American Type Culture Collection Research Articles

Luteibacter mycovicinus sp. nov., a yellow-pigmented gammaproteobacterium found as an endohyphal symbiont of endophytic Ascomycota.

We isolated and described a yellow-pigmented strain of bacteria (strain 9143T), originally characterized as an endohyphal inhabitant of an endophytic fungus in the Ascomycota. Although the full-length sequence of its 16S rRNA gene displays 99 % similarity to Luteibacter pinisoli, genomic hybridization demonstrated <30 % genomic similarity between 9143T and its closest named relatives, further supported by average nucleotide identity results. This and related endohyphal strains form a well-supported clade separate from L. pinisoli and other validly named species including the most closely related Luteibacter rhizovicinus. The name Luteibacter mycovicinus sp. nov. is proposed, with type strain 9143T (isolate DBL433), for which a genome has been sequenced and is publicly available from the American Type Culture Collection (ATCC TSD-257T) and from the Leibniz Institute DSMZ (DSM 112764T). The type strain reliably forms yellow colonies across diverse media and growth conditions (lysogeny broth agar, King's Medium B, potato dextrose agar, trypticase soy agar and Reasoner's 2A (R2A) agar). It forms colonies readily at 27 °C on agar with a pH of 6-8, and on salt (NaCl) concentrations up to 2 %. It lacks the ability to utilize sulphate as a sulphur source and thus only forms colonies on minimal media if supplemented with alternative sulphur sources. It is catalase-positive and oxidase-negative. Although it exhibits a single polar flagellum, motility was only clearly visible on R2A agar. Its host range and close relatives, which share the endohyphal lifestyle, are discussed.

Metabolic Comparison and Molecular Networking of Antimicrobials in Streptomyces Species.

Streptomyces are well-known for producing bioactive secondary metabolites, with numerous antimicrobials essential to fight against infectious diseases. Globally, multidrug-resistant (MDR) microorganisms significantly challenge human and veterinary diseases. To tackle this issue, there is an urgent need for alternative antimicrobials. In the search for potent agents, we have isolated four Streptomyces species PC1, BT1, BT2, and BT3 from soils collected from various geographical regions of the Himalayan country Nepal, which were then identified based on morphology and 16S rRNA gene sequencing. The relationship of soil microbes with different Streptomyces species has been shown in phylogenetic trees. Antimicrobial potency of isolates was carried out against Staphylococcus aureus American Type Culture Collection (ATCC) 43300, Shigella sonnei ATCC 25931, Salmonella typhi ATCC 14028, Klebsiella pneumoniae ATCC 700603, and Escherichia coli ATCC 25922. Among them, Streptomyces species PC1 showed the highest zone of inhibition against tested pathogens. Furthermore, ethyl acetate extracts of shake flask fermentation of these Streptomyces strains were subjected to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis for their metabolic comparison and Global Natural Products Social Molecular Networking (GNPS) web-based molecular networking. We found very similar metabolite composition in four strains, despite their geographical variation. In addition, we have identified thirty-seven metabolites using LC-MS/MS analysis, with the majority belonging to the diketopiperazine class. Among these, to the best of our knowledge, four metabolites, namely cyclo-(Ile-Ser), 2-n-hexyl-5-n-propylresorcinol, 3-[(6-methylpyrazin-2-yl) methyl]-1H-indole, and cyclo-(d-Leu-l-Trp), were detected for the first time in Streptomyces species. Besides these, other 23 metabolites including surfactin B, surfactin C, surfactin D, and valinomycin were identified with the help of GNPS-based molecular networking.

In vitro evaluation of the binding activity of novel mouse IgG1 opsonic monoclonal antibodies to Mycobacterium tuberculosis and other selected mycobacterial species

