Gene regulatory network analysis identifies MYL1, MDH2, GLS, and TRIM28 as the principal proteins in the response of mesenchymal stem cells to Mg2+ ions.

Magnesium (Mg)-based implants have emerged as a promising alternative for orthopedic applications, owing to their bioactive properties and biodegradability. As the implants degrade, Mg2+ ions are released, influencing all surrounding cell types, especially mesenchymal stem cells (MSCs). MSCs are vital for bone tissue regeneration, therefore, it is essential to understand their molecular response to Mg2+ ions in order to maximize the potential of Mg-based biomaterials. In this study, we conducted a gene regulatory network (GRN) analysis to examine the molecular responses of MSCs to Mg2+ ions. We used time-series proteomics data collected at 11 time points across a 21-day period for the GRN construction. We studied the impact of Mg2+ ions on the resulting networks and identified the key proteins and protein interactions affected by the application of Mg2+ ions. Our analysis highlights MYL1, MDH2, GLS, and TRIM28 as the primary targets of Mg2+ ions in the response of MSCs during 1-21 days phase. Our results also identify MDH2-MYL1, MDH2-RPS26, TRIM28-AK1, TRIM28-SOD2, and GLS-AK1 as the critical protein relationships affected by Mg2+ ions. By offering a comprehensive understanding of the regulatory role of Mg2+ ions on MSCs, our study contributes valuable insights into the molecular response of MSCs to Mg-based materials, thereby facilitating the development of innovative therapeutic strategies for orthopedic applications.

Targeting NETs using dual-active DNase1 variants.

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

Exploring the Concept of Spiritual Sensitivity from the Perspectives of Healthcare Providers in Iran.

Spiritual sensitivity (SS) is defined as attention to the available spiritual values in a conflicting situation and awareness of one's roles and responsibilities in that situation. It helps differentiate between the right and the wrong and leads to sound practice. This study explored the concept of SS from the perspectives of healthcare providers in Iran. This qualitative study was carried out in 2017-2019 using conventional content analysis. Twenty-two physicians, faculty members with clinical work experience, and healthcare providers were purposefully recruited. Data were collected using unstructured interviews and were analyzed using conventional content analysis. Participants' experiences of the concept of SS were grouped into three main themes, namely sense of value, spiritual growth and morale boosting, and SS as a motivator for purposeful service delivery. The findings of the present study will help healthcare managers develop programs for improving healthcare providers' spiritual sensitivity and also will help healthcare providers develop spirituality-based holistic care plans.

The effects of dyslipidemia on noise-induced hearing loss in petrochemical workers in the Southwest of Iran.

BACKGROUND: Research has shown that the decrease in the inner diameter of vessels caused by hyperlipidemia lowers the capacity for blood oxygen delivery to the cochlea. This leads to impaired cochlear metabolism and causes hearing problems. OBJECTIVE: The effects of dyslipidemia on noise-induced hearing loss in workers were examined. METHODS: This descriptive cross-sectional study was performed on 692 male employees in a petrochemical industry in the southwest of Iran exposed to 85 dB noise. Clinical audiometry and blood sample tests were used to evaluate the hearing and prevalence indices of dyslipidemia (cholesterol, triglyceride, HDL and LDL). The data were analyzed using SPSS software version 25 (p = 0.05). RESULTS: The results showed that the prevalence of dyslipidemia was 24.5% with abnormal relative triglyceride frequency of 49.5%, HDL of 28%, LDL of 33%, and total blood cholesterol level of 37.8%. There was no significant relationship between NIHL and dyslipidemia (p > 0.09). However, the major NIHL drops at different frequencies were in the individuals with dyslipidemia. The parameters age and dyslipidemia increased NIHL odds ratio (95% C.I.) by 1.130 (1.160-1.100) and 1.618 (2.418-1.082) respectively. CONCLUSION: The rate of hearing loss in individuals with dyslipidemia increases at different frequencies and it leads to an increase of the OR of NIHL in individuals with dyslipidemia. We can control dyslipidemia and its effective factors. The NIHL is more common in people exposed to noise.

A critical-cultural-relational approach to rupture resolution: A case illustration with a cross-racial dyad.

Ruptures are common in any therapeutic relationship and their successful resolution is associated with positive outcomes. However, therapist and client differences with regard to power, privilege, identity, and culture increase social and cultural distance, contributing to alliance ruptures and complicating the repair process. Informed by critical race theories, cultural psychological perspectives, and relational principles, we highlight how power, privilege, identity, and culture shape the development of ruptures and thus, how analyses of these dynamics should inform the process of repair. We present an expanded critical-cultural-relational approach to rupture resolution that emphasizes essential skills of critical self-awareness, wise affect, and anti-oppressive interpersonal engagement, and extends Safran and Muran's (2000) general rupture resolution model to emphasize a critical analysis of the rupture and repair processes. We illustrate our approach through a case presentation involving a rupture in a cross-racial dyad with themes of racism and classism.

