New neuroprotective derivatives of cinnamic acid by biotransformation.

Microbial transformation is extensively utilized to generate new metabolites in bulk amounts with more specificity and improved activity. As cinnamic acid was reported to exhibit several important pharmacological properties, microbial transformation was used to obtain its new derivatives with enhanced biological activities. By manipulating the 2-stage fermentation protocol of biotransformation, five metabolites were produced from cinnamic acid. Two of them were new derivatives; N-propyl cinnamamide 2̲ and 2-methyl heptyl benzoate 3̲ produced by Alternaria alternata. The other 3 metabolites, p-hydroxy benzoic acid 4̲, cinnamyl alcohol 5̲ and methyl cinnamate 6̲, were produced by Rhodotorula rubra, Rhizopus species and Penicillium chrysogeneum, respectively. Cinnamic acid and its metabolites were evaluated for their cyclooxygenase (COX) and acetylcholinesterase (AChE) inhibitory activities. Protection against H2O2 and Aß1-42 induced-neurotoxicity in human neuroblastoma (SH-SY5Y) cells was also monitored. Metabolite 4̲ was more potent as a COX-2 inhibitor than the parent compound with an IC50 value of 1.85 ± 0.07 µM. Out of the tested compounds, only metabolite 2̲ showed AChE inhibitory activity with an IC50 value of 8.27 µM. These results were further correlated with an in silico study of the binding interactions of the active metabolites with the active sites of the studied enzymes. Metabolite 3̲ was more potent as a neuroprotective agent against H2O2 and Aß1-42 induced-neurotoxicity than catechin and epigallocatechin-3-gallate as positive controls. This study suggested the two new metabolites 2̲ and 3̲ along with metabolite 4̲ as potential leads for neurodegenerative diseases associated with cholinergic deficiency, neurotoxicity or neuroinflammation.

Tissue Inhibitor Of Metalloproteinase 3 As A Potential Staging Biomarker In Hepatocellular Carcinoma.

Objectives: To assessthe potential role of tissue inhibitor of metalloproteinase 3 as a staging marker of hepatocellular carcinoma. Method: The experimental study was conducted at Faculty of Pharmacy, Kafrelsheikh University, Egypt, from December 2020 to March 2022 after approval from the Faculty of Pharmacy, Kafrelsheikh University, Egypt, and comprised male albino rats. The subjects were divided into 4 groups. The control group was administrated a single intraperitoneal injection of normal saline, while the other groups were generated post-induction of hepatocellular carcinoma. The induction was done by injecting rats intraperitoneally with a single dose of 200mg/kg diethyl nitrosamine, followed by the administration of 0.05% phenobarbital sodium in drinking water daily. Rats were euthanised at 8, 16 and 24 weeks after the injection to obtain three groups related to the 3 stages of hepatocellular carcinoma. Serum was used for measuring the alpha protein level. Liver homogenates were used for the assessment of the hepatic tissue inhibitor of metalloproteinase 3 expression, B-cell lymphoma 2-associated X protein expression, and the hepatic content of matrix metalloproteinase -9 and cyclin D1. Data was analysed using Graph Prism 8. RESULTS: Of the 24 rats with weight range 120-130g, 6(25%) were in each of the 4 groups. The relative protein and messenger ribonucleic acid tissue inhibitor of metalloproteinase 3 expressions were significantly decreased in the intervention groups compared to the control group, with more decline as the hepatocellular carcinoma stage increased. The matrix metalloproteinase -9 and cyclin D1 concentrations and the relative hepatic protein Ki67 expression were significantly raised in the intervention groups compared to the control group (p<0.05). The relative expression of hepatic B-cell lymphoma 2-associated X protein was markedly decreased in the intervention groups compared to the control group (p<0.05). CONCLUSIONS: Tissue inhibitor of metalloproteinase 3 might be a promising diagnostic and staging biomarker in hepatocellular carcinoma.

Coexistence Of Blaoxa-48, Blavim, And Blashv Genes In Klebsiella Pneumoniae And Escherichia Coli Isolated From Urinary Tract Infections: Microbiological And Epidemiological Analysis.

Objectives: To investigate antimicrobial resistance mechanisms of isolated bacterialstrains, and their correlation with virulence profile. Method: The cross-sectional study was conducted in January 2020 at outpatient health centres in Kafrelsheikh Governorate of Egypt, and comprised urine samples from patients regardless of age and gender. Midstream samples were collected into sterile swaps which were kept in ice-cooled boxes until transported to the laboratory within 5h. Antimicrobial resistance profile of the isolated Enterobacteriaceae was done using Kirby-Bauer disk diffusion method and was confirmed withVitek compact 2. The phenotypic of carbapenemases and extended-spectrum beta lactamase was determined, and polymerase chain reaction was used, as appropriate. Data was analysed using SPSS 20. RESULTS: Of the 199 patients, 101(50.7%) were females and 98(49.3%) were males. The majority 73(36.6%) were aged 30-50 years. Urinary tract infection was found in 68(34.2%) patients. In 28(41.2%) of these patients, there were 32 isolates of Enterobacterales; 21(65.62%) Klebsiella pneumoniae, 7(21.87%) Escherichia coli and 4(12.5%) Enterobacter cloacae. Of the 28(41.2%) patients, 24(85.7%) were infected with a single strain; 17(70.8%) Klebsiella pneumoniae, 4(16.7%) Escherichia coli and 3(12.5%) Enterobacter cloacae. In 3(10.7%) cases, there was co-infection with Escherichia coli and Klebsiella pneumoniae, and 1(3.6%)sample had mixed infection with Klebsiella pneumoniae and Enterobacter cloacae. The other 40(58.8%) patients had other causative agents. Housewives, agricultural workers and those aged >50 years had a higher risk of urinary tract infections(p<0.05) Among Klebsiella pneumonia isolates, 6(28.5%) possessed carbapenemase-related genes and 4(19.1%) extended-spectrum beta lactamase-related genes. The carbapenemase related genes were bla-Verona integron-encoded metallo beta lactamase 6(100%) bla-New Delhi metallo beta lactamase-1 4(66.6%) and bla-oxacillinase-48 2(33.3%). The 4(19.1%) cases of extended-spectrum beta lactamase related genes had bla-temoneira gene 3(75%) and bla-sulfhydryl variable gene 4(100%). In Escherichia coli isolates, bla-oxacillinase-48 and bla-Cefotaximase genes were observed in 2(28.5%) cases.Virulence genes uridine diphosphate glucose 4-epimerase, fimbrial adhesion and mannose-resistance adhesin of Klebsiella spp genes in Klebsiella pneumoniae isolates were positive in in 16(76.2%), 14(66.7%) and 10(47.6%) cases, respectively. All 21(100%) isolates of Klebsiella pneumoniae were negative for mucoviscosity-associated gene A. CONCLUSIONS: There was evidence of the coexistence of bla- oxacillinase-48, bla-Verona integron-encoded metallo beta lactamase and bla-sulfhydryl variable genes in Klebsiella pneumoniae and Escherichia coli isolates from mixed urinary tract infection samples.

Daidzein and Chicory Extract Arrest the Cell Cycle via Inhibition of Cyclin D/CDK4 and Cyclin A/CDK2 Gene Expression in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers, associated with a high rate of mortality. A disturbance between cell proliferation and cell death is one of the cancer hallmarks including HCC. Cell proliferation is mainly controlled by the cell cycle. The arrest of the cell cycle is one of the important targets of anticancer agents. OBJECTIVES: The present study tries to clarify the exact role of some natural products such as daidzein (DAZ) and alcoholic chicory leaf extract (CE), as possible regulators of cell cycle and apoptosis. METHODS: HCC in rats was induced using diethylnitrosamine (DENA). Ninety rats were allocated and divided equally into nine groups, treated with CE, DAZ, a combination of both, and sorafenib with non-treated control groups. RESULTS: Treatment with CE, DAZ, and their combination significantly downregulated hepatic tissue expression of cyclin D1/CDK4 axis as well as cyclin A/CDK2 axis. The suggested therapeutic protocol inhibited the proliferation and dampened Bcl-2 expression. Furthermore, the efficiency of combining CE and DAZ demonstrated a potency comparable to sorafenib in terms of cyclin D/CDK4 axis expression, as well as; this combination protocol was more potent in revealing a potentiated inhibitory effect on cyclin A and Ki-67 expression. CONCLUSION: Treatment with DAZ or CE alone, or in combination, could possess an inhibitory effect on hepatocarcinogenesis via cell cycle arrest, inhibition of proliferation through suppression of Ki-67 expression, and apoptosis induction, mediated by downregulation of Bcl-2.

Dual regulating of mitochondrial fusion and Timp-3 by leflunomide and diallyl disulfide combination suppresses diethylnitrosamine-induced hepatocellular tumorigenesis in rats.

AIMS: Hepatocellular carcinoma (HCC) is considered one of the main causes of cancer-related death globally. Combination therapy targeting different pathways can improve the efficacy of HCC management. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, and a tissue inhibitor of matrix metalloproteinase 3 (Timp-3) were found to be downregulated in various cancers, including HCC. Our study aimed to evaluate the possible antineoplastic effect of a novel combination in the treatment of HCC through targeting mitochondrial fusion and metastatic proteins. MAIN METHODS: HCC induction was performed using a single intraperitoneal dose of diethylnitrosamine (200 mg/kg), followed by adding phenobarbital sodium (0.05%) to the drinking water for successive 18 weeks. Then, leflunomide (LF, 10 mg/kg) was administered orally for 28 days. Diallyl disulfide (DADS, 50 mg/kg) was also given orally for 28 days, either alone or in combination with LF. KEY FINDINGS: Treatment with LF or DADS could alleviate the HCC- induced histological and biochemical variations, including liver enzyme activities (ALT, AST), alpha-fetoprotein, Bax, cyclin D1, Ki67, malondialdehyde, and reduced glutathione. They could shift the mitochondrial dynamics toward mitochondrial fusion through upregulating the expression of Mfn2 and also exhibited antimetastatic activity through upregulating the expression of Timp-3 and decreasing hepatic MMP9 content. SIGNIFICANCE: the treatment with a combination of LF and DADS displayed a more potent effect than the treatment with each drug alone. Our results suggest that the combined use of LF and a naturally occurring DADS can be used as a promising novel combination in managing HCC.

Targeting the HIF-1α/Cav-1 Pathway with a Chicory Extract/Daidzein Combination Plays a Potential Role in Retarding Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most rapidly growing solid cancers, that is characterized by hypoxia. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates tumor proliferation and metastasis. It induces caveolin-1 (Cav-1) expression, a glycoprotein found on the membrane surface, then Cav-1 triggers angiogenesis and metastasis in HCC. OBJECTIVE: We hypothesize that targeting HIF-1α and consequently, Cav-1 using the antioxidant natural compound such as chicoric acid and a Cav-1 inhibitor daidzein (DAZ) could be a useful approach in the management of HCC. This study was conducted to investigate the possible therapeutic efficacy of standardized chicory leaf extract (SCLE) and DAZ via modulation of HIF-1α and Cav-1 in HCC rats. METHODS: Diethyl nitrosamine (DENA) was used for HCC induction. After the induction period, four groups (10 rats for each) were treated with SCLE, DAZ, a combination of both, as well as sorafenib, all compared to the non-treated control. We assessed hepatic HIF-1α protein expression, Cav-1 gene expression, serum level of AFP, hepatic tissue content of VEGF, MMP-9, oxidative stress markers MDA and SOD. RESULTS: DAZ, SCLE, and their combination, significantly down-regulated the expression of HIF-1α, Cav-1, and consequently dampened MMP-9, VEGF, hepatic content. It has been observed that the combination treatment showed a synergistic effect compared to either treatment alone. Importantly, the combination treatment exhibited a significantly more potent effect than sorafenib. CONCLUSION: This study showed the potential role of the HIF-1α/Cav-1 pathway in HCC progression, moreover, SCLE and DAZ showed a potent efficacy in retarding HCC via modulation of this pathway.

The Shigella ProU system is required for osmotic tolerance and virulence.

To cope with hyperosmotic stress encountered in the environments and in the host, the pathogenic as well as non-pathogenic microbes use diverse transport systems to obtain osmoprotectants. To study the role of Shigella sonnei ProU system in response to hyperosmotic stress and virulence, we constructed deletion and complementation strains of proV and used an RNAi approach to silence the whole ProU operon. We compared the response between wild type and the mutants to the hyperosmotic pressure in vitro, and assessed virulence properties of the mutants using gentamicin protection assay as well as Galleria mellonella moth larvae model. In response to osmotic stress by either NaCl or KCl, S. sonnei highly up-regulates transcription of proVWX genes. Supplementation of betaine greatly elevates the growth of the wild type S. sonnei but not the proV mutants in M9 medium containing 0.2 M NaCl or 0.2 M KCl. The proV mutants are also defective in intracellular growth compared with the wild type. The moth larvae model of G. mellonella shows that either deletion of proV gene or knockdown of proVWX transcripts by RNAi significantly attenuates virulence. ProU system in S. sonnei is required to cope with osmotic stress for survival and multiplication in vitro, and for infection.

Inflammasome priming is similar for francisella species that differentially induce inflammasome activation.

Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as Francisella activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared Francisella strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed Francisella strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between Francisella strains (F. novicida, F. holarctica -LVS and F. tularensis Schu S4). This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, Francisella strains LVS and Schu S4 were impaired in inflammasome triggering compared to F. novicida. Thus, observed differences in inflammasome activation by F. novicida, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.

Inflammasome priming by lipopolysaccharide is dependent upon ERK signaling and proteasome function.

Caspase-1 activation is a central event in innate immune responses to many pathogenic infections and tissue damage. The NLRP3 inflammasome, a multiprotein scaffolding complex that assembles in response to two distinct steps, priming and activation, is required for caspase-1 activation. However, the detailed mechanisms of these steps remain poorly characterized. To investigate the process of LPS-mediated NLRP3 inflammasome priming, we used constitutively present pro-IL-18 as the caspase-1-specific substrate to allow study of the early events. We analyzed human monocyte caspase-1 activity in response to LPS priming, followed by activation with ATP. Within minutes of endotoxin priming, the NLRP3 inflammasome is licensed for ATP-induced release of processed IL-18, apoptosis-associated speck-forming complex containing CARD, and active caspase-1, independent of new mRNA or protein synthesis. Moreover, extracellular signal-regulated kinase 1 (ERK1) phosphorylation is central to the priming process. ERK inhibition and small interfering RNA-mediated ERK1 knockdown profoundly impair priming. In addition, proteasome inhibition prevents ERK phosphorylation and blocks priming. Scavenging reactive oxygen species with diphenylene iodonium also blocks both priming and ERK phosphorylation. These findings suggest that ERK1-mediated posttranslational modifications license the NLRP3 inflammasome to respond to the second signal ATP by inducing posttranslational events that are independent of new production of pro-IL-1ß and NOD-like receptor components.

Tyrosine phosphatase inhibition induces an ASC-dependent pyroptosis.

Pyroptosis is a type of cell death in which danger associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) induce mononuclear phagocytes to activate caspase-1 and release mature IL-1ß. Because the tyrosine kinase inhibitor AG126 can prevent DAMP/PAMP induced activation of caspase-1, we hypothesized that tipping the tyrosine kinase/phosphatase balance toward phosphorylation would promote caspase-1 activation and cell death. THP-1 derived macrophages were therefore treated with the potent specific tyrosine phosphatase inhibitor, sodium orthovanadate (OVN) and analyzed for caspase-1 activation and cell death. OVN induced generalized increase in phosphorylated proteins, IL-1ß release and cell death in a time and dose dependent pattern. This OVN induced pyroptosis correlated with speck formations that contained the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Culturing the cells in the presence of extracellular K(+) (known to inhibit ATP dependent pyroptosis), a caspase inhibitor (ZVAD) or down regulating the expression of ASC with stable expression of siASC prevented the OVN induced pyroptosis. These data demonstrate that pyroptotic death is linked to tyrosine phosphatase activity providing novel targets for future pharmacologic interventions.

Evaluation of immunomodulatory effect of three herbal plants growing in Egypt.

A group of medicinal plant including Silene (Silene nocturna), Black seed (Nigella sativa) and Chamomile (Matricaria chamomilla) growing in Egypt were examined for their immunomodulatory effect in Balb/c mice. Treatment (intraperitoneal injection) with five doses of methanolic extract for each plant was found to enhance the total white blood cells count (up to 1.2 × 10(4) cells/mm(3)). Bone marrow cellularity also increased significantly (P < 0.01) after the administration of the extract of each of three test plants. Furthermore, spleen weight of the treated groups was significantly increased (P < 0.01). Two groups of mice were immunosuppressed with cyclophosphamide, the one which pretreated with the plants extracts significantly (P < 0.01) restored their resistance against lethal infection with the predominately granulocyte-dependant Candida albicans. These results confirm the immunomodulatory activity of Silene, Black seed, and Chamomile extracts and may have therapeutical implications in prophylactic treatment of opportunistic infections and as supportive treatment in oncogenic cases.