the culture medium. Microalgal antibodies were tested on THP-1 cell line to evaluate Fcγ receptor interactions, which are specific cell-superficial receptor for IgG, binding to human Fcγ receptor, and inducing an immune response. Monoclonal IgG antibodies against Marburg Virus (MARV), responsible for hemorrhagic fever with high fatality rates in Western Africa has been produced [P. tricornutum, and the recombinant protein was secreted in the culture medium at concentration of 1300 ng/mL. ELISA demonstrated an efficient binding for algal antibodies [Another goal is the production of monoclonal antibodies from microalgae. Nowadays, monoclonal antibodies represent an innovative solution against cancer and other human diseases. Vanier et al.  expresseproduced . MARV setibodies .In recent years, researchers explored the possible applications of extracellular vesicles (EVs) as drug carriers ,90,91. EIt should be noted that all these studies require critical control points, in order to carefully regulate growth conditions and the activation of specific metabolic pathways. Therefore, the possibility to utilize extremophilic organisms as advanced bioreactors able to synthetize compounds upon particular conditions is a promising strategy for these aims.Chlorella to mitigate the effects of industrial pollution, by absorbing CO2; the use of Arthrospira platensis for bioremediation of contaminated effluents; the utilization of a number of microalgal species for biofuel productions [A renewed attention has been focused on organisms from extreme environments ,94. Thisductions .Chlamydomonas sp. ICE-L [Lobosphaera incise [Chlamydomonas sp. ICE-L contributes to membrane fluidity for adaptation to Antarctic extreme environment; Δ5FAD contributes to arachidonic acid synthesis, thus avoiding the risk of photodamage upon chilling. A number of snow algae were tested for their ability to produce lipid species in low temperatures [Thermophilic microalgae cultivated in large-scale open ponds are able to produce bulk amount of lipids, which can be utilised for biodiesel production technology . Particup. ICE-L  and the a incise . Both eneratures . These aeratures .A. platensis are actually produced as dye in food industries but high temperatures (62 °C) limited their use [Cyanidioschyzon merolae to overcome this problem [Cyanidioschyzon merolae were recently characterized by Yoshida et al. [Thermophillic microalgae are studied for the applications of phycobiliproteins , a groupheir use . Differe problem ,109. Thea et al. , confirma et al. .Chlamydomonas nivalis, Raphidonema sp., and Chloromonas sp. showed ability in the production of astaxanthin, α-Tocopherol, xanthophylles, and glycerol [Desmodesmus and the acidophilic red alga Galdieria sulphuraria were tested for the production of lutein and blue pigment phycocianin, respectively [A number of extromophilic microalgae were recently characterized for the ability in the production of bio-compounds with high economic interest . For exaglycerol ,111,112.ectively ,114. TheHere, we reviewed the remarkable value of photosynthetic organisms and their potential emerged in recent years. Through the last decades, there has been significant research into the exploitaion of microalgae as sustainable resources for the production of high-value products and proteins. It is clear that microalgae can play a crucial role in modern biotechnological processes role at energetic, pharmaceutical, and nutraceutical levels. It is worth to point out that even if the current knowledge about natural and engineering microalgae is broad and wide, present notions are still not sufficient, and significant efforts are required to reach the goals. The cloning technologies to obtain suitable platform or bio-factories to produce proteins or metabolites of interest are now widely accessible and at least readily implementable by modern technologies as well as synthetic biology approaches. It is now time to further expand this research and the applications of microalgae in order to develop more refined tools and strategies for an efficient sustainable and ecological breakthrough.


Staphylococcus aureus and methicillin-resistance S. aureus (MRSA) in rabbits, rabbit handlers, and rabbitry environments in Terengganu. This study aims to investigate the prevalence and antibiogram of n = 180) samples from rabbitries were collected from 10 rabbit farms in Terengganu. The associated S. aureus isolates from the swabs were isolated using phenotypic microbiology tests. The bacteria were confirmed by polymerase chain reaction targeting nuc (S. aureus) and mecA (MRSA) genes. The antibiogram of all S. aureus isolates was determined using the Kirby–Bauer test. Swab samples from 183 rabbits , 45 rabbit handlers , and environmental  could not be detected. Antibiotic susceptibility test revealed that S. aureus from rabbits showed low resistance (<20%) against 15 different antibiotics while fully susceptible to 4 antibiotics. Meanwhile, S. aureus from rabbit handlers showed high resistance against penicillin (86%), oxacillin (64%), and amoxicillin (50%). S. aureus in rabbit farms settings. Therefore, careful selection of antimicrobial agents will be essential to preserve the effectiveness of treatments toward S. aureus infections.This study suggests the emergence of antibiotic-resistant  Staphylococcus aureus is a zoonotic, opportunistic Gram-positive, commensal bacterium colonizing humans and animals [Staphylococcus aureus is capable of causing several diseases in humans, ranging from minor skin infections to life-threatening illnesses [S. aureus can be commonly found on the skin. It is one of the primary pathogens related to dermatitis, mastitis, and metritis infections, causing losses in the rabbit farming industry [S. aureus to others via direct and indirect pathways [ animals . Staphylllnesses . In rabbindustry ,3. A prepathways .S. aureus is a significant public health concern in human and veterinary medicines. Methicillin-resistant S. aureus (MRSA) is one of the critical widespread nosocomial pathogens seen worldwide. The occurrence of MRSA in animals has been reported worldwide. The apparent animal-to-human transmissions have raised concerns on the risks of animal populations as potential reservoirs for this zoonotic infection [Recently, the emergence of bacteria with antimicrobial resistance (AMR) traits such as nfection . In addinfection . S. aureus with AMR traits have been reported [S. aureus and MRSA from rabbit farms in Malaysia. Hence, this study investigates the prevalence and determines the antibiogram of S. aureus and MRSA isolated from rabbits, rabbit handlers, and farm environments in Terengganu, Malaysia. Similar to other animals, rabbits are also prone to various bacterial infections . Medicatreported . Moreovereported ,8. In MaThe sampling and experimental design method of this study were approved by UniSZA Animal and Plant Research Ethics Committee (Protocol code: UAPREC/04/18/006.) and UniSZA Human Research Ethics Committee (Protocol code: UniSZA/UHREC/2019/85). Written informed consent was obtained from each rabbit handler before obtaining swabs samples.n = 30), drinkers (n = 30), door lock (n = 30), cage wall (n = 30), and floor (n = 30) of randomly selected rabbits’ cages as well from the footwears of the rabbit handlers (n = 30). The swab samples were kept in modified transport media containing nutrient broth  supplemented with 6.5% sodium chloride (NaCl) and stored at 4°C. The samples were transported from the sampling sites to the laboratory within 24 h for analysis.Swab samples  from 183 randomly selected rabbits of various ages from 10 different rabbit farms in Terengganu were taken using sterilized cotton swabs. Ear swab samples were collected by swabbing both the external ear canals of the rabbit. Oral swab sample was collected by swapping the back of throat and tonsil of the rabbits for a few seconds. Besides, 90 swab samples  were also collected from 45 human handlers having close contact with rabbits. The oral swabs were taken by swapping the back of the throat, while nasal samples were collected by swabbing the anterior nares of the animal handlers. For environmental samples, a total of 180 swabs were taken from feeders  plates  was supplemented with 6.5% of sodium chloride (NaCl). The NA plates then underwent an incubation process at 37°C for 24 h. The growth of bacteria was then evaluated using Gram stain and biochemical tests . Bacteria isolates showing phenotypic characteristics similar to S. aureus were kept under 4°C before polymerase chain reaction (PCR) testing. The samples were inoculated onto mannitol salt agar plates  before being incubated for 48 h at 