Isolation, Plant Growth-Promoting Properties, and Whole-Genome Sequence of a Novel Paenibacillus Species.

This work aimed to isolate and characterize a novel chitin-degrading bacterium from Yok Don National Park, Vietnam, for crop production studies. Among the chitinolytic isolates, strain YSY-4.3 was selected, which grew rapidly and produced a large halo around the colony. 16S rDNA analysis indicated that the strain is a novel species in the genus paenibacillus, and an in vitro evaluation showed that the strain produced phytohormones (IAA, GA3, and zeatin), biofilms, and siderophores; possessed cellulase; and exerted antifungal activity. The whole genome of the strain was 5,628,400 bp with 49.3% GC content, 5056 coding sequences, 48 tRNA, and 1 rRNA. It shared the highest values of digital DNA-DNA hybridization (67.4%) and average nucleotide identity (89.54%) with those of Paenibacillus woosongensis B2_4 (CP126084.1), suggesting a novel species. Of the coding sequences, 4287 proteins were identified by COG, and 2561 were assigned by KEGG. The genome contained at least 51 genes involved in plant growth and resistance to heavy-metal toxicity and 359 carbohydrate-active enzymes. The chitinolytic system of the strain was composed of 15 enzymes, among them, PsChiC, which contained a GH18 catalytic domain and a GH5 catalytic domain, had not been previously reported. In addition, the genome possessed 15 gene clusters encoding antimicrobial metabolites, 10 of which are possible novel clusters. This study expands knowledge regarding novel chitinolytic bacteria from Yok Don National Park and provides a valuable gene resource for future studies.

Novel resources of chitinolytic bacteria isolated from Yok Don National Park, Vietnam.

AIMS: This study focused on the isolation and characterization of chitinolytic bacteria from Yok Don National Park, Vietnam for future studies regarding biofertilizers and biocontrol agents. METHODS AND RESULTS: Chitinolytic bacteria were isolated from soils and chitin flakes soaked in river water at the National Park. On the basis of the halo zones caused by colloidal chitin degradation and colony morphologies, 12 chitinolytic strains were chosen from 15 700 isolates for various examinations. Findings from 16S rDNA analysis indicated that among these strains, 10 could be identified as different species, and the remaining 2 showed less identity to known species and genera. The 12 bacteria possess numerous properties concerning plant growth promotion and/or phytopathogenic biocontrol. Paenibacillus chitinolyticus YSY-3.1, which exhibited the highest chitinase activity and remarkable properties for plant growth, was chosen for sequencing and draft genome analysis. The results showed that the genome is 6571 781 bp in length with 6194 coding sequences, 52.2% G + C, and 96.53% ANI value. It harbors the chitinolytic system comprising 22 enzymes. Among these enzymes, PcChiQ has a loop structure different from that of known family 19 chitinases, PcChiA contains two GH18 catalytic domains rarely found in microorganisms, and PcChiF contains three GH18 catalytic domains that have never been reported. CONCLUSIONS: The 12 identified chitinolytic bacteria exhibit great potential for further studies on plant growth-promoting and/or biocontrol properties. Among these bacteria, two strains might be good candidates for next examinations concerning novel species and/or genera, and strain YSY-3.1 could possess a novel chitinolytic system.

Changes in Ascitic Fluid Polymorphonuclear Cell Count After Antibiotics Are Associated With Mortality in Spontaneous Bacterial Peritonitis.

Spontaneous bacterial peritonitis (SBP) is a feared complication of ascites that affects 10%-30% of hospitalized patients with cirrhosis with an associated mortality rate of approximately 20%.1-3 Although efforts have been undertaken to encourage prompt evaluation and treatment of SBP, outcomes have generally remained dismal.3 There is significant interest in identifying factors that can reliably predict mortality among individuals with SBP.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

Expression, purification, and basic properties of a novel domain structure possessing chitinase from Escherichia coli carrying the family 18 chitinase gene of Bacillus velezensis strain RB.IBE29.

