Search results for: gene expression datasets

Authors: Amirreza Razzaghipour Sorkhab

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One of the most common disorders among humans is hearing impairment. This paper provides an evidence base that recovers understanding of Meniere’s disease and highlights the physical and mental health correlates of the disorder. Meniere's disease is more common in the elderly. The term idiopathic endolymphatic hydrops has been attributed to this disease by some in the previous. Meniere’s disease demonstrations a genetic tendency, and a family history is found in 10% of cases, with an autosomal dominant inheritance pattern. The COCH gene may be one of the hereditary factors contributing to Meniere’s disease, and the possibility of a COCH mutation should be considered in patients with Meniere’s disease symptoms. Should be considered Missense mutations in the COCH gene cause the autosomal dominant sensorineural hearing loss and vestibular disorder. Meniere’s disease is a complex, heterogeneous disorder of the inner ear and that is characterized by episodes of vertigo lasting from minutes to hours, fluctuating sensorineural hearing loss, tinnitus, and aural fullness. The existing evidence supports the suggestion that age and sleep disorder are risk factors for Meniere's disease. Many factors have been reported to precipitate the progress of Menier, including endolymphatic hydrops, immunology, viral infection, inheritance, vestibular migraine, and altered intra-labyrinthine fluid dynamics. Although there is currently no treatment that has a proven helpful effect on hearing levels or on the long-term evolution of the disease, however, in the primary stages, the hearing may improve among attacks, but a permanent hearing loss occurs in the majority of cases. Current publications have proposed a role for the intratympanic use of medicine, mostly aminoglycosides, for the control of vertigo. more than 85% of patients with Meniere's disease are helped by either changes in lifestyle and medical treatment or minimally aggressive surgical procedures such as intratympanic steroid therapy, intratympanic gentamicin therapy, and endolymphatic sac surgery. However, unilateral vestibular extirpation methods (intratympanic gentamicin, vestibular nerve section, or labyrinthectomy) are more predictable but invasive approaches to control the vertigo attacks. Medical therapy aimed at reducing endolymph volume, such as low-sodium diet, diuretic use, is the typical initial treatment.

Keywords: meniere's disease, endolymphatic hydrops, hearing loss, vertigo, tinnitus, COCH gene

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Authors: Reem Alajmi, Hind Al Harbi, Tahany Ayaad, Zainab Al Musawi

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Hard ticks Hyalomma spp. (family: Ixodidae) are obligate ectoparasite in their all life stages on some domestic animals mainly camels and cattle. Ticks may lead to many economic and public health problems because of their blood feeding behavior. Also, they act as vectors for many bacterial, viral and protozoan agents which may cause serious diseases such as tick-born encephalitis, Rocky-mountain spotted fever, Q-fever and Lyme disease which can affect human and/or animals. In the present study, molecular identification of ticks that attack camels in Riyadh region, Saudi Arabia based on the partial sequence of mitochondrial 16s rRNA gene was applied. Also, the present study aims to detect natural infections of collected camel ticks with Rickessia spp. and Borelia spp. using PCR/hybridization of Citrate synthase encoding gene present in bacterial cells. Hard ticks infesting camels were collected from different camels located in a farm in Riyadh region, Saudi Arabia. Results of the present study showed that the collected specimens belong to two species: Hyalomma dromedari represent 99% of the identified specimens and Hyalomma marginatum which account for 1 % of identified ticks. The molecular identification was made through blasting the obtained sequence of this study with sequences already present and identified in GeneBank. All obtained sequences of H. dromedarii specimens showed 97-100% identity with the same gene sequence of the same species (Accession # L34306.1) which was used as a reference. Meanwhile, no intraspecific variations of H. marginatum mesured because only one specimen was collected. Results also had shown that the intraspecific variability between individuals of H. dromedarii obtained in 92 % of samples ranging from 0.2- 6.6%, while the remaining 7 % of the total samples of H. dromedarii showed about 10.3 % individual differences. However, the interspecific variability between H. dromedarii and H. marginatum was approximately 18.3 %. On the other hand, by using the technique of PCR/hybridization, we could detect natural infection of camel ticks with Rickettsia spp. and Borrelia spp. Results revealed the natural presence of both bacteria in collected ticks. Rickettsial spp. infection present in 29% of collected ticks, while 35% of collected specimen were infected with Borrelia spp. The valuable results obtained from the present study are a new record for the molecular identification of camel ticks in Riyadh, Saudi Arabia and their natural infection with both Rickettsia spp. and Borrelia spp. These results may help scientists to provide a good and direct control strategy of ticks in order to protect one of the most important economic animals which are camels. Also results of this project spotlight on the disease that might be transmitted by ticks to put out a direct protective plan to prevent spreading of these dangerous agents. Further molecular studies are needed to confirm the results of the present study by using other mitochondrial and nuclear genes for tick identification.

Keywords: Camel ticks, Rickessia spp. , Borelia spp. , mitochondrial 16s rRNA gene

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Authors: Hailiang Liu, Yan Ni, Jie Zheng, Xiaoyan Jiang, Min Hong

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Allergic contact dermatitis (ACD) is a commonly clinical type IV allergic skin disease, with the pathological features of infiltration by mononuclear cells and tissue necrosis. Traditional Chinese medicine Radix Saposhnikoviae (RS) is traditionally employed to treat exogenous evils, rubella, itching, rheumatism and tetanus. Meanwhile, it is an important component of the commonly used anti-allergy compound. It’s now widely used as an immuno-modulating agent in mixed herbal decoctions to treat inflammation. However, its mechanism of anti-allergy remains unknown. RS was found to reduce ear thickness, as well as the infiltration of eosinophils. The proliferation of T lymphocytes was inhibited significantly by RS, markedly decreased IFN-γ levels in the supernatant of cells cultured and serum were detected with the treatment of RS. RS significantly decreased the amount of DCs in the mouse lymph nodes, and inhibited the expression of CD4 0 and CD86. Meanwhile, T-bet mRNA expression was down remarkably regulated by RS. These results indicate that RS cures Th1-induced allergic skin inflammation by regulating Th1/Th2 balance with decreasing Th1 differentiation, which might be associated with DCs.

