Biomolecules

Molecular fluorescence-guided surgery using near-infrared light has the potential to improve the rate of complete resection of cancer. Typically, monoclonal antibodies are being used as targeting moieties, however smaller fragments, such as single-domain antibodies (i.e., Nanobodies^®) improve tumor specificity and enable tracer injection on the same day as surgery. In this study, the feasibility of a carcinoembryonic antigen-targeting Nanobody (NbCEA5) conjugated to two zwitterionic dyes (ZW800-1 Forte [ZW800F] and ZW800-1) for visualization of pancreatic ductal adenocarcinoma (PDAC) was investigated. After site-specific conjugation of NbCEA5 to the zwitterionic dyes, binding specificity was evaluated on human PDAC cell lines with flow cytometry. A dose escalation study was performed for both NbCEA5-ZW800F and NbCEA5-ZW800-1 in mice with subcutaneously implanted pancreatic tumors. Fluorescence imaging was performed up to 24 h after intravenous injection. Furthermore, the optimal dose for NbCEA5-ZW800-1 was injected in mice with orthotopically implanted pancreatic tumors. A dose-escalation study showed superior mean fluorescence intensities for NbCEA5-ZW800-1 compared to NbCEA5-ZW800F. In the orthotopic tumor models, NbCEA5-ZW800-1 accumulated specifically in pancreatic tumors with a mean in vivo tumor-to-background ratio of 2.4 (SD = 0.23). This study demonstrated the feasibility and potential advantages of using a CEA-targeted Nanobody conjugated to ZW800-1 for intraoperative PDAC imaging. Full article

Despite recent advances in treatment and significant improvements in prognosis, thrombosis remains the major cause of death in systemic lupus erythematosus (SLE). Antiphospholipid antibodies (aPL) are the main triggers of thrombosis in patients with SLE, with a frequency of approximately 30–40%. Lupus anticoagulant, anticardiolipin, and anti-β2-glycoprotein I antibodies, which are included in the criteria for antiphospholipid syndrome, and ‘non-criteria’ aPL such as anti-phosphatidylserine/prothrombin complex antibodies, are risk factors for thrombosis in patients with SLE. Multiple positivity for aPL is also associated with an increased risk of thrombosis, and scores calculated from aPL profiles can predict the risk of developing thrombosis. Although there is insufficient evidence for treatment, aPL-positive SLE patients should/may be treated with anticoagulants and/or low-dose aspirin as appropriate. This review summarises the evidence on the clinical significance of the aPL profile as a biomarker of thrombophilia in patients with SLE. Full article

Objective: to analyze the association between blood lipid metabolism and osteoporosis (OP) in older adults with type 2 diabetes mellitus (T2DM). Methods: a total of 1158 older patients with T2DM treated by the Department of Endocrinology, Peking University International Hospital, were retrospectively analyzed, including 541 postmenopausal women and 617 men. Results: (1) Levels of low-density lipoprotein cholesterol (LDL-C) were significantly higher in the OP group, while levels of high-density lipoprotein cholesterol (HDL-C) were higher in the non-osteoporotic group (both p < 0.05). (2) Age, parathyroid hormone (PTH), total cholesterol (TC) and LDL-C were negatively linked to the patients’ bone mineral density (BMD) (all p < 0.05), while the body mass index (BMI), uric acid (UA) level, HDL-C level and glomerular filtration rate (eGFR) were positively related to their BMD (all p < 0.05). (3) In postmenopausal women, after adjustment for other indexes, raised LDL-C is an independent risk factor for OP (OR = 3.38, 95% CI 1.64, 6.98, p < 0.05) while raised HDL-C is protective (OR = 0.49, 95% CI 0.24, 0.96, p < 0.05). However, raised HDL-C was protective against OP (OR = 0.07, 95% CI 0.01, 0.53, p < 0.05). Conclusion: In older T2DM patients, the effect of blood lipid levels is related to sex. Our study conducted a detailed sex stratification. In addition to seeing the traditional risk factors of OP, such as age, sex, and BMI, we comprehensively analyzed the correlation between the blood glucose level, complications, and blood lipids with OP. HDL-C is a protective factor for OP in both men and women, while LDL-C independently predicts OP in postmenopausal women. Full article

Lowe Syndrome (LS) is a condition due to mutations in the OCRL1 gene, characterized by congenital cataracts, intellectual disability, and kidney malfunction. Unfortunately, patients succumb to renal failure after adolescence. This study is centered in investigating the biochemical and phenotypic impact of patient’s OCRL1 variants (OCRL1^VAR). Specifically, we tested the hypothesis that some OCRL1^VAR are stabilized in a non-functional conformation by focusing on missense mutations affecting the phosphatase domain, but not changing residues involved in binding/catalysis. The pathogenic and conformational characteristics of the selected variants were evaluated in silico and our results revealed some OCRL1^VAR to be benign, while others are pathogenic. Then we proceeded to monitor the enzymatic activity and function in kidney cells of the different OCRL1^VAR. Based on their enzymatic activity and presence/absence of phenotypes, the variants segregated into two categories that also correlated with the severity of the condition they induce. Overall, these two groups mapped to opposite sides of the phosphatase domain. In summary, our findings highlight that not every mutation affecting the catalytic domain impairs OCRL1′s enzymatic activity. Importantly, data support the inactive-conformation hypothesis. Finally, our results contribute to establishing the molecular and structural basis for the observed heterogeneity in severity/symptomatology displayed by patients. Full article