Antimicrobial resistance alongside other challenges in tuberculosis (TB) therapeutics have stirred renewed interest in host-directed interventions, including the role of antibodies as adjunct therapeutic agents. This study assessed the binding efficacy of two novel IgG1 opsonic monoclonal antibodies (MABs; GG9 & JG7) at 5, 10, and 25 µg/mL to live cultures of Mycobacterium tuberculosis, M. avium, M. bovis, M. fortuitum, M. intracellulare, and M. smegmatis American Type Culture Collection laboratory reference strains, as well as clinical susceptible, multi-drug resistant, and extensively drug resistant M. tuberculosis strains using indirect enzyme-linked immunosorbent assays. These three MAB concentrations were selected from a range of concentrations used in previous optimization (binding and functional) assays. Both MABs bound to all mycobacterial species and sub-types tested, albeit to varying degrees. Statistically significant differences in MAB binding activity were observed when comparing the highest and lowest MAB concentrations (p < 0.05) for both MABs GG9 and JG7, irrespective of the M. tuberculosis resistance profile. Binding affinity increased with an increase in MAB concentration, and optimal binding was observed at 25 µg/mL. JG7 showed better binding activity than GG9. Both MABs also bound to five MOTT species, albeit at varied levels. This non-selective binding to different mycobacterial species suggests a potential role for GG9 and JG7 as adjunctive agents in anti-TB chemotherapy with the aim to enhance bacterial killing.

Abstract 5892: SCUBE3 promotes therapy resistance in triple negative breast cancer

Abstract Introduction: The development of novel targeted therapies is urgently required for improving the outcome of triple negative breast cancer (TNBC) patients. Chemotherapy is the common treatment option for TNBC. However, resistance and tumor relapse remain the major obstacles hindering the effectiveness of chemotherapeutic agents in cancer patients. Therefore, identifying genes/factors that sensitize breast cancer cells to chemotherapeutic agents could improve treatment outcome in patients. Here we report Signal peptide CUB domain EGF-like 3 (SCUBE3) genes as a novel therapeutic adjuvant that can improve the efficacy of doxorubicin, a chemotherapeutic agent commonly used in treating breast cancer patients. Methods: Unbiased high-throughput loss of function genomic screen was performed on breast cancer cells in the presence or absence of doxorubicin. Breast cancer cells lines (MDA-MB-231 and MDA-MB-468) were purchased from the American Type Culture Collection and cultured in standard medium. Breast cancer cells were transfected either with SCUBE3 overexpression plasmid or shRNA specific to SCUBE3. These overexpression/knockdown breast cancer cells were analyzed for cell viability, migration, invasion, colony formation, cell cycle, apoptosis assays, western blotting, and in vivo tumor xenograft study. Results: Our finding demonstrated that SCUBE3 promotes breast cancer cell progression as knockdown of SCUBE3 inhibited the ability of breast cancer cells to form colony, migrate, and invade, while overexpression of SCUBE3 promoted tumor growth in preclinical mouse models. Our results revealed that SCUBE3 mediates its protumor effects by regulating genes involved in growth and survival in the MAPK pathway, DNA damage surveillance pathway including RAD51 and FOXM1, and apoptotic pathway including Mcl-1. We demonstrated that SCUBE3 promotes tumor growth and progression by interacting with and activating several oncoproteins. Conclusions: Overall, our data suggests that SCUBE3 can be a potent therapeutic target for treating TNBC patients. Citation Format: Deepika Singh, Daisy Medina, Benjamin Onyeagucha, Rahul Mojidra, Panneerdoss Subbarayallu, Alex Taylor, Dongwen Lyu, Santosh Timilsina, Prabhakar Pitta Venkata, Saif S R Nirzhor, Shahad M. Abdulsahib, Trong Phat Do, Yidong Chen, Ratna Vadlamudi, Manjeet K. Rao. SCUBE3 promotes therapy resistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5892.

Abstract 3156: Unveiling the functional role of HDGFRP3 in pediatric medulloblastoma: Implications for growth, progression, and radioresistance

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain cancer in children. Although long-term survival has improved in recent years, only a modest percentage of patients survive high-risk disease. The quality of life for those who survive is substantially reduced due to the high toxicity associated with the radiation and chemotherapy. Therefore, identification of safe and potent therapeutic approaches that can sensitize the therapy response in MB without inducing toxicity will lead to more efficient treatment of MB patients. We recently discovered that targeting HDGFRP3, a protein highly expressed in MB, and can block MB growth and improve the efficacy of radiation therapy. HDGFRP3 is a multifaceted protein that plays important roles in diverse cellular processes. Our investigation sheds light on the functional role of HDGFRP3 in MB, focusing on its involvement in growth, progression, and radioresistance where we report HDGFRP3 as a novel oncogenic driver of these processes. Methods: Human MB cell lines (DAOY, D556, D425, and HDMB03) were purchased from the American Type Culture Collection and cultured in standard medium. MB cancer cells were transfected with siRNA specific to HDGFRP3. These knockdown cells were analyzed for cell viability, migration, invasion, colony formation, stemness, cell cycle, apoptosis assays for functional role assessment. Real-time qPCR and western blotting were performed to validate the downstream targets of HDGFRP3 in MB models. Results: Our findings demonstrate that HDGFRP3 functions as an oncogene in MB, as its overexpression promotes cancer growth, while its depletion results in decreased tumor growth in MB models. Furthermore, we provide evidence that HDGFRP3 knockdown results in decreased migratory potential of MB cells as well as a reduction in stemness. Interestingly, HDGFRP3 depletion improved the efficacy of standard of care radiation in multiple MB models. At present, in vivo studies are validate the oncogenic role of HDGFRP3 in preclinical animal models of MB. Conclusions: In conclusion, our data suggests HDGFRP3 facilitates MB growth, progression, stemness as well as resistance to radiation therapy. Our comprehensive investigation into HDGFRP3's functional role in MB presented here provides valuable insights into its potential as a therapeutic target and opens avenues for further research into novel treatment strategies for the pediatric brain tumors. Citation Format: Saif S. Nirzhor, Prabhakar Pitta Venkata, Shahad Abdulsahib, Panneerdoss Subbarayalu, Santosh Timilsina, Manjeet K. Rao. Unveiling the functional role of HDGFRP3 in pediatric medulloblastoma: Implications for growth, progression, and radioresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3156.

Abstract 628: RNA binding protein Matrin3 acts as a tumor suppressor by inhibiting microtubule nucleation

Abstract Introduction: Microtubule-associated proteins (MAPs) regulate microtubule dynamics, which is critical for controlling cell division, proliferation, migration, and intracellular transport. Due to their critical role in maintaining cellular integrity, microtubules have served as potent therapeutic targets for cancer treatment. Here, we report “Matrin 3 (MATR3)” as a novel MAP that interacts with β- tubulin and γ-tubulin, which constitutes microtubule organizing center (MTOC) essential for proper spindle formation and chromosomal segregation. Methods: Breast cancer cell lines (MDA-MB-231, MDA-MB-468, SKBR3, MCF7, BT549, HMEC, and MCF10A) were purchased from the American Type Culture Collection and cultured in standard medium. Breast cancer cells were transfected either with MATR3 overexpression plasmid or siRNA specific to MATR3. These overexpression/knockdown breast cancer cells were analyzed for cell viability, migration, invasion, colony formation, cell cycle, apoptosis assays, RNA sequencing, RT-qPCR, western blotting, RNA immunoprecipitation, and in vivo tumor xenograft study. Results: Our findings show that MATR3 binds to RNA encoding MAPs including β-tubulin and γ-tubulin, and regulate their stabilization. MATR3 also interacted with β-tubulin protein. Our data demonstrate that MATR3 functions as a tumor suppressor, as its overexpression inhibits cancer growth, while its depletion increased tumor growth in vivo. Mechanistic studies confirmed that MATR3 mediates its tumor suppressor role by regulating the expression of the MTOC-associated protein-encoding gene MZT2B (Mitotic Spindle Organizing Protein 2B), which is overexpressed in cancers and promotes cancer growth. Further, loss of MATR3 expression or function results in dysregulated microtubule dynamics and uncontrolled expression of the oncogenic proteins including MZT2B. Conclusions: In conclusion, our data strongly suggests that MATR3 serves as a novel RNA-binding protein with tumor suppressor properties. This discovery holds significant importance as MATR3 becomes the second protein, following Adenomatous Polyposis Coli (APC), to be identified for its binding to both MAP RNA and protein. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Santosh Timilsina, Saif Nirzhor, Daisy Medina, Shahad Abdulsahib, Pitta V Prabhakar, Deepika Singh, Fu-Yang Li, Pooja Yadav, Esha Reddy, Krishna Evani, Vijay Eedunuri, Trong Phat Do, Benjamin C. Onyeagucha, Tabrez A. Mohammad, Jae-Hoon Ji, Yidong Chen, Nourhan Abdelfattah, Nicholas Dybdal-Hargreaves, Li-Ju Wang, Yu-Chiao Chiu, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi, Manjeet K. Rao. RNA binding protein Matrin3 acts as a tumor suppressor by inhibiting microtubule nucleation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 628.

