Characterization of an engineered ACE2 protein for its improved biological features and its transduction into MSCs: A novel approach to combat COVID-19 infection.

Transduced MSCs that express engineered ACE2 could be highly beneficial to combat COVID-19. Engineered ACE2 can act as decoy targets for the virus, preventing its entry into healthy lung cells. To this end, genetic engineering techniques were used to integrate the ACE2 gene into the MSCs genome. The MSCs were evaluated for proper expression and functionality. The mutated form of ACE2 was characterized using various techniques such as protein expression analysis, binding affinity against spike protein, thermal stability assessment, and enzymatic activity assays. The functionality of the mACE2 was assessed on SARS-CoV-2 using the virus-neutralizing test. The obtained results indicated that by introducing specific mutations in the ACE2 gene, the resulting mutant ACE2 had enhanced interaction with viral spike protein, its thermal stability was increased, and its enzymatic function was inhibited as a decoy receptor. Moreover, the mACE2 protein showed higher efficacy in the neutralization of the SARS-CoV-2. In conclusion, this study proposes a novel approach with potential benefits such as targeted drug delivery and reduced side effects on healthy tissues. These transduced MSCs can also be used in combination with other anti-COVID-19 treatments. Design of similar engineered biomolecules with desired properties could also be used to target other diseases.

Intrathecal injection of human placental mesenchymal stem cells derived exosomes significantly improves functional recovery in spinal cord injured rats.

BACKGROUND: Spinal cord injury (SCI) due to lack of restoration of damaged neuronal cells is associated with sensorimotor impairment. This study was focused on using the human placental mesenchymal stem cells- exosome (HPMSCs- Exosomes) in an animal model of severe SCI under myelogram procedure. METHODS AND RESULTS: Intrathecal injection of exosomes was performed in the acute phase of SCI in female rats. The improved functional recovery of the animals was followed for 6 weeks in control (saline, n = 6) and HPMSCs- EXO (HPMSCs-Exosomes, n = 6) groups. Pathological changes and glial scar size were evaluated. The Immunohistochemistry (IHC) of GFAP and NF200 factors as well as the apoptosis assay was investigated in the tissue samples from the injury site. The results demonstrated that HPMSCs-exosomes can improve motor function by attenuating apoptosis of neurons at the injury site, decreasing GFAP expression and increasing NF200 in the HPMSCs-EXO group. Also, HPMSCs-exosomes by preventing the formation of cavities causes preservation of tissue in SCI rats. CONCLUSIONS: These findings demonstrate the effectiveness of HPMSC-Exosomes as a therapeutic method to improve functional recovery, reduce pathological changes associated with injury, and prevent chronicity after SCI. The neuroprotective and anti-apoptotic potential of HPMSCs- Exosomes may be a promising therapeutic approach for SCI. Another result was the importance of intrathecal injection of exosomes in the acute phase, which accelerated the healing process. Furthermore, the myelogram can be a feasible and suitable method to confirm the accuracy of intrathecal injection and examine the subarachnoid space in the laboratory animals.

A Recombinant RBD-Based Phage Vaccine Report: A Solution to the Prevention of New Diseases?

The safety, inherent immunogenicity, stability, and low-cost production of bacteriophages make them an ideal platform for vaccine development. Most vaccination strategies against COVID-19 have targeted the spike protein of SARS-CoV-2 to generate neutralizing antibodies. P1, a truncated RBD-derived spike protein, has been shown to induce virus-neutralizing antibodies in preclinical studies. In this study, we first investigated whether recombinant phages displaying P1 on the M13 major protein could immunize mice against COVID-19, and second, whether inoculation with 50 µg of purified P1 in addition to the recombinant phages would stimulate the immune systems of the animals. The results showed that the mice that received recombinant phages were immunized against the phage particles, but did not have anti-P1 IgG. In contrast, compared with the negative control, the group that received a combination of P1 protein and recombinant phage was immunized against the P1 protein. In both groups, CD4+ and CD8+ T cells appeared in the lung tissue. These results suggest that the number of antigens on the phage body plays a crucial role in stimulating the immune system against the bacteriophage, although it is immunogenic enough to function as a phage vaccine.

Recombinant highly antigenic truncated fusion-based protein as a diagnostic antigen for anti-SARS-CoV-2 nucleocapsid antibody ELISA.

Among the main structural protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), nucleocapsid phosphoprotein (NP) exhibits high immunogenicity and is the most abundant viral protein produced and shed during infection. Detection of antibodies against NP may help assess the number of individuals exposed to SARS-COV-2 or vaccinated against it. Based on these findings and other structural and antigenic evaluations, we designed a recombinant truncated fusion NP-based protein for application in an immunoassay for detecting immunoglobulins in patients who have recovered from COVID-19. In this research, we aligned the NPs from SARS-CoV and SARS-CoV-2 and selected highly antigenic parts of the SARS-CoV-2 sequences based on in-silico studies. The protein was expressed under optimum conditions in the bacterial host BL21 and purified by nickel immobilized metal affinity chromatography. Moreover, the purity level was assessed by SDS-PAGE and Western blotting whereas the folding of the protein was evaluated by circular dichroism. Ultimately, we used the purified recombinant protein in ELISA development in which 42 samples from convalescent patients were compared with 20 samples of the past 2019 patients who had attended laboratories for various clinical check-ups. The sensitivity and specificity were determined as 71% and 90%, respectively, in the optimum cut-off point measured by the receiver operating characteristic curve.

Tandem Mass Analysis of Amino Acids and Acylcarnitine Profiles in Neonates with Congenital Hypothyroidism.

BACKGROUND: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder. This study aimed to investigate whether disturbances in amino acid metabolism and fatty acid oxidation existed in neonates with CH compared to healthy neonates. METHODS: In this case-control study, we evaluated the metabolomics of neonates with newly diagnosed CH and healthy neonates. Forty-three metabolites, including 13 amino acids and 30 acylcarnitines, were investigated. RESULTS: Two hundred neonates with CH and 209 healthy children were enrolled. The mean age of males and females was 4.8 ± 2.4 and 5.52 ± 3.2 days in the case group and 5.1 ± 2.6 and 4.7 ± 3.6 days in the control group, respectively. Of the metabolites, 34 were significantly different between the two groups. Five amino acids and four acylcarnitines did not differ significantly between groups. CONCLUSION: These findings pave the way for a better understanding of the relationship between alterations and the clinical manifestation of CH, which has the potential for identifying novel therapeutics.

The Ability of Antibody against Truncated Matrix Metalloproteinase 9 Peptide to Evaluate the Native Protein on Tear Drops of Dry Eye Disease Patients by a Point-of-Care Diagnostic System.

Purpose: To obtain a reactive and specific antibody against truncated matrix metalloproteinase 9 (MMP-9), that has reactivity toward the native protein. Precision, accuracy, specificity, and sensitivity were evaluated using a point-of-care test. Methods: An in silico study was used to confirm the anti peptide truncated MMP-9 is against native MMP-9. After an antibody titer assessment, purification, and characterization, the anti MMP-9 was assessed. The cut-off value was determined using the purified gelatinases of the supernatant HCT 116 cell line. The supernatant was purified by preparative native-polyacrylamide gel electrophoresis based on charge and size of the proteins. Furthermore, quality control (QC) of the results were performed following standard densitometry methods. Results: A truncated MMP-9 is the major epitope peptide that can trigger the immune system to scavenge for a specific and reactive antibody against the native MMP-9. The MMP-9 native protein is purified from the supernatant of the HCT 116 cell line and the commercially available, full-length MMP-9. The cut-off value was estimated at 30 µg/mL. QC results indicated that the specificity was 80%, sensitivity was 96.7%, accuracy was 91%, and precision was 91.66%. The area under curve was 0.827 (P < 0.001). The positive predictive value was 83%, and the negative predictive value was 96%. Conclusions: The antibody against the synthetic epitope peptide can detect the native MMP-9. Native MMP-9 is considered the main biomarker in an immunoassay POCT and is used to diagnose dry eye disease (DED). In accordance with QC results, MMP-9 point of care test can be utilized for screening patients suffering from DED.

Preparation and preclinical characterization of 111In-DTPA-Anti-MUC1 as a radioimmunoconjugate for diagnosis of breast cancer by single-photon emission computed tomography.