Differential regulation of extracellular matrix proteins in three recurrent liver metastases of a single patient with colorectal cancer.

Colorectal cancer (CRC) patients suffer from the second highest mortality among all cancer entities. In half of all CRC patients, colorectal cancer liver metastases (CRLM) can be observed. Metastatic colorectal cancer is associated with poor overall survival and limited treatment options. Even after successful surgical resection of the primary tumor, metachronous liver metastases occur in one out of eight cases. The only available curative intended treatment is hepatic resection, but metachronous CRLM frequently recur after approximately 1 year. In this study, we performed a proteome analysis of three recurrent liver metastases of a single CRC patient by mass spectrometry. Despite surgical resection of the primary CRC and adjuvant chemotherapy plus cetuximab treatment, the patient developed three metachronous CRLM which occurred consecutively after 9, 21 and 31 months. We identified a set of 1132 proteins expressed in the three metachronous CRLM, of which 481 were differentially regulated, including 81 proteins that were associated with the extracellular matrix (ECM). 56 ECM associated proteins were identified as upregulated in the third metastasis, 26 (46%) of which were previously described as negative prognostic markers in CRC, including tenascin C, nidogen 1, fibulin 1 and vitronectin. These data may reflect an ascending trend of malignancy from the first to the third metachronous colorectal cancer liver metastasis. Additionally, the results indicate different ECM phenotypes for recurrent metachronous metastasis, associated with different grades of malignancy and highlights the importance of individual analysis of molecular features in different, consecutive metastatic events in a single patient.

Mass Spectrometric Comparison of HPV-Positive and HPV-Negative Oropharyngeal Cancer.

Squamous cell carcinoma of the head and neck (HNSCC) consist of two distinct biological entities. While the numbers of classical, tobacco-induced HNSCC are declining, tumors caused by human papillomavirus (HPV) infection are increasing in many countries. HPV-positive HNSCC mostly arise in the oropharynx and are characterized by an enhanced sensitivity towards radiotherapy and a favorable prognosis. To identify molecular differences between both entities on the protein level, we conducted a mass spectrometric comparison of eight HPV-positive and nine HPV-negative oropharyngeal tumors (OPSCC). Overall, we identified 2051 proteins, of which 31 were found to be differentially expressed. Seventeen of these can be assorted to three functional groups, namely DNA replication, nuclear architecture and cytoskeleton regulation, with the differences in the last group potentially reflecting an enhanced migratory and invasive capacity. Furthermore, a number of identified proteins have been described to directly impact on DNA double-strand break repair or radiation sensitivity (e.g., SLC3A2, cortactin, RBBP4, Numa1), offering explanations for the differential prognosis. The unequal expression of three proteins (SLC3A2, MCM2 and lamin B1) was confirmed by immunohistochemical staining using a tissue microarray containing 205 OPSCC samples. The expression levels of SLC3A2 and lamin B1 were found be of prognostic relevance in patients with HPV-positive and HPV-negative OPSCC, respectively.

Understanding Protein Networks Using Vester's Sensitivity Model.

Magnesium-based biomaterials belong to the third generation of biomaterials that are also bioactive. These smart materials combine bioactivity and biodegradability, and elicit specific cellular responses at the molecular level. In fact, osteoinductive properties have been observed in mesenchymal stem cells in the presence of Magnesium. The mechanistic understanding of the physiological effects however, remains a difficult task as Mg is involved in a multitude of biological reactions. The study of protein interactions may shed light on the molecular processes in Mg-stimulated cells, therefore, suitable data mining tools are required to analyze the large amount data generated via proteomics. Protein compositions over time between two conditions (human mesenchymal stem cells cultured with and without Mg degradation products) were analyzed using Vester's Sensitivity Model. Proteins whose dynamics significantly change from one setup to the other were classified into four categories: passive, active, critical, and buffering according to their regulatory activity. In this work, we demonstrated the use of Vester's Sensitivity Model as an appropriate data mining tool. Protein network analyses highlighted the primary role of Mg-based implant degradation on cell metabolism without deleterious effect on cell viability. Furthermore, key proteins involved in calcium-dependant cellular activities were emphasized leading to further studies.