Establishment of Big Bone chicken fibroblast cell bank and study of its biological characteristics

A fibroblast bank of Big Bone chicken was established using tissue adherent culture method. This cell bank included 29 embryo samples, had stocks of 147 cryogenically-preserved vials each containing 2~3Å~106 cells, and met all the cell line quality control standards established by the American Type Culture Collection (ATCC). The cells cultured in vitro showed the typical morphology of fibroblasts. The growth curve assumed the “S” shape, and the cell population doubling time (PDT) was about 30h. Cell viability was 98.1% before cryopreservation and 96.6% after recovery. All tests for microbial contamination were negative. The isoenzyme pattern was of species specificity. The frequency of diploid cells was 91%. The transfection efficiency of three fluorescent protein genes fluctuated between 10.6% and 26.5%. These results showed that the cells cultured in vitro grew well and had stable genetic properties. This research thus does not only preserve the poultry genetic resources of Big Bone chicken at the cell level, but also opens new ways for preserving important genetic resources of endangered animals in the form of somatic cells.

ISOLATION, CHARACTERIZATION AND ANTIMYCOBACTERIAL POTENCY OF A FURAN DERIVATIVE FROM THE CHLOROFORM CRUDE EXTRACT OF Icacina trichantha oliv TUBER.

Spectroscopic analysis of a pure isolate (OAU 2, a furan derivative) by column chromatography from the chloroform crude extract of Icacina trichantha tuber was done using FT-IR, 1HNMR, 13CNMR, COSY and was established by literature as well as the investigation of antimycobacterial potency. This anitmycobacterial activity revealed that this isolate actually has activity against DR-TB Strain and H37RV Strain which are American type culture collection strain and multi-drug resistant strains of M. tuberculosis being the test organisms. Hence, confirming the folk believe of the use of I. trichantha tuber in the treatment of “serious cough”

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the "Big Six." The "Big Six" strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain's individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this "Big Six" reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.

Machine Learning-Based Virtual Screening of Antibacterial Agents against Methicillin-Susceptible and Resistant Staphylococcus aureus.

The application of computer-aided drug discovery (CADD) approaches has enabled the discovery of new antimicrobial therapeutic agents in the past. The high prevalence of methicillin-resistantStaphylococcus aureus(MRSA) strains promoted this pathogen to a high-priority pathogen for drug development. In this sense, modern CADD techniques can be valuable tools for the search for new antimicrobial agents. We employed a combination of a series of machine learning (ML) techniques to select and evaluate potential compounds with antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA strains. In the present study, we describe the antibacterial activity of six compounds against MSSA and MRSA reference (American Type Culture Collection (ATCC)) strains as well as two clinical strains of MRSA. These compounds showed minimal inhibitory concentrations (MIC) in the range from 12.5 to 200 μM against the different bacterial strains evaluated. Our results constitute relevant proven ML-workflow models to distinctively screen for novel MRSA antibiotics.

Epigallocatechin Gallate (EGCG) ve Yeşil Çay (Camellia Sinensis) Yağının Çeşitli Patojenler Üzerinde İn Vitro Antimikrobiyal Etkinliğinin Değerlendirilmesi

Aim: EGCG, the polyphenolic component of Camellia sinesis catechins, and Camellia sinesis extract have broad antimicrobial activity. To investigate the invitro antibacterial and antifungal activity of the Epigallocatechin gallate and Camellia sinesis extract. Method: The present study tested the antibacterial and antifungal activity of Epigallocatechin gallate and Camellia sinesis extract against some gram-negative, gram-positive, and fungal isolates of American Type Culture Collection (ATCC). The minimum inhibitory concentration (MIC) of Epigallocatechin gallate (EGCG) and Camellia sinensis extract were determined for each test microorganism. In general, EGCG and Camellia sinensis extract results were found to be compatible. Epigallocatechin gallate and Camellia sinesis extract exhibited antibacterial and antifungal activity against all test organisms. MIC was determined visually after 16-20 hours of incubation at 37°C according to broth microdilution methods recommended by the Clinical and Laboratory Standards Institute (CLSI). Result: The Camellia sinesis extract and EGCG MIC for gram-positive bacteria was found to be lower than the MIC for gram-negative bacteria and the Camellia sinesis extract MIC for C. albicans was found to be higher than the EGCG MIC. Conclusions: However, EGCG was found to be more effective on C. albicans isolate than Camellia sinensis extract. Camellia sinesis extract Epigallocatechin gallate and Camellia sinesis extract may be a promising sources of antibacterial and antifungal for further studies.