The dynamic mechanism of cell uptake and genomic integration of exogenous linear DNA still has to be completely clarified, especially within each phase of the cell cycle. We present a study of integration events of double-stranded linear DNA molecules harboring at their ends sequence homologies to the host’s genome, all throughout the cell cycle of the model organism Saccharomyces cerevisiae, comparing the efficiency of chromosomal integration of two types of DNA cassettes tailored for site-specific integration and bridge-induced translocation. Transformability increases in S phase regardless of the sequence homologies, while the efficiency of chromosomal integration during a specific cycle phase depends upon the genomic targets. Moreover, the frequency of a specific translocation between chromosomes XV and VIII strongly increased during DNA synthesis under the control of Pol32 polymerase. Finally, in the null POL32 double mutant, different pathways drove the integration in the various phases of the cell cycle and bridge-induced translocation was possible outside the S phase even without Pol32. The discovery of this cell-cycle dependent regulation of specific pathways of DNA integration, associated with an increase of ROS levels following translocation events, is a further demonstration of a sensing ability of the yeast cell in determining a cell-cycle-related choice of DNA repair pathways under stress. Full article

Multidrug resistance is a significant barrier that makes anticancer therapies less effective. Glutathione transferases (GSTs) are involved in multidrug resistance mechanisms and play a significant part in the metabolism of alkylating anticancer drugs. The purpose of this study was to screen and select a lead compound with high inhibitory potency against the isoenzyme GSTP1-1 from Mus musculus (MmGSTP1-1). The lead compound was selected following the screening of a library of currently approved and registered pesticides that belong to different chemical classes. The results showed that the fungicide iprodione [3-(3,5-dichlorophenyl)-2,4-dioxo-N-propan-2-ylimidazolidine-1-carboxamide] exhibited the highest inhibition potency (ΙC₅₀ = 11.3 ± 0.5 μΜ) towards MmGSTP1-1. Kinetics analysis revealed that iprodione functions as a mixed-type inhibitor towards glutathione (GSH) and non-competitive inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). X-ray crystallography was used to determine the crystal structure of MmGSTP1-1 at 1.28 Å resolution as a complex with S-(p-nitrobenzyl)glutathione (Nb-GSH). The crystal structure was used to map the ligand-binding site of MmGSTP1-1 and to provide structural data of the interaction of the enzyme with iprodione using molecular docking. The results of this study shed light on the inhibition mechanism of MmGSTP1-1 and provide a new compound as a potential lead structure for future drug/inhibitor development. Full article

Mutations in the multidomain protein Leucine-rich-repeat kinase 2 (LRRK2) have been identified as a genetic risk factor for both sporadic and familial Parkinson’s disease (PD). LRRK2 has two enzymatic domains: a RocCOR tandem with GTPase activity and a kinase domain. In addition, LRRK2 has three N-terminal domains: ARM (Armadillo repeat), ANK (Ankyrin repeat), and LRR (Leucine-rich-repeat), and a C-terminal WD40 domain, all of which are involved in mediating protein–protein interactions (PPIs) and regulation of the LRRK2 catalytic core. The PD-related mutations have been found in nearly all LRRK2 domains, and most of them have increased kinase activity and/or decreased GTPase activity. The complex activation mechanism of LRRK2 includes at least intramolecular regulation, dimerization, and membrane recruitment. In this review, we highlight the recent developments in the structural characterization of LRRK2 and discuss these developments from the perspective of the LRRK2 activation mechanism, the pathological role of the PD mutants, and therapeutic targeting. Full article

Single-cell transcriptomics is rapidly advancing our understanding of the composition of complex tissues and biological cells, and single-cell RNA sequencing (scRNA-seq) holds great potential for identifying and characterizing the cell composition of complex tissues. Cell type identification by analyzing scRNA-seq data is mostly limited by time-consuming and irreproducible manual annotation. As scRNA-seq technology scales to thousands of cells per experiment, the exponential increase in the number of cell samples makes manual annotation more difficult. On the other hand, the sparsity of gene transcriptome data remains a major challenge. This paper applied the idea of the transformer to single-cell classification tasks based on scRNA-seq data. We propose scTransSort, a cell-type annotation method pretrained with single-cell transcriptomics data. The scTransSort incorporates a method of representing genes as gene expression embedding blocks to reduce the sparsity of data used for cell type identification and reduce the computational complexity. The feature of scTransSort is that its implementation of intelligent information extraction for unordered data, automatically extracting valid features of cell types without the need for manually labeled features and additional references. In experiments on cells from 35 human and 26 mouse tissues, scTransSort successfully elucidated its high accuracy and high performance for cell type identification, and demonstrated its own high robustness and generalization ability. