RT-PCR based detection of Pepper mild mottle virus from capsicum seeds and seed transmission assay.

Pepper mild mottle virus (PMMoV), a Tobamovirus from Virgaviridae family, is highly contagious and transmitted by seeds as well as soil in nature. PMMoV has become a greater threat to capsicum cultivation worldwide. To develop an indigenous, rapid, and sensitive protocol for routine detection of PMMoV from seeds, the sensitivity of DAS-ELISA and RT-PCR was compared in the present study. The infected seeds of California Wonder were included in the study. Through DAS-ELISA the virus was successfully detected from 20 mg of seeds. However, using RT-PCR, we were able to detect the virus even from one infected seed with reproducibility. In the present study, vertical seed transmission of the test virus was investigated by employing a grow-out test under greenhouse conditions as well as directly through RT-PCR omitting the grow-out test in three capsicum cultivars. Based on symptoms observations in grow out test, seed transmission was observed in the 3 capsicum cultivars viz., California Wonder (63.04%), Yolo Wonder (33.80%) and Doux des LAndes (33.30%). Through RT-PCR it was estimated to be 55.56% (California Wonder), 28.96% (Yolo Wonder), and 40.64% (Doux des Landes), respectively. Thus, indicating 100% seed-to-seedling PMMoV transmission and reliability of RT-PCR in direct PMMoV detection from seeds. Even a small percentage of infected seed has the potential to greatly increase the PMMoV inoculum in the field and result in 100% plant infection. Therefore, we suggest using the established procedure for PMMoV detection right from the seed. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00807-0.

Electric Fields Regulate In Vitro Surface Phosphatidylserine Exposure of Cancer Cells via a Calcium-Dependent Pathway.

Cancer is the second leading cause of death worldwide after heart disease. The current treatment options to fight cancer are limited, and there is a critical need for better treatment strategies. During the last several decades, several electric field (EF)-based approaches for anti-cancer therapies have been introduced, such as electroporation and tumor-treating fields; still, they are far from optimal due to their invasive nature, limited efficacy and significant side effects. In this study, we developed a non-contact EF stimulation system to investigate the in vitro effects of a novel EF modality on cancer biomarkers in normal (human astrocytes, human pancreatic ductal epithelial -HDPE-cells) and cancer cell lines (glioblastoma U87-GBM, human pancreatic cancer cfPac-1, and MiaPaCa-2). Our results demonstrate that this EF modality can successfully modulate an important cancer cell biomarker-cell surface phosphatidylserine (PS). Our results further suggest that moderate, but not low, amplitude EF induces p38 mitogen-activated protein kinase (MAPK), actin polymerization, and cell cycle arrest in cancer cell lines. Based on our results, we propose a mechanism for EF-mediated PS exposure in cancer cells, where the magnitude of induced EF on the cell surface can differentially regulate intracellular calcium (Ca2+) levels, thereby modulating surface PS exposure.

Heterologous expression of pepper mild mottle virus coat protein encoding region and its application in immuno-diagnostics.

Pepper mild mottle virus (PMMoV), a tobamovirus of family Virgaviridae affects the quality and quantity of Capsicum. PMMoV is highly contagious, capable of transmitting through infected seeds and soil. Symptoms are more severe when crop is infected at young stage but remain unnoticed when infection takes place at maturity. Therefore, cost effective diagnostic techniques are required for timely and accurate detection of virus. In present study, coat protein encoding region of PMMoV-HP1 isolate was cloned into expression vector system, pET28a and expressed in BL21, a protease deficient strain of Escherichia coli. The PMMoV-HP1 pathotype was identified as PMMoV-P12 on the basis of coat protein amino acid sequence analysis in our previous study. The overexpression of recombinant coat protein of 26 kDa, corresponding to the expected 6X Histidine tag fused recombinant protein was purified using Ni-NTA columns from insoluble fraction. For antisera production, the purified recombinant protein was dialyzed ~ 24 h to remove urea and then used for raising polyclonal antisera. The specificity and sensitivity of antiserum obtained was demonstrated using different dilutions of antiserum for western blot assay and direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). In Western blot assay, the test antiserum reacted strongly both with PMMoV-CP in purified protein and native CP in crude sap from PMMoV infected pepper plants, whereas no reaction was observed with healthy plant sap. In DAC-ELISA antiserum dilution up to 1:1000 was capable of detecting the virus in infected sample. The absence of any cross reactivity of test antiserum was confirmed against tobacco mosaic virus, cucumber mosaic virus, tomato spotted wilt virus, pepper veinal mottle virus, potato virus Y and tomato yellow leaf curl virus antigen, known to infect capsicum.

SapC-DOPS - a Phosphatidylserine-targeted Nanovesicle for selective Cancer therapy.

Phosphatidylserine (PS) is normally located in the inner leaflet of the membrane bilayer of healthy cells, however it is expressed at high levels on the surface of cancer cells. This has allowed for the development of selective therapeutic agents against cancer cells (without affecting healthy cells). SapC-DOPS is a PS-targeting nanovesicle which effectively targets and kills several cancer types including pancreatic, lung, brain, and pediatric tumors. Our studies have demonstrated that SapC-DOPS selectively induces apoptotic cell death in malignant and metastatic cells, whereas untransformed cells remain unaffected due to low surface PS expression. Furthermore, SapC-DOPS can be used in combination with standard therapies such as irradiation and chemotherapeutic drugs to significantly enhance the antitumor efficacy of these treatments. While the PS-targeting nanovesicles are a promising selective therapeutic option for the treatment of cancers, more preclinical studies are needed to fully understand the mechanisms leading to non-apoptotic PS expression on the surface of viable cancer cells and to determine the effectiveness of SapC-DOPS in advanced metastatic disease. In addition, the completion of clinical studies will determine therapeutic effects and drug safety in patients. A phase I clinical trial using SapC-DOPS has been completed on patients with solid tumors and has demonstrated compelling patient outcomes with a strong safety profile. Results from this study are informing future studies with SapC-DOPS. Abstract video.