Application of biochemical biomarkers to aid precision diagnosis

Biochemistry is a ubiquitous speciality that crosses many specialities in medicine. Biomarkers are the foundation on which testing is done in this discipline. The identification and assessment of biomarkers has been well established, but the interpretation of these biomarkers against a reference population is impacted by various factors. There are pre-analytical, analytical and post-analytical factors that need to be taken into consideration as well as the population the biomarker is validated from. The current need for precision medicine has driven the need for precision diagnosis. The current processes to identify and interpret biomarkers using large population data, which opposes this new requirement. This thesis presents seven papers that provide data for three areas where precision diagnosis is challenged due to the health burden of each disease. The first area is the detection of disease in high-risk patients, the second is the detection of biomarkers that detect poor prognosis in patients with chronic disease and the third is to optimise pre- and post-analytical to improve biomarker applications. The papers here demonstrate that they meet these aims are provide important data to each individual field, ultimately, improving the clinical use of the biomarkers in improve detection of disease and initiating treatment that will reduce morbidity and mortality. As stated earlier these papers span different specialities where Biochemistry plays an important role

Is There a Link between Bisphenol A (BPA), a Key Endocrine Disruptor, and the Risk for SARS-CoV-2 Infection and Severe COVID-19?

Infection by the severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) is the causative agent of a new disease (COVID-19). The risk of severe COVID-19 is increased by certain underlying comorbidities, including asthma, cancer, cardiovascular disease, hypertension, diabetes, and obesity. Notably, exposure to hormonally active chemicals called endocrine-disrupting chemicals (EDCs) can promote such cardio-metabolic diseases, endocrine-related cancers, and immune system dysregulation and thus, may also be linked to higher risk of severe COVID-19. Bisphenol A (BPA) is among the most common EDCs and exerts its effects via receptors which are widely distributed in human tissues, including nuclear oestrogen receptors (ERα and ERβ), membrane-bound oestrogen receptor (G protein-coupled receptor 30; GPR30), and human nuclear receptor oestrogen-related receptor gamma. As such, this paper focuses on the potential role of BPA in promoting comorbidities associated with severe COVID-19, as well as on potential BPA-induced effects on key SARS-CoV-2 infection mediators, such as angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Interestingly, GPR30 appears to exhibit greater co-localisation with TMPRSS2 in key tissues like lung and prostate, suggesting that BPA exposure may impact on the local expression of these SARS-CoV-2 infection mediators. Overall, the potential role of BPA on the risk and severity of COVID-19 merits further investigation

Cardiovascular Biomarkers: Lessons of the Past and Prospects for the Future

Cardiovascular diseases (CVDs) are a major healthcare burden on the population worldwide. Early detection of this disease is important in prevention and treatment to minimise morbidity and mortality. Biomarkers are a critical tool to either diagnose, screen, or provide prognostic information for pathological conditions. This review discusses the historical cardiac biomarkers used to detect these conditions, discussing their application and their limitations. Identification of new biomarkers have since replaced these and are now in use in routine clinical practice, but still do not detect all disease. Future cardiac biomarkers are showing promise in early studies, but further studies are required to show their value in improving detection of CVD above the current biomarkers. Additionally, the analytical platforms that would allow them to be adopted in healthcare are yet to be established. There is also the need to identify whether these biomarkers can be used for diagnostic, prognostic, or screening purposes, which will impact their implementation in routine clinical practice

Methylation of the imprinted GNAS1 gene in cell-free plasma DNA : equal steady-state quantities of methylated and unmethylated DNA in plasma

Background Genomic DNA sequences in cell-free plasma are biomarkers of cancer prognosis, where characteristic changes in methylation of tumour suppressor or oncogene DNA regions are indicative of changes in gene activity. Also, cell-free fetal DNA can be distinguished, by its methylation status, from the maternal DNA in the plasma of pregnant women, hence providing DNA biomarkers for the proposed minimally-invasive diagnosis of fetal aneuploidies, including Down's syndrome. However, the production and clearance of cell-free DNA from plasma in relation to its methylation status, are poorly understood processes. Methods We studied the methylation status of DNA derived from the imprinted GNAS1 locus, in cell-free plasma DNA of healthy adults. Heterozygotes were identified that carried the SNP rs1800905 in the imprinted region. The parent-of-origin-dependent DNA methylation was analysed by bisulfite conversion, followed by cloning and sequencing. Results Genomic DNA molecules derived from both the methylated, maternal, allele and the unmethylated, paternal, allele were found in plasma. Methylated and unmethylated DNA molecules were present in equal numbers. Conclusions Our data indicate that the methylation status of a DNA sequence has no effect on its steady state concentration in the cell-free DNA component of plasma, in healthy adults

