Structure-based engineering of an antiangiogenic scFv antibody for soluble production in E. coli without loss of activity.

Development of monoclonal antibody therapeutics against vascular endothelial growth factor receptor 2 (VEGFR-2) protein, which is the main regulator in angiogenesis, has been a major challenge for years. In the current study, we engineer an inclusion body forming single-chain variable fragment (scFv) against VEGFR-2 by using complementarity determining regions (CDR) grafting technique to improve its solubility and investigate the activity of the engineered molecule. CDR sequences of the target scFv were grafted into the framework of another intrinsically soluble scFv molecule. Based on the computational results, CDR grafting has increased the solubility of the grafted scFv molecule. Results confirmed that the grafting approach increased in vivo folding properties of the target scFv molecule compared with the original scFv molecule. Similar binding affinities to the VEGFR-2 were observed for the original and the grafted scFv by surface plasmon resonance assays. Biological activity assays, including human umbilical vein endothelial cells proliferation and wound healing assays, showed that grafted scFv molecule has an antiangiogenic property. This study suggests that an antiangiogenic scFv fully expressed as an inclusion body can be rescued by grafting its CDR regions to a scFv expressed in a soluble form without any loss in its binding property and its activity.

Altered kidney function induced by SARS-CoV-2 infection and acute kidney damage markers predict survival outcomes of COVID-19 patients: a prospective pilot study.

BACKGROUND: Literature with regard to coronavirus disease 2019 (COVID-19) associated morbidities and the risk factors for death are still emerging. In this study, we investigated the presence of kidney damage markers and their predictive value for survival among hospitalized subjects with COVID-19. METHODS: Forty-seven participants was included and grouped as: 'COVID-19 patients before treatment', 'COVID-19 patients after treatment', 'COVID-19 patients under treatment in intensive care unit (ICU)', and 'controls'. Kidney function tests and several kidney injury biomarkers were compared between the groups. Cumulative rates of death from COVID-19 were determined using the Kaplan-Meier method. The associations between covariates including kidney injury markers and death from COVID-19 were examined, as well. RESULTS: Serum creatinine and cystatin C levels, urine Kidney Injury Molecule-1 (KIM-1)/creatinine ratio, and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), CKD-EPI cystatin C, and CKD-EPI creatinine-cystatin C levels demonstrated significant difference among the groups. The most significant difference was noted between the groups 'COVID-19 patients before treatment' and 'COVID-19 patients under treatment in ICU'. Advancing age, proteinuria, elevated serum cystatin C, and urine KIM-1/creatinine ratio were all significant univariate correlates of death (p < 0.05, for all). However, only elevated urine KIM-1/creatinine ratio retained significance in an age, sex, and comorbidities adjusted multivariable Cox regression (OR 6.11; 95% CI: 1.22-30.53; p = 0.02), whereas serum cystatin C showing only a statistically non-significant trend (OR 1.42; 95% CI: 0.00-2.52; p = 0.09). CONCLUSIONS: Our findings clearly demonstrated the acute kidney injury related to COVID-19. Moreover, urine KIM-1/creatinine ratio was associated with COVID-19 specific death.

Germline pathogenic variant spectrum in 25 cancer susceptibility genes in Turkish breast and colorectal cancer patients and elderly controls.