The Psychiatric Drug Lithium Increases DNA Damage and Decreases Cell Survival in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines Expos ed to Ionizing Radiation.

BACKGROUND: Breast cancer is the most common cancer among women. Radiation therapy is used for treating almost every stage of breast cancer. A strategy to reduce irradiation side effects and to decrease the recurrence of cancer is concurrent use of radiation and radiosensitizers. We studied the effect of the antimanic drug lithium on radiosensitivity of estrogen-receptor (ER)-positive MCF-7 and ER-negative, invasive, and radioresistant MDA-MB-231 breast cancer cell lines. METHODS: MCF-7 and MDA-MB-231 breast cancer cell lines were treated with 30 mM and 20 mM concentrations of lithium chloride (LiCl), respectively. These concentrations were determined by MTT viability assay. Growth curves were depicted and comet assay was performed for control and LiCl-treated cells after exposure to X-ray. Total and phosphorylated inactive levels of glycogen synthase kinase-3beta (GSK-3ß) protein were determined by ELISA assay for control and treated cells. RESULTS: Treatment with LiCl decreased cell proliferation after exposure to X-ray as indicated by growth curves of MCF-7 and MDA-MB-231 cell lines within six days following radiation. Such treatment increased the amount of DNA damages represented by percent DNA in Tails of comets at 0, 1, 4, and even 24 hours after radiation in both studied cell lines. The amount of active GSK-3ß was increased in LiCl-treated cells in ER-positive and ER-negative breast cancer cell lines. CONCLUSION: Treatment with LiCl that increased the active GSK-3ß protein, increased DNA damages and decreased survival independent of estrogen receptor status in breast cancer cells exposed to ionizing radiation.

Proteome analysis of human mesenchymal stem cells undergoing chondrogenesis when exposed to the products of various magnesium-based materials degradation.

Treatment of physeal fractures (15%-30% of all paediatric fractures) remains a challenge as in approximately 10% of the cases, significant growth disturbance may occur. Bioresorbable Magnesium-based implants represent a strategy to minimize damage (i.e., load support until bone healing without second surgery). Nevertheless, the absence of harmful effects of magnesium-implants and their degradation products on the growth plate should be confirmed. Here, the proteome of human mesenchymal stem cells undergoing chondrogenesis was evaluated when exposed to the products of various Magnesium-based materials degradation. The results of this study indicate that the materials induced regulation of proteins associated with cell chondrogenesis and cartilage formation, which should be beneficial for cartilage regeneration.

Ultrafast extraction of proteins from tissues using desorption by impulsive vibrational excitation.

A picosecond IR laser (PIRL) can be used to blast proteins out of tissues through desorption by impulsive excitation (DIVE) of intramolecular vibrational states of water molecules in the cell in less than a millisecond. With PIRL-DIVE proteins covering a range of a few kDa up to several MDa are extracted in high quantities compared to conventional approaches. The chemical composition of extracted proteins remains unaltered and even enzymatic activities are maintained.

MALDI imaging-based identification of prognostically relevant signals in bladder cancer using large-scale tissue microarrays.

OBJECTIVE: Although most patients with urinary bladder cancer present with noninvasive and low-malignant stages of the disease, about 20% eventually develop life-threatening metastatic tumors. This study was designed to evaluate the potential of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to identify molecular markers predicting the clinical course of bladder cancer. MATERIALS AND METHODS: We employed MALDI-MSI to a bladder cancer tissue microarray including paraffin-embedded tissue samples from 697 patients with clinical follow-up data to search for prognostically relevant associations. RESULTS: Analysis of our MALDI imaging data revealed 40 signals in the mass spectra (m/z signals) associated with epithelial structures. The presence of numerous m/z signals was statistically related to one or several phenotypical findings including tumor aggressiveness (stage, grade, or nodal status; 30 signals), solid (5 signals) or papillary (3 signals) growth patterns, and increased (6 signals) or decreased (12 signals) cell proliferation, as determined by Ki-67 immunohistochemistry. Two signals were linked with tumor recurrence in noninvasive (pTa category) tumors, of which one was also related to progression from pTa-category to pT1-category disease. The absence of one m/z signal was linked with decreased survival in the subset of 102 muscle-invasive cancers. CONCLUSION: Our data demonstrate the suitability of combining MSI and large-scale tissue microarrays to simultaneously identify and validate clinically useful molecular markers in urinary bladder cancer.

MALDI imaging on large-scale tissue microarrays identifies molecular features associated with tumour phenotype in oesophageal cancer.