BACKGROUND: Bacillus velezensis possesses numerous chitinolytic enzymes; however, not much is known about the role of its chitinase molecules. METHODS AND RESULTS: In this study, the chiA gene, which encodes a novel domain structure possessing family 18 chitinase from B. velezensis RB.IBE29, was expressed successfully in Escherichia coli BL21-CodonPlus (DE3)-RIPL using the pColdII expression vector. The recombinant protein, rBvChiA, was purified using the HisTrap FF column. Purified rBvChiA showed hydrolytic activity against insoluble chitin and bound to chitinous substrates. In addition, the purified recombinant enzyme displayed remarkable inhibition effects on the spore germination of Fusarium falciforme and the egg hatch of root-knot nematodes (Meloidogyne spp.), which are the main causes of black pepper diseases in the Central Highlands region, Vietnam. CONCLUSION: The current work results might enable further studies to develop novel chitinase A and strain RB.IBE29 as a natural fungicide and nematicide for sustainable black pepper production and other crops in the Central Highlands, Vietnam. This is the second report describing chitinase from B. velezensis based on the experimental data.

Efficacy of the incorporation between self-encapsulation and cryoprotectants on improving the freeze-dried survival of probiotic bacteria.

AIMS: This study aimed to improve the viability of probiotic bacteria during freeze-drying by the combination of self-encapsulation and cryoprotectants. METHODS AND RESULTS: Lactiplantibacillus plantarum VAL6 and Lactobacillus acidophilus VAR1 were exposed to environmental stresses including temperature, pH and increased CO2 concentration before performing freeze-drying with the addition of cryoprotectants. The results proved that tested stresses can stimulate the bacteria to synthesize more extracellular polymeric substances to form self-encapsulation that increases their freeze-dried viability. In combination with cryoprotectants to form double-layered microencapsulation, L. plantarum VAL6 stressed at pH 3.5 in combination with whey protein isolate could achieve the highest Improving Cell Viability of 4361-fold, while L. acidophilus VAR1 stressed at 25o C in combination with alginate gave a maximum Improving Cell Viability of 73.33-fold. CONCLUSIONS: The combination of self-encapsulation and cryoprotectants significantly improves the freeze-dried viability of probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that uses environmental stress to stimulate extracellular polymeric substance synthesis for self-encapsulation formation combined with the addition of cryoprotectants to enhance the freeze-dried survival of probiotics. This could be a novel approach in improving the viability of probiotic strains for various applications.

Molecular analysis of genes involved in chitin degradation from the chitinolytic bacterium Bacillus velezensis.

Bacillus velezensis RB.IBE29 is a potent biocontrol agent with high chitinase activity isolated from the rhizosphere of black pepper cultivated in the Central Highlands, Vietnam. Genome sequences revealed that this species possesses some GH18 chitinases and AA10 protein(s); however, these enzymes have not been experimentally characterized. In this work, three genes were identified from the genomic DNA of this bacterium and cloned in Escherichia coli. Sequence analysis exhibited that the ORF of chiA consists of 1,203 bp and encodes deduced 45.46 kDa-chitinase A of 400 aa. The domain structure of chitinase A is composed of a CBM 50 domain at the N-terminus and a catalytic domain at the C-terminus. The ORF of chiB includes 1,263 bp and encodes deduced 47.59 kDa-chitinase B of 420 aa. Chitinase B consists of two CBM50 domains at the N-terminus and a catalytic domain at the C-terminus. The ORF of lpmo10 is 621 bp and encodes a deduced 22.44 kDa-AA10 protein, BvLPMO10 of 206 aa. BvLPMO10 contains a signal peptide and an AA10 catalytic domain. Chitinases A and B were grouped into subfamily A of family 18 chitinases. Amino acid sequences in their catalytic domains lack aromatic residues (Trp, Phe, Tyr) probably involved in processivity and substrate binding compared with well-known bacterial GH18 chitinases. chiB was successfully expressed in E. coli. Purified rBvChiB degraded insoluble chitin and was responsible for inhibition of fungal spore-germination and egg hatching of plant-parasitic nematode. This is the first report describing the analysis of the chitinase system from B. velezensis.

Correlation Between the Amount of Extracellular Polymeric Substances and the Survival Rate to Freeze-Drying of Probiotics.

