Cloning and Production of Antigen 85A Mycobacterium tuberculosis for Diagnostic Latent Tuberculosis: a Preliminary Study

The main challenge in the management of Tuberculosis (TB) is diagnosing quickly and accurately, especially Latent Tuberculosis Infection (LTBI). LTBI detection was carried out using the Tuberculin Skin Test (TST) and Interferon Gamma Release Assay (IGRA). In TB endemic areas, these two examinations have limitations, so current research is directed at finding specific antigens for the diagnosis of LTBI. One  of the potential proteins is Antigen 85A (Ag85A) Mycobacterium tuberculosis (Mtb) encoded by Fibronectin-binding protein A (FbpA). The Ag85 complex induces the proliferation of T-cells and interferon-gamma in most healthy individuals infected with Mycobacterium tuberculosis, Mycobacterium leprae, and BCG-vaccinated mice, making it a potential antigen. This study aims to clone and produce recombinant protein Ag85A from Mtb in Escherichia coli BL21. The methods used were ligation to the pET-32a expression vector, transformation to Escherichia coli BL21, and production of protein by IPTG induction. Characterization of recombinant clones by colony PCR and sequencing. The results obtained were that the fbpA gene isolated from Mtb clinical isolate had been amplified, and the PCR product was 900 bp. The production of Antigen 85A has been successfully carried out and produces 44 kDa

Untargeted LC-QTOF-MS/MS Based Metabolomic Profile Approach of Bacterial Ferment Lysates and Skin Commensal Bacterial Cocktail Ferment Lysates

Microbial therapy has been increasingly developed in the medical and health fields and has triggered advances in the process of formulating skincare products. The skin microbiota becomes the target in the development of active ingredients to produce an optimal effect in the balance of its composition which leads to its usefulness in maintaining skin health and providing protection. Postbiotic bacteria can maintain homeostasis of the skin microbiome so that it has the potential to be used as an active ingredient in Active Pharmaceutical Ingredient (API) in skincare products and have broad benefits due to its various active substances. The aim of this study was to examine the metabolites profile contained in the bacterial fermented lysate fraction, which is also served as a marker in identifying the metabolite variations of the lysate fractions and their API dosage forms. Ferment lysate API preparations were prepared in the form of freeze dried and spray dried. The metabolite profile analysis was carried out using the untargeted LC-QTOF-MS/MS metabolomic approach and multivariate analysis. Result revealed 30 differential features of the putative metabolites, and by performing metabolites annotationfor their bioactivities through intensive literature research, such as antimicrobial, antioxidant, and anti-inflammatory, we elucidated these compounds are discovered in dry form of lysates

Microscopical Evaluation and TLC Analysis of Pluchea indica (L.) Less: Leaf, Stem, and Root

Pluchea indica (L.) Less is traditionally utilized to treat postpartum women in Indonesia. The plant has many pharmacological properties, so that it can be further developed as herbal medicine. In that development process, plant authentication is needed to ensure the quality of raw materials. A simple microscopical and thin-layer chromatography (TLC) analysis might be a way to authenticate the plant, but it has never been reported. So, this study evaluates the microscopical and TLC analysis for P. indica authentication in standardized herbal medicines production. Plant microscopic observation, fluorescence analysis, and polyphenol screening were conducted. n-Hexane, ethyl acetate, and methanol extracts of plant organs were then analyzed by TLC. Here, we reported that in microscopical analysis the simplicia of P. indica contains trichomes and tannin-containing cells. In addition, chlorogenic acid as a marker was present in TLC analysis by ethyl acetate-water-formic acid-acetic acid (8.5:1.5:1:1, v/v). The results of this evaluation might provide additional information in the identification, authentication, and quality control of P. indica as a raw material for herbal medicine

Development of Few Significant SNP Markers from Transcriptomic Data for Selection of Sengon (Falcataria falcata (L.) Greuter & R. Rankin) Resistant to Boktor Stem Borer and Gall Rust Disease

Sengon (Falcataria falcata (L.) Greuter & R. Rankin) plantations in Indonesia are threatened by attacks from Boktor stem borers and gall rust disease. Controlling pests and diseases is difficult; therefore, planting resistant trees obtained from tree selection programs is necessary. Currently, genomic breeding often incorporates GWAS, which uses thousands of SNP markers to identify markers with significant associations with the traits studied. This study aimed to bypass such expensive studies by identifying and developing SNP markers from sequences of putative resistance genes to Boktor stem borer and gall rust disease, identified from sengon transcriptomic data analysis. A total of 496,194 putative SNP sites were identified from transcriptomic sequences using the SAMtools and BFCtools programs, of which 119 SNP sites were associated with resistance genes. Of the 101 non-synonymous SNPs selected, only 12 were located in the conserved domain of each gene and were used for primer design. Of the 13 primers designed, only 10 were successfully amplified. Validation of 10 developed SNP markers on 100 sengon accessions using the HRM method confirmed a significant association between SNP markers and resistance traits, with a -log 10 (P-value) between 10.49 and 16.63. A few SNPs markers developed from putative resistance gene sequences are associated with resistance traits in sengon. Therefore, the SNP markers could be applied in selection programs for sengon trees resistant to Boktor stem borers and gall rust disease