Keywords: allergic contact dermatitis, Radix saposhnikoviae, dendritic cells, T-bet, GATA-3, CD4+ CD25+ Foxp3+ treg cells

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

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Authors: A. Vijayendra Chary, R. Hemalatha

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Background: Vitamin D is a potent modulator of the immune system and is involved in regulating cell proliferation and differentiation. Vitamin D deficiency has been linked to increased severity of asthma in children. Asthma has dramatically increased in past decades, particular in developing countries and affects up to 20% of the population. IgE and its receptors, CD23 (FcεRII) and CD 21, play an essential role in all allergic conditions. Methods: A case control study was conducted on asthma and age and sex matched control children. 25 hydroxyvitamin D3 was quantified by HPLC; CD23; and CD21 expression on B cells were performed by flow cytometry. Total Histamine, total IGE and IL-5 and IFN-γ cytokines were determined by ELISA in blood samples of bronchial asthma (n=45) and control children (n=45). Results: The mean ± SE of vitamin D was significantly (p<0.05) low in asthma children (13.6±0.54 ng/mL) than in controls (17.4 ± 0.37 ng/mL). The mean (%) ± SE of CD23 and CD21 expression on B cells were significantly (p<0.01) high in asthma (1.02±0.09; 1.67± 0.13), when compared to controls (0.24±0.01; 0.94±0.03) respectively. The mean± SE of Serum IgE and blood histamine levels in asthma children (354.52 ± 17.33 IU/mL; 53.27 ± 2.54 nM/mL) were increased (P<0.05) when compared to controls (183.12±17.62 IU/mL 39.34±4.16 nM/mL) respectively and IFN-γ (Th1 cytokine) was lower (P<0.01) (16.37±1.27 pg/mL) than in controls (43.34±6.21 pg/mL). Conclusion: Our study provides evidence that low vitamin D levels are associated with increased IgE receptors CD23 and CD21 on B cells. In addition, there was preferential activation of Th2 (IL-5) and suppression of Th1 (IFN-γ) cytokines in children with asthma.

Keywords: bronchial asthma, CD23, IgE, vitamin D

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Authors: Alan S. Hoback

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Most people see human faces in car front and back ends because of the process of pareidolia. 96 people were surveyed to see how many of them saw a face in the vehicle styling. Participants were aged 18 to 72 years. 94% of the participants saw faces in the front-end design of production models. All participants that recognized faces indicated that most styles showed some degree of an angry expression. It was found that women were more likely to see faces in inanimate objects. However, with respect to whether women were more likely to perceive anger in the vehicle design, the results need further clarification. Survey responses were correlated to the design features of vehicles to determine what cues the respondents were likely looking at when responding. Whether the features looked anthropomorphic was key to anger perception. Features such as the headlights which could represent eyes and the air intake that could represent a mouth had high correlations to trends in scores. Results are compared among models, makers, by groupings of body styles classifications for the top 12 brands sold in the US, and by year for the top 20 models sold in the US in 2016. All of the top models sold increased in perception of an angry expression over the last 20 years or since the model was introduced, but the relative change varied by body style grouping.

Keywords: aggressive driving, face recognition, road rage, vehicle styling

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Authors: Wenxing Chen, Guangying Chen, Yin Lu, Shile Huang

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Cryptotanshinone (CPT), a fat-soluble tanshinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit mTOR pathway, resulting in inhibition of cancer cell proliferation. However, the molecular mechanism how CPT acts on mTOR is unknown. Here, cancer cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while phosphorylation of AMPK and TSC2 was activated, suggesting that CPT inhibition of mTOR maybe due to activating upstream of mTOR, AMPK, but not directly binding to and inhibiting mTOR. Further results indicated that Compound C, inhibitor of AMPK, could partially reversed CPT inhibition effect on cancer cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4EBP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with MEF cells with AMPK positive, MEF cells with AMPK knock out are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively much. Furthermore, downexpression of TSC2 slightly recovered expression of 4EBP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not affect expression of TSC1 by CPT. Collectively, the above-mentioned results suggest that CPT inhibited mTOR pathway mostly was due to activation of AMPK-TSC2 pathway rather than specific inhibition of mTOR and then induction of subsequent lethal cellular effect.

Keywords: cryptotanshinone, AMPK, TSC2, mTOR, cancer cells

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Authors: Aditya Arya, Hairin Taha, Ataul Karim Khan, Nayiar Shahid, Hapipah Mohd Ali, Mustafa Ali Mohd

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This study has investigated the antidiabetic and antioxidant potential of Pseudovaria macrophylla bark extract on streptozotocin–nicotinamide induced type 2 diabetic rats. LCMS-QTOF and NMR experiments were done to determine the chemical composition in the methanolic bark extract. For in vivo experiments, the STZ (60 mg/kg/b.w, 15 min after 120 mg/kg/1 nicotinamide, i.p.) induced diabetic rats were treated with methanolic extract of Pseuduvaria macrophylla (200 and 400 mg/kg∙bw) and glibenclamide (2.5 mg/kg) as positive control respectively. Biochemical parameters were assayed in the blood samples of all groups of rats. The pro-inflammatory cytokines, antioxidant status and plasma transforming growth factor βeta-1 (TGF-β1) were evaluated. The histological study of the pancreas was examined and its expression level of insulin was observed by immunohistochemistry. In addition, the expression of glucose transporters (GLUT 1, 2 and 4) were assessed in pancreas tissue by western blot analysis. The outcomes of the study displayed that the bark methanol extract of Pseuduvaria macrophylla has potentially normalized the elevated blood glucose levels and improved serum insulin and C-peptide levels with significant increase in the antioxidant enzyme, reduced glutathione (GSH) and decrease in the level of lipid peroxidation (LPO). Additionally, the extract has markedly decreased the levels of serum pro-inflammatory cytokines and transforming growth factor beta-1 (TGF-β1). Histopathology analysis demonstrated that Pseuduvaria macrophylla has the potential to protect the pancreas of diabetic rats against peroxidation damage by downregulating oxidative stress and elevated hyperglycaemia. Furthermore, the expression of insulin protein, GLUT-1, GLUT-2 and GLUT-4 in pancreatic cells was enhanced. The findings of this study support the anti-diabetic claims of Pseudovaria macrophylla bark.

