Objective: Artemisia annua is the chief source of artemisinin, a potent antimalarial agent, in which other bioactive phytochemicals are also present. Due to low levels of bioactive compounds including artemisinin and flavonoids, it is necessary to increase the level of the secondary metabolites by regulating the expression of rol genes in the plant. Methods: A hybrid variety of A. annua (Hyb1209r, Shennong)developed by the Centre for Novel Agricultural Products, University of York, UK, was selected to produce transgenics of rolB and rolC genes. Genetic transformation was carried out via Agrobacterium tumefaciens GV3101 harboring rolB and rolC genes of Agrobacterium rhizogenes cloned separately. HPLC was used for the qualitative and quantitative analysis of flavonoids and artemisinin. Furthermore, thin layer chromatography (TLC)was also used to analyze artemisinin content. Results: Comparative analysis via HPLC revealed considerable enhancement in the phytochemical content of transgenic A. annua plants as compared to the wild type plant. Transgenics of rolB gene showed an average increase of 321% in rutin, 97.2% in caffeic acid, and 218.4% in myricetin, respectively. In the case of rolC gene transgenics, an average increase of 197.5% in rutin, 76.3% in caffeic acid, and 209.3% in myricetin was observed. Transgenics of rolB and rolC genes showed a 14.3%–28.6% and 2.8%–12.7% increase in artemisinin content respectively by HPLC analysis. TLC analysis showed that an average 142.2% and 110.2% enhancement in artemisinin for rolB and rolC transgenics respectively, compared with the wild type. An enhanced production of total flavonoids (average 30.2% and 25.5% increase in rolB and rolC transgenics, respectively)and total phenolics (average 34.3% and 25.8% increase in rolB and rolC transgenics, respectively)was observed as a result of transformation. Transformed A. annua plants showed improved free radical scavenging activity (average 46.5% and 29.1% increase in rolB and rolC transgenics, respectively)and total reducing power (average 32.7% and 26.4% increase in rolB and rolC transgenics, respectively)compared with untransformed plant. Conclusion: rolB and rolC genes were effective for developing A. annua plants with an enhanced level of phytochemicals.

BACKGROUND: Chronic urticaria is termed as idiopathic if there is an absence of any identifiable causes of mast cell and basophil degranulation. Various cytokines have been found to be involved in inflammatory processes associated with chronic idiopathic urticaria, including interleukin (IL) 18 and IL-6. OBJECTIVE: To evaluate any possible correlation of IL-18 and IL-6 cytokines with the clinical disease severity in chronic idiopathic urticaria (CIU). METHODS: IL-18 and IL-6 levels of CIU patients (n = 62) and healthy controls (n = 27) were assessed by commercially available enzyme linked immunosorbent assay kits following the manufacturer's protocols. RESULTS: Serum IL-18 concentration (mean ± standard deviation [SD], 62.95 ± 36.09 pg/mL) in CIU patients and in healthy controls (54.35 ± 18.45 pg/mL) showed no statistical significance (p > 0.05). No statistically significant difference (p > 0.05) was observed between autologous serum skin test (ASST) positive and ASST negative patients with regard to the serum IL-18 levels either. Similarly, serum IL-6 concentration (0.82 ± 4.6 pg/mL) in CIU patients and in healthy controls (0.12 ± 1.7 pg/mL), showed no statistical significance (p > 0.05). Also, comparison between positive and ASST negative patients with regard to the serum IL-6 levels was statistically nonsignificant (p > 0.05). However, statistical significance was found both in IL-18 and IL-6 concentrations in certain grades with regard to the clinical disease severity of urticaria. CONCLUSION: There is no significant association as such found between IL-18 and IL-6 levels with CIU, however, these cytokines may help in predicting the clinical disease severity in CIU. Hence, these cytokines may indicate a potential role as a biomarker to assess the disease severity in CIU.
Basophils ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-18 ; Interleukin-6 ; Interleukins ; Mast Cells ; Skin Tests ; Urticaria

