Antioxidant and Antibacterial Properties of 1,3-Dihydroxy-,2', 2'-Dimethylpyrano-(5,6)-Xanthone from Streptomyces sp. SU84.

<b>Background and Objective:</b> The SU84 was isolated from the rhizosphere of <i>Curcuma longa</i> and identified to be <i>Streptomyces</i> sp. via analysis of its 16S rDNA sequence, chemotaxonomy and morphology. This study aimed to isolate major compounds from the extract culture of strain SU84 and evaluate their antibacterial activity. <b>Materials and Methods:</b> The TLC and silica gel column chromatography were used to purify major compounds, elucidate 1,3-dihydroxy-,2',2'-dimethylpyrano-(5,6)-xanthone (compound <b>1</b>) and lupeol (compound <b>2</b>) using mass spectrometry and nuclear magnetic resonance. One new chemical, compound <b>1</b>, was first isolated from microbial sources. Antibacterial, antioxidant and cytotoxic properties of these compounds were carried out. <b>Results:</b> Various bioassays showed that compound <b>1</b> displayed antibacterial property against Gram-positive bacteria, with a minimum inhibitory concentration of 8-32 µg/mL and minimum bactericidal concentration of 32-128 µg/mL. In addition, the purified compounds were tested against normal cell lines using tetrazolium assay. The results did not show cytotoxic property against L929 and Vero cells, with IC₅₀ values of >512.00 µg/mL. Compounds <b>1</b> and <b>2</b> have also antioxidant properties, with IC₅₀ values of 16.67±7.48 and 38.86±8.45 µg/mL, respectively. <b>Conclusion:</b> The findings suggested that compounds of <i>Streptomyces</i> sp. SU84 displayed antibacterial and antioxidant properties without cytotoxic activity. Extensive studies of compound <b>1</b> may be useful for the advancement of improved methods for avoidance, control and management of bacterial infections and metabolic-related free radical contribution.

Apoptotic Potential and Molecular Docking of 3,4-Dihydro-lactucin, a Compound With Anticancer Properties Derived from Microbispora rosea AL22.

BACKGROUND/AIM: Tetrazolium-based cell proliferation assays using MDA-MB-231 and HeLa cells revealed that 3,4-dihydro-lactucin (3,4-DHL), a compound isolated from Microbispora rosea AL22, possesses anticancer properties. Apoptotic cell death was observed in 3,4-DHL-treated cells. Lactucopicrin, a related compound, reportedly exerts anticancer activity against different cancer types. However, data on the anticancer mechanism of lactucins are limited. This study aimed to investigate apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL. MATERIALS AND METHODS: Morphological changes, changes in mitochondrial membrane potential, and apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL were investigated. Furthermore, molecular docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of anti-apoptotic proteins were performed to determine the effector mechanism of 3,4-DHL. RESULTS: 3,4-DHL induced cytotoxicity at a half-maximal inhibitory concentration of 37.62 µg/ml, along with various morphological alterations in apoptotic and viable cells. Furthermore, 3,4-DHL-treated cells showed mitochondrial membrane potential depolarization, intense annexin V-fluorescein isothiocyanate staining, and increased caspase 3 and 8 activities. Molecular-docking studies demonstrated that 3,4-DHL should bind to the active site of various anti-apoptotic proteins, forming stable complexes. CONCLUSION: Our findings revealed that 3,4-DHL has great potential to be used as an apoptosis-inducing agent in cancer therapy. However, further in-vivo confirmation is required in evaluation of 3,4-DHL as an anticancer agent in cancer chemotherapy.

Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation.

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+.

Antibacterial and Anticancer Properties of Microbispora sp., AL22: An Endophyte of Alpinia galanga (L.) Willd.