Inherited pathogenic variants account for 5% to 10% of all breast cancer (BC) and colorectal cancer (CRC) cases. Here, we sought to profile the pathogenic variants in 25 cancer susceptibility genes in Turkish population. Germline pathogenic variants were screened in 732 BC patients, 189 CRC patients and 490 cancer-free elderly controls, using next-generation sequencing-based multigene panel testing and multiplex ligation-dependent probe amplification testing. Pathogenic variants were detected in 17.2% of high-risk BC patients and 26.4% of high-risk CRC patients. More than 95% of these variants were clinically actionable. BRCA1/2 and mismatch repair genes (MLH1, MSH2 and MSH6) accounted for two-thirds of all pathogenic variants detected in high-risk BC and CRC patients, respectively. Pathogenic variants in PALB2, CHEK2, ATM and TP53 were also prevalent in high-risk BC patients (4.5%). BRCA1 exons 17-18 deletion and CHEK2 c.592+3A>T were the most common variants predisposing to BC, and they are likely to be founder variants. Three frequent MUTYH pathogenic variants (c.884C>T, c.1437_1439delGGA and c.1187G>A) were responsible for all MUTYH biallelic cases (4.4% of high-risk CRC patients). The total pathogenic variant frequency was very low in controls (2.4%) and in low-risk BC (3.9%) and CRC (6.1%) patients. Our study depicts the pathogenic variant spectrum and prevalence in Turkish BC and CRC patients, guiding clinicians and health authorities for genetic testing applications and variant classification in Turkish population.

Recent Developments in Targeting RAS Downstream Effectors for RAS-Driven Cancer Therapy.

Aberrant activity of oncogenic rat sarcoma virus (RAS) protein promotes tumor growth and progression. RAS-driven cancers comprise more than 30% of all human cancers and are refractory to frontline treatment strategies. Since direct targeting of RAS has proven challenging, efforts have been centered on the exploration of inhibitors for RAS downstream effector kinases. Two major RAS downstream signaling pathways, including the Raf/MEK/Erk cascade and the phosphatidylinositol-3-kinase (PI3K) pathway, have become compelling targets for RAS-driven cancer therapy. However, the main drawback in the blockade of a single RAS effector is the multiple levels of crosstalk and compensatory mechanisms between these two pathways that contribute to drug resistance against monotherapies. A growing body of evidence reveals that the sequential or synergistic inhibition of multiple RAS effectors is a more convenient route for the efficacy of cancer therapy. Herein, we revisit the recent developments and discuss the most promising modalities targeting canonical RAS downstream effectors for the treatment of RAS-driven cancers.

Co-Chaperone Bag-1 Plays a Role in the Autophagy-Dependent Cell Survival through Beclin 1 Interaction.

Expression levels of the major mammalian autophagy regulator Beclin 1 and its interaction with Bcl-2 regulate the switch between autophagic cell survival and apoptotic cell death pathways. However, some of the regulators and the precise mechanisms of these processes still remain elusive. Bag-1 (Bcl-2 associated athanogene-1), a member of BAG family proteins, is a multifunctional pro-survival molecule that possesses critical functions in vital cellular pathways. Herein, we report the role of Bag-1 on Bcl-2/Beclin 1 crosstalk through indirectly interacting with Beclin 1. Pull-down experiments suggested a molecular interaction between Bag-1 and Beclin 1 in breast cancer cell lines. On the other hand, in vitro binding assays showed that Bag-1/Beclin 1 interaction does not occur directly but occurs through a mediator molecule. Bag-1 interaction with p-Beclin 1 (T119), indicator of early autophagy, is increased during nutrient starvation suggesting involvement of Bag-1 in the autophagic regulation. Furthermore, CRISPR/Cas9-mediated Bag-1 knock-out in MCF-7 cells hampered cell survival and proliferation and resulted in decreased levels of total LC3 under starvation. Collectively, we suggest that Bag-1 modulates cell survival/death decision through maintaining macroautophagy as a component of Beclin 1-associated complexes.

Bag-1 stimulates Bad phosphorylation through activation of Akt and Raf kinases to mediate cell survival in breast cancer.