Neuropilin‑1 as a new potential SARS‑CoV‑2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID‑19

Infection by the severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) is the cause of the new viral infectious disease (coronavirus disease 2019; COVID-19). Emerging evidence indicates that COVID-19 may be associated with a wide spectrum of neurological symptoms and complications with central nervous system (CNS) involvement. It is now well-established that entry of SARS-CoV-2 into host cells is facilitated by its spike proteins mainly through binding to the angiotensin-converting enzyme 2 (ACE-2). Preclinical studies have suggested that neuropilin-1 (NRP1), which is a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in COVID-19 by enhancing the entry of SARS-CoV-2 into the brain through the olfactory epithelium. In the present study, we expand on these findings and demonstrate that the NRP1 is also expressed in the CNS, including olfactory-related regions such as the olfactory tubercles and paraolfactory gyri. This furthers supports the potential role of NRP1 as an additional SARS-CoV-2 infection mediator implicated in the neurologic manifestations of COVID-19. Accordingly, the neurotropism of SARS-CoV-2 via NRP1-expressing cells in the CNS merits further investigation

Anti-osteoporotic treatments in the era of non-alcoholic fatty liver disease: friend or foe

Over the last years non-alcoholic fatty liver disease (NAFLD) has grown into the most common chronic liver disease globally, affecting 17-38% of the general population and 50-75% of patients with obesity and/or type 2 diabetes mellitus (T2DM). NAFLD encompasses a spectrum of chronic liver diseases, ranging from simple steatosis (non-alcoholic fatty liver, NAFL) and non-alcoholic steatohepatitis (NASH; or metabolic dysfunction-associated steatohepatitis, MASH) to fibrosis and cirrhosis with liver failure or/and hepatocellular carcinoma. Due to its increasing prevalence and associated morbidity and mortality, the disease-related and broader socioeconomic burden of NAFLD is substantial. Of note, currently there is no globally approved pharmacotherapy for NAFLD. Similar to NAFLD, osteoporosis constitutes also a silent disease, until an osteoporotic fracture occurs, which poses a markedly significant disease and socioeconomic burden. Increasing emerging data have recently highlighted links between NAFLD and osteoporosis, linking the pathogenesis of NAFLD with the process of bone remodeling. However, clinical studies are still limited demonstrating this associative relationship, while more evidence is needed towards discovering potential causative links. Since these two chronic diseases frequently co-exist, there are data suggesting that anti-osteoporosis treatments may affect NAFLD progression by impacting on its pathogenetic mechanisms. In the present review, we present on overview of the current understanding of the liver-bone cross talk and summarize the experimental and clinical evidence correlating NAFLD and osteoporosis, focusing on the possible effects of anti-osteoporotic drugs on NAFLD

Pan‑cancer analysis of transmembrane protease serine 2 and cathepsin L that mediate cellular SARS‑CoV‑2 infection leading to COVID-19

Severe acute respiratory syndrome (SARS) coronavirus‑2 (SARS‑CoV2) is the cause of a new disease (COVID‑19) which has evolved into a pandemic during the first half of 2020. Older age, male sex and certain underlying diseases, including cancer, appear to significantly increase the risk for severe COVID‑19. SARS‑CoV‑2 infection of host cells is facilitated by the angiotensin‑converting enzyme 2 (ACE‑2), and by transmembrane protease serine 2 (TMPRSS2) and other host cell proteases such as cathepsin L (CTSL). With the exception of ACE‑2, a systematic analysis of these two other SARS‑CoV2 infection mediators in malignancies is lacking. Here, we analysed genetic alteration, RNA expression, and DNA methylation of TMPRSS2 and CTSL across a wide spectrum of tumors and controls. TMPRSS2 was overexpressed in cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), uterine corpus endometrial carcinoma and uterine carcinosarcoma, with PRAD and READ exhibiting the highest expression of all cancers. CTSL was upregulated in lymphoid neoplasm diffuse large B‑cell lymphoma, oesophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, lower grade glioma, pancreatic adenocarcinoma, skin cutaneous melanoma, stomach adenocarcinoma, and thymoma. Hypo‑methylation of both genes was evident in most cases where they have been highly upregulated. We have expanded on our observations by including data relating to mutations and copy number alterations at pan‑cancer level. The novel hypotheses that are stemming out of these data need to be further investigated and validated in large clinical studies

