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Hemavathi Dhandapani,Abirami Seetharaman,Hascitha Jayakumar,Selvaluxmy Ganeshrajah,Shirley Sunder Singh,Rajkumar Thangarajan,Priya Ramanathan 대한부인종양학회 2021 Journal of Gynecologic Oncology Vol.32 No.4
Objective: Dendritic cells (DCs) are administered as immunotherapeutic adjuvants after the completion of standard treatment in most settings. However, our Phase I trial indicated that one patient out of four, who received autologous tumor lysate-pulsed dendritic cell (TLDC) also received cisplatin chemotherapy and experienced complete regression of her lung lesion, continuing to be disease free till date. Hence, the objective of our current study is to evaluate the sustenance or augmentation of immune responses when autologous human papillomavirus positive cervical tumor lysate pulsed DC- are combined with cisplatin, using co-culture assays in vitro. Methods: Before treatment, peripheral blood and punch biopsy samples were collected from 23 cervical cancer patients after obtaining an informed consent. DC functionality was confirmed through phenotypic and functional assays using autologous peripheral blood mononuclear cells as responders. For cisplatin experiments, the drug was added at 150, 200 (clinical dose equivalent), and 400 µM concentrations to DCs alone or DC-T cell co- cultures. Phenotypic assessment and functional characterization of DCs was done using flow cytometry. Cytokine enzyme-linked immunosorbent assay and interferon (IFN)-γ enzyme- linked immune absorbent spot assays were also performed. Results: The functionality of TLDCs was not compromised upon cisplatin treatment in vitro even at the highest (400 μM) concentration. Even though cisplatin treatment reduced the secretion of IFN-γ and interleukin (IL)-12p40 in co-cultures stimulated with TLDCs, this effect was not significant (p>0.05). A doubling of IFN-γ secretion following cisplatin treatment was observed in at least one of three independent experiments. Additional experiments showed a reduction in both FOXP3+ regulatory T cells and IL-10 levels. Conclusion: Our results provide evidence that cisplatin treatment may be given after autologous TLDC administration to maintain or improve a productive anti-tumor response in vaccinated patients.
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RNA Interference Silencing in Root-knot Nematodes
Hemavathi Ajjappala,Joon-Soo Sim,Bum-Soo Hahn 한국국제농업개발학회 2012 韓國國際農業開發學會誌 Vol.24 No.4
RNAi 기법은 예쁜꼬마선충을 비롯하여 초파리, 생쥐, 인체및 식물들을 포함한 다양한 생물종에서 작용기작이 잘 알려져 있고, 특정 유전자들의 발현을 제어하기 위해 사용되어 왔다.본 논문은 RNAi 기법을 통해 구축된 뿌리혹선충 저항성 형질전환식물체들의 개발현황, 발전 및 응용 가능성에 대해 고찰하고자 하였다. 지난 10여년에 걸쳐 진행된 연구들을 통해 다양한 분화단계의 뿌리혹선충으로부터 분석된 79,978개의 EST가 GenBank에등록되었고, 고구마뿌리혹선충(M. incognita)과 당근뿌리혹선충(M. hapla)의 전체 게놈 염기서열이 해독됨으로써 뿌리혹선충의 기생에 관련된 단백질들 및 식물세포벽 분해 관련 효소들에 대한 정보를 이용할 수 있게 되었다. RNAi의 기본 기작은 모든 진핵생물종에서 잘 보존되어 있고, 최근 식물기생선충들에서 RNAi 효과에 대한 연구결과들이 많이 발표되었다. 서로 다른 뿌리혹선충들에서 현재까지 22종 이상의 RNAi를위한 목표 유전자들이 보고되었다. 제2령 식물기생선충(second-stage juvenile of plant parasitic nematodes)에게dsRNA의 섭취를 유도하는 octopamine, resorcinol, serotonin등의 화합물들이 발견되었고, 이를 이용하여 선충 유전자의 발현제어를 쉽게 판별할 수 있는 새로운 기술의 활용이 가능하게 되었다. 최근에 RNAi 기술의 응용 및 발전을 통해 형질전환식물체에서 발현된 dsRNA에 의한 식물기생선충 유전자들의 발현제어가 증명되었고, 이를 위한 핵심 요소들로서 적절한 선충 표적 유전자의 선택, 식물체 내 높은 함량의 dsRNA의 발현 및선충이 섭취할 수 있는 충분한 양의 dsRNA의 운반 등이 중요하다는 것이 밝혀졌다. 특히, 비표적 유전자서열(off-target gene sequence)의 발현제어를 피하기 위해 다음과 같은 사항들이 고려되어야 한다. 1)비표적 유전자서열을 확인할 수 있는 소프트웨어의 개발 및 이를 통한 비표적 유전자서열을 제거한 RNAi 벡터를 제작하여야 한다. 2)식물과 동물에서 상동성이 높은 표적 유전자의 발현을 피해야 한다. 3)전사해석틀(open reading frames)의 염기서열들 보다 상동성이 낮은 5' 혹은 3'-비해석부위(untranslated regions)로부터 표적 유전자를설계하여야 한다. The root-knot nematodes (RKN) belonging to the genus Meloidogyne are obligate and sedentary endoparasites that infect wide range of plant species. By parasitizing the root system, they disrupt the water and nutrient uptake thus affecting the whole plant causing significant reduction in the crop yield. They pose huge losses of several million dollars worldwide. Use of chemical nematicide, including soil fumigants such as methyl bromide was the most reliable strategy of controlling the rootknot nematodes. Recently, molecular strategies such as RNA interference (RNAi) mediated nematode gene silencing, R gene transformation, expression of proteins detrimental to RKN are gaining importance. RNAi is used for downregulating the expression of specific gene in several organisms, particularly free-living nematode Caenorhabditis elegans. RNAi strategy has been widely used for silencing