Endocytosis Involved d-Oligopeptide of Tryptophan and Arginine Displays Ordered Nanostructures and Cancer Cell Stereoselective Toxicity by Autophagy.

Owing to their self-aggregation propensity and selective interaction with the anionic membranes, the peptides rich in tryptophan (Trp) and arginine (Arg) are considered for the development of novel anticancer therapeutics. However, the structural insights from the perspective of backbone chirality and spatial orientation of side chains into the selective toxicity of peptides are limited. Here, we investigated the selectivity and cellular uptake of HHC36, a Trp/Arg-rich nonapeptide, and its d-enantiomer (allDHHC36) and a retroinverso analogue in the lung A549 and breast MDA-MB-231 cancer cells. We realized that the d-peptides can specifically induce autophagy at nontoxic concentrations only in the A549 cells supported from the LC 3-II immunostaining expression in the vicinity of the nucleus and the ultrastructural analysis revealing the autophagosome formation. The autophagic flux was also remarkable in the cells exposed to d-peptides at a far lower concentration in synergism with doxorubicin (DOX). In marked contrast, nonselective cell death was observed only if a high amount of HHC36 was applied. HHC36 tended to irregular collagen-like fibrils relative to allDHHC36 that distinctly formed higher-order coiled nanostructures. Interestingly, the short d-peptide fragments were generated in a harsh oxidative condition. Compared with the direct membrane transduction of HHC36, the entry of d-peptides into the lung cancer cells was controlled by endocytosis through the contribution of heparan sulfate proteoglycans (HSPGs) and cholesterol (CHO). However, both l- and d-peptides feasibly crossed the membrane and localized inside the S-phase-arrested cell nucleus. This suggested the likelihood of peptide intercalation with DNA that might differently appear in selective and/or nonselective deaths. These results unraveled the d-handedness-selective toxicity of a self-assembling Trp/Arg-rich sequence that is dependent on the cell type from the aspects of the density of anionic charges and CHO in the outer leaflet of the plasma membrane, as well as the intracellular redox imbalance that may drive the formation of toxic peptide nanostructure fragments.

Safety and potency of BIV1-CovIran inactivated vaccine candidate for SARS-CoV-2: A preclinical study.

The development of effective and safe COVID-19 vaccines is a major move forward in our global effort to control the SARS-CoV-2 pandemic. The aims of this study were (1) to develop an inactivated whole-virus SARS-CoV-2 candidate vaccine named BIV1-CovIran and (2) to determine the safety and potency of BIV1-CovIran inactivated vaccine candidate against SARS-CoV-2. Infectious virus was isolated from nasopharyngeal swab specimen and propagated in Vero cells with clear cytopathic effects in a biosafety level-3 facility using the World Health Organization's laboratory biosafety guidance related to COVID-19. After characterisation of viral seed stocks, the virus working seed was scaled-up in Vero cells. After chemical inactivation and purification, it was formulated with alum adjuvant. Finally, different animal species were used to determine the toxicity and immunogenicity of the vaccine candidate. The study showed the safety profile in studied animals including guinea pig, rabbit, mice and monkeys. Immunisation at two different doses (3 or 5 µg per dose) elicited a high level of SARS-CoV-2 specific and neutralising antibodies in mice, rabbits and nonhuman primates. Rhesus macaques were immunised with the two-dose schedule of 5 or 3 µg of the BIV1-CovIran vaccine and showed highly efficient protection against 104 TCID50 of SARS-CoV-2 intratracheal challenge compared with the control group. These results highlight the BIV1-CovIran vaccine as a potential candidate to induce a strong and potent immune response that may be a promising and feasible vaccine to protect against SARS-CoV-2 infection.

Turn-folded magainin lipopeptide analog induces cytoplasmic vacuoles in MDA-MB-231 cells through G2-phase arrest.

Selective induced non-canonical programmed deaths in the lipid raft type 1-enriched MDA-MB-231 is a promising treatment approach. Cationic amphiphilic peptides conjugated to relatively long fatty acyl chains that tend to self-aggregate are prone to upregulate necroptotic and paraptotic signaling. We investigated the toxic effects of an N-terminally palmitoylated magainin derivate (P1MK5E) in the MDA-MB-231 cells in relation to its structure at molecular level. The modeling showed that the palmitoylation reinforces a turn-like structural motif in the lipopeptide which is likely required for its activity. P1MK5E triggered intracellular generation of reactive oxygen species (ROS), G2-phase arrest, mitochondrial membrane potential (ΔΨmt) disturbance and presumable flopping of phosphatidylserine (PtdSer) to the cancer cell membrane outer surface in a comparable manner to doxorubicin (DOX) that induces apoptotic signaling. Despite forming extensive congregates of different sizes at the cell surface, P1MK5E had little impacts on the MDA-MB-231 membrane integrity. The cell death upon exposure to the lipopeptide was, however, caspase 3 independent and characterized by cytoplasmic vacuolation and no distinct nuclear fragmentation that is to be privileged in the treatment of apoptotic resistance pathways in triple-negative breast cancers (TNBCs).

