Marine Bioactive Molecules as Inhibitors of the Janus Kinases: A Comparative Molecular Docking and Molecular Dynamics Simulation Approach.

A treasure trove of naturally occurring biomolecules can be obtained from sea living organisms to be used as potential antioxidant and anti-inflammatory agents. These bioactive molecules can target signaling molecules involved in the severity of chronic autoimmune diseases such as rheumatoid arthritis (RA). The intracellular tyrosine kinases family, Janus kinases (JAKs, includes JAK1, JAK2, and JAK3), is implicated in the pathogenesis of RA through regulating several cytokines and inflammatory processes. In the present study, we conducted molecular docking and structural analysis investigations to explore the role of a set of bioactive molecules from marine sources that can be used as JAKs' specific inhibitors. Around 200 antioxidants and anti-inflammatory molecules out of thousands of marine molecules found at the Comprehensive Marine Natural Products Database (CMNPD) website, were used in that analysis. The details of the interacting residues were compared to the recent FDA approved inhibitors tofacitinib and baricitinib for data validation. The shortlisted critical amino acids residues of our pharmacophore-based virtual screening were LYS905, GLU957, LEU959, and ASP1003 at JAK1, GLU930 and LEU932 at JAK2, and GLU905 and CYS909 of JAK3. Interestingly, marine biomolecules such as Sargachromanol G, Isopseudopterosin E, Seco-Pseudopterosin, and CID 10071610 showed specific binding and significantly higher binding energy to JAK1 active/potential sites when being compared with the approved inhibitors. In addition, Zoanthoxanthin and Fuscoside E bind to JAK2's critical residues, GLU930 and LEU932. Moreover, Phorbaketal and Fuscoside E appear to be potential candidates that can inhibit JAK3 activity. These results were validated using molecular dynamics simulation for the docked complexes, JAK1(6sm8)/SG, JAK2 (3jy9)/ZAX, and JAK3 (6pjc)/Fuscoside E, where stable and lower binding energy were found based on analyzing set of parameters, discussed below (videos are attached). A promising role of these marine bioactive molecules can be confirmed in prospective preclinical/clinical investigations using rheumatoid arthritis models.

Flavipin from fungi as a potential inhibitor of rheumatoid arthritis signaling molecules.

Flavipin, a fungal lower molecular weight biomolecule (MW 196.16 g/mol), has not been yet extensively studied for beneficial preclinical and clinical applications. In recent years, various preclinical mouse models including adjuvant-induced arthritis (AIA) were employed to understand mechanisms associated with Rheumatoid arthritis (RA) and to develop new therapeutic drugs. In the current study, we studied the inhibitory effect of Flavipin on major signaling molecules involved in the inflammatory response during RA using both in-silico virtual interaction and in vivo mouse model of AIA. Our in-silico results clarified that Flavipin interacts with the tumor necrosis factor alpha (TNF-α) through conventional hydrogen binding (H-H) at one of TNF-α critical amino acids tyrosine residues, Tyr119, with binding energy (b.e.) -5.9. In addition, Flavipin binds to ATP-binging sites of the Jesus kinases, JAK1, JAK2 and JAK3, through H-H (b. e. between -5.8 and -6.1) and then it may inhibit JAKs, regulators of RA signaling molecules. Moreover, our molecular dynamics stimulation for the docked TNF-α/Flavipin complex confirmed the specificity and the stability of the interaction. In vitro, Flavipin is not toxic to normal cells at doses below 50 µM (its IC50 in normal fibroblast cell line was above 100 µM). However, in vivo, the arthritis score and hind paw oedema parameters were modulated in Flavipin treated mice. Consistent with the in-silico results the levels of the TNF-α, the nuclear transcription factor kappaB (NF-κB) and the signal transduction and activator of transcription (STAT3, downstream of JAKs) were modulated at joint tissues of the hind-paw of Flavipin/AIA treated mice. Our data suggest Flavipin as a potential therapeutic agent for arthritis can inhibit RA major signaling molecules.

Protective effect of 5,4'-dihydroxy-6,8-dimethoxy7-O-rhamnosylflavone from Indigofera aspalathoides Vahl on lipopolysaccharide-induced intestinal injury in mice.

