Molecular docking of the pentapeptide derived from rice bran protein as anticancer agent inhibiting both receptor and non-receptor tyrosine kinases.

The cationic pentapeptide Glu-Gln-Arg-Pro-Arg (EQRPR) belongs to the family of anti-cancer peptides with significant anti-cancer activity. However, the mechanism by which the peptide performs this activity is unknown. In this study, we explored the pharmaceutical profile of Glu-Gln-Arg-Pro-Arg pentapeptide and revealed its anticancer properties by in silico docking studies. Moreover, the effect of EQRPR behavior of the DPPC membrane was investigated by means of Langmuir monolayer technique and the results were discussed in terms of mutual interactions. To evaluate the binding mechanisms, the pentapeptide and its various D-amino acid substituted analogs were docked to both epidermal growth factor receptor (EGFR) tyrosine kinase and proto-oncogene tyrosine-protein kinase, Fyn. Simultaneous binding of the pentapeptides to both EGFR and Fyn proteins, which are receptor- and non-receptor-kinases, respectively, suggest that these peptides can be an effective agent for cancer treatment. Moreover, to show the potential of the investigated pentapeptides to overcome the generated mutation-related drug resistance to EGFR targeted therapies, molecular docking investigations of EQRPR and all its D-analogs were performed against the prospective targets: Wild type EGFRWT and mutant EGFRT790M. Erlotinib and TAK-285 were used as reference molecules. The strong interaction of the peptide with EGFRWT (from -9.24 to -9.75 kcal/mol) and the secondary mutant EGFRT790M (from -9.28 to -9.64 kcal/mol) observed in most cancer recurrence cases indicates its good potential to overcome drug resistance in cancer therapy. In addition, the pharmacological properties of the investigated pentapeptides were revealed by in silico ADME (Absorption, Distribution, Metabolism, Excretion) and toxicity analysis.Communicated by Ramaswamy H. Sarma.

Sodium fusidate prevents protein aggregation of silk fibroin and offers new perspectives for human lens material disaggregation.

Silk fibroin (SF) is a non-pathological amyloidogenic protein prone, in solution, to the formation of amyloid-like aggregated species, displaying similarities in fibrillation kinetics with pathological amyloids, as widely reported in the literature. We show here, on the basis of different biophysical approaches (turbidity, Congo Red assays, CD, DLS and fluorescence), that fusidic acid (FA), a well-known antibiotic, acts on SF as an anti-aggregating agent in a dose-dependent manner, being also able to revert SF aggregation. FA binds to SF inducing changes in the environment of SF aromatic residues. We further provide the proof of principle that FA, already approved as drug on humans and used in ophthalmic preparations, displays its anti-aggregation properties also on lens material derived from cataract surgery and is capable of reducing aggregation. Thus it is suggested that FA can be foreseen as a therapeutic treatment for cataract and other protein aggregation disorders.

Evaluation of anti-cancer and anti-covid-19 properties of cationic pentapeptide Glu-Gln-Arg-Pro-Arg, from rice bran protein and its d-isomer analogs through molecular docking simulations.

Bioactive peptides derived from food proteins are becoming increasingly popular due to the growing awareness of their health-promoting properties. The structure and mechanism of anti-cancer action of pentapeptide Glu-Gln-Arg-Pro-Arg (EQRPR) derived from a rice bran protein are not known. Theoretical and experimental methods were employed to fill this gap. The conformation analysis of the EQRPR pentapeptide was performed first and the obtained lowest energy conformer was optimized. The experimental structural data obtained by FTIR and CD spectroscopies agree well with the theoretical results. d-isomer introduced one-by-one to each position and all D-isomers of the peptide were also examined for its possible anti-proteolytic and activity enhancement properties. The molecular docking revealed avid binding of the pentapeptide to the integrins α5ß1 and αIIbß3, with Kd values of 90 nM and 180 nM, respectively. Moreover, the EQRPR and its D-isomers showed strong binding affinities to apo- and holo-forms of Mpro, spike glycoprotein, ACE2, and dACE2. The predicted results indicate that the pentapeptide may significantly inhibit SARS-CoV-2 infection. Thus, the peptide has the potential to be the leading molecule in the drug discovery process as having multifunctional with diverse biological activities.