<b>Background and Objective:</b> The AL22 strain was isolated from the rhizosphere soil of <i>Alpinia galanga</i> (L.) Willd (Zingiberaceae) and identified as <i>Microbispora</i> sp., by analysing its morphology, chemotaxonomy and 16S rDNA sequence. Previous studies demonstrated the bactericidal effects of its crude extract against <i>Bacillus cereus</i>, <i>Bacillus subtilis</i>, <i>Staphylococcus aureus</i> and methicillin-resistant <i>Staphylococcus aureus</i>. The present study aimed to isolate the major compounds and evaluate their biological properties. <b>Materials and Methods:</b> Silica gel column chromatography and thin-layer chromatography were used for the purification and identification of 3,4-dihydro-lactucin (compound <b>1</b>) and umbelliferone (compound <b>2</b>) by NMR and mass spectrometry, respectively. Antibacterial and anticancer activities were carried out. <b>Results:</b> The bioassay studies illustrated that compound <b>1</b> had antibacterial activity against gram-positive bacteria, with its minimum inhibitory concentration and minimum bactericidal concentration of 16-32 and 64-128 µg mL¹, respectively. The crude extract and purified compounds showed weak cytotoxic activity on the L929 and Vero cells with IC₅₀ values >512.00 µg mL¹. The cytotoxicity of compound <b>1</b> was observed in the MDA-MB-231 and HeLa cells with IC₅₀ values of 37.62 and 75.34 µg mL¹, respectively, while its IC₅₀ value against the HepG2 cells was 456.67 µg mL¹. <b>Conclusion:</b> These findings showed that compound <b>1</b> of <i>Microbispora</i> sp., AL22 exhibited antibacterial and anticancer activities. Extensive studies on 3,4-dihydro-lactucin could lead to the development of beneficial approaches for managing bacterial infections and cancer.

Synergistic Antibacterial Activity of 1-Methyl Ester-Nigericin and Methyl 5-(Hydroxymethyl) Furan-2-Carboxylate Against Proteus spp.

<b>Background and Objective:</b> Synergistic combinations of antimicrobial agents with different mechanisms of action are successful approaches for combating bacterial infections. This study aimed to evaluate the synergistic effect of 1-methyl ester-nigericin <b>(1)</b> and methyl 5-(hydroxymethyl) furan-2-carboxylate <b>(2)</b> against <i>Proteus</i> spp., isolates. <b>Materials and Methods:</b> The synergistic antimicrobial activity of the compounds was tested by the checkerboard method and time-kill curves. To estimate the interaction between the compounds, the Fractional Inhibitory Concentration Index (FICI) of the combination was calculated. The cytotoxic activity of the compounds in combination was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on LLC-MK2 cell lines. The reduction percentage of biofilms was obtained using the colourimetric method. <b>Results:</b> The MIC values for compounds <b>1</b> and <b>2</b> against test bacteria ranged from 39.06-78.12 µg mL¹ and from 78.12-156.25 µg mL¹, respectively. The MIC was reduced to 1-8th as a result of the combination of compounds <b>1</b> and <b>2</b>. After 4-24 hrs of treatment with ½ MIC of compounds <b>1</b> and <b>2</b>, the killing rate (in CFU mL¹) increased to a greater degree than observed with either test compound alone. The combination of compounds <b>1</b> and <b>2</b> showed a synergistic effect with FICI of 0.50 and 0.28. The synergistic combination of compounds <b>1</b> and <b>2</b> was effective on the biofilm reduction of <i>Proteus</i> <i>vulgaris</i> NP16 (85.72%) and NP47 (89.14%). <b>Conclusion:</b> This study recommends compounds <b>1</b> and <b>2</b> in combination as a potential alternative treatment agent for <i>Proteus</i> spp. infections.

Antibiotic-Resistant Bacteria in Green Turtle (Chelonia mydas) Rearing Seawater.

Antibiotic resistance of microorganisms is a serious health problem for both humans and animals. Infection of these bacteria may result in therapy failure, leading to high mortality rates. During an early intervention program process, the Sea Turtle Conservation Center of Thailand (STCCT) has faced high mortality rates due to bacterial infection. Previously, investigation of juvenile turtle carcasses found etiological agents in tissue lesions. Further determination of sea water in the turtle holding tanks revealed a prevalence of these causative agents in water samples, implying association of bacterial isolates in rearing water and infection in captive turtles. In this study, we examined the antibiotic resistance of bacteria in seawater from the turtle holding tank for a management plan of juvenile turtles with bacterial infection. The examination was carried out in three periods: 2015 to 2016, 2018, and 2019. The highest isolate numbers were resistant to beta-lactam, whilst low aminoglycoside resistance rates were observed. No gentamicin-resistant isolate was detected. Seventy-nine isolates (71.17%) were resistant to at least one antibiotic. Consideration of resistant bacterial and antibiotic numbers over three sampling periods indicated increased risk of antibiotic-resistant bacteria to sea turtle health. Essentially, this study emphasizes the importance of antibiotic-resistant bacterial assessment in rearing seawater for sea turtle husbandry.

The Occurrence of Vibrionaceae, Staphylococcaceae, and Enterobacteriaceae in Green Turtle Chelonia mydas Rearing Seawater.