Moraxella oculi sp. nov., isolated from a cow with infectious bovine keratoconjunctivitis.

A novel species of the genus Moraxella was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was Moraxella bovis (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related Moraxella species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name Moraxella oculi sp. nov. for this new species. The type strain is Tifton1T and has been deposited into the American Type Culture Collection (TSD-373T) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942T).

Antioxidant and antimicrobial effect of sodium alginate nanoemulsion coating enriched with oregano essential oil (Origanum vulgare L.) and Trachyspermum ammi oil (Carum cupticum) on food pathogenic bacteria.

Today, microbial contamination in food is one of the major problems of the food industry and public health in general around the world. Foodborne illnesses, such as diarrheal diseases, kill many people around the world each year. The general objective of this study was to evaluate the antioxidant and antibacterial activity of sodium alginate nanoemulsion coating incorporated with oregano essential oil (Origanum vulgare L.) and Trachyspermum ammi oil (Carum cupticum) on Escherichia coli, and Listeria monocytogenes. To achieve this study, fresh chicken meat was used for this experiment. Listeria monocytogenes ATCC 19111 and Escherichia coli ATCC 35218 were obtained from the American Type Culture Collection (Manassas, VA, USA). After the preparation of the essential oil, the chemical composition of this essential oil was determined by using (GC-MS). The physicochemical properties of the nanoemulsion essential oil prepared were characterized and their antimicrobial activity was evaluated. The results showed that the GC-MS analysis of the volatile constituents of the Origanum vulgare essential oil compounds allowed the identification of 19 compounds representing 93.72% of the total oil. The major components detected in Origanum vulgare essential oil were pulegone (49.25%), eucalyptol (18.23%), and menthone (12.37%). About the Carum cupticum essential oil, 21 compounds representing 98.5% of the total oil were identified. The major components detected in Origanum vulgare essential oil were thymol (23.3%), p-cymene (17.5%), and γ-terpinene (16.8%). The best z-average (d.nm) is 483.4 nm (Carum cupticum essential oil + nano) followed by 470.1 nm (nanochitosan). The results of the antimicrobial test showed that the different preparations have a good inhibitory activity for the invitro growth of Escherichia coli and Listeria monocytogenes. According to the MIC and MBC results of this study, the nanoemulsion also presented a good bacteriostatic activity against the two pathogenic bacteria tested in this study.

Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells.

In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. Human larynx epidermoid carcinoma (HEp-2) (ATCC® CCL-23™) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse™ MitoPotential kit manufacturer instructions. MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 µM). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.

Building in vitro tools for livestock genomics: chromosomal variation within the PK15 cell line

BackgroundCultured porcine cell lines are powerful tools for functional genomics and in vitro phenotypic testing of candidate causal variants. However, to be utilised for genomic or variant interrogation assays, the genome sequence and structure of cultured cell lines must be realised. In this work, we called variants and used read coverage in combination with within-sample allele frequency to detect potential aneuploidy in two immortalised porcine kidney epithelial (PK15) cell lines and in a pig embryonic fibroblast line.ResultsWe compared two PK15 cultured cells samples: a new American Type Culture Collection (ATCC) sample and one that has been utilised and passaged within the laboratory for an extended period (> 10 years). Read coverage and within-sample allele frequencies showed that several chromosomes are fully or partially aneuploid in both PK15 lines, including potential trisomy of chromosome 4 and tetrasomy of chromosome 17. The older PK15 line showed evidence of additional structural variation and potentially clonal variation. By comparison, the pig embryonic fibroblast line was free from the gross aneuploidies seen in the PK15s.ConclusionsOur results show that the PK15 cell lines examined have aneuploidies and complex structural variants in their genomes. We propose that screening for aneuploidy should be considered for cell lines, and discuss implications for livestock genomics.

Disinfection Ability of Aqueous Ocimum sanctum (Tulsi) Extract against Candida albicans and Staphylococcus aureus Inoculated on Maxillofacial Silicone - An In Vitro Study

Abstract Background: Patients who use maxillofacial prostheses often encounter difficulties cleaning them thoroughly daily. Traditional cleaning methods may not work and could change the prostheses’ physical properties. Therefore, it is essential to look for alternative disinfection methods. Aim: The aim of this study was to evaluate the efficacy of aqueous extract of Ocimum sanctum (tulsi) against Candida albicans and Staphylococcus aureus inoculated on maxillofacial silicone. Materials and Methods: One hundred and twenty silicone discs, 5 mm diameter, 2 mm thick, were made with maxillofacial Factor II A-2000. Samples were divided into four main groups, each with three subgroups of 10 samples. Each subset was inoculated with C. albicans, S. aureus clinical strains, and the American Type Culture Collection (ATCC) strains. The specimens were incubated at 37°C for 72 h and then soaked in three disinfectant solutions: a neutral soap solution for 30 s, 0.2% chlorhexidine for 10 min, and aqueous tulsi for 10 min. The disinfection ability was determined based on the number of surviving cells by methoxy-nitro-sulfophenyl-tetrazolium carboxanilide assay, and the results were analyzed using a one-way analysis of variance and the post hoc Tukey’s test. Results: There were no significant differences in the ability to disinfect between neutral soap, 0.2% chlorhexidine, and tulsi extract when tested against clinical and ATCC C. albicans and clinical S. aureus. The P &gt; 0.05 indicates that the differences between the disinfection abilities were not statistically significant. However, the 0.2% chlorhexidine disinfectant showed better results when used on Staphylococcus ATCC strains, with a P &lt; 0.001. Conclusion: A 10% aqueous extract of tulsi can be used as a disinfectant for clinical and ATCC strains of C. albicans and the clinical strain of S. aureus.

Anti-tuberculosis activity of morusin: a promising flavonoid from white mulberry.

BACKGROUND: TB has remained a significant public health concern from historical times to the present day. Each year, growing drug resistance problems necessitate the discovery of new drugs and drug precursors for TB treatment. Morusin is an important flavone found in the bark of white mulberry (Morus alba L.) with anti-oxidant, antimicrobial, anti-tumour, anti-inflammatory and antiallergic activity.OBJECTIVE: To determine the anti-TB efficacy of morusin on Mycobacterium tuberculosis strains.DESIGN: Anti-TB efficacy of morusin was tested on H37Ra (American Type Culture Collection [ATCC] 25177), H37Rv (ATCC 27294), ATCC 35822 (isoniazid [INH] resistant), ATCC 35838 (rifampicin [RIF] resistant), and ATCC 35820 (streptomycin [SM] resistant) standard strains and its efficacy was determined using nitrate reductase assay (NRA).RESULTS: The minimum inhibitory concentration (MIC) of morusin was tested in the range of 53.83â-"0.21 μg/ml. The MIC for H37Ra (ATCC 25177), H37Rv (ATCC 27294) and ATCC 35838 (RIF-resistant) strains were found to be 6.72 μg/ml, and this was 13.45 μg/ml for the ATCC 35822 (INHresistant) and ATCC 35820 (SM-resistant) strains.CONCLUSION: To consider morusin as a viable alternative or precursor drug for TB treatment, it is imperative to conduct an exhaustive examination of its mechanism of action and conduct in vitro studies using clinical isolates.

Exosomal miR-4645-5p from hypoxic bone marrow mesenchymal stem cells facilitates diabetic wound healing by restoring keratinocyte autophagy.