Individual-oocyte transcriptomic analysis shows that genotoxic chemotherapy depletes human primordial follicle reserve in vivo by triggering proapoptotic pathways without growth activation.

Gonadotoxic chemotherapeutics, such as cyclophosphamide, can cause early menopause and infertility in women. Earlier histological studies showed ovarian reserve depletion via severe DNA damage and apoptosis, but others suggested activation of PI3K/PTEN/Akt pathway and follicle 'burn-out' as a cause. Using a human ovarian xenograft model, we performed single-cell RNA-sequencing on laser-captured individual primordial follicle oocytes 12 h after a single cyclophosphamide injection to determine the mechanisms of acute follicle loss after gonadotoxic chemotherapy. RNA-sequencing showed 190 differentially expressed genes between the cyclophosphamide- and vehicle-exposed oocytes. Ingenuity Pathway Analysis predicted a significant decrease in the expression of anti-apoptotic pro-Akt PECAM1 (p = 2.13E-09), IKBKE (p = 0.0001), and ANGPT1 (p = 0.003), and reduced activation of PI3K/PTEN/Akt after cyclophosphamide. The qRT-PCR and immunostaining confirmed that in primordial follicle oocytes, cyclophosphamide did not change the expressions of Akt (p = 0.9), rpS6 (p = 0.3), Foxo3a (p = 0.12) and anti-apoptotic Bcl2 (p = 0.17), nor affect their phosphorylation status. There was significantly increased DNA damage by γH2AX (p = 0.0002) and apoptosis by active-caspase-3 (p = 0.0001) staining in the primordial follicles and no change in the growing follicles 12 h after chemotherapy. These data support that the mechanism of acute follicle loss by cyclophosphamide is via apoptosis, rather than growth activation of primordial follicle oocytes in the human ovary.

Comparison of 1-year results of single transforaminal epidural steroid injection among patients with different spinal pathologies-related radicular pain.

AIMS: This study aims to investigate the effectiveness of transforaminal epidural steroid injection (TFESI) in patients with lumbar radicular pain or radiculopathy caused by different spinal pathologies. METHODS: One hundred and seventy seven patients who underwent single transforaminal epidural steroid injection were included in the study group and divided into 3 subgroups (central spinal stenosis + lateral recess stenosis, foraminal stenosis, lumbar disc herniation) according to existing spinal pathology. Patients' visuel analogue scale (VAS) measures and Oswestry Disability Index (ODI) scores were recorded and the patients who give favourable response to treatment were called respondents and who were not called as non-respondents. Subgroups were compared statistically at the end of 12 months. RESULTS: Sixty patients (33.9%) were considered as respondents and 117 patients (66.1%) were non-respondents in the entire study group. Patients with foraminal stenosis included the vast majority of the respondents and showed better results of pain relief as opposed to patients of other groups at the end of 12 months (P < 0.001). CONCLUSION: TFESI was an effective treatment modality for pain relief and functional improvement in patients with foraminal stenosis. However, it could not produce the same results in patients with central spinal stenosis and lumbar disc herniations.

Oocyte cryopreservation for fertility preservation in postpubertal female children at risk for premature ovarian failure due to accelerated follicle loss in Turner syndrome or cancer treatments.

OBJECTIVE: To preliminarily study the feasibility of oocyte cryopreservation in postpubertal girls aged between 13 and 15 years who were at risk for premature ovarian failure due to the accelerated follicle loss associated with Turner syndrome or cancer treatments. DESIGN: Retrospective cohort and review of literature. SETTING: Academic fertility preservation unit. PARTICIPANTS: Three girls diagnosed with Turner syndrome, 1 girl diagnosed with germ-cell tumor. and 1 girl diagnosed with lymphoblastic leukemia. INTERVENTIONS: Assessment of ovarian reserve, ovarian stimulation, oocyte retrieval, in vitro maturation, and mature oocyte cryopreservation. MAIN OUTCOME MEASURE: Response to ovarian stimulation, number of mature oocytes cryopreserved and complications, if any. RESULTS: Mean anti-müllerian hormone, baseline follical stimulating hormone, estradiol, and antral follicle counts were 1.30 ± 0.39, 6.08 ± 2.63, 41.39 ± 24.68, 8.0 ± 3.2; respectively. In Turner girls the ovarian reserve assessment indicated already diminished ovarian reserve. Ovarian stimulation and oocyte cryopreservation was successfully performed in all female children referred for fertility preservation. A range of 4-11 mature oocytes (mean 8.1 ± 3.4) was cryopreserved without any complications. All girls tolerated the procedure well. CONCLUSIONS: Oocyte cryopreservation is a feasible technique in selected female children at risk for premature ovarian failure. Further studies would be beneficial to test the success of oocyte cryopreservation in young girls.

Fertility preservation options in women with endometriosis.

Endometriosis is a common, chronic condition in reproductive age women. Although some women may be asymptomatic, most women present with dysmenorrhea, dyspareunia, pelvic pain and/or infertility. Despite the fact that a causal relationship between endometriosis and infertility has not been clearly established, the fecundity rate of untreated women with endometriosis is lower than normal couples. However, suppressive medical therapy for endometriosis has not been shown to improve fecundity rates and may only result in a delay in the use of more effective treatments to achieve pregnancy. In the other hand, surgery for severe endometriosis can be useful to treat infertile women, but several studies reported a lower ovarian reserve after excision of ovarian endometriomas, due to incidental excision of normal ovarian tissue together with the endometrioma wall. Therefore, fertility preservation procedures should be considered to reproductive-age women at risk of impaired fertility related to endometriosis progression or endometriosis surgical treatment. The purpose of this document was to review the current literature regarding fertility preservation techniques for patients diagnosed with endometriosis.

Ovarian cryopreservation.

Cryopreservation of ovarian tissue and future autotransplantation are new promising strategies for fertility preservation in various malignant and non-malignant diseases facing the risk of ovarian failure. Ovarian cortrical tissue pieces or intact whole ovary can be removed by laparoscopy without any significant delay in chemotherapy. Slow freezing and vitrification methods are developed to avoid damage to follicles. Ovarian tissue can be transplanted in an orthotopic or heterotopic location when the patient is cured from the disease. Autotransplantation can be performed if absence of malignant cells in the graft is confirmed. Although the procedures are still experimental, ovarian cryopreservation is the single option in prepubertal girls who have not sexual maturity. Earlier team approach of oncologists and reproductive endocrinologists may provide a more successful and professional way of fertility preservation.

In vitro maturation improves oocyte or embryo cryopreservation outcome in breast cancer patients undergoing ovarian stimulation for fertility preservation.

This study tested in-vitro maturation (IVM) as a complementary strategy to improve the mature oocyte yield of breast cancer patients undergoing ovarian stimulation for fertility preservation. Secondary analysis of prospectively collected data is performed for 32 breast cancer patients undergoing oocyte or embryo cryopreservation before chemotherapy. Total number of oocytes and/or embryos cryopreserved following IVM is compared with the total number cryopreserved before IVM. Overall, 464 oocytes were retrieved, of which 274 were mature. Following IVM, the number of total mature oocytes increased to 399 (45% increase in mature oocyte yield, P<0.0001). Fertilization rate after IVM was statistically significantly higher than the fertilization of already mature oocytes at retrieval (86% versus 73%, respectively, P<0.05). The total number of oocytes and embryos frozen before IVM was 207 (45% of all oocytes retrieved). This number increased to 320 (69% of all oocytes retrieved) following IVM (P<0.0001). IVM is a useful strategy to improve the mature oocyte yield of fertility preservation cycles. Immature oocytes retrieved during oocyte/embryo cryopreservation cycles should not be discarded to improve the future potential of fertility.