the target genes in plant parasitic nematodes. Nearly 79,978 ESTs analysed from various developmental stages of Meloidogyne species has gained entry into the GenBank. The complete genome sequencing of M. incognita and M. hapla provides information regarding the parasitism genes and their success in parasitizing the plants. Recent RNAi approach has resulted in successful reports suggesting suppression of genes essential for nematode development, survival and parasitism in plants. These studies may further serve as a foundation for identification of novel genes and their role in parasitism and could be used as target genes for silencing thus conferring resistance to plants. This review focuses on the progress made towards the development of transgenic plants resistant to RKN through RNAi approach.
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연구보문 : 자연과학 ; RNAi 기술을 이용한 뿌리혹선충 저항성 식물 개발 동향
Hemavathi Ajjappala,심준수,한범수 韓國國際農業開發學會 2012 韓國國際農業開發學會誌 Vol.24 No.4
The root-knot nematodes (RKN) belonging to the genus Meloidogyne are obligate and sedentary endoparasites that infect wide range of plant species. By parasitizing the root system, they disrupt the water and nutrient uptake thus affecting the whole plant causing significant reduction in the crop yield. They pose huge losses of several million dollars worldwide. Use of chemical nematicide, including soil fumigants such as methyl bromide was the most reliable strategy of controlling the rootknot nematodes. Recently, molecular strategies such as RNA interference (RNAi) mediated nematode gene silencing, R gene transformation, expression of proteins detrimental to RKN are gaining importance. RNAi is used for downregulating the expression of specific gene in several organisms, particularly free-living nematode Caenorhabditis elegans. RNAi strategy has been widely used for silencing the target genes in plant parasitic nematodes. Nearly 79,978 ESTs analysed from various developmental stages of Meloidogyne species has gained entry into the GenBank. The complete genome sequencing of M. incognita and M. hapla provides information regarding the parasitism genes and their success in parasitizing the plants. Recent RNAi approach has resulted in successful reports suggesting suppression of genes essential for nematode development, survival and parasitism in plants. These studies may further serve as a foundation for identification of novel genes and their role in parasitism and could be used as target genes for silencing thus conferring resistance to plants. This review focuses on the progress made towards the development of transgenic plants resistant to RKN through RNAi approach.
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RNA Interference Silencing in Root-knot Nematodes
Hemavathi Ajjappala,심준수,한범수 한국국제농업개발학회 2012 韓國國際農業開發學會誌 Vol.24 No.4
The root-knot nematodes (RKN) belonging to the genus Meloidogyne are obligate and sedentary endoparasites that infect wide range of plant species. By parasitizing the root system, they disrupt the water and nutrient uptake thus affecting the whole plant causing significant reduction in the crop yield. They pose huge losses of several million dollars worldwide. Use of chemical nematicide, including soil fumigants such as methyl bromide was the most reliable strategy of controlling the rootknot nematodes. Recently, molecular strategies such as RNA interference (RNAi) mediated nematode gene silencing, R gene transformation, expression of proteins detrimental to RKN are gaining importance. RNAi is used for downregulating the expression of specific gene in several organisms, particularly free-living nematode Caenorhabditis elegans. RNAi strategy has been widely used for silencing the target genes in plant parasitic nematodes. Nearly 79,978 ESTs analysed from various developmental stages of Meloidogyne species has gained entry into the GenBank. The complete genome sequencing of M. incognita and M. hapla provides information regarding the parasitism genes and their success in parasitizing the plants. Recent RNAi approach has resulted in successful reports suggesting suppression of genes essential for nematode development, survival and parasitism in plants. These studies may further serve as a foundation for identification of novel genes and their role in parasitism and could be used as target genes for silencing thus conferring resistance to plants. This review focuses on the progress made towards the development of transgenic plants resistant to RKN through RNAi approach.
