Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation

Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9

Seven days ex vivo perfusion of whole ewe ovaries with follicular maturation and oocyte retrieval: towards the development of an alternative fertility preservation method

Fertility preservation methods for prepubertal women about to undergo gonadotoxic chemo and/or radiation therapy are limited. Therefore, the aim of this study was to investigate the feasibility to develop an alternative fertility preservation method based on an ex vivo perfusion platform for whole ewe ovaries. Thirteen ewe ovaries were divided into two groups (group 1 and 2) that were perfused in a bioreactor for up to 7days. Group 1 (n =3) were stimulated with human menopausal gonadotropin (hMG) administered in single daily dose, while group 2 (n =10) were stimulated continuously for 24h. The perfused ovaries in group 1 showed no significant differences in follicular density, sub-follicular morphology and oocyte quality after ischaemia and after ex vivo perfusion compared with non-perfused control ovaries. The perfused ovaries in group 2 showed a significant decrease in the follicular reserve and oocyte quality compared with the control group. In total, 16 GV-MI oocytes were retrieved from both groups. This study describes for the first time the ex vivo maintenance of viable follicles of ewe ovaries with oocyte integrity and the retrieval of oocytes after ex vivo hormonal perfusion with two different protocols for up to 7days

The SARS‐CoV‐2/COVID‐19 pandemic and challenges in stroke care in India

Stroke care in India has evolved rapidly in the last decade with a focus on stroke awareness, prevention, rapid triage, treatment, and rehabilitation. But acute stroke care and poststroke rehabilitation in the country have limitations owing to the economic constraints and poor access to health care. The SARS-CoV-2/COVID-19 pandemic has made stroke care even more challenging. We outline the unfavorable circumstances in stroke care induced by the pandemic; propose mitigating measures; crisis management; and provide a comparative evaluation of stroke care between India and the United States during the pandemic. There is a need for public health systems in both developed and developing countries to improve awareness, implement proper strategies of triage, acute treatment, well-defined rehabilitation plans, telemedicine services, and virtual check-ins

P–459 Ex vivo perfusion of whole ewe ovaries with follicular maturation for up to seven days: towards the development of an alternative fertility preservation method

Abstract Study question To develop an alternative fertility preservation method for young female cancer patients based on an ex vivo perfusion of whole ovaries serving as a platform for future ovarian stimulation studies. Summary answer It is possible to maintain viable follicles and to retrieve oocytes after ex vivo perfusion of ewe ovaries for up to 7 days. What is known already Some progress has been made in terms of follicular growth and the isolation of mature oocytes in vitro. However, full development, from early follicular stages to a viable offspring, has only been described in rodent models. The complex events controlling follicular expansion and the long time required for folliculogenesis and oocyte maturity in large mammalian species creating challenges and limitations for in vitro studies. Ex vivo perfusion of a whole ovary could potentially be a solution by exploiting the intact ovarian architecture to support folliculogenesis and oocyte maturation. Study design, size, duration Thirty-one ewe ovaries were divided into 4 groups and ex vivo perfused in a bioreactor. Group 1 (n = 14) perfusion for 48 hours with no hormone supplementation; Group 2 (n = 4) perfusion 96–101 hours with follicle stimulating hormone (FSH); Group 3 (n = 3) perfusion 120–168 hours with human menopausal gonadotropin (hMG); Group 4 (n = 10) perfusion 72–144 hours with hMG. Participants/materials, setting, methods Ewe ovaries from sexually mature ewes were ex vivo perfused in a bioreactor under normothermic conditions for up to 7 days (max total 168 hours). Histomorphological, immunohistochemical, hormonal and biochemical analyses were performed to assess ovarian structure and viability after cold ischemia and after perfusion which was subsequently compared to control ovaries. Main results and the role of chance The perfused ovaries in group 2 and 3 showed no significant differences in follicular density, viability and oocyte quality after ischemia and perfusion compared to control ovaries. Estradiol and progesterone levels did not increase during the perfusion. The perfused ovaries in group 1 and 4 showed a significant decrease in the ovarian reserve and oocyte quality. In total, 16 GV-MI oocytes were retrieved from groups 3 and 4. Limitations, reasons for caution 1. Ovaries were retrieved from ewes of unknown cycle and reproductive history. 2. The perfusion medium was changed after 24 hours from perfusion start to remove detrimental metabolites and this could affect the measured concentrations of hormones and metabolites in the perfusion medium. Wider implications of the findings: These results pave the way to propose ex vivo perfusion as a good platform for fertility preservation studies on whole mammalian and human ovaries to retrieve fully mature oocytes. Trial registration number Not applicabl

SRL histochemistry to normal and cancerous human breast tissues.

<p>SRL histochemistry was performed in human breast (normal, primary cancer and metastatic) tissue samples. SRL shows weak binding to normal human breast tissues but strong binding to primary cancer and metastatic breast tissues. All the images were obtained with 100X magnification. Arrows point to SRL binding to apical surface of the secretory gland epithelia. Representative images of both Haematoxylin-Eosin and Biotin-SRL staining are shown. SRL binding was evaluated through optical analysis by measuring the mean area of stained cells scored arbitrarily as intense (+++), moderate (++), light (+) and no staining (−).</p

Effect of SRL on proliferation of non-tumorigenic and normal human breast epithelial cells.

<p>(<b>A and B</b>) Non-tumorigenic MCF-10A and normal HMECs were incubated with different concentrations of SRL in serum free media for different time intervals (24, 48 and 72 h for MCF-10A) and 48 h (HMECs). Cell proliferation was measured using Calcein AM and MTT for MCF-10A and HMEC respectively. The experiments were carried out in triplicate, and the data were expressed as Mean ± SD percentage of control. *p<0.05; ***p<0.001.</p

SRL induces cell apoptosis.

<p>(<b>A</b>) Annexin V cell surface binding in MCF-7 cells after incubation with SRL (20 µg/ml) for 24, 36 or 48 h. Percentages of cells in each quadrant are shown as inserts. The graph indicates percentage of the cell population in different phases. (<b>B</b>) Effect of SRL on cell cycle. MCF-7 cells were exposed to SRL (20 µg/ml) for 24, 36 and 48 h before the cellswere stained with propidium iodide and DNA content was measured by flow cytometry. The graph indicates percentage of cells in subG1, G1, S, and G2–M phases of the cell cycle.</p

Sepharose-conjugated SRL induced growth inhibition of breast cancer cells.

<p>(<b>A</b>) SRL shows strong binding to MCF-7 cells and very weak binding to (<b>B</b>) HMEC. (<b>C</b>) MCF-7 cells were incubated without or with SRL or Sepharose-SRL (C-SRL), at equivalent concentrations to SRL with 128 and 256 hemagglutination units (HAU) activity for 72 h before the cell proliferation was assessed by Calcein AM. Data represent Mean ±SD of triplicateexperiments. *** p<0.0001 when compared to control.</p