Development of a Simple and Reproducible Method for Removal of Contaminants from Ginseng Protein Samples Prior to Proteomics Analysis

Ravi Gupta(굽타 라비),So Wun Kim(김소운),Chul Woo Min(민철우),Gi-Ho Sung(성기호),Ganesh Kumar Agrawal(아그라왈 가네시 쿠마르),Randeep Rakwal(락왈 랜딥),Ick Hyun Jo(조익현),Kyong Hwan Bang(방경환),Young-Chang Kim(김영창),Kee-Hong Kim(김기홍 한국생명과학회 2015 생명과학회지 Vol.25 No.7

본 연구는 인삼의 잎과 뿌리 단백질 추출물에서 활성탄을 이용하여 염, 계면활성제, 색소를 제거하여 단백질체 분석 연구에 미치는 잠재적인 효과에 대한 평가를 기술하고 있다. 5%(w/v) 활성탄(100-400 mesh)과 함께 단백질 추출물을 30분간 4°C에서 반응시켜 염과 계면활성제를 제거한 후 SDS-PAGE를 분석하여 단백질의 양상을 관찰하였다. 엽록소 함량의 분석은 활성탄 처리 후 엽록소의 상당한 양(~33%)이 제거되는 것을 보여주었고, 이 분석은 염, 계면활성제 제거만이 아닌 색소의 제거에서도 활성탄의 잠재적 효과가 있음을 보여 주고 있다. 활성탄을 처리한 단백질 시료를 이용하여 이차원 전기영동과 PCA 통계분석을 시행한 결과 단백질은 gel에서 더 나은 해상도를 보여주었으며 단백질 시료의 정제에서도 활성탄의 효과가 있음을 확인하였다. 종합적으로, 이 결과들은 활성탄을 이용한 간단한 방법으로 다양한 식물 조직의 단백질 추출물에서 염, 계면활성제, 색소를 제거함으로써 고해상도의 단백질체 분석에 적용될 수 있을 것으로 기대된다. This study describes the effects of activated charcoal on the removal of salts, detergents, and pigments from protein extracts of ginseng leaves and roots. Incubation of protein extracts with 5% (w/v) activated charcoal (100-400 mesh) for 30 min at 4°C almost removed the salts and detergents including NP-40 as can be observed on SDS-PAGE. In addition, analysis of chlorophyll content showed significant depletion of chlorophyll (~33%) after activated charcoal treatment, suggesting potential effect of activated charcoal on removal of pigments too along with the salts and detergents. 2-DE analysis of activated charcoal treated protein samples showed better resolution of proteins, further indicating the efficacy of activated charcoal in clearing of protein samples. In case of root proteins, although not major differences were observed on SDS-PAGE, 2-DE gels showed better resolution of spots after charcoal treatment. In addition, both Hierarchical clustering (HCL) and Principle component analysis (PCA) clearly separated acetone sample from rest of the samples. Phenol and AC-phenol samples almost overlapped each other suggesting no major differences between these samples. Overall, these results showed that activated charcoal can be used in a simple manner to remove the salts, detergents and pigments from the protein extracts of various plant tissues.

Comparative phosphoproteome analysis upon ethylene and abscisic acid treatment in <i>Glycine max</i> leaves

Gupta, Ravi,Min, Cheol Woo,Meng, Qingfeng,Agrawal, Ganesh Kumar,Rakwal, Randeep,Kim, Sun Tae Elsevier 2018 Vol. No.

<P><B>Abstract</B></P> <P>Abscisic acid (ABA) and ethylene play key roles in growth and development of plants. Several attempts have been made to investigate the ABA and ethylene-induced signaling in plants, however, the involvement of phosphorylation and dephosphorylation in fine-tuning of the induced response has not been investigated much. Here, a phosphoproteomic analysis was carried out to identify the phosphoproteins in response to ABA, ethylene (ET) and combined ABA + ET treatments in soybean leaves. Phosphoproteome analysis led to the identification of 802 phosphopeptides, representing 422 unique protein groups. A comparative analysis led to the identification of 40 phosphosites that significantly changed in response to given hormone treatments. Functional annotation of the identified phosphoproteins showed that these were majorly involved in nucleic acid binding, signaling, transport and stress response. Localization prediction showed that 67% of the identified phosphoproteins were nuclear, indicating their potential involvement in gene regulation. Taken together, these results provide an overview of the ABA, ET and combined ABA + ET signaling in soybean leaves at phosphoproteome level.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A total of 802 phosphopeptides representing 422 unique protein groups were identified. </LI> <LI> Of these, 40 phosphosites showed significant change upon hormone treatments. </LI> <LI> Significant (67%) proportion of the identified phosphoproteins were nuclear. </LI> <LI> ABA alters phosphorylation of plasma membrane localized transporters. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Graft tunnel integration occurs early in the tibial tunnel compared with the femoral tunnel after anterior cruciate ligament reconstruction with preserved insertion hamstring tendon graft

( Ravi Gupta ),( Sandeep Singh ),( Anil Kapoor ),( Ashwani Soni ),( Ravinder Kaur ),( Narinder Kaur ) 대한슬관절학회 2021 대한슬관절학회지 Vol.33 No.-

Background: Preservation of hamstring tendon insertion at the time of anterior cruciate ligament (ACL) reconstruction is a well-known technique; however, its effect on graft integration is not well studied. The present study was conducted to study the graft integration inside the tibial and femoral tunnels, respectively, after ACL reconstruction using hamstring tendon graft with preserved insertion. Methods: Twenty-five professional athletes who underwent ACL reconstruction using hamstring tendon graft with preserved tibia insertion were enrolled in the study. Functional outcomes were checked at final follow-up using Lysholm score and Tegner activity scale. Magnetic resonance imaging (MRI) was done at 8 months and 14 months follow-up to study the graft tunnel integration of the ACL graft at both tibial and femoral tunnels. Results: The mean Fibrous interzone (FI) score (tibial tunnel) decreased from 2.61 (1-5) at 8 months to 2.04 (1-4) at 14 months follow-up (p = 0.02). The mean FI score (femoral side) decreased from 3.04 (2-5) at 8 months to 2.57 (2-4) at 14 months (p = 0.02). Conclusions: Graft integration occurs early in the tibial tunnel as compared with the femur tunnel with preserved insertion hamstring tendon autograft. Trial registration CTRI/2019/07/020320 [registered on 22/07/2019]; http:// www. ctri. nic. in/ Clini caltr ials/ pdf_ gener ate. php? trial id= 33884 & EncHid= & modid= & compi d=% 27,% 27338 84det% 27

Peroneal Nerve Dysfunction in Patients with Clubfoot Deformity: Evaluation of Clinical Presentation and Treatment

Parmanand Gupta,Bharath Patil,Prakash Gupta,Rohil Mehta,Ravi Gupta 대한정형외과학회 2021 Clinics in Orthopedic Surgery Vol.13 No.4

Background: Complete peroneal nerve dysfunction associated with congenital clubfoot is uncommonly reported. Our retrospective study highlights the recognition of clinical presentation and mid-term outcomes of treatment in these patients. Methods: Eight out of 658 patients undergoing treatment for clubfoot were identified with unilateral complete peroneal nerve dysfunction associated with congenital clubfoot. Three patients presented primarily to our center; 5 were treated elsewhere initially. All patients were treated with Ponseti casts, Achilles tenotomy, and subsequent foot abduction bracing. Diagnosis of complete peroneal nerve dysfunction was confirmed using nerve conduction velocity studies in all patients. After full-time bracing, an insole polythene molded ankle foot orthosis was given. Three patients underwent tibialis posterior transfer to improve foot dorsiflexor power. Results: The mean age at presentation was 1.3 years (range, 1 week–5 years). All patients had prominence of lateral 3 metatarsal heads and dimpling of intermetatarsal spaces. At a mean follow-up of 5.1 years, mean shortening of 1.2 cm in tibia (range, 1–2.5 cm) and mean calf wasting of 4.4 cm were observed. There was no relapse of any clubfoot deformity till the final follow-up. Conclusions: Prominence of lateral metatarsal heads and dimpling of intermetatarsal spaces should raise early suspicion of peroneal nerve dysfunction. Standard Ponseti protocol is useful in treatment of these patients. Tibialis posterior transfer to dorsum partially restores the ankle dorsiflexion.

A review on change in plant proteome following biotic stress.

R. Krishna,Ravi Gupta,Chul Woo Min,So Wun Kim,Sun Tae Kim 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07

Different biotic agents such as bacteria, fungi, nematode and virus interact with plants, and causes significant annual crop loss. The plants interact with these pathogen and undergo various changes at physiological, biochemical and molecular levels. The omics technique is a powerful way which provides important information related to molecular changes occurring during plant-pathogen interaction. Several studies have been conducted and revealed either up or down-regulation of many genes involved in metabolism, energy, photosynthesis, signaling, defense and ROS upon pathogen interaction. In this review, we highlight recent progress in proteomic studies of plant-pathogen interaction, which could be useful for controlling disease and development of molecular markers for early detection of different diseases.