Context: Targeting MUC1 antigens which are overexpressed in 80% of breast cancers can be widely used in the field of radioimmunoscintigraphy (RIS) of breast cancer. Aims: The aim of this study was to develop a new diagnostic labeled compound for breast cancer RIS. Settings and Design: In this study, an efficient indirect labeling method of PR81 with Indium-111 was developed and preliminary preclinical qualifications were reported. Subjects and Methods: 111In-DTPA-PR81 was prepared and its radiochemical purity and stabilities in human serum and in phosphate-buffered saline (PBS) buffer were surveyed. Furthermore, cellular studies including complex reactivity, binding specificity, cell toxicity, etc., were examined. Finally, biodistribution and scintigraphy of the complex were studied in normal and tumoral animals. Statistical Analysis Used: Statistical analyses were performed using SPSS 10.0. Results: 111In-DTPA-PR81 was prepared with a radiochemical purity of >99% at optimized conditions. Stability studies showed the radiochemical purity of >90% in PBS buffer after 96 h, while the stability in human serum showed decrement to 81% after 96 h. Reactivity of the complex with MUC1 was significantly (P < 0.005) higher than bovine serum albumin (BSA) (about 7-8 times), even though BSA concentration was about twice the MUC1. The binding specificity of the complex to the MUC1 antigen was confirmed by means of immunoreactivity assay. Cell toxicity examination showed no significant lethal effect of the radiolabeled compound on the cells. Biodistribution studies of the complex in normal rats were consistent with the biodistribution of antibodies and high accumulation was observed in the tissues expressing MUC1 antigen. The results of 111In-DTPA-PR81 scintigraphy in tumoral female BALB/c mice at 24 and 48 h after injection showed an increasement of the accumulation in the tumor site. Conclusions: 111In-DTPA-PR81 can be considered as a potential agent for imaging of the MUC1 +breast tumors.

Different combinations of monoclonal antibodies and polyclonal antibodies in the design of neonatal hypothyroidism diagnostic kit.

Neonatal hypothyroidism is a deficiency of thyroid hormones at birth that can cause lifelong mental and physical disorders in humans. Lack of timely detection could lead to irreversible damage by neonatal hypothyroidism. However, it could be managed quickly and efficiently via timely diagnosis. The screening programs rely on immunoassays to diagnose neonatal hypothyroidism in most countries. This method is time-consuming, needs laboratory equipment, and should be performed by trained and skilled technicians. Given these circumstances, the ELISA method is not a preferable method for the diagnosing of neonatal hypothyroidism. However, it can be used as a confirmatory method in infants with suspected and unknown neonatal hypothyroidism. In the present study, the homemade SR95-1, SR95-2, and SR95-3 anti-ß-TSH polyclonal and the commercially available monoclonal antibodies were used to detect ß-TSH in a rapid assay kit design hypothyroidism screening. To design the kit, the different combinations of the antibodies were used to establish a sandwich immune-chromatography method. The designed rapid neonatal hypothyroidism tests were used to measure neonatal ß-TSH in 100 dry blood samples. This study showed that the best antibody pair in terms of sensitivity is the SR95-1 antibody as capture antibody and the SR95-2 as a conjugated antibody. Using 100 clinical samples, the designed assay was shown to have 94% sensitivity, 83% specificity, and 94% accuracy. The results showed that polyclonal antibodies (SR95-1 as capture) and SR95-2 (as detector) antibodies can detect the reference range of ß-TSH in dried blood samples and can be used in the screening of neonatal hypothyroidism.

Recombinant subunits of SARS-CoV-2 spike protein as vaccine candidates to elicit neutralizing antibodies.

OBJECTIVES: The spike protein has been reported as one of the most critical targets for vaccine design strategies against the SARS-CoV-2 infection. Hence, we have designed, produced, and evaluated the potential use of three truncated recombinant proteins derived from spike protein as vaccine candidates capable of neutralizing SARS-CoV-2 virus. METHODS: In silico tools were used to design spike-based subunit recombinant proteins (RBD (P1 ), fusion peptide (P2 ), and S1/S2 cleavage site (P3 )). These proteins were checked for their ability to be identified by the anti-SARS-CoV-2 antibodies by exposing them to COVID-19 serum samples. The proteins were also injected into mice and rabbit, and the antibody titers were measured for 390 days to assess their neutralization efficiency. RESULTS: The antibodies that existed in the serum of COVID-19 patients were identified by designed proteins. The anti-spike antibody titer was increased in the animals injected with recombinant proteins. The VNT results revealed that the produced antibodies could neutralize the cultured live virus. CONCLUSION: Truncated subunit vaccines could also be considered as robust tools for effective vaccination against COVID-19. Using a combination of in silico, in vitro, and in vivo experiments, it was shown that the injection of spike-based truncated recombinant proteins could stimulate long-lasting and neutralizing antibody responses.

Downregulation of fatty acid oxidation by involvement of HIF-1α and PPARγ in human gastric adenocarcinoma and related clinical significance.

Lipid metabolism rewiring in gastric adenocarcinoma (GA) pathogenesis is still not clearly elucidated. This study aimed to describe the role of lipid catabolism in GA patient outcomes and possible therapeutic targets by analyzing the effect of hypoxia-inducible factor-1α (HIF-1α) on fatty acid oxidation (FAO). AGS cell line was cultured in normoxic and hypoxic conditions, and FAO-related genes were analyzed by real-time-PCR and Western-blot. The study group comprised 108 newly diagnosed GA patients and 152 control cases. Serum concentrations of medium and long-chain acyl-CoA dehydrogenases (MCAD and LCAD) proteins were measured using ELISA, and local expression of HIF-1α, carnitine palmitoyl transferase 1 (CPT1A) and peroxisome proliferator-activated receptor γ (PPARγ) was evaluated by immunohistochemistry. In addition, gene expression of PPARγ, CPT1A, LCAD, and MCAD was assessed by real-time-PCR. In vitro findings indicate HIF-1α upregulation and FAO-related genes and proteins reduction in the hypoxic culture of AGS cells. GA patients had significantly lower circulating levels of LCAD compared to controls. Higher protein expression of HIF-1α and downregulated CPT1A and PPARγ were observed in GA tissues versus controls. Gene expression of CPT1A, PPARγ, LCAD, and MCAD were repressed in GA tissues compared to controls. Moreover, reduced expression of CPT1A, PPARγ, and MCAD were correlated with HIF-1α upregulation in GA. Poor patient outcome was associated with lower PPARγ and LCAD expression in GA. HIF-1α upregulation in human GA patients and AGS cells was paralleled by downregulation of lipid catabolism genes potentially via reduced PPARγ-mediated FAO. This metabolic adaptation to hypoxic condition may play a role in GA pathogenesis and might have clinical and therapeutic value in GA patients.

Inhibition of matrix metalloproteinase-9 for the treatment of dry eye syndrome; a review study.

Dry eye syndrome (DES) and tear dysfunction are multifactorial conditions affecting meibomian glands, lacrimal glands, and ocular surface. This ocular disorder can cause eye irritation, irregular cornea, corneal barrier disruption, and blurred vision. Uncontrolled increase in matrix metalloproteinase-9 (MMP-9) level and activity has been detected in the tears and ocular surface in the patients with DES, which has been proved to be related to disruption of tight junctions in apical corneal epithelium associated with severe signs of DES. These uncontrolled activities of MMP-9 lead to desquamation of ocular surface epithelia. Therefore, this review study was conducted to summarize the evidence regarding MMP-9 contribution in DES, and inhibition of MMP-9, as a therapeutic target for treatment of DES. For this purpose, herein, the related studies designed novel pharmaceutical compounds for direct and indirect inhibition of MMP-9 as treatment approaches for DES were reviewed. These compounds were designed to improve corneal barrier function, reduce inflammation on ocular surface, and restore tear production.

Novel anti-PD-L1 peptide selected from combinatorial phage library inhibits tumor cell growth and restores T-cell activity.

PD-L1 overexpression on tumour cells forms a protective shield against cytotoxic T-cell killing, which consequently leads to immune evasion. Engagement of PD-1 in tumour infiltrating T cells with PD-L1 results in an exhausted T-cell phenotype, thus preventing an effective immune response against tumour cells. In the present study, we employed phage display combinatorial peptide library to discover anti-PD-L1 peptides. The peptides discovered here, could computationally exhibit specific interactions with PD-L1 at residues with which PD-1 also interacts. Binding affinity and specificity of the peptides were examined by flow cytometry. Anti- tumour activity of peptides was also investigated using several cell-based assays. Surprisingly, we demonstrated that Pep-39 can inhibit PDL-1, and reduce MDA-MB-231, CT-26, and DU-145 cells survival. In co-culture experiments, Pep-39 restored proliferation of Jurkat cells cultured in the presence of MDA-MB-231 cells. In addition, Jurkat cells apoptosis was impeded, indicating blocking potential of Pep-39 against PD-1/PD-L1 interaction.

Isolation and characterization of a novel nanobody for detection of GRP78 expressing cancer cells.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone that has been shown that is overexpressed in cancer cells. Overexpression of GRP78 on cancer cells makes this molecule a suitable candidate for cancer detection and targeted therapy. VHH is the binding fragment of camelid heavy-chain antibodies also known as "nanobody." The aim of this study is to isolate and produce a new recombinant nanobody using phage display technique to detect cancer cells. Using the c-terminal domain of GRP78 (CGRP) as an antigen, four rounds of biopanning were performed, and high-affinity binders were selected by ELISA. Their affinity and functionality were characterized by surface plasmon resonance (SPR) cell ELISA and immunocytochemistry. A unique nanobody named V80 was purified. ELISA and SPR showed that this antibody had high specificity and affinity to the GRP78. Immunofluorescence analysis showed that V80 could specifically bind to the HepG2 and A549 cancer cell lines. This novel recombinant nanobody could bind to the cell surface of different cancer cells. After further evaluation, this nanobody can be used as a new tool for cancer detection and tumor therapy.

In Vivo Effect of RSH-12, a Novel Selective MMP-9 Inhibitor Peptide, in the Treatment of Experimentally Induced Dry Eye Model.

PURPOSE: To investigate the efficacy of RSH-12, a novel selective matrix metalloproteinase 9 (MMP-9) inhibitor peptide in rabbit models of dry eye syndrome (DES). METHODS: In vitro toxicity of RSH-12 on cultured human corneal fibroblasts was investigated with MTT. Ocular toxicity of RSH-12 was investigated by clinical examinations, histology, and TUNEL assay. Experimental model of dry eye was induced by 1.0% atropine sulfate administration followed after 15 min by treatment with PBS, RSH-12, and Restasis in individual groups, three times a day for 7 days. In addition to performing Schirmer's test for evaluating basic tear secretion and tear break-up time test for investigating tear stability, the occurrence of superficial punctate keratopathy was also investigated in the study groups. RESULTS: MTT assay demonstrated that RSH-12 was not toxic to human corneal fibroblasts in different concentrations. During the administration of atropine, TBUT values and tear volume were decreased in vehicle group while these indices improved significantly in groups treated with RSH-12 in a promising manner. RSH-12 increased the mean value of tear volume from 4.85 to 10.75 mm (P = .0001) and mean of TBUT values from 20.3 s to 34.5 s (P = .0001) compared with the vehicle. In contrast to the presence of severe superficial punctate keratopathy in the controls, no significant dotted staining was observed in the RSH-12 and Restasis groups. CONCLUSIONS: These outcomes propose that RSH-12 has a therapeutic effect in the rabbit model of dry eye and might be a potential treatment for severe DES.

Preparation, quality control, and biodistribution assessment of [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors.

In this study, [111 In]In-DOTA-PR81 was developed, and its preliminary preclinical qualifications were assessed for single photon emission computed tomography (SPECT) imaging of breast cancer. DOTA-NHS-ester was practiced and successively purified by molecular filtration. The chelate:mAb ratio was determined by spectrophotometry. DOTA-PR81 was radiolabeled with In-111 and its radiochemical yield, in vitro stability, in vitro internalization, and immunoreactivity tests were performed. SPECT imaging and tissue counting were applied to evaluate the tissue distribution of [111 In]In-DOTA-hIgG and [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors. The radiochemical yield of [111 In]In-DOTA-PR81 complex was >95.0 ± 0.5% (ITLC), and the specific activity was 170 ± 44 MBq/mg. Conjugation reaction resulted in the average number of chelators attached to a mAb (c/a) of 3.4 ± 0.3:1. The radioimmunoconjugate showed immunoreactivity towards MCF7 cell line and MUC1 antigen while its significant in vitro and in vivo stability were investigated over 48 h, respectively (93.0 ± 1.2% in phosphate-buffered saline (PBS) and 84.0 ± 1.3% in human serum). The peak concentration of internalized activity of [111 In]In-DOTA-PR81 was between 4 to 6 h. In comparison with control probes, the complex was accumulated with high specificity and sensitivity at the tumor site. Achieved results indicated that [111 In]In-DOTA-PR81 could be contemplated as an appropriate radiotracer for prognostic imaging of antigens in oncology.

Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR.

Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays.

Design of an engineered ACE2 as a novel therapeutics against COVID-19.

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.