BACKGROUND: Bag-1 (Bcl-2-associated athanogene) is a multifunctional anti-apoptotic protein frequently overexpressed in cancer. Bag-1 interacts with a variety of cellular targets including Hsp70/Hsc70 chaperones, Bcl-2, nuclear hormone receptors, Akt and Raf kinases. In this study, we investigated in detail the effects of Bag-1 on major cell survival pathways associated with breast cancer. METHODS: Using immunoblot analysis, we examined Bag-1 expression profiles in tumor and normal tissues of breast cancer patients with different receptor status. We investigated the effects of Bag-1 on cell proliferation, apoptosis, Akt and Raf kinase pathways, and Bad phosphorylation by implementing ectopic expression or knockdown of Bag-1 in MCF-7, BT-474, MDA-MB-231 and MCF-10A breast cell lines. We also tested these in tumor and normal tissues from breast cancer patients. We investigated the interactions between Bag-1, Akt and Raf kinases in cell lines and tumor tissues by co-immunoprecipitation, and their subcellular localization by immunocytochemistry and immunohistochemistry. RESULTS: We observed that Bag-1 is overexpressed in breast tumors in all molecular subtypes, i.e., regardless of their ER, PR and Her2 expression profile. Ectopic expression of Bag-1 in breast cancer cell lines results in the activation of B-Raf, C-Raf and Akt kinases, which are also upregulated in breast tumors. Bag-1 forms complexes with B-Raf, C-Raf and Akt in breast cancer cells, enhancing their phosphorylation and activation, and ultimately leading to phosphorylation of the pro-apoptotic Bad protein at Ser112 and Ser136. This causes Bad's re-localization to the nucleus, and inhibits apoptosis in favor of cell survival. CONCLUSIONS: Overall, Bad inhibition by Bag-1 through activation of Raf and Akt kinases is an effective survival and growth strategy exploited by breast cancer cells. Therefore, targeting the molecular interactions between Bag-1 and these kinases might prove an effective anticancer therapy.

Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization.

Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.

Time-dependent leaf proteome alterations of Brachypodium distachyon in response to drought stress.

KEY MESSAGE: For the first time, a comprehensive proteome analysis was conducted on Brachypodium leaves under drought stress. Gradual changes in response to drought stress were monitored. Drought is one of the major stress factors that dramatically affect the agricultural productivity worldwide. Improving the yield under drought is an urgent challenge in agriculture. Brachypodium distachyon is a model species for monocot plants such as wheat, barley and several potential biofuel grasses. In the current study, a comprehensive proteome analysis was conducted on Brachypodium leaves under different levels of drought application. To screen gradual changes upon drought, Brachypodium leaves subjected to drought for 4, 8 and 12 days were collected for each treatment day and relative water content of the leaves was measured for each time point. Cellular responses of Brachypodium were investigated through a proteomic approach involving two dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Among 497 distinct spots in Brachypodium protein repertoire, a total of 13 differentially expressed proteins (DEPs) were identified as responsive to drought by mass spectrometry and classified according to their functions using bioinformatics tools. The biological functions of DEPs included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, which responded differentially at each time point of drought treatment. To examine further transcriptional expression of the genes that code identified protein, quantitative real time PCR (qRT-PCR) was performed. Identified proteins will contribute to the studies involving development of drought-resistant crop species and lead to the delineation of molecular mechanisms in drought response.

Proteomic profiling of HBV infected liver biopsies with different fibrotic stages.

BACKGROUND: Hepatitis B virus (HBV) is a global health problem, and infected patients if left untreated may develop cirrhosis and eventually hepatocellular carcinoma. This study aims to enlighten pathways associated with HBV related liver fibrosis for delineation of potential new therapeutic targets and biomarkers. METHODS: Tissue samples from 47 HBV infected patients with different fibrotic stages (F1 to F6) were enrolled for 2D-DIGE proteomic screening. Differentially expressed proteins were identified by mass spectrometry and verified by western blotting. Functional proteomic associations were analyzed by EnrichNet application. RESULTS: Fibrotic stage variations were observed for apolipoprotein A1 (APOA1), pyruvate kinase PKM (KPYM), glyceraldehyde 3-phospahate dehydrogenase (GAPDH), glutamate dehydrogenase (DHE3), aldehyde dehydrogenase (ALDH2), alcohol dehydrogenase (ALDH1A1), transferrin (TRFE), peroxiredoxin 3 (PRDX3), phenazine biosynthesis-like domain-containing protein (PBLD), immuglobulin kappa chain C region (IGKC), annexin A4 (ANXA4), keratin 5 (KRT5). Enrichment analysis with Reactome and Kegg databases highlighted the possible involvement of platelet release, glycolysis and HDL mediated lipid transport pathways. Moreover, string analysis revealed that HIF-1α (Hypoxia-inducible factor 1-alpha), one of the interacting partners of HBx (Hepatitis B X protein), may play a role in the altered glycolytic response and oxidative stress observed in liver fibrosis. CONCLUSIONS: To our knowledge, this is the first protomic research that studies HBV infected fibrotic human liver tissues to investigate alterations in protein levels and affected pathways among different fibrotic stages. Observed changes in the glycolytic pathway caused by HBx presence and therefore its interactions with HIF-1α can be a target pathway for novel therapeutic purposes.