Intestinal inflammation is one of the main health challenges affecting the quality of life of millions of people worldwide. Accumulating evidence introduces several flavonoids with multifaceted therapeutic properties in inflammatory diseases including intestinal inflammation. Herein, we examined potential anti-inflammatory properties of 5,4'-dihydroxy-6,8-dimethoxy7-O-rhamnosylflavone (DDR) flavone derived from Indigofera aspalathoides Vahl (I. aspalathoides Vahl) on lipopolysaccharide (LPS)-induced intestinal inflammation and injury in mice. Oral DDR treatment decreased serum levels of pro-inflammatory cytokines including TNF-α, IL-6, and IL-1ß. It reduced oxidative stress through augmenting the activities of catalase (CAT) and superoxide dismutase (SOD) and reducing the level of malondialdehyde (MDA) in the duodenum and colon tissues. Moreover, DDR enhanced the activities of digestive enzymes including trypsin, pancreatic lipase, and amylase, and increased the production of short-chain fatty acids (SCFAs) by colon microbiota. Histopathological investigation of duodenum and colon revealed that DDR inhibited inflammatory infiltration and largely restored mucosal architecture and protected lining integrity. Importantly, DDR suppressed activation of nuclear factor-κB (NF-κB) signaling pathway through reduced expression of Toll-like receptor 4 (TLR4) and expression and phosphorylation of P65. The current study identified DDR as anti-inflammatory flavonoid capable of ameliorating LPS-induced intestinal inflammation through suppression of NF-κB signaling.

SOXC Transcription Factors as Diagnostic Biomarkers and Therapeutic Targets for Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are two common disorders that disrupt the quality of life of millions of people. These two chronic diseases cause damage to the joint cartilage and surrounding tissues of more than 220 million people worldwide. Sex-determining region Y-related (SRY) high-mobility group (HMG) box C, SOXC, is a superfamily of transcription factors that have been recently shown to be involved in various physiological and pathological processes. These include embryonic development, cell differentiation, fate determination, and autoimmune diseases, as well as carcinogenesis and tumor progression. The SOXC superfamily includes SOX4, SOX11, and SOX12, all have a similar DNA-binding domain, i.e., HMG. Herein, we summarize the current knowledge about the role of SOXC transcription factors during arthritis progression and their potential utilization as diagnostic biomarkers and therapeutic targets. The involved mechanistic processes and signaling molecules are discussed. SOX12 appears to have no role in arthritis, however SOX11 is dysregulated and promotes arthritic progression according to some studies but supports joint maintenance and protects cartilage and bone cells according to others. On the other hand, SOX4 upregulation during OA and RA was documented in almost all studies including preclinical and clinical models. Molecular details have indicated that SOX4 can autoregulate its own expression besides regulating the expression of SOX11, a characteristic associated with the transcription factors that protects their abundance and activity. From analyzing the currently available data, SOX4 seems to be a potential diagnostic biomarker and therapeutic target of arthritis.

Effects of polyphenols on ncRNAs in cancer-An update.

In recent years, oncotherapy has received considerable attention concerning plant polyphenols. Increasing evidence suggests that because of the efficiency of polyphenols, they may have anti-tumour effects in various cancers. However, their regulatory structures remain elusive. Long non-coding RNAs (lncRNAs) have been identified in the regulation of various forms of tumorigenesis and tumour development. Long non-coding RNAs have recently emerged as regulatory eukaryotic transcripts and therapeutic targets with important and diverse functions in health and diseases. LncRNAs may be associated with the initiation, development, and progression of cancer. This review summarizes the research on the modulatory effects of IncRNAs and their roles in mediating cellular processes. The mechanisms of action of polyphenols underlying their therapeutic effects on cancers are also discussed. Based on our review, polyphenols might facilitate a significant epigenetic modification as part of their tissue- and/or cell-related biological effects. This finding may be attributed to their interaction with cellular signalling pathways involved in chronic diseases. Certain lncRNAs might be the target of specific polyphenols, and some critical signalling processes involved in the intervention of cancers might mediate the therapeutic roles of polyphenols.

The microRNA-202 as a Diagnostic Biomarker and a Potential Tumor Suppressor.

MicroRNA-202 (miR-202) is a member of the highly conserved let-7 family that was discovered in Caenorhabditis elegans and recently reported to be involved in cell differentiation and tumor biology. In humans, miR-202 was initially identified in the testis where it was suggested to play a role in spermatogenesis. Subsequent research showed that miR-202 is one of the micro-RNAs that are dysregulated in different types of cancer. During the last decade, a large number of investigations has fortified a role for miR-202 in cancer. However, its functions can be double-edged, depending on context they may be tumor suppressive or oncogenic. In this review, we highlight miR-202 as a potential diagnostic biomarker and as a suppressor of tumorigenesis and metastasis in several types of tumors. We link miR-202 expression levels in tumor types to its involved upstream and downstream signaling molecules and highlight its potential roles in carcinogenesis. Three well-known upstream long non-coding-RNAs (lncRNAs); MALAT1, NORAD, and NEAT1 target miR-202 and inhibit its tumor suppressive function thus fueling cancer progression. Studies on the downstream targets of miR-202 revealed PTEN, AKT, and various oncogenes such as metadherin (MTDH), MYCN, Forkhead box protein R2 (FOXR2) and Kirsten rat sarcoma virus (KRAS). Interestingly, an upregulated level of miR-202 was shown by most of the studies that estimated its expression level in blood or serum of cancer patients, especially in breast cancer. Reduced expression levels of miR-202 in tumor tissues were found to be associated with progression of different types of cancer. It seems likely that miR-202 is embedded in a complex regulatory network related to the nature and the sensitivity of the tumor type and therapeutic (pre)treatments. Its variable roles in tumorigenesis are mediated in part thought its oncogene effectors. However, the currently available data suggest that the involved signaling pathways determine the anti- or pro-tumorigenic outcomes of miR-202's dysregulation and its value as a diagnostic biomarker.

Atropine Is a Suppressor of Epithelial-Mesenchymal Transition (EMT) That Reduces Stemness in Drug-Resistant Breast Cancer Cells.

Atropine (ATR) is extracted from a belladonna plant that belongs to a class of anticholinergic drugs and is therefore involved in the treatment of the overdose of cholinergic drugs or mushroom poisoning. It is a well-known blocker of muscarinic acetylcholine receptors (mAChRs) that are expressed in various tumor cells, including breast tumors from animal and human origin, but it has yet to be recommended as an anticancer drug. Our in silico docking analysis indicates that atropine has a roust virtual binding, with a stable binding energy, to two major signaling molecules involved in EMT regulation: E-cad and ZEB-2. For both, the gene and the protein expression level results show that atropine is an effective molecule in reducing epithelial-mesenchymal transition (EMT) and colony formation induced by TGF-B or carboplatin in both the mesenchymal-like cell line MDA-MB-231 and the epithelial-like cell line T47D. We conclude that atropine as a potential suppressor of EMT could be co-administrated with other chemotherapeutic drugs to reduce stemness in drug-resistant breast tumor cells.

Polyphenols as Potent Epigenetics Agents for Cancer.

Human diseases such as cancer can be caused by aberrant epigenetic regulation. Polyphenols play a major role in mammalian epigenome regulation through mechanisms and proteins that remodel chromatin. In fruits, seeds, and vegetables, as well as food supplements, polyphenols are found. Compounds such as these ones are powerful anticancer agents and antioxidants. Gallic acid, kaempferol, curcumin, quercetin, and resveratrol, among others, have potent anti-tumor effects by helping reverse epigenetic changes associated with oncogene activation and tumor suppressor gene inactivation. The role dietary polyphenols plays in restoring epigenetic alterations in cancer cells with a particular focus on DNA methylation and histone modifications was summarized. We also discussed how these natural compounds modulate gene expression at the epigenetic level and described their molecular targets in cancer. It highlights the potential of polyphenols as an alternative therapeutic approach in cancer since they modulate epigenetic activity.

Beta-Caryophyllene Enhances the Anti-Tumor Activity of Cisplatin in Lung Cancer Cell Lines through Regulating Cell Cycle and Apoptosis Signaling Molecules.