In this study, levels of Vibrionaceae, Staphylococcaceae, and Enterobacteriaceae were observed in seawater from juvenile green turtle Chelonia mydas rearing tanks and in the incoming coastal seawater (the water supply). Bacterial loads were compared between the incoming coastal seawater and two different rearing conditions: in cement tanks at a low stocking density and in fiberglass tanks at a high stocking density. The total bacterial counts in seawater from fiberglass tanks were statistically greater than those in cement tanks. The nonlactose and lactose fermenting enterobacteria, tellurite-reducing bacteria, and total plate counts in water from all rearing containers were greater than those in coastal seaweater by a logarithmic fold change of 2--3. Differences in bacterial population structure of the incoming coastal seawater and rearing water were also addressed. The results from biochemical identification of 344 isolates revealed that the bacteria that were commonly found in water samples were Citrobacter spp., Enterobacteria spp., Edwardsiella spp., Staphylococcus spp., Staphylococcus aureus, Photobacterium spp., Vibrio alginolyticus, and Vibrio spp. Conclusively, the microbiological monitoring of rearing water provides important and essential information on the management of aquatic animal health and husbandry.

Effects of porcine follicle stimulating hormone, luteinizing hormone and estradiol supplementation in culture medium on ultrastructures of porcine cumulus oocyte complexes (pCOCs).

Inhibition of primary oocyte developing to secondary oocyte results from oocyte maturation inhibitor (OMI) which is secreted from oocyte-surrounding cumulus cells (CCs) during forming complexes with an oocyte. Development of primary oocyte occurs when the CCs are dissociated from the oocyte. This research studied the effects of pFSH, LH, and estradiol supplementation in culture medium on ultrastructures of porcine cumulus oocyte complexes (pCOCs) using transmission electron microcopy (TEM) and inverted microscopy. A total of 880 oocytes were isolated from 110 ovaries: an average of 8 oocytes per ovary. The oocytes were round in shape and surrounded by zona pellucida with layers of cumulus cells (CCs), at a diameter ranging between 90 and 150 µm and more than 400 µm. Based on the CCs surrounding an oocyte, pCOCs were classified into 5 types, which were intact-, multi-, partial cumulus cell layers, completely denuded oocyte, and expanded cumulus cell layer, which were found at the percentage of 53.86%, 14.32%, 4.32%, 19.20%, and 8.30%, respectively. The Types I and II pCOCs (intact- and multi-CCs layers) were further cultured at 37°C with 5% CO(2), 95% air atmosphere, and high humidity for 24-48 h to investigate their morphological changes after hormonal induction. For the pCOCs cultured without hormonal induction, the CCs were still round in shape and remained in contact with an oocyte via a process of granular end point sticking into the zona pellucida. In contrast, for the hormone supplemented groups, morphological alteration of pCOCs were seen after culture of 24-48 h. The CCs shape was changed from round into elongated or columnar in an opposite direction from an oocyte as well as no communication between microvilli of CCs observed. This led into ceasing of OMI secretion. Therefore, changes of CCs morphology were a sign of the beginning of oocyte maturation. Further study is to characterize the granular substance at the end point of CCs that stick into the zona pellucida.

RNA splicing factors regulated by HPV16 during cervical tumour progression.

The most prevalent human papillomaviruses (HPVs) causing cervical disease are the 'high-risk' HPV types 16 and 18. All papillomaviruses express a transcription factor, E2, that can regulate viral and cellular gene expression. Recently, we demonstrated high-risk HPV E2-mediated transcriptional transactivation of SF2/ASF. This essential oncoprotein is a key member of a family of proteins, the SR proteins, that regulate constitutive and alternative splicing. Tight control of RNA splicing is necessary for the production of wild-type proteins. So, aberrant expression of SR proteins is involved in the aetiology of a range of human diseases, including cancer. Here we demonstrate epithelial differentiation-specific control of SF2/ASF in HPV16-infected keratinocytes in organotypic raft culture and in low-grade cervical lesions (CIN1). Further, we demonstrate HPV16 infection/differentiation-induced up-regulation of a specific subset of SR proteins and present evidence that HPV16 E2 controls expression of SRp20, SC35 and SRp75. Using a series of cell lines that model cervical tumour progression, we show that SF2/ASF, SRp20 and SC35 are specifically up-regulated in a model of cervical tumour progression. These SR proteins are also over-expressed in high-grade cervical lesions, indicating that they may all have oncogenic functions. SR proteins could be useful biomarkers for HPV-associated disease.