To demonstrate that the amount of extracellular polymeric substances (EPS) and the freeze-dried viability of probiotics are correlated. Three strains of probiotics including Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Bifidobacterium bifidum were subjected to environmental challenges, such as temperature, pH, and carbon dioxide. The results indicated that the challenges could stimulate the EPS synthesis of the probiotics. The experimental correlation between the amount of synthesized EPS and the freeze-dried survival rate was also analyzed, and the viability of each of the three strains was represented by the following functions in which the equation of L. plantarum is y = - 0.0336x2 + 2.7059x - 14.849 with R2 = 0.9699, the B. bifidum's equation is y = - 0.0554x2 + 2.6243x - 13.654 with R2 = 0.9554, and the L. acidophilus's one was y = 0.0346x2 + 0.5862x - 9.1339 with R2 = 0.9733. This could be a new approach to determining the freeze-dried viability of probiotic strains based on the measured EPS content.

Expression of genes involved in exopolysaccharide synthesis in Lactiplantibacillus plantarum VAL6 under environmental stresses.

Environmental factors can alter exopolysaccharide biosynthesis in lactic acid bacteria (LAB). To further clarify this potential relationship, the mRNA expression of genes involved in exopolysaccharide synthesis such as glmU, pgmB1, cps4E, cps4F, cps4J, and cps4H in Lactiplantibacillus plantarum VAL6 under different conditions including temperature, pH, sodium chloride (NaCl), and carbon dioxide (CO2) intensification culture was studied. The transcriptomic data revealed that the exposure of L. plantarum VAL6 at pH 3 increased the expression level of cps4H but decreased the expression levels of pgmB1 and cps4E. Under pH 8, cps4F and cps4E were significantly upregulated, whereas pgmB1 was downregulated. Similarly, the expression levels of cps4F, cps4E, and cps4J increased sharply under stresses at 42 or 47 °C. In the case of NaCl stress, glmU, pgmB1, cps4J, and cps4H were downregulated in exposure to NaCl at 7 and 10% concentrations while cps4E and cps4F were upregulated at 1 h of 10%-NaCl treatment and at 5 h of 4%-NaCl treatment. Remarkably, CO2 intensification culture stimulated the expression of all tested genes. In addition, simultaneous changes in expression of cps4E and cps4F under environmental challenges may elicit the possibility of an association between the two genes. These findings indicated that the expression level of eps genes is responsible for changes in the yield and monosaccharide composition of exopolysaccharides under environmental stresses.

Urinary 1-hydroxypyrene levels among office workers living in industrial areas.

We examined exposure to polycyclic aromatic hydrocarbons (PAHs) among non-smoking office workers in 2 countries living in the vicinity of a coke-oven factory by measuring their levels of urinary 1-OHP, a known metabolite of PAHs. Subjects included 10 non-smoking office workers in Kitakyushu city (Japan) and 20 workers in Thai Nguyen city (Vietnam). Measurement was optimized by using the high-performance liquid chromatography (HPLC) method developed by Jongeneelen et al. This method required only a small amount of urine and had a short incubation time, and its detection limit was very low (0.00448 ng/ml), which was practical and highly sensitive.The median urinary 1-OHP concentration in the Vietnamese subjects (0.417 ng/mg creatinine) was six times as high as that in the Japanese subjects (0.069 ng/mg creatinine) (P < 0.001). However, both concentrations were significantly below the guideline level, below which there is no genotoxic effect, implying a low probability of any adverse health effects.Our measurements from both countries showed higher urinary 1-OHP concentrations than in previous studies from locations without factories, indicating that ambient air pollution from industrial emissions is an important source of PAH exposure. Finally, the urinary 1-OHP concentrations did not correlate with gender or lifestyle factors.

Wilson Disease and the COVID-19 pandemic: exploring patients' mental health and vaccination attitudes in a longitudinal study.

Introduction: The COVID-19 pandemic significantly impacted the mental health of individuals with chronic conditions such as Wilson's Disease (WD). This study investigates stress, anxiety, depression, quality of life, cognitive function, vaccination rates, infection rates, and perceptions related to the pandemic and vaccines among WD patients. Methods: The study analyzed COVID-19 perceptions and vaccine attitudes of 62 adult WD patients enrolled in the international multisite WD Registry. A subgroup of 33 participants completed a series of mental health scales. The effect of working essentially, income loss, wellness activity initiation, and infection of COVID-19 during the pandemic was observed. Results: Results indicate that, overall, the pandemic did not exacerbate anxiety or cognitive function in WD patients but did lead to increased depression among essential workers. Patients experiencing income loss exhibited higher levels of stress and anxiety. Despite these challenges, WD patients showed high vaccination rates and positive attitudes towards vaccines. Discussion: The findings underscore the significant impact of the pandemic on the mental health of WD patients.