Exfoliative epitheliopathy of bullous keratopathy with breaches in the MUC16 Glycocalyx.

PURPOSE: Expression of cellular adhesion molecules is altered in bullous keratopathy. The hypothesis that epithelial alterations in bullous keratopathy compromise the surface of the cornea and its glycocalyx was tested. METHODS: Studies were performed on eight cases each of pseudophakic bullous keratopathy and healthy corneas. The number of epithelial cell layers was determined with a stereological method of point counting. The minimum distance between points was established by estimates of cell size with variable pressure scanning electron microscopy performed in backscatter mode. The mean number of cell layers with mucin expression was identified by immunohistochemistry with mouse monoclonal antibodies for MUC1 and MUC16. Data were analyzed by Student's t-test if values showed a normal distribution or, alternatively, by the Wilcoxon rank-sum test. RESULTS: Mean numbers of wing cell and superficial cell layers were lower in bullous keratopathy specimens (1.6 vs. 2.0; P < 0.0001) than in controls (1.1 vs. 1.8; P < 0.000001). The number of exfoliated cell layers evident in sections was increased in the bullous keratopathy specimens compared with controls (0.36 vs. 0.03; P < 0.0001). The number of cell layers decorated with antibodies to MUC16 was lower in bullous keratopathy specimens than in controls (0.5 vs. 1.2; P < 0.025). The reduction of layers expressing MUC1 in bullous keratopathy was not statistically significant. CONCLUSIONS: Pseudophakic bullous keratopathy manifests an abnormal corneal ocular surface in which superficial cell layers are exfoliated, leaving breaches in the protective MUC16 glycocalyx. The results provide a morphologic correlate for the surface epithelial abnormalities noted clinically in these patients.

Tear lipocalin: evidence for a scavenging function to remove lipids from the human corneal surface.

PURPOSE: Lipid contamination of the cornea may create an unwettable surface and result in desiccation of the corneal epithelium. Tear lipocalin (TL), also known as lipocalin-1, is the principal lipid-binding protein in tears. TL has been shown to scavenge lipids from hydrophobic surfaces. The hypothesis that TL can remove contaminating fatty acids and phospholipids from the human corneal surface was tested. METHODS: TL was purified from pooled human tear samples by size exclusion and ion exchange chromatographies. Tears depleted of TL were reconstituted from fractions eluted by size exclusion chromatography that did not contain TL. Fresh and formalin-fixed human corneas were obtained from exenteration specimens. Fluorescent analogs of either palmitic acid or phosphatidylcholine were applied to the corneal epithelial surface. Tears, TL, or tears depleted of TL were applied over the corneas, and spectrofluorometry and fluorescent stereomicroscopy were used to monitor the removal of fluorescent lipids. Tears used in the experiments were then fractionated by size exclusion chromatography to determine the component of tears associated with fluorescent lipids. RESULTS: Significant enhancement of fluorescence for 16AP and NBD C(6)-HPC was evident in solutions incubated with whole tears and purified TL but not with tears depleted of TL for fixed and unfixed corneas. After the experiment, size exclusion fractions of tears showed that the fluorescence component coeluted with TL. CONCLUSIONS: TL scavenges lipids from the human corneal surface and delivers them into the aqueous phase of tears. TL may have an important role in removing lipids from the corneal surface to maintain the wettability and integrity of the ocular surface.

Fertility preservation in breast cancer patients: IVF and embryo cryopreservation after ovarian stimulation with tamoxifen.