37°C. The appearances of the bacteria colonies were monitored and recorded at 24 and 48 h. Bacterial colonies with the yellow round appearance (yellow colored as a result of mannitol fermentation) similar to nuc (detection of S. aureus) and mecA (detection of MRSA) genes were then carried out using PCR [nuc gene) and 533 base pair (bp) (mecA gene) were considered MRSA. Bacterial isolates that only harbored nuc genes were referred to as methicillin-susceptible S. aureus (MSSA).The genomic DNA of the isolated bacteria was extracted via the heat lysis method described by Suhaili et al. . The DNAsing PCR . PCR proStaphylococcus aureus isolated from rabbits and rabbit handlers were subjected to Kirby–Bauer test to determine their antibiogram profile [S. aureus cultures. The antibiotic disks used in this study include penicillin (10 units), oxacillin (1 μg), amoxicillin/clavulanate (10 μg), tetracycline (30 μg), erythromycin (15 μg), clindamycin (2 μg), cefotaxime (30 μg), trimethoprim–sulfamethoxazole (25 μg), amikacin (30 μg), cefoxitin (30 μg), chloramphenicol (30 μg), cephalothin (30 μg), ciprofloxacin (5 μg), doxycycline (30 μg), gentamicin (30 μg), kanamycin (30 μg), linezolid (30 μg), norfloxacin (10 μg), and quinupristin-dalfopristin (15 μg). The plates were then incubated at 37°C for 24 h. The inhibition zones of each antibiotic were measured and interpreted according to the disc diffusion breakpoints given by CLSI [Staphylococcus aureus isolates that showed resistance to three and more classes of antibiotics were categorized as multidrug-resistance S. aureus isolates [ profile . The tur profile . The Kir profile . Ninetee by CLSI . Staphylisolates . S. aureus and MRSA were counted and presented in percentages. The number of S. aureus that showed resistance against all antibiotics was calculated and presented as antibiotic resistance rate (%). Categorical data were compared and analyzed using the Pearson Chi-square test or Fisher’s exact test , with a 95% confidence interval (p < 0.05) was set to indicate the significant difference. The prevalence of antibiotic resistance was presented as the proportion of isolates tested with an inhibition zone (diameter) below the respective antibiotic breakpoint. The relationships between antibiotic exposure and overall antibiotic resistance in S. aureus isolates were assessed using a multiple antimicrobial resistance index (MARI). The MARI was calculated as the proportion of antibiotics tested to which the isolate was phenotypically resistant. A dendrogram based on the phenotypic antibiotic resistance profile was generated using unweighted pair group method with arithmetic mean method (UPGMA) in BioNumerics 8.0 software  to visualize the relatedness of the S. aureus isolated from rabbit and rabbit handlers. The number of nuc gene  as mentioned in S. aureus isolates were from the ear while the remaining 22  isolates came from the oral swabs of rabbits. The prevalence result was lower than previous studies that reported 41%–63% of S. aureus occurrence rate in lesion swab samples [S. aureus, where most isolates were oral. This finding agrees with the statement given by Kozajda et al. [S. aureus carriers. However, Agnoletti et al. [S. aureus among 58.3% of individuals related to rabbit farms. The differences in S. aureus prevalence rates in both rabbits and handlers may be due to geographical factors, different sampling sites, and sampling size. Post-screening of the nuc gene  had resu samples ,14. Genoa et al. , where 2S. aureus-positive, indicating a low S. aureus contamination rate at the surface of feeder, drinker, door lock, wall and floor of cages, and feet wear of farmers and their families , suggesting that drinkers and drinking water may play a role in the transmission and dispersion of S. aureus among the rabbits. Staphylococcus aureus isolated from the drinker surfaces may be originated from the rabbits or from the water itself. However, as the water sources from the farms were not tested, the possibility of S. aureus originated from the drinking water remained unclear. Apart from drinkers, S. aureus was also found on boots or slippers (13.3%), door lock (10%), wall of the cage (6.7%), the floor of the cage (3.3%), and feeder (3.3%). Previous studies also reported S. aureus on the surface of various farm objects, suggesting that various environmental surfaces in rabbit farms may act as a vehicle of transmission for S. aureus [In this study, only 8.8% 16/180) of the environmental swabs were families . Further. aureus ,6,16./180 of tS. aureus samples isolated from