Beta-Caryophyllene (BCP), a natural bicyclic sesquiterpenes, is an abundant biomolecule in red pepper and other plants. Recently, it was reported to reduce the growth and the proliferation as well as enhance the apoptosis in numerous cancer cells, including colorectal, ovarian, bladder cancer and lung cancer. On the other hand, the combination therapy of cisplatin (CDDP) with other phytochemical compounds has synergistically enhanced the killing effect of CDDP on several types of cancer. In the current model, we have tested the role of BCP in enhancing the anti-tumor activity of CDDP on lung cancer cell lines. The results showed that BCP is not toxic at moderate doses and it can prevent lung cancer progression in doses above 75 µM. However, when being combined with CDDP, BCP improved the former chemotherapeutic function through regulating cell cycle, apoptosis and EMT signaling molecules. Gene and protein expression analysis showed that the combined treatment of CDDP and BCP significantly upregulated the level of the cyclin-dependent kinase inhibitor, CDKN1A, and the inhibitor of the apoptosis, BCL-xl2. In addition, the combination treatment reduced the protein level of the apoptosis regulator, BCL-2. Moreover, BCP appears to prohibit the EMT process that is associated with CDDP chemotherapy since the combination treatment induced a significant increase in the level of the epithelial cell marker E-cad that was reduced in CDDP-treated cells. In agreement with that, the combined treatment managed to modulate the effect of CDDP on the mesenchymal transcription factor ZEB-2. Additionally, molecular docking has been conducted to check the virtual interaction of BCP with these and other signaling molecules, but only cyclin-dependent kinase CDK6 was found to virtually bind with BCP, and at four sites with higher and stable biding energy (-7.8). Together, these data indicate that BCP enhances CDDP chemotherapeutic function through regulating the cell cycle, the apoptosis and EMT signaling molecules.

Thidiazuron, a phenyl-urea cytokinin, inhibits ergosterol synthesis and attenuates biofilm formation of Candida albicans.

Candida albicans is a common human fungal pathogen that colonizes mucosa and develops biofilm in the oral cavity that causes oral candidiasis. It has been reported that cytochrome P450 enzyme (CYP51), a vital part of the ergosterol synthesis cascade, is associated with Candida infections and its biofilm formation. Thidiazuron, a phenyl-urea cytokinin, exhibits anti-senescence and elicitor activity against fungal infection in plants. However, how Thidiazuron impacts C. albicans biofilm formation is still uncertain. Here, we aimed to investigate the effects of a Thidiazuron against the growth and biofilm formation properties of C. albicans using in silico and in vitro experimental approaches. A preliminary molecular docking study revealed potential interaction between Thidiazuron and amino acid residues of CYP51. Further in vitro antifungal susceptibility test, scanning electron microscopy (SEM) and time kill analysis revealed the anti-fungal activity of Thidiazuron in both dose and time-dependent manner. Crystal violet staining, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay revealed 50% inhibition in C. albicans biofilm by Thidiazuron at concentrations 11 and 19 µM respectively. Acridine orange staining assay visually confirmed the biofilm inhibitory potential of Thidiazuron. The gene expression study showed that Thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis (ERG3, ERG11, ERG25), cell adhesion (ASL3, EAP1), and hyphae development (EFG1, HWP1, SAP5) in C. albicans. Wherease, the expression of negative transcription regulator of hyphae (NRG1) was upregulated (5.7-fold) by Thidiazuron treatment. Collectively, our data suggest that Thidiazuron is a robust antifungal compound and an outstanding biofilm inhibitor, which may promise further therapeutic development due to CYP51 binding and inhibition of ergosterol formation against C. albicans.

SOX4: Epigenetic regulation and role in tumorigenesis.

Sex-determining region Y-related (SRY) high-mobility group box 4 (SOX4) is a member of the group C subfamily of SOX transcription factors and promotes tumorigenesis by endowing cancer cells with survival, migratory, and invasive capacities. Emerging evidence has highlighted an unequivocal role for this transcription factor in mediating various signaling pathways involved in tumorigenesis, epithelial-to-mesenchymal transition (EMT), and tumor progression. During the last decade, numerous studies have highlighted the epigenetic interplay between SOX4-targeting microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and SOX4 and the subsequent modulation of tumorigenesis, invasion and metastasis. In this review, we summarize the current state of knowledge about the role of SOX4 in cancer development and progression, the epigenetic regulation of SOX4, and the potential utilization of SOX4 as a diagnostic and prognostic biomarker and its depletion as a therapeutic target.

Thidiazuron suppresses breast cancer via targeting miR-132 and dysregulation of the PI3K-Akt signaling pathway mediated by the miR-202-5p-PTEN axis.

Chemo-resistance and metastasis are the most common causes of breast cancer recurrence and death. Thidiazuron (TDZ) is a plant growth regulator (phytohormone) whose biological effects on humans and animals has not yet been determined. In this study, we investigated the anticancer activity of this phytohormone on the drug resistant-triple negative breast cancer cell line MDA-MB-231. Treatment of the breast cancer cells with TDZ (1-50 µmol/L) caused more stressful environment and induced a significant increase in active caspase-positive cells. In addition, TDZ treatment (5 and 10 µmol/L) significantly attenuated the migration and the invasiveness of these highly metastatic cancer cells. Mechanistically, TDZ reduces cancer progression and invasiveness by targeting miR-202-5p, which stimulates the expression of phosphatase and tensin homolog (PTEN), the tumor suppressor that downregulates the PI3K-Akt signaling pathway. Treatment with TDZ significantly upregulates miRNA-132, the suppressor of breast cancer proliferation, which is also implicated in dysregulation of the TEN-Akt-NFκB signaling pathway. Interestingly, our molecular docking analysis revealed a potential non-covalent interaction between TDZ and Akt, PTEN, and PI3K. These findings suggest that TDZ suppresses breast cancer metastasis by targeting miRNA-132, the miR-202-5p-PTEN axis, and the PI3K-Akt signaling pathway downstream.

S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates.

We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

Uranium exposure increases spermatocytes metaphase apoptosis in rats: inhibitory effect of thymoquinone and N-acetylcysteine.

Uranyl acetate (UA), a commercial stock from depleted uranium (DU), has a combined effect of chemical toxicity and mild radioactivity. Here, we investigated the potential antioxidant, antiapoptotic and cytoprotective effects of thymoquinone (TQ) and N-acetylcysteine (NAC) against UA-induced testicular damage in rats. UA reduced testicular superoxide dismutase (SOD) activity and nitric oxide (NO) and glutathione (GSH) levels relative to the control group. Interestingly, the testicular SOD activity and NO and GSH levels of UA/TQ- and UA/NAC-treated groups were also significantly lower relative to the control. A marked increase in spermatocytes metaphase apoptosis was found (stage XIII) in UA-treated rats, which is probably due to difficulties in segregation of homologous-chromosomes. This may clarify why UA exposure decreased round spermatids numbers and fertility in previous studies. To check the reason of partial metaphase arrest, the presence of DNA-damage-related γ-H2AX foci in late spermatocytes of all groups was checked, but only insignificant increase was found in UA-treated group. TQ or NAC supplementation reduced the apoptosis and improved the testicular histological alterations. Thus, TQ and NAC attenuate UA adverse effects on the testicular microenvironement through anti-apoptotic and cytoprotective but not antioxidant effects.

DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts.

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G1-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

Camel Whey Protein Protects B and T Cells from Apoptosis by Suppressing Activating Transcription Factor-3 (ATF-3)-Mediated Oxidative Stress and Enhancing Phosphorylation of AKT and IκB-α in Type I Diabetic Mice.

BACKGROUND: Diabetes mellitus (DM) is associated with severe immune system complications. Camel whey protein (CWP) decreases free radicals (ROS) and modulates immune functions, but its effect on DM-impaired immune systems has not been studied. We investigated the impact of CWP on the immune system in a Type 1 diabetes mouse model. METHODS: Three experimental groups were used: (1) non-diabetic control; (2) diabetic; and (3) CWP-treated diabetic mice. RESULTS: Induction of diabetes by streptozotocin was associated with reduction of body weight and insulin level, increase in glucose level and pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), and reduction in IL-2 and IL-4 levels. Upregulated ATF-3 expression was followed by a marked elevation in ROS levels. Lymphocytes from diabetic mice exhibited increased apoptosis through decreased phosphorylation of AKT and IκB-α, increased infiltration of T cells in the spleen and thymus, and decreased B cell numbers in the spleen. Supplementation with CWP decreased the levels of proinflammatory cytokines, ROS, and ATF-3 expression, and increased the levels of IL-4. Treatment with CWP decreased apoptosis by enhancing the phosphorylation of AKT and IκB-α as well as T-cell and B-cell distribution in the spleen and thymus. CONCLUSIONS: Our findings suggest the beneficial effects of CWP supplementation during diabetes on decreasing and orchestrating the redox status and subsequently rescuing the immune cells from exhaustion.

Ku70 and non-homologous end joining protect testicular cells from DNA damage.

Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(Δ/Δ) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of γH2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Cytotoxic activity of bimetallic Ag@Se green synthesized nanoparticles using Jerusalem Thorn (Parkinsonia aculeata).

Introduction: The process of green synthesis of metal nanoparticles is considered to be eco-friendly and cost-effective. Methods: In this study, bimetallic Ag@Se-P and Ag@Se-S nanoparticles were synthesized successfully using Parkinsonia aculeata aerial parts and seed extracts. The phytochemical contents in P. aculeata aerial parts and seed aqueous extract serve as reducing and stabilizing capping agents without the need for any chemical stabilization additive in the synthesis of bimetallic nanoparticles. Result and Discussion: The obtained results from UV-vis spectrophotometry, scanning electron microscopy (SEM), X-ray powder diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FT-IR) confirmed the successful synthesis of bimetallic nanoparticles with cluster irregular spherical morphology, crystalline nature, and average particle sizes of 17.65 and 24.36 nm for Ag@Se-S and Ag@Se-P, respectively. The cytotoxicity assessment of greenly synthesized nanomaterials using seed and plant extracts showed cell inhibition >50 µg/mL. Ag@Se-S and Ag@Se-P seed and plant extracts significantly reduced LPS-induced inflammation, which was assessed by NO and cytokines IL-1ß, IL-6, and TNF-α. The mRNA and protein expression levels of phosphoinositide 3 kinase (PI3K) and nuclear factor kappa B (NFkB) were significantly overexpressed in LPS-induced RAW 264.7 cell lines. Ag@Se-S and Ag@Se-P downregulated the expression of PI3K and NFkB in LPS-induced cell models.

Geraniol attenuates oxidative stress and neuroinflammation-mediated cognitive impairment in D galactose-induced mouse aging model.

D-galactose (D-gal) administration was proven to induce cognitive impairment and aging in rodents' models. Geraniol (GNL) belongs to the acyclic isoprenoid monoterpenes. GNL reduces inflammation by changing important signaling pathways and cytokines, and thus it is plausible to be used as a medicine for treating disorders linked to inflammation. Herein, we examined the therapeutic effects of GNL on D-gal-induced oxidative stress and neuroinflammation-mediated memory loss in mice. The study was conducted using six groups of mice (6 mice per group). The first group received normal saline, then D-gal (150 mg/wt) dissolved in normal saline solution (0.9%, w/v) was given orally for 9 weeks to the second group. In the III group, from the second week until the 10th week, mice were treated orally (without anesthesia) with D-gal (150 mg/kg body wt) and GNL weekly twice (40 mg/kg body wt) four hours later. Mice in Group IV were treated with GNL from the second week up until the end of the experiment. For comparison of young versus elderly mice, 4 month old (Group V) and 16-month-old (Group VI) control mice were used. We evaluated the changes in antioxidant levels, PI3K/Akt levels, and Nrf2 levels. We also examined how D-gal and GNL treated pathological aging changes. Administration of GNL induced a significant increase in spatial learning and memory with spontaneously altered behavior. Enhancing anti-oxidant and anti-inflammatory effects and activating PI3K/Akt were the mechanisms that mediated this effect. Further, GNL treatment upregulated Nrf2 and HO-1 to reduce oxidative stress and apoptosis. This was confirmed using 99mTc-HMPAO brain flow gamma bioassays. Thus, our data suggested GNL as a promising agent for treating neuroinflammation-induced cognitive impairment.