BACKGROUND: Breast cancer chemotherapy commonly causes premature ovarian failure and infertility. Because increased estrogen levels are thought to be potentially risky in breast cancer patients, natural cycle IVF (NCIVF) has been used to preserve fertility and treat infertility in these women. METHODS: Twelve women with breast cancer received 40-60 mg tamoxifen for 6.9 +/- 0.6 days beginning on days 2-3 of their menstrual cycle (15 cycles), and had IVF (TamIVF) with either fresh embryo transfer (six cycles) or cryopreservation (nine cycles). They were compared to a retrospective control group (n = 5) who had natural cycle IVF (NCIVF, nine cycles). RESULTS: Cycle cancellation was significantly less frequent in TamIVF, compared with NCIVF (1/15 versus 4/9, P < 0.05). Compared with NCIVF, TamIVF patients had a greater number of mature oocytes (1.6 +/- 0.3 versus 0.7 +/- 0.2, P = 0.03) and embryos (1.6 +/- 0.3 versus 0.6 +/- 0.2, P = 0.02) per initiated cycle. TamIVF resulted in the generation of embryo(s) in every patient (12/12) while only three out of five patients had an embryo following NCIVF. Two out of six patients in TamIVF, and 2/5 in NCIVF conceived. One patient in the TamIVF group delivered a set of twins. After a mean follow up of 15 +/- 3.6 months (range 3-54), none of the patients had a recurrence of cancer. CONCLUSIONS: Tamoxifen stimulation appears to result in a higher number of embryos and may provide a safe method of IVF and fertility preservation in breast cancer patients.

RET and anisotropy measurements establish the proximity of the conserved Trp17 to Ile98 and Phe99 of tear lipocalin.

Previous studies suggest that the conserved Trp17 on strand A of TL has a role in lipocalin stability and interacts, directly or indirectly, with Ile98 and Phe99 on strand G to influence ligand binding. Here, we determined the proximity of Trp17 to Ile98 and Phe99. Time-resolved fluorescence experiments showed resonance energy transfer between tryptophans at positions 17 and 98. In addition, an exciton effect was discovered in CD experiments resulting from interactions of the excited states of these tryptophans. Fluorescence anisotropy values of mutants containing two tryptophans (positions 99/17 and 98/17) were lower than expected in the absence of RET, confirming that these residues are proximate in tear lipocalin. The data support a model of tear lipocalin in which Trp17 and Phe99 are close together deep in the cavity and participate in an internal hydrophobic cluster. Ile98 is proximate to Trp17 but faces toward the outside of the cavity and in the model is part of an external hydrophobic patch. Comparison with beta-lactoglobulin suggests that these motifs may have an important influence on protein stability and ligand binding in other members of the lipocalin family.

Ovarian tissue cryopreservation and transplantation: preliminary findings and implications for cancer patients.

Cancer treatment modalities are increasingly more effective in achieving complete remission and cure. Aggressive chemotherapy and radiotherapy, as well as bone marrow transplantation, results in >90% cure in many cancers of children and young women. As a result of this success however, a new problem has arisen. Many young women survive to live the rest of their lives in menopause, and have no chance of conceiving on their own. Oocyte cryopreservation has resulted in a handful of pregnancies, but the technique may not be applicable to young women and children. Ovarian tissue cryopreservation and transplantation has emerged against this background, first in successful rodent studies, and then in sheep and human ovarian xenograft studies. Because of the encouraging results with animals and xenografts, a human ovarian transplantation trial was launched. Pelvic auto-transplantation of frozen-banked ovarian tissue resulted in ovulation in one patient. Several other patients received fresh grafts subcutaneously, and preliminary results indicated antral follicle development at least in one patient. With the addition of promising data from humans, ovarian tissue cryopreservation from selected patients before cancer treatment, and in those requiring oophorectomy for benign causes, is now advocated.

Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm.