Bag-1 promotes cell survival through c-Myc-mediated ODC upregulation that is not preferred under apoptotic stimuli in MCF-7 cells.

Bag-1, Bcl-2 associated athanogene-1, is a multifunctional protein that can regulate a wide variety of cellular processes: proliferation, cell survival, transcription, apoptosis and motility. Bag-1 interacts with various targets in the modulation of these pathways; yet molecular details of Bag-1's involvement in each cellular event are still unclear. We first showed that forced Bag-1 expression promotes cell survival and prevents drug-induced apoptosis in MCF-7 breast cancer cells. Increased mRNA expressions of c-myc protooncogene and ornithine decarboxylase (ODC), biosynthetic enzyme of polyamines, were detected in Bag-1L+ cells, and western blots against the protein product of c-Myc and ODC confirmed these findings. Once ODC, a c-Myc target, gets activated, polyamine biosynthesis increases. We observed enhanced polyamine content in the Bag-1L+ cells. On the contrary, when polyamine catabolic mechanisms were investigated, Bag-1 silencing suppressed biosynthesis of polyamines because of the downregulation of ODC and upregulation of PAO. Exposure of cells to apoptotic inducers enhances the cell death mechanism by producing toxic products such as H2 O2 and aldehydes. Bag-1L+ cells prevented drug-induced PAO activation leading to a decrease in H2 O2 production following cisplatin or paclitaxel treatment. In this line, our results suggested that Bag-1 indirectly affects cell survival through c-Myc activated signalling that causes elevation of ODC levels, leading to an increase of the polyamine content.

A two-step purification platform for efficient removal of Fab-related impurities: A case study for Ranibizumab.

Antibodies (mAbs) and antibody fragments (Fabs) constitute one of the largest and most rapidly expanding groups of protein pharmaceuticals. In particular, antibody fragments have certain advantages over mAbs in some therapeutic settings. However, due to their greater chemical diversity, they are more challenging to purify for large-scale production using a standard purification platform. Besides, the removal of Fab-related byproducts poses a difficult purification challenge. Alternative Fab purification platforms could expedite their commercialization and reduce the cost and time invested. Accordingly, we employed a strong cation exchanger using a pH-based, highly linear gradient elution mode following Protein L affinity purification and developed a robust two-step purification platform for an antibody fragment. The optimized pH gradient elution conditions were determined on the basis of purity level, yield, and the abundance of Fab-related impurities, particularly free light chain. The purified Fab molecule Ranibizumab possessed a high degree of similarity to its originator Lucentis. The developed purification platform highly intensified the process and provided successful clearance of formulated Fab- and process-related impurities (∼98 %) with an overall process recovery of 50 % and, thus, might be a new option for Fab purification for both academic and industrial purposes.

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source "Turkish DeLight".

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g., proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryocrystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

Pectin-Zeolite-Based Wound Dressings with Controlled Albumin Release.