Keywords: diabetes mellitus, Pseuduvaria macrophylla, alkaloids, caffeic acid

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Authors: Anjali Roy, Mirza S. Baig

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Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid rich plaque enriched with necrotic core, modified lipid accumulation, smooth muscle cells, endothelial cells, leucocytes and macrophages. Macrophage foam cells play a critical role in the occurrence and development of inflammatory atherosclerotic plaque. Foam cells are the fat-laden macrophages in the initial stage atherosclerotic lesion formation. Foam cells are an indication of plaque build-up, or atherosclerosis, which is commonly associated with increased risk of heart attack and stroke as a result of arterial narrowing and hardening. The mechanisms that drive atherosclerotic plaque progression remain largely unknown. Dissecting the molecular mechanism involved in process of macrophage foam cell formation will help to develop therapeutic interventions for atherosclerosis. To investigate the mechanism, we studied the role of nitric oxide synthase 1(NOS1)-mediated nitric oxide (NO) on low-density lipoprotein (LDL) uptake by bone marrow derived macrophages (BMDM). Using confocal microscopy, we found that incubation of macrophages with NOS1 inhibitor, TRIM (1-(2-Trifluoromethylphenyl) imidazole) or L-NAME (N omega-nitro-L-arginine methyl ester) prior to LDL treatment significantly reduces the LDL uptake by BMDM. Further, addition of NO donor (DEA NONOate) in NOS1 inhibitor treated macrophages recovers the LDL uptake. Our data strongly suggest that NOS1 derived NO regulates LDL uptake by macrophages and foam cell formation. Moreover, we also checked proinflammatory cytokine mRNA expression through real time PCR in BMDM treated with LDL and copper oxidized LDL (OxLDL) in presences and absences of inhibitor. Normal LDL does not evoke cytokine expression whereas OxLDL induced proinflammatory cytokine expression which significantly reduced in presences of NOS1 inhibitor. Rapid NOS-1-derived NO and its stable derivative formation act as signaling agents for inducible NOS-2 expression in endothelial cells, leading to endothelial vascular wall lining disruption and dysfunctioning. This study highlights the role of NOS1 as critical players of foam cell formation and would reveal much about the key molecular proteins involved in atherosclerosis. Thus, targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages at early stage of disease and hence dampening the atherosclerosis progression.

Keywords: atherosclerosis, NOS1, inflammation, oxidized LDL

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Authors: Michael Josef Schwerer

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Background: Analyzing any aircraft accident is mandatory based on the regulations of the International Civil Aviation Organization and the respective country’s criminal prosecution authorities. Legal medicine investigations are unavoidable when fatalities involve the flight crew or when doubts arise concerning the pilot’s aeromedical health status before the event. As a result of frequently tremendous blunt and sharp force trauma along with the impact of the aircraft to the ground, consecutive blast or fire exposition of the occupants or putrefaction of the dead bodies in cases of delayed recovery, relevant findings can be masked or destroyed and therefor being inaccessible in standard pathology practice comprising just forensic autopsy and histopathology. Such cases are of considerable risk of remaining unsolved without legal consequences for those responsible. Further, no lessons can be drawn from these scenarios to improve flight safety and prevent future mishaps. Aims and Methods: To learn from previously unsolved aircraft accidents, re-evaluations of the investigation files and modern molecular pathology studies were performed. Genetic testing involved predominantly PCR-based analysis of gene regulation, studying DNA promotor methylations, RNA transcription and posttranscriptional regulation. In addition, the presence or absence of infective agents, particularly DNA- and RNA-viruses, was studied. Technical adjustments of molecular genetic procedures when working with archived sample material were necessary. Standards for the proper interpretation of the respective findings had to be settled. Results and Discussion: Additional molecular genetic testing significantly contributes to the quality of forensic pathology assessment in aviation mishaps. Previously undetected cardiotropic viruses potentially explain e.g., a pilot’s sudden incapacitation resulting from cardiac failure or myocardial arrhythmia. In contrast, negative results for infective agents participate in ruling out concerns about an accident pilot’s fitness to fly and the aeromedical examiner’s precedent decision to issue him or her an aeromedical certificate. Care must be taken in the interpretation of genetic testing for pre-existing diseases such as hypertrophic cardiomyopathy or ischemic heart disease. Molecular markers such as mRNAs or miRNAs, which can establish these diagnoses in clinical patients, might be misleading in-flight crew members because of adaptive changes in their tissues resulting from repeated mild hypoxia during flight, for instance. Military pilots especially demonstrate significant physiological adjustments to their somatic burdens in flight, such as cardiocirculatory stress and air combat maneuvers. Their non-pathogenic alterations in gene regulation and expression will likely be misinterpreted for genuine disease by inexperienced investigators. Conclusions: The growing influence of molecular pathology on legal medicine practice has found its way into aircraft accident investigation. As appropriate quality standards for laboratory work and data interpretation are provided, forensic genetic testing supports the medico-legal analysis of aviation mishaps and potentially reduces the number of unsolved events in the future.

Keywords: aviation medicine, aircraft accident investigation, forensic pathology, molecular pathology

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Authors: Magdalena Rusady Goey, Gordon Strathdee, Neil Perkins

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Background: Acute lymphoblastic leukaemia (ALL) is the most frequent type of childhood malignancy, accounting for 25% of all cases. TWIST2, a basic helix-loop-helix transcription factor, has been implicated in ALL development. Prior studies found that TWIST2 undergoes epigenetic silencing in more than 50% cases of ALL through promoter hypermethylation and suggested that re-expression of TWIST2 may inhibit cell growth/survival of leukaemia cell lines. TWIST2 has also been implicated as a regulator of NF-kappaB activity, which is constitutively active in leukaemia. Here, we use a lentiviral transductions system to confirm the importance of TWIST2 in controlling leukaemia cell growth and to investigate whether this is achieved through altered regulation of NF-kappaB activity. Method: Re-expression of TWIST2 in leukaemia cell lines was achieved using lentiviral-based transduction. The lentiviral vector also expresses enhanced green fluorescent protein (eGFP), allowing transduced cells to be tracked using flow cytometry. Analysis of apoptosis and cell proliferation were done using annexinV and VPD450 staining, respectively. Result and Discussion: TWIST2-expressing cells were rapidly depleted from a mixed population in ALL cell lines (NALM6 and Reh), indicating that TWIST2 inhibited cell growth/survival of ALL cells. In contrast, myeloid cell lines (HL60 and K562) were comparatively insensitive to TWIST2 re-expression. Analysis of apoptosis and cell proliferation found no significant induction of apoptosis, but did find a rapid induction of proliferation arrest in TWIST2-expressing Reh and NALM6 cells. Initial experiment with NF-kappaB inhibitor demonstrated that inhibition of NF-kappaB has similar impact on cell proliferation in the ALL cell lines, suggesting that TWITST2 may induce cell proliferation arrest through inhibition of NF-kappaB. Conclusion: The results of this study suggest that epigenetic inactivation of TWIST2 in primary ALL leads to increased proliferation, potentially by altering the regulation of NF-kappaB.

Keywords: leukaemia, acute lymphoblastic leukaemia, NF-kappaB, TWIST2, lentivirus

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Authors: Maryam Parsian, Pelin Mutlu, Ufuk Gunduz

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Two-dimensional cell culture affords simplicity and low cost, but it has serious limitations; lacking cell-cell and cell-matrix interactions that are present in tissues. Cancer cells grown in 3D culture systems have distinct phenotypes of adhesion, growth, migration, invasion as well as profiles of gene and protein expression. These interactions cause the 3D-cultured cells to acquire morphological and cellular characteristics relevant to in vivo tumors. Palbociclib is a chemotherapeutic agent for the treatment of ER-positive and HER-negative metastatic breast cancer. Poly-amidoamine (PAMAM) dendrimer is a well-defined, special three-dimensional structure and has a multivalent surface and internal cavities that can play an essential role in drug delivery systems. In this study, palbociclib is loaded onto the magnetic PAMAM dendrimer. Hanging droplet method was used in order to form 3D spheroids. The possible toxic effects of both free drug and drug loaded nanoparticles were evaluated in 2D and 3D MCF-7, MD-MB-231 and SKBR-3 breast cancer cell culture models by performing MTT cell viability and Alamar Blue assays. MTT analysis was performed with six different doses from 1000 µg/ml to 25 µg/ml. Drug unloaded PAMAM dendrimer did not demonstrate significant toxicity on all breast cancer cell lines. The results showed that 3D spheroids are clearly less sensitive than 2D cell cultures to free palbociclib. Also, palbociclib loaded PAMAM dendrimers showed more toxic effect than free palbociclib in all cell lines at 2D and 3D cultures. The results suggest that the traditional cell culture method (2D) is insufficient for mimicking the actual tumor tissue. The response of the cancer cells to anticancer drugs is different in the 2D and 3D culture conditions. This study showed that breast cancer cells are more resistant to free palbociclib in 3D cultures than in 2D cultures. However, nanoparticle loaded drugs can be more cytotoxic when compared to free drug.

Keywords: 2D and 3D cell culture, breast cancer, palbociclibe, PAMAM magnetic nanoparticles

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Authors: Sadaf Ilyas, Saba Riaz

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The extensive use of antibiotics has led to increases emergence of antibiotic-resistant organisms. Pseudomonas is a notorious opportunistic pathogen involoved in nosocomial infections and exhibit innate resistance to many antibiotics. The present study was conducted to assess the prevalence, levels of antimicrobial susceptibility and resistance mechanisms of Pseudomonas. A total of thirty clinical strains of Pseudomonas were isolated from different clinical sites of infection. All clinical specimens were collected from Chughtais Lahore Lab. Jail road, during 8-07-2010 to 11-01-2011. Biochemical characterization was done using routine biochemical tests. Antimicrobial susceptibility was determined by Kirby-Baeur method. The plasmids were isolated from all the strains and digested with restriction enzyme PstI and EcoRI. Transfer of Multi-resistance plasmid was checked via transformation and conjugation to confirm the plasmid mediated resistance to antibiotics. The prevalence of Pseudomonas in clinical specimens was found out to be 14% of all bacterial infections. IPM has shown to be the most effective drug against Pseudomonas followed by CES, PTB and meropenem, wheareas most of the Pseudomonas strains have developed significant resistance against Penicillins and some Cephalasporins. Antibiotic resistance determinants were carried by plasmids, as they conferred resistance to transformed K1 strains. The isolates readily undergo conjugation, transferring the resistant genes to other strains, illustrating the high rates of cross infection and nosocomial infection in the immunocompromised patients.

Keywords: pseudomonas, antibiotics, drug resistance, horizontal gene transfer

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Authors: Lubna Azmi, Ila Shukla, Shyam Sundar Gupta, Padam Kant, Ch. V. Rao

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Gastric ulcer is a major global health threat, and it is the leading cause of stomach cancer death worldwide. Helicobacter pylori bacteriumis the most important etiologic factor for gastric ulcer. This infection is highly pervasive in South Asian developing countries, especially in India, Nepal, Srilanka etc. due to diversification in geographic area. Pathophysiology of gastric mucosal damage associated with non-invasive bacterium has not justified in detail, but it leads to change in histopathology, immunochemistry of the gastric and duodenal reason of host. The mechanism responsible for bacteria tissue tropism and mucosal damage in stomach variance during the disease is not clearly described and understood scientifically in treatment and control of pathogenic organisms. Polyphenols are secondary metabolites of plants and are generally involved in defense against aggression by pathogens. 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one and 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde are polyphenolic compound obtained from popular Indian medicinal plants ghavpatta (ArgeriaspeciosaLinn.f) andBael (Aeglemarmelos) have long been used in traditional Ayurvedic Indian medicine for various diseases. They have promising effects on ulcer, as detailed investigation has made in our laboratory. Therefore, the aim of present study is to explore membrane –dependent morphogenesis of H. pylori and associated apoptosis-mediated cell death. Based on this we analyzed immune gene expression in stomach of experimental animals with H. pylori, using quantitative reverse transcription polymerase chain reaction(q RT-PCR). This revealed rapid induction of prostaglandin, interferon I (INF-I), interferon II (INF-II) and INF-I associated genes in the infected animal. Ultrastructural changes associated with H. pylori will be taken for advanced studies. This investigation shows that the biomarkers eradicate H. pylori bacterium caused gastric ulcer which is a major risk factor for gastric cancer.

Keywords: gastric ulcer, Helicobacter pylori, immunochemistry, polyphenols

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Authors: Laura Gantley, Vanessa M. Conn, Stuart Webb, Kirsty Kirk, Marta Gabryelska, Duncan Holds, Brett W. Stringer, Simon J. Conn

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Trinucleotide repeat disorders comprise ~20 severe, inherited human neuromuscular and neurodegenerative disorders, which are a result of an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington’s disease, results from the expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Non-coding RNAs have been implicated in the initiation and progression of many diseases; thus, we focus on one circular RNA (circRNA) molecule arising from non-canonical splicing (back splicing) of HTT pre-mRNA. This circRNA and its mouse orthologue were transgenically overexpressed in human cells (SHSY-5Y and HEK293T) and mouse cells (Mb1), respectively. High-content imaging and flow cytometry demonstrated the overexpression of this circRNA reduces cell proliferation, reduces nuclear size independent of cellular size, and alters cell cycle progression. Analysis of protein by western blot and immunofluorescence demonstrated no change to HTT protein levels but altered nuclear-cytoplasmic distribution without impacting the expansion of the HTT repeat region. As these phenotypic and genotypic changes are found in Huntington’s disease patients, these results may suggest that this circRNA may play a functional role in the progression of Huntington’s disease.

Keywords: cell biology, circular RNAs, Huntington’s disease, molecular biology, neurodegenerative disorders

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Authors: Swati Gautam, Salman Akhtar, S. P. Jaiswar, Amita Jain

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Background: FGTB is now a major global health problem mostly in developing countries including India. In humans, Mycobacterium Tuberculosis (M.tb) is a causating agent of infection. High index of suspicion is required for early diagnosis due to asymptomatic presentation of FGTB disease. In macrophages Toll Like Receptor-2 (TLR-2) is one which mediated host’s immune response to M.tb. The expression of TLR-2 on macrophages is important to determine the fate of innate immune responses to M.tb. TLR-2 have two work. First its high expression on macrophages worsen the outer of infection and another side, it maintains M.tb to its dormant stage avoids activation of M.tb from latent phase. Single Nucleotide Polymorphism (SNP) of TLR-2 gene plays an important role in susceptibility to TB among different populations and subsequently, in the development of infertility. Methodology: This Case-Control study was done in the Department of Obs and Gynae and Department of Microbiology at King George’s Medical University, U.P, Lucknow, India. Total 300 subjects (150 Cases and 150 Controls) were enrolled in the study. All subjects were enrolled only after fulfilling the given inclusion and exclusion criteria. Inclusion criteria: Age 20-35 years, menstrual-irregularities, positive on Acid-Fast Bacilli (AFB), TB-PCR, (LJ/MGIT) culture in Endometrial Aspiration (EA). Exclusion criteria: Koch’s active, on ATT, PCOS, and Endometriosis fibroid women, positive on Gonococal and Chlamydia. Blood samples were collected in EDTA tubes from cases and healthy control women (HCW) and genomic DNA extraction was carried out by salting-out method. Genotyping of TLR2 genetic variants (Arg753Gln and Arg677Trp) were performed by using single amplification refractory mutation system (ARMS) PCR technique. PCR products were analyzed by electrophoresis on 1.2% agarose gel and visualized by gel-doc. Statistical analysis of the data was performed using the SPSS 16.3 software and computing odds ratio (OR) with 95% CI. Linkage Disequiliribium (LD) analysis was done by SNP stats online software. Results: In TLR-2 (Arg753Gln) polymorphism significant risk of FGTB observed with GG homozygous mutant genotype (OR=13, CI=0.71-237.7, p=0.05), AG heterozygous mutant genotype (OR=13.7, CI=0.76-248.06, p=0.03) however, G allele (OR=1.09, CI=0.78-1.52, p=0.67) individually was not associated with FGTB. In TLR-2 (Arg677Trp) polymorphism a significant risk of FGTB observed with TT homozygous mutant genotype (OR= 0.020, CI=0.001-0.341, p < 0.001), CT heterozygous mutant genotype (OR=0.53, CI=0.33-0.86, p=0.014) and T allele (OR=0.463, CI=0.32-0.66, p < 0.001). TT mutant genotype was only found in FGTB cases and frequency of CT heterozygous more in control group as compared to FGTB group. So, CT genotype worked as protective mutation for FGTB susceptibility group. In haplotype analysis of TLR-2 genetic variants, four possible combinations, i.e. (G-T, A-C, G-C, and A-T) were obtained. The frequency of haplotype A-C was significantly higher in FGTB cases (0.32). Control group did not show A-C haplotype and only found in FGTB cases. Conclusion: In conclusion, study showed a significant association with both genetic variants of TLR-2 of FGTB disease. Moreover, the presence of specific associated genotype/alleles suggest the possibility of disease severity and clinical approach aimed to prevent extensive damage by disease and also helpful for early detection of disease.

Keywords: ARMS, EDTA, FGTB, TLR

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Authors: Denise Schrama, Claudia Raposo, Pedro Rodrigues

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Background: Food allergies are conducted by a hypersensitive response of the immune system. These allergies are a global concern for the public health. Consumption of fish is increasing worldwide as it is a healthy meat with high nutritional value. Unfortunately, fish can cause adverse immune-mediate reactions, affecting part of the population with higher incidence in children. β-parvalbumin, a small, highly conserved stable, calcium or magnesium binding muscle protein is the main fish allergen. In fish-allergic patients, cross-reactivity between different fish species exist due to recognition of highly identical protein regions. Enolases, aldolases, or fish gelatin are other identified fish allergens in some fish species. With no available cure for fish allergies, clinical management is only based on an avoidance diet aiming at the total exclusion of offending food. Methods: Mediterranean fish (S. aurata and D. labrax) were fed specifically designed diets, enriched in components that target the expression or inactivation of parvalbumin (creatine and EDTA, respectively). After 90 days fish were sampled and biological tissues were excised. Proteomics was used to access fish allergens characterization and expression in muscle while IgE assays to confirm the lower allergenic potential are conducted in patients with history of fish allergies. Fish welfare and quality of flesh were established with biochemical, texture and sensorial analysis. Results: Fish welfare shows no major impact between diets. In case of creatine supplementation in D. labrax proteomic analysis show a slight decrease in parvalbumin expression. No accumulation of this compound was found in muscle. For EDTA supplementation in S. aurata IgE assay show a slight decrease in allergenicity when using sera of fish allergic patients. Conclusion: Supplementation with these two compounds seems to change slightly the allergenicity of the two mean Mediterranean species.

Keywords: fish allergies, fish nutrition, proteomics, aquaculture

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Authors: Feng Zhang, Li Chen, Desong Kong, Xiaoping Zhang, Xiaojing Zhu, Yin Lu, Shizhong Zheng

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Sinusoidal pathological angiogenesis is a novel therapeutic target for liver fibrosis. We demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells (LSECs) were not impaired by curcumin. Further investigations showed that curcumin inhibited VEGF expression in hepatic stellate cells (HSCs) by disrupting PDGF-βR/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin via blocking PDGF-βR/FAK/RhoA cascade. Gain- or loss-of-function analyses revealed that activation of PPARγ was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPARγ activation-dependent mechanism. PPARγ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.

Keywords: angiogenesis, hepatic stellate cell, curcumin, peroxisome proliferator-activated receptor-γ

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Authors: Katie Strong, Brian P. O'Connor, Jacqueline M. Kanippayoor

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To date, self-report measures of psychopathy have largely been conceptualized with a male-focused understanding of the disorder, with the presumption that psychopathy expression is uniform across genders. However, generalizing this understanding to the female population may be misleading. The objective of this research was to explore gender differences in the expression of psychopathy and to assess current self-report psychopathy measures for gender bias. It was hypothesized that some items in commonly used measures of psychopathy may show gender bias and that existing measures may not contain enough items that are relevant to the manifestation of psychopathy in women. An exploratory investigation was conducted on statistical bias in common measures of psychopathy, and novel, relevant, but previously neglected items and measures were included in a new data collection. The participant pool included a sample of 403 university students and 354 participants recruited using Amazon Mechanical Turk. Item Response Theory methods - including Differential Item Functioning - were used to assess for the item- and test- level bias across several common self-report measures of psychopathy. Analyses indicated occasional and modest levels of item-level bias, and that some additional female-relevant items merit consideration for inclusion in measures of psychopathy. These findings suggest that current self-report measures of psychopathy may be demonstrating gender-bias and warrant further examination.

Keywords: gender, measurement bias, personality, psychopathy

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Authors: Phani Kiran, Prabodha Manas Yarlagadda

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This paper attempts to treat Shakespeare’s tragic hero, Hamlet as an existential hero who faces many dilemmas in the process of taking revenge for his father’s murder. Hamlet can be considered as a predecessor of existentialism, and Shakespeare, as a pioneer, focused on some serious existential issues in the play much before they were fully developed in 20th century. Hamlet's internal struggles reflect existential themes such as alienation, despair, and the quest for authenticity. Hamlet’s famous soliloquy, "To be, or not to be," is a quintessential expression of existential ponderings, contemplating the choice between life and death and the uncertainty of what lies beyond. Hamlet grapples with existential questions like the purpose and meaninglessness of life, the nature of morality, the inevitability of death, and the existence of an afterlife. He doubts the authenticity of appearance and the reliability of his own perceptions, highlighting the inherent ambiguity and uncertainty of existence. Overall, "Hamlet" aligns with existential philosophy by exploring the complexities of human existence, the search for meaning, and the individual's struggle to find their place in an inherently uncertain and perplexing world. The character of Hamlet and the play's exploration of existential themes continue to resonate with audiences and provoke contemplation on the nature of life and the human experience.

Keywords: to be or not to be, death, dilemmas, illusion and reality

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Authors: Fan Zhang

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In the process of urbanization in China, the new rural construction is in the ascendant, which is becoming more and more popular. Under the driving effect of rural urbanization, the house pattern and tectonic methods of traditional vernacular houses have shown great differences from the family structure and values of contemporary peasant families. Therefore, it is particularly important to find a prototype, form and strategy, to make a balance between the traditional memory and modern functional requirements. In order for research to combine the regional culture with modern life, under the situation of the current batch production of new rural residence, Badie village, in Linhai, Zhejiang province, is taken as the case. This paper aims to put forward a prototype which can not only meet the demand of modern life but also ensure the continuation of traditional culture and historical context for the new rural dwellings design. This research not only helps to extend the local context in the construction of the new site but also contributes to the fusion of old and new rural dwellings in the old site construction. Through the study and research of this case, the research methodology and results can be drawn as reference for the new rural construction in other areas.

Keywords: badie village, design strategy, new rural dwellings, regional context, regional expression

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Authors: Aswini M. S.

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Tomato (Solanum lycopersicum L.) is one of the most significant vegetable crops in terms of its economic benefits. Both fresh and processed tomatoes are consumed. Tomatoes have a limited genetic base, which makes breeding extremely challenging. Plant breeding has become much simpler and more effective with genome editing tools of CRISPR and CRISPR-associated 9 protein (CRISPR/Cas9), which address the problems with traditional breeding, chemical/physical mutagenesis, and transgenics. With the use of CRISPR/Cas9, a number of tomato traits have been functionally distinguished and edited. These traits include plant architecture as well as flower characters (leaf, flower, male sterility, and parthenocarpy), fruit ripening, quality and nutrition (lycopene, carotenoid, GABA, TSS, and shelf-life), disease resistance (late blight, TYLCV, and powdery mildew), tolerance to abiotic stress (heat, drought, and salinity) and resistance to herbicides. This study explores the potential of CRISPR/Cas9 genome editing for enhancing yield in tomato plants. The study utilized the CRISPR/Cas9 genome editing technology to functionally edit various traits in tomatoes. The de novo domestication of elite features from wild cousins to cultivated tomatoes and vice versa has been demonstrated by the introgression of CRISPR/Cas9. The CycB (Lycopene beta someri) gene-mediated Cas9 editing increased the lycopene content in tomato. Also, Cas9-mediated editing of the AGL6 (Agamous-like 6) gene resulted in parthenocarpic fruit development under heat-stress conditions. The advent of CRISPR/Cas has rendered it possible to use digital resources for single guide RNA design and multiplexing, cloning (such as Golden Gate cloning, GoldenBraid, etc.), creating robust CRISPR/Cas constructs, and implementing effective transformation protocols like the Agrobacterium and DNA free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex. Additionally, homologous recombination (HR)-based gene knock-in (HKI) via geminivirus replicon and base/prime editing (Target-AID technology) remains possible. Hence, CRISPR/Cas facilitates fast and efficient breeding in the improvement of tomatoes.

Keywords: CRISPR-Cas, biotic and abiotic stress, flower and fruit traits, genome editing, polygenic trait, tomato and trait introgression

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Authors: Hani M. M. Alothaid, Louise Robson, Richmond Muimo

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This study will investigate cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) dependent calcineurin (Cn), known as protein phosphatase 2 B (PP2B) as well, regulation of chloride ion (Cl⁻) secretion and the release of pro-inflammatory molecules in immune cells such as cytokines. THP-1-derived monocytes, primary human monocytes and the bronchial epithelial cell line (16HBE14o-) were used in this study. The 16HBE14o- cells were chosen as positive control. Hence, to further confirm the expression of cystic fibrosis transmembrane conductance regulator (CFTR), calcium binding protein (S100A10), annexin A2 (AnxA2) and calcineurin A subunit (CnA) in all three cell types, cell lysate was probed against corresponding primary antibodies by immunoblotting. Western blot analyses show the expression of CFTR, AnxA2, CnA and S100A10 in THP-1-derived monocytes and primary human monocytes. In conclusion, CFTR, S100A10, CnA and AnxA2 are expressed in THP-1-derived monocytes and primary human monocytes and regulate Cl⁻ secretion. Also, they may play a role in the pro-inflammatory molecules release. The ongoing work will confirm interaction between these proteins in the cell lines.

Keywords: annexin A2, calcineurin, CFTR, chloride, monocytes, pro-inflammatory molecules, S100A10

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Authors: O. Ubani, H. I. Atagana, M. S. Thantsha, R. Adeleke

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The oil sludge components (polycyclic aromatic hydrocarbons, PAHs) have been found to be cytotoxic, mutagenic and potentially carcinogenic and microorganisms such as bacteria and fungi can degrade the oil sludge to less toxic compounds such as carbon dioxide, water and salts. In the present study, we isolated different bacteria with PAH-degrading potentials from the co-composting of oil sludge and different animal manure. These bacteria were isolated on the mineral base medium and mineral salt agar plates as a growth control. A total of 31 morphologically distinct isolates were carefully selected from 5 different compost treatments for identification using polymerase chain reaction (PCR) of the 16S rDNA gene with specific primers (16S-P1 PCR and 16S-P2 PCR). The amplicons were sequenced and sequences were compared with the known nucleotides from the gene bank database. The phylogenetical analyses of the isolates showed that they belong to 3 different clades namely Firmicutes, Proteobacteria and Actinobacteria. These bacteria identified were closely related to genera Bacillus, Arthrobacter, Staphylococcus, Brevibacterium, Variovorax, Paenibacillus, Ralstonia and Geobacillus species. The results showed that Bacillus species were more dominant in all treated compost piles. Based on their characteristics these bacterial isolates have high potential to utilise PAHs of different molecular weights as carbon and energy sources. These identified bacteria are of special significance in their capacity to emulsify the PAHs and their ability to utilize them. Thus, they could be potentially useful for bioremediation of oil sludge and composting processes.

Keywords: bioaugmentation, biodegradation, bioremediation, composting, oil sludge, PAHs, animal manures

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Authors: Chaudhary Muhammad Aqdus Ilyas, Matthias Rehm, Kamal Nasrollahi, Thomas B. Moeslund

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This paper presents the investigation of data collection procedures, associated with robots when placed with traumatic brain injured (TBI) patients for rehabilitation purposes through facial expression and mood analysis. Rehabilitation after TBI is very crucial due to nature of injury and variation in recovery time. It is advantageous to analyze these emotional signals in a contactless manner, due to the non-supportive behavior of patients, limited muscle movements and increase in negative emotional expressions. This work aims at the development of framework where robots can recognize TBI emotions through facial expressions to perform rehabilitation tasks by physical, cognitive or interactive activities. The result of these studies shows that with customized data collection strategies, proposed framework identify facial and emotional expressions more accurately that can be utilized in enhancing recovery treatment and social interaction in robotic context.

Keywords: computer vision, convolution neural network- long short term memory network (CNN-LSTM), facial expression and mood recognition, multimodal (RGB-thermal) analysis, rehabilitation, robots, traumatic brain injured patients

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Authors: Reihane Mehrparvar

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Human age could exceed further by altering gene expression through food intaking, although as a consequence of recent century eating patterns, human life-span getting shorter by emerging irregulating in autophagy mechanism, insulin, leptin, gut microbiota which are important etiological factors of type-2 diabetes, obesity, infertility, cancer, metabolic and autoimmune diseases. However, restricted calorie intake and vigorous exercise might be beneficial for losing weight and metabolic regulation in a short period but could not be implementable in the long term as a way of life. Therefore, the lack of a dietary program that is compatible with the genes of the body is essential. Sweet and high-glycemic-index (HGI) foods were associated with type-2 diabetes and cancer morbidity. The neuropsychological perspective characterizes the inclination of sweet and HGI-food consumption as addictive behavior; hence this process engages preference of gut microbiota, neural node, and dopaminergic functions. Moreover, meal composition is not the only factor that affects body hemostasis. In this narrative review, it is believed to attempt to investigate how the body responded to different food intakes and represent an accurate model based on current evidence. Eating frequently and ingesting unassimilable protein and carbohydrates may not be compatible with human genes and could cause impairments in the self-renovation mechanism. This trajectory indicates our body is more adapted to starvation and eating animal meat and marrow. Here has been recommended a model that takes into account three important factors: frequent eating, meal composition, and circadian rhythm, which may offer a promising intervention for obesity, inflammation, cardiovascular, autoimmune disorder, type-2 diabetes, insulin resistance, infertility, and cancer through intensifying autophagy-mechanism and eliminate medical costs.

Keywords: metabolic disease, anti-aging, type-2 diabetes, autophagy

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Authors: Loubna Safar, Lama Youssef, Majd Aljamali

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Age-related macular degeneration (AMD) is one of the leading causes of blindness worldwide. Complement factor H polymorphism (Y402H) is thought to play a potential role in the predisposition to AMD and response of patients with exudative AMD to treatment with anti-Vascular Endothelial Growth Factor (anti-VEGF). This study aimed to investigate the frequency of Y402H among Syrians, its impact on their susceptibility to AMD, and the hypothesized role of Y402H in patients' response to intravitreal anti-VEGF (i.e.,, bevacizumab). Our case-control study encompassed unrelated 54 AMD cases and 44 controls. Genotyping was determined by standard sequencing of PCR products. Frequency was compared between patients and controls, and correlation between genotype and response to treatment was assessed in 20 patients with wet AMD who received a therapeutic course of three intravitreal bevacizumab injections (once monthly). Our results revealed a significantly higher prevalence of the risk allele C among AMD cases (51.9%) in comparison with controls (37.5%) (P= 0.04, OR= 1.386, CI= 0.999- 1.923). Patients with the TT genotype (no risk allele) exhibited a significantly better primary response rate, reached 87.5% compared to only 41.7% in patients carrying the risk allele C (TC + CC), (P= 0.04, OR= 9.8, CI=0.899- 106.84). The findings of this study prove the importance of investigating Y402H polymorphism as a prognostic marker for predicting response to bevacizumab in AMD patients.

Keywords: age-related macular degeneration, bevacizumab, complement factor H gene, polymorphism, Y402H

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Authors: Chiara Colarusso, Michela Terlizzi, Aldo Pinto, Rosalinda Sorrentino

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Cigarette smoking is the main cause and the most common risk factor for both COPD and lung cancer. In our previous studies, we proved that caspase-11 in mice and its human analogue, caspase-4, are involved in lung carcinogenesis and that AIM2 inflammasome might play a pro-cancerous role in lung cancer. Therefore, the aim of this study was to investigate potential crosstalk between COPD and lung cancer, focusing on AIM2 and caspase-11-dependent inflammasome signaling pathway. To mimic COPD, we took advantage of an experimental first-hand smoking mouse model and, to confirm what was observed in mice, we used human samples of lung adenocarcinoma patients stratified according to the smoking and COPD status. We demonstrated that smoke exposure led to emphysema-like features, bronchial tone impairment, and release of IL-1-like cytokines (IL-1α, IL-1β, IL-33, IL-18) in a caspase-1 independent manner in C57Bl/6N. Rather, a dysfunctional caspase-11 in smoke-exposed 129Sv mice was associated to lower bronchial inflammation, collagen deposition, and IL-1-like inflammation. In addition, for the first time, we found that AIM2 inflammasome is involved in lung inflammation in smoking and COPD, in that its expression was higher in smoke-exposed C57Bl/6N compared to 129Sv smoking mice, who instead did not show any alteration of AIM2 in both macrophages and dendritic cells. Moreover, we found that AIM2 expression in the cancerous tissue, albeit higher than non-cancerous tissue, was not statistically different according to the COPD and smoking status. Instead, the higher expression of AIM2 in non-cancerous tissue of smoker COPD patients than smokers who did not have COPD was correlated to a higher hazard ratio of poor survival rate than patients who presented lower levels of AIM2. In conclusion, our data highlight that caspase-11 in mice is associated to smoke-induced lung latent inflammation which could drive the establishment of lung cancer, and that AIM2 inflammasome plays a role at the crosstalk between smoking/COPD and lung adenocarcinoma in that its higher presence is correlated to lower survival rate of smoker COPD adenocarcinoma.

Keywords: COPD, inflammasome, lung cancer, lung inflammation, smoke

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Authors: Soniya Nityanand, Ekta Minocha, Manali Jain, Rohit Anthony Sinha, Chandra Prakash Chaturvedi

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Amniotic fluid stem cells (AFSC) have been shown to contribute towards the amelioration of Acute Renal Failure (ARF), but the mechanisms underlying the renoprotective effect are largely unknown. Therefore, the main goal of the current study was to evaluate the therapeutic efficacy of AFSC in a cisplatin-induced rat model of ARF and to investigate the underlying mechanisms responsible for its renoprotective effect. To study the therapeutic efficacy of AFSC, ARF was induced in Wistar rats by an intra-peritoneal injection of cisplatin, and five days after administration, the rats were randomized into two groups and injected with either AFSC or normal saline intravenously. On day 8 and 12 after cisplatin injection, i.e., day 3 and day7 post-therapy respectively, the blood biochemical parameters, histopathological changes, apoptosis and expression of pro-apoptotic, anti-apoptotic and autophagy-related proteins in renal tissues were studied in both groups of rats. Administration of AFSC in ARF rats resulted in improvement of renal function and attenuation of renal damage as reflected by significant decrease in blood urea nitrogen, serum creatinine levels, tubular cell apoptosis as assessed by Bax/Bcl2 ratio, and expression of the pro-apoptotic proteins viz. PUMA, Bax, cleaved caspase-3 and cleaved caspase-9 as compared to saline-treated group. Furthermore, in the AFSC-treated group as compared to saline-treated group, there was a significant increase in the activation of autophagy as evident by increased expression of LC3-II, ATG5, ATG7, Beclin1 and phospho-AMPK levels with a concomitant decrease in phospho-p70S6K and p62 expression levels. To further confirm whether the protective effects of AFSC on cisplatin-induced apoptosis were dependent on autophagy, chloroquine, an autophagy inhibitor was administered by the intra-peritoneal route. Chloroquine administration led to significant reduction in the anti-apoptotic effects of the AFSC therapy and further deterioration in the renal structure and function caused by cisplatin. Collectively, our results put forth that AFSC ameliorates cisplatin-induced ARF through induction of autophagy and inhibition of apoptosis. Furthermore, the protective effects of AFSC were blunted by chloroquine, highlighting that activation of autophagy is an important mechanism of action for the protective role of AFSC in cisplatin-induced renal injury.

Keywords: amniotic fluid stem cells, acute renal failure, autophagy, cisplatin

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Authors: D. P. Houhoula, J. Papaparaskevas, S. Konteles, A. Dargenta, A. Farka, C. Spyrou, M. Ziaka, S. Koussisis, E. Charvalos

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Objectives: Nanotechnology is providing revolutionary opportunities for the rapid and simple diagnosis of many infectious diseases. Staphylococcus aureus, Listeria monocytogenes and Salmonella enteritis are important human pathogens. Diagnostic assays for bacterial culture and identification are time consuming and laborious. There is an urgent need to develop rapid, sensitive, and inexpensive diagnostic tests. In this study, a gold nanoprobe strategy developed and relies on the colorimetric differentiation of specific DNA sequences based approach on differential aggregation profiles in the presence or absence of specific target hybridization. Method: Gold nanoparticles (AuNPs) were purchased from Nanopartz. They were conjugated with thiolated oligonucleotides specific for the femA gene for the identification of members of Staphylococcus aureus, the mecA gene for the differentiation of Staphylococcus aureus and MRSA Staphylococcus aureus, hly gene encoding the pore-forming cytolysin listeriolysin for the identification of Listeria monocytogenes and the invA sequence for the identification of Salmonella enteritis. DNA isolation from Staphylococcus aureus Listeria monocytogenes and Salmonella enteritis cultures was performed using the commercial kit Nucleospin Tissue (Macherey Nagel). Specifically 20μl of DNA was diluted in 10mMPBS (pH5). After the denaturation of 10min, 20μl of AuNPs was added followed by the annealing step at 58oC. The presence of a complementary target prevents aggregation with the addition of acid and the solution remains pink, whereas in the opposite event it turns to purple. The color could be detected visually and it was confirmed with an absorption spectrum. Results: Specifically, 0.123 μg/μl DNA of St. aureus, L.monocytogenes and Salmonella enteritis was serially diluted from 1:10 to 1:100. Blanks containing PBS buffer instead of DNA were used. The application of the proposed method on isolated bacteria produced positive results with all the species of St. aureus and L. monocytogenes and Salmonella enteritis using the femA, mecA, hly and invA genes respectively. The minimum detection limit of the assay was defined at 0.2 ng/μL of DNA. Below of 0.2 ng/μL of bacterial DNA the solution turned purple after addition of HCl, defining the minimum detection limit of the assay. None of the blank samples was positive. The specificity was 100%. The application of the proposed method produced exactly the same results every time (n = 4) the evaluation was repeated (100% repeatability) using the femA, hly and invA genes. Using the gene mecA for the differentiation of Staphylococcus aureus and MRSA Staphylococcus aureus the method had a repeatability 50%. Conclusion: The proposed method could be used as a highly specific and sensitive screening tool for the detection and differentiation of Staphylococcus aureus Listeria monocytogenes and Salmonella enteritis. The use AuNPs for the colorimetric detection of DNA targets represents an inexpensive and easy-to-perform alternative to common molecular assays. The technology described here, may develop into a platform that could accommodate detection of many bacterial species.

Keywords: gold nanoparticles, pathogens, nanotechnology, bacteria

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