CONTEXT: In reproductive-age women, one of the common adverse effects of chemotherapy and radiotherapy is premature ovarian failure. In addition, a significant number of women experience early menopause due to oophorectomy performed for benign indications. OBJECTIVE: To develop an ovarian transplantation technique to preserve endocrine function in women undergoing sterilizing radiotherapy and/or chemotherapy, or oophorectomy. DESIGN AND SETTING: Case study of 2 patients in New York who received autologous ovarian transplantation (patient A, November 1999; patient B, April 2000) to the forearm prior to pelvic radiotherapy or after oophorectomy. PARTICIPANTS: Patient A is a 35-year-old woman with stage IIIB squamous cell cervical carcinoma and patient B is a 37-year-old woman with recurrent benign ovarian serous cysts. MAIN OUTCOME MEASURES: Follicular development evident by ultrasound examination; cyclical production of estradiol and progesterone; restoration of serum follicle-stimulating hormone, luteinizing hormone, and testosterone levels to nonmenopausal range; and disappearance of menopausal symptoms. RESULTS: Menopause was confirmed immediately after the transplantation in both patients by serum follicle-stimulating hormone measurements (patient A, 47 mIU/mL; patient B, 50.7 mIU/mL). In patient A, follicle development was noted by physical and ultrasound examinations approximately 10 weeks after the transplantation. The mean (SE) follicle-stimulating hormone and luteinizing hormone levels decreased to 8.6 (0.4) mIU/mL and 12.8 (0.8) mIU/mL, respectively. The peripheral estradiol levels showed cyclical variation (mean [SE], 115 [9.2] pg/mL [422 (33.8) pmol/L), and during the 18-month follow-up, a dominant follicle developed each month. The estradiol levels from the right cubital vein were consistent with ovarian vein measurements (mean [SE], 1069 [269] pg/mL [3924 (987.5) pmol/L]). Percutaneous oocyte aspirations yielded a mature oocyte. In patient B, ovarian function was demonstrated by ultrasound visualization of a 9-mm follicle by 6 months after transplantation. Thereafter, the patient had spontaneous menstruation every 25 to 28 days. Ovulation was further confirmed by midluteal progesterone measurements (range, 7-10.1 ng/mL; mean [SE], 8.5 [0.9] ng/mL). Patient B's ovarian graft was still functional 10 months after the transplantation. CONCLUSIONS: Subcutaneous ovarian transplantation appears to be a relatively simple, novel technique to preserve endocrine function in women undergoing sterilizing cancer therapy or surgery.

A technique for laparoscopic transplantation of frozen-banked ovarian tissue.

OBJECTIVE: To describe a laparoscopic technique for transplanting frozen-banked ovarian tissue. DESIGN: Case study. SETTING: Academic medical center. PATIENT(S): A patient whose ovarian tissue was previously frozen with the slow-freeze technique. INTERVENTION(S): Microsurgical reconstruction of ovarian cortex and its laparoscopic transplantation to the ovarian fossa. MAIN OUTCOME MEASURE(S): Blood flow to the grafts by doppler, follicle development and ovulation by ultrasound, E(2) and progesterone production, and resumption of spontaneous menses. RESULT(S): The patient ovulated and menstruated 4 months after the transplant in response to ovarian stimulation with menopausal gonadotropins. CONCLUSION(S): Laparoscopic transplantation of frozen-banked ovarian tissue beneath the pelvic peritoneum can restore ovarian function.

Recent progress in oocyte and ovarian tissue cryopreservation and transplantation.

Ovarian tissue cryopreservation and transplantation is an experimental technique that has been developed to sustain the reproductive function of women and children who are faced with sterilizing chemotherapy, radiotherapy, or radical reproductive surgery. Oocyte cryopreservation, on the other hand, is less feasible in the context of cancer because there is usually inadequate time to complete an ovarian stimulation cycle. The main promise of oocyte cryopreservation is that it offers an alternative when embryo freezing is not possible for technical, regulatory, or religious reasons. Oocyte freezing is more suitable for a single woman when the concern is age-related decline in fecundity. There have been significant scientific advances in the field of cryopreservation of ovarian tissue and oocytes, especially within the past few years. Ovarian function has been reported after the first cases of ovarian transplantation, and the number of pregnancies from cryopreserved oocytes has grown. Ovarian tissue and oocyte freezing can now be recommended in a carefully selected group of patients, provided that these options are offered under protocols that are approved by an institutional review board.