Chemical Composition, Antioxidant, Antibacterial, Antibiofilm, and Cytotoxic Activities of Robusta Coffee Extract (Coffea canephora)

Coffee extracts are a rich source of potential compounds with numerous biological activities. This study aimed to investigate the potential of chemical compounds derived from robusta coffee extract (Coffea canephora) and its dominant compound, caffeine, to scavenge free radicals, inhibit bacterial growth and biofilm formation, and cytotoxic properties in human breast adenocarcinoma cell line. Chemical constituents of coffee extract were analysed quantitively for total phenolic, flavonoid, and alkaloid contents, along with Liquid Chromatography Quadrupole-Mass Spectrometry (LC/MS–MS) analysis. Antioxidant activity was measured by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis; 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. Antibacterial pair with antibiofilm properties against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus were also determined, as well as cytotoxic activity on the MCF7 cell line model. The LC/MS–MS analysis of coffee extracts revealed high levels of caffeine; thus, the caffeine standard is used in all subsequent assays. Notably, robusta coffee extract showed remarkable antioxidant activity and selective inhibition of the growth against gram-positive and negative bacteria, along with the best inhibition of P. aeruginosa biofilm. However, compared with the caffeine standard, robusta coffee extract had lower cytotoxic activity and different bacterial targets in antibacterial and antibiofilm properties. Our results indicate that Robusta coffee extract is potentially a functional food due to its high alkaloid, phenolic, and flavonoid content and antioxidant activity, besides being used for natural compounds against bacterial infections

Detection of Avirulence Gene AvrPi9 in Magnaporthe oryzae, a Rice Blast Fungus, Using a Combination of RPA and CRISPR-Cas12a Techniques

Rice blast disease is one of the most devastating diseases of rice production worldwide, which causes by an ascomycete fungus, Magnaporthe oryzae. The virulence of the rice blast fungus is determined by avirulence genes (Avr genes). Therefore, the identification of Avr genes is important for rice resistance variety improvement. Avr genes are currently identified using the pathogenicity assay with rice near-isogenic lines (NILs) or PCR amplification and gene sequencing, both of which are time-consuming and labor-intensive methods. This study aims to develop a simple method for Avr gene identification using AvrPi9 as a model. A recombinase polymerase amplification (RPA) technique was carried out to amplify AvrPi9 by incubating rice blast fungus genomic DNA with gene-specific primers at 37°C for 20 min. Cas12a-based AvrPi9 detection was performed by incubating at 37°C for 5 min. The fluorescence signal was visualized by the naked eye under an LED transilluminator. The study found that AvrPi9 can be amplified and detected using RPA and a Cas12a-based method. AvrPi9_crRNA2 has a higher efficiency than AvrPi9_crRNA1. The sensitivity of the method was 3.8 ng of DNA target for AvrPi9_crRNA1 and 1.9 ng of DNA target for AvrPi9_crRNA2. This RPA and Cas12a combination technique is a newer method for Avr gene detection in plants and has several advantages over traditional methods. It is considered easier to use and more efficient in terms of time and labor, making it a potentially useful tool for plant breeders and pathologists

Distribution Analysis of Asiatic Palm Weevil Rhynchophorus vulneratus Panzer (Coleoptera: Dryphthoridae) using GIS Technique and the Interaction with Coconut Beetle Oryctes rhinoceros L. (Coleoptera: Scarabaeidae)

Asiatic palm weevil Rhynchophorus vulneratus Panzer is one of the key pests of coconut in Indonesia. Information regarding the spatial distribution of R. vulneratus is needed to support Integrated Pest Management and can be analyzed using Geographic Information System (GIS). Initial studies on the interaction between R. vulneratus and Oryctes rhinoceros were also studied in this study. This research aimed to analyze the spatial distribution of R. vulneratus using GIS and its interaction with the Coconut beetle Oryctes rhinoceros. The research was conducted in Yogyakarta as a habitat model (0-1,000 masl). R. vulneratus and O. rhinoceros were catched by installing aggregation pheromone traps hanging to coconut plants at 1.7 m above ground. The research was conducted during the rainy and dry seasons. The spatial distribution of R. vulneratus was analyzed by IDW interpolation using QGIS 3.22, whereas its interaction with O. rhinoceros was analyzed by the Pearson correlation test using SPSS 22. The results showed low number of R. vulneratus captured in the Yogyakarta area during the rainy season at 0-1,000 masl. However, the number of R. vulneratus during the dry season has increased, and the distribution rate was dominated by medium to very high levels, especially at an altitude of 0-300 masl. The study also showed that the number of R. vulneratus was not influenced by the number of O. rhinoceros, which suggested that the abundance of O. rhinoceros cannot accurately predict the abundance of R. vulneratus

The Facial Width-To-Height Ratio is Unrelated to Aggressive Behaviour in Indonesian People

As a channel of non-verbal communication, faces can give information such as mate attraction, intelligence, and aggressivity. Aggressivity is a character to dominate, protect position, and fight over resources. Several aggressive behaviours in humans are, for example, anger, hostility, physical aggression, and verbal aggression. Previous studies in western society showed that aggressivity could be perceived from the faces. We tested 100 Indonesian males ranging from 19-51 years old to fill out the Buss-Perry Aggression Questionnaire (BPAQ) to measure the aggression scale. The mean of their BPAQ scales (total aggression, anger, hostility, physical aggression, and verbal aggression) were 72.44±10.84, 17.37±3.97, 21.38±4.53, 18.97±4.65, 14.72±2.68, respectively. The average facial photograph was generated based on the min-Q1 (Low Aggressivity (LA)) and Q3-max (High Aggressivity (HA)) BPAQ scale. Next, the aggressivity of averaged LA and HA faces was evaluated by raters. The raters consisted of 145 males and 213 females randomly recruited, ranging from 17 to 67 years old. The facial width-to-height (fWHR) ratio between the average faces of the Low-Aggression face and the High-Aggression face was insignificant. This study concluded that Indonesian people could not perceive aggressivity in their faces

Effect of the Supplementation of Endogenous Probiotics in Feed on Growth Performance, Digestive Enzyme Activity, and Non-Specific Immunity Gene Expression of Sandfish, Holothuria scabra Juveniles

The present study aimed to evaluate the effects of endogenous compared to commercial probiotics on the growth, digestive enzyme activity, and non-specific immunity of sandfish juveniles. Treatments were: A. diet without probiotic; B. diet supplemented with endogenous probiotics Gamma proteobacterium strain M-4, Bacillus subtilis strain Q-1, Bacillus sp. strain E-2; C. diet supplemented with a commercial probiotic containing B. subtilis, B. licheniformis, and B. pumilus. The research was conducted for 4 months using 12 plastic containers, 30 juveniles each (initial weight of 2.72±0.37 g, mean ± standard deviation). From each container, ten samples were taken randomly every 14 days and weighed and three intestine samples at initial and every following month to analysis enzyme activities. At the end the study, all juveniles were counted and weighed individually, and performed a challenge test by injected a 106 CFU ml-1 Vibrio azureus strain 4C-1 at 0.1 ml/ind. Immune response observed were: total coelomate and gene expression (SOD, CAT, LZM) by Quantitative real-time PCR. The specific growth rate of juveniles fed by diet B was significantly different from treatment A (P<0.05), its final weight was higher compared to diets A and C (P<0.05). Protease, lipase, and amylase activity in treatment B was higher compared to C and A (P<0.05). The survival rate, total coelomocyte count, gene expression after being challenged were higher in treatment B compared to C and A (P<0.05). The present study showed endogenous probiotics promote growth, improve digestion enzyme activity, and stimulate non-specific immune responses

Bananas and Their Wild Relatives in Pandeglang, Indonesia

Pandeglang is one of the banana producer regencies of Banten, Indonesia, that produce 924 tons per year. However, information regarding the diversity of banana germplasm in this region is unavailable. This study explored districts Cadasari, Banjar, Jiput, Labuan, and Menes. Morphological characters were noted and used to identify the cultivars, subspecies, or varieties. In total, 22 accessions were collected from 22 collection sites. Based on morphological characters, we described 14 banana accessions of 10 cultivars, three accessions of wild Musa acuminata and one of Musa balbisiana. Considering its genome composition, five genome types we described among these banana cultivars, i.e., AA (divided into AA and AA wild), BB, AAA, AAB, and ABB. In this study, we did a principal component analysis and saw that AA wild and AAB were clearly separated, but the other genome types were clustered partially or included in one cluster. Our study expands the information on banana diversity in Indonesia. Further study on the potential of this banana germplasm to be used as a genetic resource to find resistance to banana diseases is needed