Skin Cancer Concerns in People of Color: Risk Factors and Prevention

Gupta, Alpana K,Bharadwaj, Mausumi,Mehrotra, Ravi Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.12

Background: Though people of color (POC) are less likely to become afflicted with skin cancer, they are much more likely to die from it due to delay in detection or presentation. Very often, skin cancer is diagnosed at a more advanced stage in POC, making treatment difficult.The purpose of this research was to improve awareness regarding skin cancers in people of color by providing recommendations to clinicians and the general public for early detection and photo protection preventive measures. Methods: Data on different types of skin cancers were presented to POC. Due to limited research, there are few resources providing insights for evaluating darkly pigmented lesions in POC. Diagnostic features for different types of skin cancers were recorded and various possible risk factors were considered. Results: This study provided directions for the prevention and early detection of skin cancer in POC based on a comprehensive review of available data. Conclusions: The increased morbidity and mortality rate associated with skin cancer in POC is due to lack of awareness, diagnosis at a more advanced stage and socioeconomic barriers hindering access to care. Raising public health concerns for skin cancer prevention strategies for all people, regardless of ethnic background and socioeconomic status, is the key to timely diagnosis and treatment.

Global Transcriptome Profiling of Xanthomonas oryzae pv. oryzae under in planta Growth and in vitro Culture Conditions

이소의,Ravi Gupta,Ramesha H. Jayaramaiah,이서현,Yiming Wang,박상렬,김선태 한국식물병리학회 2017 Plant Pathology Journal Vol.33 No.5

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight, is a major threat to rice productivity. Here, we performed RNA-Seq based transcriptomic analysis of Xoo transcripts isolated under in planta growth (on both susceptible and resistant hosts) and in vitro culture conditions. Our in planta extraction method resulted in successful enrichment of Xoo cells and provided RNA samples of high quality. A total of 4,619 differentially expressed genes were identified between in planta and in vitro growth conditions. The majority of the differentially expressed genes identified under in planta growth conditions were related to the nutrient transport, protease activity, stress tolerance, and pathogenicity. Among them, over 1,300 differentially expressed genes were determined to be secretory, including 184 putative type III effectors that may be involved in Xoo pathogenicity. Expression pattern of some of these identified genes were further validated by semi-quantitative RT-PCR. Taken together, these results provide a transcriptome overview of Xoo under in planta and in vitro growth conditions with a focus on its pathogenic processes, deepening our understanding of the behavior and pathogenicity of Xoo.

Proteome Analysis of Disease Resistance against Ralstonia solanacearum in Potato Cultivar CT206-10

Park, Sangryeol,Gupta, Ravi,Krishna, R.,Kim, Sun Tae,Lee, Dong Yeol,Hwang, Duk-ju,Bae, Shin-Chul,Ahn, Il-Pyung The Korean Society of Plant Pathology 2016 Plant Pathology Journal Vol.32 No.1

Potato is one of the most important crops worldwide. Its commercial cultivars are highly susceptible to many fungal and bacterial diseases. Among these, bacterial wilt caused by Ralstonia solanacearum causes significant yield loss. In the present study, integrated proteomics and genomics approaches were used in order to identify bacterial wilt resistant genes from Rs resistance potato cultivar CT-206-10. 2-DE and MALDI-TOF/TOF-MS analysis identified eight differentially abundant proteins including glycine-rich RNA binding protein (GRP), tomato stress induced-1 (TSI-1) protein, pathogenesis-related (STH-2) protein and pentatricopeptide repeat containing (PPR) protein in response to Rs infection. Further, semi-quantitative RT-PCR identified up-regulation in transcript levels of all these genes upon Rs infection. Taken together, our results showed the involvement of the identified proteins in the Rs stress tolerance in potato. In the future, it would be interesting to raise the transgenic plants to further validate their involvement in resistance against Rs in potato.

Glutathione S-transferase M1 and T1 Polymorphisms, Cigarette Smoking and HPV Infection in Precancerous and Cancerous Lesions of the Uterine Cervix

Sharma, Anita,Gupta, Sanjay,Sodhani, Pushpa,Singh, Veena,Sehgal, Ashok,Sardana, Sarita,Mehrotra, Ravi,Sharma, Joginder Kumar Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.15

Glutathione S-transferases (GSTs) play an important role in detoxification of carcinogenic electrophiles. The null genotypes in GSTM1 and GSTT1 have been implicated in carcinogenesis. Present study was planned to evaluate the influence of genetic polymorphisms of GSTM1 and GSTT1 gene loci in cervical carcinogenesis. The study was conducted in Lok Nayak hospital, New Delhi. DNA from clinical scrapes of 482 women with minor gynaecologic complaints attending Gynaecology OPD and tumor biopsies of 135 cervical cancer cases attending the cancer clinic was extracted. HPV DNA was detected by standard polymerase chain reaction (PCR) using L1 consensus primer pair. Polymorphisms of GSTM1 and GSTT1 were analysed by multiplex PCR procedures. Differences in proportions were tested using Pearson's Chi-square test with Odds ratio (OR) and 95% confidence interval (CI). The risk of cervical cancer was almost three times in women with GSTM1 homozygous null genotype (OR-2.62, 95%CI, 1.77-3.88; p<0.0001). No association of GSTM1 or GSTT1 homozygous null genotypes was observed in women with normal, precancerous and cervical cancerous lesions among ${\leq}35$ or >35 years of age groups. Smokers with null GSTT1 genotype had a higher risk of cervical cancer as compared to non-smokers (OR-3.01, 95% CI, 1.10-8.23; p=0.03). The results further showed that a significant increased risk of cervical cancer was observed in HPV positive smoker women with GSTT1 (OR-4.36, 95% CI, 1.27-15.03; p=0.02) and GSTM1T1 (OR-3.87, 95% CI, 1.05-14.23; p=0.04) homozygous null genotypes as compared to HPV positive non smokers. The results demonstrate that the GST null genotypes were alone not associated with the development of cervical cancer, but interacted with smoking and HPV to exert effects in our Delhi population.

Label-free quantitative secretome analysis of <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> highlights the involvement of a novel cysteine protease in its pathogenicity

Wang, Yiming,Gupta, Ravi,Song, Wei,Huh, Hyun-Hye,Lee, So Eui,Wu, Jingni,Agrawal, Ganesh Kumar,Rakwal, Randeep,Kang, Kyu Young,Park, Sang-Ryeol,Kim, Sun Tae Elsevier 2017 Journal of proteomics Vol.169 No.-

<P><B>Abstract</B></P> <P>Bacterial blight, caused by <I>Xanthomonas oryzae</I> pv. <I>oryzae</I> (<I>Xoo</I>), is one of the most devastating diseases resulting in a huge loss of the total rice productivity. The initial interaction between rice and <I>Xoo</I> takes place in the host apoplast and is mediated primarily by secretion of various proteins from both partners. Yet, such secretory proteins remain to be largely identified and characterized. This study employed a label-free quantitative proteomics approach and identified 404 and 323 <I>Xoo</I>-secreted proteins from <I>in vitro</I> suspension-cultured cells and <I>in planta</I> systems, respectively. Gene Ontology analysis showed their involvement primarily in catalytic, transporter, and ATPase activities. Of a particular interest was a <I>Xoo</I> cysteine protease (<I>Xo</I>CP), which showed dramatic increase in its protein abundance <I>in planta</I> upon <I>Xoo</I> interaction with a susceptible rice cultivar. Knock-out mutants of <I>XoCP</I> showed reduced pathogenicity on rice, highlighting its potential involvement in <I>Xoo</I> virulence. Besides, a parallel analysis of <I>in planta</I> rice-secreted proteins resulted in identification of 186 secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. Identified secretory proteins were exploited to shed light on their possible role in the rice-<I>Xoo</I> interaction, and that further deepen our understanding of such interaction.</P> <P><B>Biological significance</B></P> <P> <I>Xanthomonas oryzae</I> pv. <I>oryzae</I> (<I>Xoo</I>), causative agent of bacterial blight disease, results in a huge loss of the total rice productivity. Using a label-free quantitative proteomics approach, we identified 727 <I>Xoo</I>- and 186 rice-secreted proteins. Functional annotation showed <I>Xoo</I> secreted proteins were mainly associated with the catalytic, transporter, and ATPase activities while the rice secreted proteins were mainly associated with the catalytic, antioxidant, and electron carrier activities. A novel <I>Xoo</I> cysteine protease (<I>Xo</I>CP) was identified, showing dramatic increase in its protein abundance <I>in planta</I> upon <I>Xoo</I> interaction with a susceptible rice cultivar. Knock-out mutants of <I>XoCP</I> showed reduced pathogenicity on rice, highlighting its potential involvement in <I>Xoo</I> virulence.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A label-free quantitative proteomics approach was utilized for identification of <I>Xoo</I> secreted proteins. </LI> <LI> A total of 404 and 323 <I>Xoo</I>-secreted proteins were identified and quantified from <I>in vitro</I> and <I>in planta</I> systems respectively. </LI> <LI> A novel cysteine protease (<I>Xo</I>CP) was identified to be involved in <I>Xoo</I> pathogenicity. </LI> <LI> Moreover, 186 secretory proteins were also identified from <I>in-planta</I> secretome. </LI> <LI> Rice secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>