Refractory diabetic wounds are a common occurrence in patients with diabetes and epidermis-specific macroautophagy/autophagy impairment has been implicated in their pathogenesis. Therefore, identifying and developing treatment strategies capable of normalizing epidermis-specific macroautophagy/autophagy could facilitate diabetic wound healing. The study aims to investigate the potential of bone marrow mesenchymal stem cell-derived exosomes (BMSC-exos) from hypoxic conditions as a treatment to normalize epidermis-specific autophagy for diabetic wound healing. We compared the effects of bone marrow mesenchymal stem cell (BMSC)-sourced exosomes (BMSC-Exos) from hypoxic conditions to those of BMSC in normoxic conditions (noBMSC-Exos). Our studies involved morphometric assessment of the exosomes, identification of the microRNA (miRNA) responsible for the effects, evaluation of keratinocyte functions and examination of effects of the exosomes on several molecules involved in the autophagy pathway such as microtubule-associated protein 1 light chain 3 beta, beclin 1, sequestosome 1, autophagy-related 5 and autophagy-related 5. The experiments used human BMSCs from the American Type Culture Collection, an in vivo mouse model of diabetes (db/db) to assess wound healing, as well as the human keratinocyte HaCaT cell line. In the methodology, the authors utilized an array of approaches that included electron microscopy, small interfering RNA (siRNA) studies, RNA in situ hybridization, quantitative real-time reverse transcription PCR (qRT-PCR), the isolation, sequencing and differential expression of miRNAs, as well as the use of miR-4645-5p-specific knockdown with an inhibitor. Hypoxia affected the release of exosomes from hypoxic BMSCs (hy-BMSCs) and influenced the size and morphology of the exosomes. Moreover, hyBMSC-Exo treatment markedly improved keratinocyte function, including keratinocyte autophagy, proliferation and migration. miRNA microarray and bioinformatics analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in 'autophagy' and 'process utilizing autophagic mechanism' in the 'biological process' category and miR-4645-5p as a major contributor to the pro-autophagy effect of hyBMSC-Exos. Moreover, mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) was identified as a potential target of exosomal miR-4645-5p; this was confirmed using a dual luciferase assay. Exosomal miR-4645-5p mediates the inactivation of the MAPKAPK2-induced AKT kinase group (comprising AKT1, AKT2, and AKT3), which in turn suppresses AKT-mTORC1 signaling, thereby facilitating miR-4645-5p-mediated autophagy. Overall, the results of this study showed that hyBMSC-Exo-mediated transfer of miR-4645-5p inactivated MAPKAPK2-induced AKT-mTORC1 signaling in keratinocytes, which activated keratinocyte autophagy, proliferation and migration, resulting in diabetic wound healing in mice. Collectively, the findings could aid in the development of a novel therapeutic strategy for diabetic wounds.

Synthesis and Antibacterial Activity of Mono- and Bi-Cationic Pyridinium 1,2,4-Oxadiazoles and Triazoles.

One of the main causes of mortality in humans continues to be infectious diseases. Scientists are searching for new alternatives due to the fast increase in resistance of some harmful bacteria to the frontline antibiotics. To effectively treat pathogenic infections, it is crucial to design antibiotics that can prevent the development of pathogenic resistance. For this purpose, a set of 39 quaternary pyridinium and bis-pyridinium salts with different lengths of side alkyl or fluorinated chains, heterocyclic spacers, and counter ions were tested on diverse reference bacterial ATCC (American Type Culture Collection) strains, such as S. aureus and E. coli. Subsequently, 6 out of the 39 pyridinium salts showing relevant MIC (Minimum Inhibitory Concentration) values were tested on clinically isolated, resistant strains of S. aureus, S. epidermids, S. haemolyticus, K. pneumoniae, A. baumannii, and P. aeruginosa. Additional tests have been performed to assess if the minimum concentration detected through MIC assay may limit the growth of biofilms.

Identification of isothiazolones analogues as potent bactericidal agents against antibiotic resistant CRE and MRSA strains

Carbapenem-resistant Enterobacterales (CRE) has emerged as a worldwide spread nosocomial superbug exhibiting antimicrobial resistance (AMR) to all current antibiotics, leaving limited options for treating its infection. To discovery novel antibiotics against CRE, we designed and synthesized a series of 14 isothiazol-3(2H)-one analogues subjected to antibacterial activity evaluation against Escherichia coli (E. coli) BL21 (NDM-1) and clinical strain E. coli HN88 for investigating their structure–activity relationships (SAR). The results suggested that 5-chloroisothiazolone core with an N-(4-chlorophenyl) substitution 5a was the most potent antibacterial activity against the E. coli BL21 (NDM-1) with MIC value of less than 0.032 μg/mL, which was at least 8000-fold higher than the positive control Meropenem (MRM). It also displayed 2048-fold potent than the positive control MRM against E. coli HN88. Additionally, SAR analysis supported the conclusion that compounds with a chloro-group substituted on the 5-position of the heterocyclic ring was much more potent than other positions. The board spectrum analysis suggested that compound 5a showed a promising antimicrobial activity on MRSA and CRE pathogens. Meanwhile, cytotoxicity study of compound 5a suggested that it had a therapeutic index value of 875, suggesting future therapeutic potential. In vivo efficacy study declared that compound 5a could also protect the BALB/c mice against American type culture collection (ATCC) 43,300. Further screening of our compounds against a collection of CRE strains isolated from patients indicated that compound 5 g displayed much stronger antibacterial activity compared with MRM. In conclusion, our studies indicated that isothiazolones analogues could be potent bactericidal agents against CRE and MRSA pathogens.