AIMS: Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) and tissue microarray (TMA) technologies were jointly utilized to search for molecular features associated with clinicopathological parameters in oesophageal cancer. METHODS AND RESULTS: Two TMAs from formalin-fixed tissue samples, including 300 adenocarcinomas and 177 squamous cell carcinomas with clinical follow-up data, were analysed. MALDI-MSI analysis revealed 72 distinct mass per charge (m/z) signals associated with tumour cells, 48 of which were found in squamous cell carcinomas only, and 12 of which were specific for adenocarcinomas. In adenocarcinomas, six signals were linked to early-stage (pT1-T2) tumours (two signals) and the presence (one signal) or absence (three signals) of lymph node metastasis. In squamous cell carcinomas, 24 signals were strongly linked to different phenotypic features, including tumour stage (four signals), histological grade (four signals), and lymph node metastasis (three signals). CONCLUSIONS: The high number of m/z signals that were found to be significantly linked to one or more phenotypic features of oesophageal cancer highlights the power of MALDI-MSI in the analysis of high-density TMAs. The data also emphasise substantial biological differences between adenocarcinomas and squamous cell carcinomas.

MALDI mass spectrometric imaging based identification of clinically relevant signals in prostate cancer using large-scale tissue microarrays.

To identify molecular features associated with clinico-pathological parameters and TMPRSS2-ERG fusion status in prostate cancer, we employed MALDI mass spectrometric imaging (MSI) to a prostate cancer tissue microarray (TMA) containing formalin-fixed, paraffin-embedded tissues samples from 1,044 patients for which clinical follow-up data were available. MSI analysis revealed 15 distinct mass per charge (m/z)-signals associated to epithelial structures. A comparison of these signals with clinico-pathological features revealed statistical association with favorable tumor phenotype such as low Gleason grade, early pT stage or low Ki67 labeling Index (LI) for four signals (m/z 700, m/z 1,502, m/z 1,199 and m/z 3,577), a link between high Ki67LI for one signal (m/z 1,013) and a relationship with prolonged time to PSA recurrence for one signal (m/z 1,502; p = 0.0145). Multiple signals were associated with the ERG-fusion status of our cancers. Two of 15 epithelium-associated signals including m/z 1,013 and m/z 1,502 were associated with detectable ERG expression and five signals (m/z 644, 678, 1,044, 3,086 and 3,577) were associated with ERG negativity. These observations are in line with substantial molecular differences between fusion-type and non-fusion type prostate cancer. The signals observed in this study may characterize molecules that play a role in the development of TMPRSS2-ERG fusions, or alternatively reflect pathways that are activated as a consequence of ERG-activation. The combination of MSI and large-scale TMAs reflects a powerful approach enabling immediate prioritization of MSI signals based on associations with clinico-pathological and molecular data.

Expression of a novel chimeric truncated t-PA in CHO cells based on in silico experiments.

Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.

Cloning and expression of Leishmania major superoxide dismutase B1: A potential target antigen for serodiagnosis of Leishmaniasis.

BACKGROUND: Leishmaniasis- a neglected public health problem- is a group of diseases affecting an estimated 12 million people worldwide. OBJECTIVE: In the present study, recombinant Leishmania major superoxide dismutase B1 (rLmSODB1) has been utilized as a potential antigen for the serodiagnosis of human cutaneous (CL) and visceral leishmaniasis (VL) in the endemic regions of southern part of Iran. Additionally, the sensitivity and specificity of ELISA-based serodiagnosis using rLmSODB1 and the soluble Leishmania antigen (SLA) were compared. METHODS: For the first time, rLmSODB1 has been cloned successfully and used for ELISA-based serodiagnosis. Sera from 30 CL and 24 VL cases were included in this study. Additional studies were also done for the evaluation of cross-reactivity using sera from 41 endemic controls including normal endemic donors (n=20), systemic lupus erythematosus patients (n=5), rheumatoid arthritis patients (n=5), and patients with tuberculosis (n=11). RESULTS: Analysis indicated that rLmSODB1 was recognized by 62.5% and 13.3% of sera from patients with VL and CL, showing a sensitivity of 72.7% and 53.6%, respectively. However 95.8% of VL and 30% of CL sera reacted with SLA, revealing sensitivities of 96% and 58.8%, respectively. Additionally, from 41 sera collected either from healthy subjects or patients affected with other diseases, 97.5% were negative with SLA or rLmSODB1 (specificity 97.6%). CONCLUSION: These results show that rLmSODB1 almost does not react with sera from patients with tuberculosis and autoimmune diseases and may be considered as a candidate antigen for the specific immunodiagnosis of visceral leishmaniasis.