Halophilic lactic acid bacteria - Play a vital role in the fermented food industry.

Halophilic lactic acid bacteria have been widely found in various high-salt fermented foods. The distribution of these species in salt-fermented foods contributes significantly to the development of the product's flavor. Besides, these bacteria also have the ability to biosynthesize bioactive components which potentially apply to different areas. In this review, insights into the metabolic properties, salt stress responses, and potential applications of these bacteria have been have been elucidated. The purpose of this review highlights the important role of halophilic lactic acid bacteria in improving the quality and safety of salt-fermented products and explores the potential application of these bacteria.

Data on gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from Bacillus velezensis.

Bacillus velezensis RB.IBE29 is a chitinolytic bacterium originally isolated from the rhizospheric soil of black pepper grown in Vietnam. This bacterium is a strong biocontrol agent against plant pathogens and possesses a novel chitinase system. Genome sequences available in CAZy database revealed B. velezensis possesses one gene encoding xylanase belonging to glycoside hydrolase family 11; however, this enzyme has yet to be un-experimentally characterized. In this work, xyA gene was isolated from the genomic DNA of strain RB.IBE29 and cloned in Escherichia coli DH5α cells using the pUC19 vector. Sequencing analysis showed that the ORF of xyA contains 642 bp and encodes the deduced xylanase with 213 aa and 23.27 kDa. The domain structure of the enzyme has a signal peptide and a family 11 catalytic domain. xyA (without peptide sequence) was successfully expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells using the pColdII vector and purified using the HisTrap FF column. Purified recombinant xylanase degraded xylan substrates, had the highest hydrolytic activity at 55°C in 20 mM sodium phosphate buffer (pH 6.0), and MgCl2, CoCl2, and MnCl2 enhanced the enzymatic activity. Nucleotide sequence of xyA was submitted to the DDBJ/GenBank/EMBL under accession number LC779040. This is the first data on the gene cloning, expression, purification, and characterization of the glycoside hydrolase family 11 from B. velezensis.

Supplementary Respiratory Therapy Improves Pulmonary Function in Pediatric Patients with Cerebral Palsy: A Systematic Review and Meta-Analysis.

BACKGROUND: Despite medical advances, individuals with cerebral palsy (CP) face significant respiratory challenges, leading to heightened hospitalization rates and early mortality among this population. We hypothesize that integrating supplementary respiratory therapy into standard rehabilitation will result in significant improvements in pulmonary function, enhanced respiratory muscle strength, and an overall increase in the quality of life among pediatric patients with CP. METHODS: A systematic search of literature across five databases was conducted, and random-effects meta-analyses were performed to assess the impact of supplementary respiratory therapy on (a) pulmonary function: forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), FVC/FEV1 ratio, peak expiratory flow (PEF), and (b) respiratory muscle strength: maximal inspiratory and expiratory pressure (MIP, MEP), and (c) quality of life. Certainty of evidence was determined by the GRADE assessment. RESULTS: Analysis of data from 11 eligible randomized controlled trials revealed clinically meaningful changes in pulmonary function. We found a relevant mean difference (MD) in absolute PEF of 0.50 L/s (95% confidence interval (CI): 0.19; 0.82 p = 0.0107). The certainty of the evidence ranged from moderate to high. CONCLUSIONS: This study presents current evidence on the impact of various supplementary respiratory therapies for CP patients classified under gross motor function classification level I-IV, demonstrating clinically meaningful improvements in pulmonary function and respiratory muscle strength. These improvements suggest the potential for an enhanced quality of life. Our findings hold the promise of serving as a foundational reference for potential revisions to conventional rehabilitation care, incorporating supplementary respiratory therapy.

"Because it is a rare disease it needs to be brought to attention that there are things out of the norm": a qualitative study of patient and physician experiences of Wilson disease diagnosis and management in the US.

BACKGROUND: Wilson disease (WD) is a genetic disorder of copper metabolism that leads to copper accumulation in various organs, primarily the liver and brain, resulting in heterogenous hepatic, neurologic, and psychiatric symptoms. Diagnosis can occur at any age, requiring lifelong treatment, which can involve liver transplantation. This qualitative study aims to understand the wider patient and physician experience of the diagnosis and management of WD in the US. METHODS: Primary data were collected from 1:1 semi structured interviews with US-based patients and physicians and thematically analyzed with NVivo. RESULTS: Twelve WD patients and 7 specialist WD physicians (hepatologists and neurologists) were interviewed. Analysis of the interviews revealed 18 themes, which were organized into 5 overarching categories: (1) Diagnosis journey, (2) Multidisciplinary approach, (3) Medication, (4) The role of insurance, and (5) Education, awareness, and support. Patients who presented with psychiatric or neurological symptoms reported longer diagnostic journeys (range 1 to 16 years) than those presenting with hepatic symptoms or through genetic screening (range 2 weeks to 3 years). All were also affected by geographical proximity to WD specialists and access to comprehensive insurance. Exploratory testing was often burdensome for patients, but receipt of a definitive diagnosis led to relief for some. Physicians emphasized the importance of multidisciplinary teams beyond hepatology, neurology, and psychiatry and recommended a combination of chelation, zinc, and a low-copper diet; however, only half the patients in this sample were on a chelator, and some struggled to access prescription zinc due to insurance issues. Caregivers often advocated for and supported adolescents with their medication and dietary regimen. Patients and physicians recommended more education and awareness for the healthcare community. CONCLUSIONS: WD requires the coordination of care and medication among several specialists due to its complex nature, but many patients do not have access to multiple specialties due to geographical or insurance barriers. Because some patients cannot be treated in Centers of Excellence, easy access to reliable and up-to-date information is important to empower physicians, patients, and their caregivers in managing the condition, along with general community outreach programs.

Major Depressive Disorder in an International Multisite Wilson Disease Registry.

BACKGROUND AND AIMS: Psychiatric symptoms are frequently reported in Wilson disease (WD); however, systematic assessments with validated measures are lacking. OBJECTIVE: We aim to report the prevalence and clinical correlates for major depressive disorder (MDD) as resulting from a multisite international WD registry. METHODS: All patients enrolled in the WD registry received structured psychiatric evaluations (Mini International Neuropsychiatric Interview, Patient Health Questionnaire-9, Generalized Anxiety Disorder-7 scale, Perceived Stress Scale), laboratory tests, hepatology, and neurological assessments. We present the analysis of the data collected at enrollment for the first 3 years (N = 62). RESULTS: Thirty-seven percent (23) had a lifetime history (MDD), and 6% (4) met the criteria for an active major depressive episode. Depression was self-reported in 30.51% (19) at WD diagnosis. Patients with MDD had worse mental health quality-of-life (QOL) scores (median 43 vs 53.6, P = 0.006), higher severe anxiety (13.04% vs 0), higher perceived stress (median 18 vs 9, P < 0.003), and higher levels of neuroticism (median 8 vs 5.0, P = 0.002). We found no significant difference in physical health QOL and severity of neurological or liver disease. There was no significant difference in copper parameters or liver tests in those with MDD and without. The limitations of our study consist of the small sample size, the cross-sectional report, and the lack of brain copper measurements. CONCLUSIONS: Lifetime MDD is highly prevalent in WD and associated with worse mental health QOL. We did not find a significant association among liver disease, neurological disease laboratory tests, and MDD. Screening for depression should be considered in patients with WD.

Chemical composition, anti-inflammatory and cytotoxic activity of essential oils from two Luvunga species (L. scandens and L. hongiaoensis) from Vietnam.

New essential oils (EOs) extracted from different parts of two Luvunga species (L. scandens and L. hongiaoensis) from Vietnam were investigated for their chemical composition, anti-inflammatory and cytotoxic activity. Sixty-nine total compounds were identified in the EOs by GC/MS. The major constituent of the leaf, fruit, and root EOs from L. scandens was ß-caryophyllene (71.5%, 63.0%, and 31.5% respectively). The main compounds in L. hongiaoensis EOs were ß-elemene (34.3% in leaf oil) and caryophyllene oxide (21.2% in root oil, 19.4% in stem oil). The EO from L. scandens fruits significantly inhibited nitric oxide production on LPS-induced RAW264.7 cells (IC50 = 37.95 ± 2.76 µg/mL). The EOs from L. hongiaoensis roots and L. scandens leaves and fruits exhibited cytotoxic activity against MCF-7, SK-LU-1, and HepG2 (IC50 from 49.74 ± 3.36 to 97.82 ± 8.61 µg/mL). This is the first report on L. hongiaoensis EOs and significantly complements the composition and bioactivity of L. scandens EOs.

Optimizing Ultrasonic-Assisted and Microwave-Assisted Extraction Processes to Recover Phenolics and Flavonoids from Passion Fruit Peels.

This study optimized the ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE) processes to acquire phenolics and flavonoids from passion fruit peels using a mixture of ethanol, acetone, and water. An augmented simplex-centroid design was employed to find the suitable volume ratio among solvent ingredients to attain the highest extraction yield of phenolics and flavonoids. One-factor experiments were conducted to investigate the influence of UAE and MAE parameters on the recovery yield of phenolics and flavonoids before the two processes were optimized using Box-Behnken Design (BBD) models. The optimal UAE conditions for recovering phenolics and flavonoids from passion fruit peel powder (PFP) were 28 mL/g of liquid-to-solid ratio (LSR), 608 W of ultrasonic power, and 63 °C for 20 min to acquire total phenolic content (TPC) and total flavonoid content (TFC) at 39.38 mg of gallic acid equivalents per gram of dried basis (mg GAE/g db) and 25.79 mg of rutin equivalents per gram of dried basis (mg RE/g db), respectively. MAE conditions for attaining phenolics and flavonoids from PFP were 26 mL/g of LSR and 606 W of microwave power for 2 min to recover TPC and TFC at 17.74 mg GAE/g db and 8.11 mg RE/g db, respectively. The second-order kinetic model was employed to determine the UAE and MAE mechanism of TPC and TFC and the thermodynamic parameters of the extraction processes. The antioxidant activities of passion fruit peel extracts at optimal conditions were examined to compare the efficiency of UAE and MAE. This study establishes an effective approach for obtaining phenolics and flavonoids from passion fruit peels.

Two new halogenated sesquiterpene lactones from Palisada intermedia.

Two new sesquiterpene lactones, laurenolide A (1) and laurenolide B (2), along with six known compounds, palmitic acid (3), (R,R)-hexahydrofarnesyl acetone (4), trans-phytol acetate (5), cholesterol (6), cholesteryl acetate (7), cholesteryl heptadecanoate (8) were isolated from Palisada intermedia. The chemical structures of all compounds were elucidated by 1D and 2D-NMR spectroscopy and HR-ESI-MS analysis as well as compared with data in the literature. The petroleum ether, chloroform, ethyl acetate, methanol extracts and compounds 1, 2 were tested for the inhibition of two cancer cell lines MCF-7, NCI-H460 and they showed weak or none activities.

Zinc Finger Nuclease induced DNA double stranded breaks and rearrangements in MLL.

Radiation treatment or chemotherapy has been linked with a higher risk of secondary cancers such as therapy related Acute Myeloid Leukemia (tAML). Several of these cancers have been shown to be correlated to the introduction of double stranded breaks (DSB) and rearrangements within the Mixed Lineage Leukemia (MLL) gene. We used Zinc Finger Nucleases (ZFNs) to introduce precise cuts within MLL to examine how a single DNA DSB might lead to chromosomal rearrangements. A ZFN targeting exon 13 within the Breakpoint Cluster Region of MLL was transiently expressed in a human lymphoblast cell line originating from a CML patient. Although FISH analysis showed ZFN DSB at this region increased the rate of MLL fragmentation, we were unable to detect leukemogenic rearrangements or translocations via inverse PCR. Interestingly, gene fragmentation as well as small interstitial deletions, insertions and base substitutions increased with the inhibition of DNA-PK, suggesting repair of this particular DSB is linked to non-homologous end joining (NHEJ). Although mis-repair of DSBs may be necessary for the initiation of leukemogenic translocations, a MLL targeted DNA break alone is insufficient.