Delayed effects of sulfur mustard on autophagy suppression in chemically-injured lung tissue.

BACKGROUND: Autophagy is an intracellular hemostasis mechanism, responding to extracellular or intracellular stresses. Sulfur mustard (SM) induces cellular stress. Iranian soldiers exposed to SM gas, during the Iraq-Iran war, suffer from delayed complications even 30 years after exposure. In this study, for exploring the SM effect on autophagy pathway, gene and protein expression of autophagy markers are evaluated in the lung of SM-exposed people. METHODS: 52 FFPE lung tissues of SM-exposed people and 33 lung paraffin blocks of non-exposed patients to SM were selected. LC3 and Beclin-1 mRNA expressions were evaluated by QRT-PCR. LC3-B protein and LC3II/LC3I proteins ratio were detected by Immunohistochemistry and immunoblotting method. The collected data were analyzed in SPSS, and P value ≤ 0.05 was considered significant. RESULTS: LC3 gene expression in SM-exposed subjects (median CT value = 4.97) increased about 4 fold compared with the control group (median CT value = 0.46, P = 0.025). Beclin-1 mRNA expression had not significant difference between two groups. After adjusting the confounding variables such as drug usage, LC3-B protein (P = 0.041) and LC3II/LC3I ratio (P = 0.044) were found significantly lower in the lung cells of SM-exposed group. CONCLUSION: Upon exposure to SM gas, the lung cells are affected by acute cellular stress such as oxidative stress. The study results show that LC3 mRNA level increases in these patients, but, surprisingly, LC3-B protein via unknown mechanism has been down-regulated. N-acetyl cysteine and salbutamol drugs could induce the autophagy, and help to reduce the SM effects and improve the clinical condition of SM-injured patients.

Serum and sputum levels of IL-17, IL-21, TNFα and mRNA expression of IL-17 in sulfur mustard lung tissue with long term pulmonary complications (28 years after sulfur mustard exposure).

BACKGROUND: Iranian veterans who had exposed to Sulfur Mustard (SM) suffer from long term complications such as Chronic Obstructive Pulmonary Disease (COPD) and bronchiolitis obliterate (BO). Th17 cells product IL-17A, IL-17F, IL-21, and IL-22. They have important roles in chronic inflammatory diseases. Also, TNFα has a major part in pathobiological processes of COPD. In this study, we evaluate the serum and sputum levels of IL-17, IL-21, TNF-α, and mRNA expression of IL-17 in the lung tissue of the patients 28 years after SM exposure. MATERIAL AND METHOD: The cytokine levels of IL-17, IL-21 and TNFα were measured by ELISA method in serum and sputum of 455 SM-exposed and 123 unexposed people participated in Sardasht-Iran Cohort Study (SICS) of chemical victims. The mRNA expression of IL-17 was evaluated with qRT-PCR in lung biopsies (SM-exposed =52, control =33). Analyses of all data were accomplished with the SPSS software with P value ≤05. RESULT: The results show the sputum level of IL-17 in the exposed group decreased significantly compared to control group (P = 0.007) and Veterans and Martyrs Affair Foundation (VMAF) assessment was significantly lower in abnormal/exposed than normal/exposed group (P = 0.042). There were no significant differences between control and exposed groups in serum level of IL-17; also serum and sputum levels of IL-21, TNF-α, and IL-17 mRNA expression. CONCLUSION: Conclusively, The IL-17 level decreased in the exposed group. This decline could cause by mutation on transcription factors like Signal transducer and activator of transcription 3 gene (STAT3) or CCL20 as a chemokine.

Evaluation of mRNA Expression Levels of TNFα, TNFR1 and IL1ß in Lung Tissue 20 Years after Sulfur-mustard Exposure.

Despite many years having passed since exposure to sulfur mustard (SM) gas, there are many exposed subjects who are still suffering from delayed pulmonary complications. The levels of pro-inflammatory cytokines in the lung of these subjects have not been investigated in delay phase. In this study, we evaluated mRNA expression of pro-inflammatory  cytokines (tumor necrosis factor alpha (TNFα), tumor necrosis factor receptor type 1 (TNFR1), and interleukin 1 beta (IL-1ß)  ) in lung biopsy of SM-exposed subjects and compared them with control (non-exposed) subjects. We used formalin-fixed, paraffin-embedded (FFPE) tissue for this purpose. Lung FFPE blocks of SM-exposed subjects (30 samples) and a control group (30 samples) were collected from archival pathology department. The total mRNA of FFPE tissues were extracted and the mRNA expression of pro-inflammatory  cytokines were determined by quantitative Real Time PCR (RT-qPCR). The obtained results from two groups were compared to each other and non-parametric statistical analyses were carried out on them. Our studies showed that the mRNA expression of TNFα, TNFR1 and IL-1ß  in lung tissue of SM injured and control people have no significant difference (p-value= 0.159, 0.832 and 0.314 respectivly). TNFR1 showed direct correlation with TNFα (r=0.867, p=0.002) and IL-1ß (r= 0.65, p=0.006). The evaluation of mRNA expression in pro-inflammatory cytokines in lung of SM-exposed subjects after 20 years showed that these mediators are similar to those of non-exposed group and there was no acute inflammation in lung of these patients.

The delayed effect of mustard gas on housekeeping gene expression in lung biopsy of chemical injuries.

OBJECTIVE: Sulfur mustard (SM) was used as a chemical weapon in Iraq-Iran war. Exposed people have major complications in important organs such as pulmonary system. Some studies have shown that SM could affect the expression of endogenous genes and non-housekeeping genes, time dependently. To understand the accurate molecular mechanism of the delayed effect of SM, the identification of the gene expression pattern in these patients is essential. Hence, we have evaluated mRNA expression of four common housekeeping genes (ACTIN, PGK1, ß2m, GAPDH) in SM-exposed and non-exposed (control) formalin-fixed, paraffin-embedded (FFPE) human lung tissues. METHOD: Paraffin block of lung biopsy of SM-exposed people (11 cases) and people without exposure to SM as control group (9 cases) have been selected. The mRNA expression of four endogenous control genes has been evaluated by qRT-PCR. The stability value of each gene was calculated by different methods. RESULT: It was found that ACTIN mRNA has the highest expression (30.26±2.87) and PGK1 has the lowest standard deviation (SD) (30.885±2.215) between pooled groups. The best correlation was between ACTIN and PGK1 expressions. The M value has shown that ACTIN and then PGK1 are the most stable housekeeping genes among. The results obtained from the GeNorm and NormFinder have indicated that the pair ACTIN- PGK1 is the most suitable choice for endogenous control genes. CONCLUSION: ACTIN and PGK1 genes are stable in studied lung tissues and are the better than two other housekeeping genes. In addition, mustard gas does not affect their expression in long term.

The effect of MS14 on innate and cellular immune responses in BALB/c mice.

MS14 is an Iranian natural preparation of herbal-marine source with no obvious toxicity in oral administration, which possesses anti-inflammatory and immunomodulatory effects. In this study, the effect of oral administration of MS14 on nitric oxide (NO) production of peritoneal macrophages and lymphocyte Th1 cytokines and delayed-type hypersensitivity (DTH) test in BALB/c mice were investigated. Peritoneal macrophages were cultured and NO production was measured by Griess method. Viability of macrophages was assayed by MTT (3-(4,5-dimethy-2-lthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) test. Interleukin-2 (IL-2) and INFγ levels in supernatant of spleen lymphocytes culture were assayed by enzyme-linked immunosorbent assay kit. For DTH test the mice were immunized with sheep red blood cell and DTH was measured 24 h after the last immunization of mice. NO production of macrophages has been diminished significantly in MS14 treated group (about 40%) at the presence or absence of stimulators. Macrophage viability had no significant alteration after MS14 administration. However, interferon-γ production of lymphocytes was significantly decreased in MS14 group both at the absence or presence of concanavalin A (ConA; about 50%); IL-2 production declined about 20% at the presence of ConA. In comparison with the control group, MS14 had no statistically significant effect on DTH test. The results have pointed that MS14 may have immunomodulatory potentials at least through its decreasing effect on NO production of macrophages and level of Th1 cytokine pattern.

The effect of MS14 on Th2 cytokines pattern in Balb/C mice.

The tendency of immune response toward either Th1 or Th2 cytokine pattern can cause a number of pathologic conditions. Multiple sclerosis is postulated to be a Th1-type cell-mediated autoimmune disease. MS14--an Iranian natural product--seems to possess anti-inflammatory properties. Thus, we studied the effect of orally administered MS14 on Th2 cytokines (IL-5 and IL-10) in normal Balb/C mice (100 mg/kg; 5 days). The result indicated that activated splenocytes of MS14 group produced significantly more IL-5 and IL-10 (3-4 times in comparison with control group mice). MS14 could upregulate Th2 cytokine and thereby it may possess immunoregulatory properties probably useful in treatment of some diseases.