rabbit handlers, rabbits, and environments were categorized as MSSA as they did not carry any mecA gene. Briefly, mecA is a chromosomally derived gene responsible for producing penicillin binding protein, which helps MRSA develop resistance against antibiotics from the beta-lactam class. The mecA gene is common in MRSA but absent in methicillin-susceptible Staphylococci isolates. Today, many have considered detecting the mecA gene using PCR as the most reliable and “gold standard” technique to identify MRSA [S. aureus harbored the mecA gene, indicating the absence of MRSA. The finding agrees with the study of Attili et al. [In the present study, all ify MRSA . In thisi et al. , which ri et al. . Furtheri et al. .S. aureus from rabbit and rabbit handlers showed different resistances against 18 selected antibiotics. However, the antibiogram of S. aureus from humans (S. aureus showed a relatively low level of resistance (below 20%) against 15 antibiotics. All the rabbit farm owners claimed that no antibiotics were used during the rearing process, which might explain the low antibiotic resistance rate displayed by S. aureus. A similar result was also reported by Wang et al. [S. aureus against penicillin (11%), kanamycin (19.6%), gentamicin (10%), and ciprofloxacin (3.9%) were less than 20%. However, a previous study conducted in Italy reported that most of the S. aureus isolated from rabbits were highly resistant against tetracyclines (96%) and macrolides (94%) [The emergence of AMR in important pathogens of humans and animals is a significant concern. In the present study, m humans  and animm humans  differedg et al. , where tfloxacin .9% were S. aureus from rabbit showed the highest resistance rate against chloramphenicol . The presence of chloramphenicol resistant S. aureus in rabbits is surprising as chloramphenicol usage in the livestock industry is banned [Streptomyces venezuelae in the soil [S. aureus from rabbits remained uncertain. Nonetheless, s banned . Howeverthe soil . HoweverS. aureus isolated from rabbit handlers showed resistance against five different antibiotics, including penicillin , oxacillin , amoxicillin/clavulanate , tetracycline , and erythromycin . This finding was not surprising as -lactam antibiotics such as penicillin are often prescribed to treat bacterial infections in humans. According to Che Hamzah et al. [S. aureus from rabbit handlers in this study was higher than the 5.5% resistance prevalence rate given by the previous study [S. aureus infections in humans may no longer be a viable choice shortly. Nonetheless, all S. aureus isolated from rabbits and their handlers were fully susceptible to cephalothin, cefoxitin, and kanamycin, suggesting that these antibiotics can be used to treat persistent S. aureus treatment for both rabbit and rabbit handlers in the selected rabbit farms. Meanwhile, h et al. , 84.4% ous study . Thus, tS. aureus isolates (one from humans and two from rabbits) were found to be multidrug-resistant S. aureus (MDRSA) [S. aureus from rabbits had MARI values of 0.2 and above, indicating the isolates originated from environments with frequent antibiotic usage. In addition, a dendrogram  . This re (MDRSA) . Another (MDRSA)  revealed (MDRSA) ,23. Thusndrogram  was geneS. aureus had been successfully isolated from rabbit, rabbit handlers, and the environment. No MRSA was identified, but the presence of MDRSA was detected in both rabbits and rabbit handlers. Staphylococcus aureus from rabbit handlers showed resistance against penicillin, oxacillin, amoxicillin/clavulanate, tetracycline, and erythromycin. Meanwhile, S. aureus from rabbits showed resistance against 15 different antibiotics. These findings suggest the emergence of antibiotic-resistant S. aureus that is present in the rabbit farms settings. Therefore, antibiotic usage in rabbits needed to be regulated to prevent the further emergence of AMR. Antimicrobial stewardship programs need to be conducted to educate the rabbit farm owners and workers regarding the risk of AMR. More surveillance programs investigating the AMR in rabbits from other states of Malaysia should be conducted to better understand the AMR issues in the Malaysian rabbit farming industry.This study showed that the Staphylococcus aureus; MARI, multiple antibiotic resistance index; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin susceptible Staphylococcus aureus; NaCl, sodium chloride; S. aureus, Staphylococcus aureus.°C, degree Celsius; %, percentage; μg, microgram; AMR, antimicrobial resistance; bp, base pair; MDRSA, multidrug-resistant