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연구보문 : 참외모상근 내에 인체 조직형 플라스미노겐 활성화인자 변이체들의 발현
헤마바티 ( Hemavathi Ajjappala ),심준수 ( Joon Soo Sim ),김용환 ( Yong Hwan Kim ),한범수 ( Bum Soo Hahn ) 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.2
본 연구는 뇌졸중, 심근경색과 폐색전증에 사용되는 t-PA 단백질 변이체를 참외모상근에서 생산하고자 수행되었다. t-PA 변이체 유전자가 함유된 A. rhizogene으로 형질전환된 참외모상근에서 t-PA 변이체 단백질들이 성공적으로 발현되는 것을 확인하였으며, 발현된 t-PA변이체 단백질들은 동물세포에서 발현되어 상업적으로 판매되는 t-PA와 시험관 내의 인공 혈전 용해 활성이 동등함을 확인할 수 있었다. 1. t-PA 변이체 유전자들의 참외모상근 genomic DNA내의 삽입은 PCR법으로 t-PA 유전자의 PCR 산물(1.6 kb)을 관찰하여 참외 모상근 염색체 내에 삽입되어 있음을 확인하였다. 2. t-PA 변이체 유전자들의 전사는 RT-PCR법을 활용하여 t-PA transcript 크기에 해당하는 1.6 kb의 PCR 산물을 관찰하여 참외모상근에 정상적인 전사가 이뤄지고 있음을 확인하였다. 3. 형질전환된 참외모상근 추출물 내에 존재하는 t-PA 변이체 단백질들의 피브린 분해 활성은 상업적으로 판매되는 재조합의 t-PA 단백질과 동등하였다. 4. 형질전환된 참외모상근의 전체 수용성 단백질 중 t-PA 변이체 단백질의 평균 발현량은 전체 수용성 단백질 mg 당 0.47(t-PAer)과 0.57(t-PAhis6)로 측정되었으며, 가장 높은 발현량은 t-PAhis6로 형질전환된 모상근에서 전체 수용성 단백질 mg 당 0.65 μg으로 관찰되었다. 5. 형질전환된 모상근에서 발현된 재조합 t-PA 변이체 단백질들의 분자량은 동물세포에서 발현된 t-PA와 유사한 59 kDa으로 확인하였다. Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots in the blood vessels by activating plasminogen to plasmin. Human tissueplasminogen activator, its derivatives and synthetic genes were expressed as enzymatically active form in the hairy roots of Cucumis melo L. cv. Geumssaragi-euncheon (Oriental melon) infected by Agrobacterium rhizogene harboring binary vectors. Hairy roots were produced from the wounded surface of the cotyledon explants of Oriental melon on MS selective medium containing 300 μg/ml kanamycin and 500μg/ml carbenicillin. PCR analysis revealed the insertion of the t-PA genes in genomic DNA of transgenic hairy roots. The presence of the t-PA-specific transcripts in the total RNAs of transgenic hairy roots was confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single distinct band of 59-kDa of recombinant t-PAs. ELISA experiments demonstrated the maximum level of recombinant t-PA expression up to 0.065% of the total soluble protein in hairy roots transformed by plasmid p221a-t- PAhis6. The highest fibrinolysis of recombinant t-PAs was detected from the hairy roots expressing plasmid p221a-t-PAhis6, respectively. These studies demonstrated that hairy roots could be successfully employed for the mass production of an enzymatically active t-PA.
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A. B. Hemavathi,K. S. M. S. Raghavarao 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.2
Tangential flow ultrafiltration with a polyethersulfone (100 kDa) membrane was used for the purification and concentration of β-glycosidases (β-galactosidase and β-glucosidase) from aqueous extract of barley. The performances of mode 1 (concentration followed by diafiltration)and mode 2 (diafiltration followed by concentration) were compared. In mode 1 activity recoveries of 91.44 and 88%as well as purifications of 1.84 and 1.77-fold for β-galactosidase and β-glucosidase, respectively, were obtained in a total processing time of about 9 h. In mode 2 activity recoveries of 95.68 and 91.76% with purifications of 4.56and 4.38-fold for β-galactosidase and β-glucosidase,respectively, were obtained in a total processing time of about 6 h. The removal of total carbohydrates and protein was 56.74 and 50.73%, respectively, in mode 1, whereas it was 81.46 and 79.04%, respectively, in mode 2. The diafiltration volume and volume concentration of 3 were maintained in both mode 1 and 2. Flux decline was severe in mode 1 and led to a long processing time of about 9 h. These results indicate that mode 2 was better than mode 1for purification of β-glycosidases.
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Study on line graph of some graph operators of chemical structures via $F$ and $M_1$ indices
P.S. Hemavathi,Manjunath Muddalapuram,Pralahad Mahagaonkar,S.M. Veeresh 한국전산응용수학회 2024 Journal of applied mathematics & informatics Vol.42 No.1
The Topological indices are known as Mathematical characterization of molecules. In this paper, we have studied line graph of subdivision and semi-total point graph of triangular benzenoid, polynomino chains of 8-cycles and graphene sheet through forgotten and first Zagreb indices.
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