PURPOSE: The p15(Ink4b) gene exerts its influence as an inhibitor of cyclin-dependent kinases and is frequently associated with hematological malignancies. Inactivation of this gene through DNA methylation has been found to be the most prevalent epigenetic alteration reported, with a high frequency in all French-American-British subtypes of acute myeloid leukemias, including acute promyelocytic leukemia (APL). In this study,we investigated the prognostic significance of p15 gene promoter hypermethylation and its expression in APL patients of Kashmir (North India). MATERIALS AND METHODS: p15 gene promoter hypermethylation was conducted by methylation-specific polymerase chain reaction, while its subsequent expression analysis was carried out by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Of the 37 patients, 16 (43.2%) were found to have methylated p15 genes. Of these 16 cases, seven (43.8%) were methylated partially and nine (56.2%) were found to have complete methylation. Moreover, nine of the 37 patients (24.3%) who presented with leukocytosis at their baseline had complete p15 gene methylation as well (p < 0.05). Semiquantitative RT-PCR showed a complete loss of p15 expression in nine patients with complete methylation coupled with leukocytosis (p=0.031), while seven patients with partial methylation showed decreased p15 expression. Six patients relapsed during the maintenance phase of treatment and were found to have a completely methylated p15 gene and no p15 mRNA. CONCLUSION: Complete methylation and loss of p15 gene expression causes susceptibility to relapse and decreased survival in APL patients. Thus, p15 promoter hypermethylation is a prospective prognostic indicator and a reliable clinical aid in assessment of patients with APL.
Cyclin-Dependent Kinases ; DNA Methylation ; Epigenomics ; Gene Expression ; Hematologic Neoplasms ; Humans ; Leukemia, Myeloid, Acute ; Leukemia, Promyelocytic, Acute* ; Leukocytosis ; Methylation ; Polymerase Chain Reaction ; Prognosis* ; Prospective Studies ; Recurrence ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger

PURPOSE: The p15(Ink4b) gene exerts its influence as an inhibitor of cyclin-dependent kinases and is frequently associated with hematological malignancies. Inactivation of this gene through DNA methylation has been found to be the most prevalent epigenetic alteration reported, with a high frequency in all French-American-British subtypes of acute myeloid leukemias, including acute promyelocytic leukemia (APL). In this study,we investigated the prognostic significance of p15 gene promoter hypermethylation and its expression in APL patients of Kashmir (North India). MATERIALS AND METHODS: p15 gene promoter hypermethylation was conducted by methylation-specific polymerase chain reaction, while its subsequent expression analysis was carried out by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Of the 37 patients, 16 (43.2%) were found to have methylated p15 genes. Of these 16 cases, seven (43.8%) were methylated partially and nine (56.2%) were found to have complete methylation. Moreover, nine of the 37 patients (24.3%) who presented with leukocytosis at their baseline had complete p15 gene methylation as well (p < 0.05). Semiquantitative RT-PCR showed a complete loss of p15 expression in nine patients with complete methylation coupled with leukocytosis (p=0.031), while seven patients with partial methylation showed decreased p15 expression. Six patients relapsed during the maintenance phase of treatment and were found to have a completely methylated p15 gene and no p15 mRNA. CONCLUSION: Complete methylation and loss of p15 gene expression causes susceptibility to relapse and decreased survival in APL patients. Thus, p15 promoter hypermethylation is a prospective prognostic indicator and a reliable clinical aid in assessment of patients with APL.
Cyclin-Dependent Kinases ; DNA Methylation ; Epigenomics ; Gene Expression ; Hematologic Neoplasms ; Humans ; Leukemia, Myeloid, Acute ; Leukemia, Promyelocytic, Acute* ; Leukocytosis ; Methylation ; Polymerase Chain Reaction ; Prognosis* ; Prospective Studies ; Recurrence ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger

In tropical regions, numerous tick-borne pathogens (TBPs) play a crucial role as causative agents of infectious diseases in humans and animals. Recently, the population of companion and pet dogs has significantly increased in Vietnam; however, information on the occurrence of TBPs is still limited. The objectives of this investigation were to determine the occurrence rate, risk factors, and phylogenetic characteristics of TBPs in dogs from northern Vietnam. Of 341 blood samples tested by PCR, the total infection of TBPs was 73.9% (252/341). Babesia vogeli (18SrRNA gene – 30.5%) was detected most frequently in studied dogs followed by Rickettsia spp. (OmpA gene – 27%), Anaplasma platys (groEL gene – 22%), Bartonella spp. (16SrRNA – 18.8%), Mycoplasma haemocanis (16SrRNA – 9.4%) and Hepatozoon canis (18SrRNA gene – 1.2%), respectively. All samples were negative for Ehrlichia canis and Anaplasma phagocytophylum. Co-infection was detected in 31.4% of the samples (107/341) of which, A. platys/Bartonella spp. (34/94,10%), Rickettsia spp./B. vogeli (19/94, 5.6%), and M. haemocanis/B. vogeli (19/94, 5.6%) were recorded as the three most frequent two species of co-infection types. Statistical analysis revealed a significant correlation between TBP infection and several host variables regarding age, breed, and living area in the current study. The recent findings reported herein, for the first time in Vietnam, are essential for local veterinarians when considering the appropriate approaches for diagnosing these diseases. Furthermore, this data can be used to establish control measures for future surveillance and prevention strategies against canine TBPs in Vietnam.