Hypoalbuminemia can lead to poor and delayed wound healing, while it is also associated with acute myocardial infarction, heart failure, malignancies, and COVID-19. In elective surgery, patients with low albumin have high risks of postoperative wound complications. Here, we propose a novel cost-effective wound dressing material based on low-methoxy pectin and NaA-zeolite particles with controlled albumin release properties. We focused on both albumin adsorption and release phenomena for wounds with excess exudate. Firstly, we investigated albumin dynamics and calculated electrostatic surfaces at experimental pH values in water by using molecular dynamics methods. Then, we studied in detail pectin-zeolite hydrogels with both adsorption and diffusion into membrane methods using different pH values and albumin concentrations. To understand if uploaded albumin molecules preserved their secondary conformation in different formulations, we monitored the effect of pH and albumin concentration on the conformational changes in albumin after it was released from the hydrogels by using CD-UV spectroscopy analyses. Our results indicate that at pH 6.4, BSA-containing films preserved the protein's folded structure while the protein was being released to the external buffer solutions. In vitro wound healing assay indicated that albumin-loaded hydrogels showed no toxic effects on the fibroblast cells.

CRISPR/Cas9-mediated Bag-1 knockout increased mesenchymal characteristics of MCF-7 cells via Akt hyperactivation-mediated actin cytoskeleton remodeling.

Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation.

The comparison of differentially expressed microRNAs in Bag-1 deficient and wild type MCF-7 breast cancer cells by small RNA sequencing.

The multifunctional BAG-1 (Bcl-2 athanogene-1) protein promotes breast cancer survival through direct or indirect interaction partners. The number of the interacting partners determines its cellular role in different conditions. As well as interaction partner variability, the amount of BAG-1 protein in the cells could cause dramatic alterations. According to previous studies, while the transient silencing of Bag-1 enhanced drug-induced apoptosis, deletion of BAG-1 could induce stemness properties and Akt-mediated actin remodeling in MCF-7 breast cancer cells. Considering the heterogeneity of breast cancer and the variability of BAG-1 -mediated cell response, it has become essential to determine microRNA (miRNA) functions in breast cancer depending on Bag-1 expression level. This study aims to compare microRNA expression levels in wt and Bag-1 knockout (KO) MCF-7 breast cancer cells. hsa-miR-429 was selected as a potential miRNA in BAG-1KO MCF-7 cells because of the downregulation both in bioinformatics and validation qRT-PCR assay. According to predicted mRNA targets and functional enrichment analysis the ten hub proteins that are phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3CA), kinase insert domain receptor (KDR), GRB2 associated binding protein 1 (GAB1), Rac family small GTPase1 (RAC1), vascular endothelial growth factor A (VEGFA), Cbl proto-oncogene (CBL), syndecan 2 (SDC2), phospholipase C gamma 1 (PLCG1), E1A binding protein p300 (EP300), and CRK like proto-oncogene, adaptor protein (CRKL) were identified as targets of hsa-miR-429. The functional enrichment analysis showed that the most significant proteins were enriched in PI3K/Akt, focal adhesion, cytoskeleton regulation, proteoglycans in cancer, and Ras signaling pathways. It was determined that hsa-miR-429 targeted these pathways in Bag-1 deficient conditions and could be used as a potential therapeutic target in future studies.

COVID-19 PCR test performance on samples stored at ambient temperature.

The WHO-named Coronavirus Disease 2019 (COVID-19) infection had become a pandemic within a short time period since it was detected in Wuhan. The outbreak required the screening of millions of samples daily and overwhelmed diagnostic laboratories worldwide. During this pandemic, the handling of patient specimens according to the universal guidelines was extremely difficult as the WHO, CDC and ECDC required cold chain compliance during transport and storage of the swab samples. The aim of this study was to compare the effects of two different storage conditions on the COVID-19 real-time PCR assay on 30 positive nasopharyngeal and/or oropharyngeal samples stored at both ambient temperature (22 ± 2 °C) and +4 °C. The results revealed that all the samples stored at ambient temperature remain PCR positive for at least six days without any false-negative result. In conclusion, transporting and storing these types of swab samples at ambient temperature for six days under resource-limited conditions during the COVID-19 pandemics are acceptable.

Determining the accuracy of next generation sequencing based copy number variation analysis in Hereditary Breast and Ovarian Cancer.

BACKGROUND: Copy number variations (CNVs) are commonly associated with malignancies, including hereditary breast and ovarian cancers. Next generation sequencing (NGS) provides solutions for CNV detection in a single run. This study aimed to compare the accuracy of CNV detection by NGS analyzing tool against Multiplex Ligation Dependent Probe Amplification (MLPA). RESEARCH DESIGN AND METHODS: In total, 1276 cases were studied by targeted NGS panels and 691 cases (61 calls in 58 NGS-CNV positive and 633 NGS-CNV negative cases) were validated by MLPA. RESULTS: Twenty-eight (46%) NGS-CNV positive calls were consistent, whereas 33 (54%) calls showed discordance with MLPA. Two cases were detected as SNV by the NGS and CNV by the MLPA analysis. In total, 2% of the cases showed an MLPA confirmed CNV region in BRCA1/2. The results of this study showed that despite the high false positive call rate of the NGS-CNV algorithm, there were no false negative calls. The cases that were determined to be negative by the NGS and positive by the MLPA were actually carrying SNVs that were located on the MLPA probe binding sites. CONCLUSION: The diagnostic performance of NGS-CNV analysis is promising; however, the need for confirmation by different methods remains.

In vitro labeling of hydroxyapatite minerals by an engineered protein.

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.

Combined Metabolic Activators Accelerates Recovery in Mild-to-Moderate COVID-19.

COVID-19 is associated with mitochondrial dysfunction and metabolic abnormalities, including the deficiencies in nicotinamide adenine dinucleotide (NAD+ ) and glutathione metabolism. Here it is investigated if administration of a mixture of combined metabolic activators (CMAs) consisting of glutathione and NAD+ precursors can restore metabolic function and thus aid the recovery of COVID-19 patients. CMAs include l-serine, N-acetyl-l-cysteine, nicotinamide riboside, and l-carnitine tartrate, salt form of l-carnitine. Placebo-controlled, open-label phase 2 study and double-blinded phase 3 clinical trials are conducted to investigate the time of symptom-free recovery on ambulatory patients using CMAs. The results of both studies show that the time to complete recovery is significantly shorter in the CMA group (6.6 vs 9.3 d) in phase 2 and (5.7 vs 9.2 d) in phase 3 trials compared to placebo group. A comprehensive analysis of the plasma metabolome and proteome reveals major metabolic changes. Plasma levels of proteins and metabolites associated with inflammation and antioxidant metabolism are significantly improved in patients treated with CMAs as compared to placebo. The results show that treating patients infected with COVID-19 with CMAs lead to a more rapid symptom-free recovery, suggesting a role for such a therapeutic regime in the treatment of infections leading to respiratory problems.

Interactome analysis of Bag-1 isoforms reveals novel interaction partners in endoplasmic reticulum-associated degradation.

Bag-1 is a multifunctional protein that regulates Hsp70 chaperone activity, apoptosis, and proliferation. The three major Bag-1 isoforms have different subcellular localizations and partly non-overlapping functions. To identify the detailed interaction network of each isoform, we utilized mass spectrometry-based proteomics and found that interactomes of Bag-1 isoforms contained many common proteins, with variations in their abundances. Bag-1 interactomes were enriched with proteins involved in protein processing and degradation pathways. Novel interaction partners included VCP/p97; a transitional ER ATPase, Rad23B; a shuttling factor for ubiquitinated proteins, proteasome components, and ER-resident proteins, suggesting a role for Bag-1 also in ER-associated protein degradation (ERAD). Bag-1 pull-down from cells and tissues from breast cancer patients validated these interactions and showed cancer-related prominence. Using in silico predictions we detected hotspot residues of Bag-1. Mutations of these residues caused loss of binding to protein quality control elements and impaired proteasomal activity in MCF-7 cells. Following CD147 glycosylation pattern, we showed that Bag-1 downregulated VCP/p97-dependent ERAD. Overall, our data extends the interaction map of Bag-1, and broadens its role in protein homeostasis. Targeting the interaction surfaces revealed in this study might be an effective strategy in the treatment of cancer.