Retinol-binding protein 4 (RBP4) circulating levels and gestational diabetes mellitus:a systematic review and meta-analysis

Background: Gestational diabetes mellitus (GDM) is a prevalent condition where diabetes is diagnosed during pregnancy, affecting both maternal and fetal outcomes. Retinol-binding protein 4 (RBP4) is a circulating adipokine which belongs to the lipocalin family and acts as a specific carrier protein that delivers retinol (vitamin A) from the liver to the peripheral tissues. Growing data indicate that circulating RBP4 levels may positively correlate with GDM. Thus, this systematic review and meta-analysis aimed to investigate the potential relationship between circulating RBP4 levels and GDM when measured at various stages of pregnancy. Methods: MEDLINE, CINAHL, EMCARE, EMBASE, Scopus, and Web of Science databases were searched to identify studies comparing pregnant women with and without GDM, whose circulating RBP4 levels were measured in at least one pregnancy trimester. Findings were reported using standardized mean difference (SMD) and random-effects models were used to account for variability among studies. Furthermore, the risk of bias was assessed using the RoBANS tool. Results: Out of the 34 studies identified, 32 were included in the meta-analysis (seven with circulating RBP4 levels measured in the first trimester, 19 at 24–28 weeks, and 14 at >28 weeks of pregnancy). RBP4 levels were statistically higher in the GDM group than in controls when measured during all these pregnancy stages, with the noted RBP4 SMD being 0.322 in the first trimester (95% CI: 0.126–0.517; p 28 weeks of gestation (95% CI: 0.252–1.498; p = 0.006; 870 GDM cases vs. 1942 non-GDM controls). Significant study heterogeneity was noted for all three pregnancy timepoints. Conclusion: The present findings indicate consistently higher circulating RBP4 levels in GDM cases compared to non-GDM controls, suggesting the potential relevance of RBP4 as a biomarker for GDM. However, the documented substantial study heterogeneity, alongside imprecision in effect estimates, underscores the need for further research and standardization of measurement methods to elucidate whether RBP4 can be utilized in clinical practice as a potential GDM biomarker. Systematic review registration: PROSPERO (CRD42022340097: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022340097)

Anti-osteoporotic treatments in the era of non-alcoholic fatty liver disease:friend or foe

Over the last years non-alcoholic fatty liver disease (NAFLD) has grown into the most common chronic liver disease globally, affecting 17-38% of the general population and 50-75% of patients with obesity and/or type 2 diabetes mellitus (T2DM). NAFLD encompasses a spectrum of chronic liver diseases, ranging from simple steatosis (non-alcoholic fatty liver, NAFL) and non-alcoholic steatohepatitis (NASH; or metabolic dysfunction-associated steatohepatitis, MASH) to fibrosis and cirrhosis with liver failure or/and hepatocellular carcinoma. Due to its increasing prevalence and associated morbidity and mortality, the disease-related and broader socioeconomic burden of NAFLD is substantial. Of note, currently there is no globally approved pharmacotherapy for NAFLD. Similar to NAFLD, osteoporosis constitutes also a silent disease, until an osteoporotic fracture occurs, which poses a markedly significant disease and socioeconomic burden. Increasing emerging data have recently highlighted links between NAFLD and osteoporosis, linking the pathogenesis of NAFLD with the process of bone remodeling. However, clinical studies are still limited demonstrating this associative relationship, while more evidence is needed towards discovering potential causative links. Since these two chronic diseases frequently co-exist, there are data suggesting that anti-osteoporosis treatments may affect NAFLD progression by impacting on its pathogenetic mechanisms. In the present review, we present on overview of the current understanding of the liver-bone cross talk and summarize the experimental and clinical evidence correlating NAFLD and osteoporosis, focusing on the possible effects of anti-osteoporotic drugs on NAFLD

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis

Aims/hypothesis: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. Methods: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. Results: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. Conclusion: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis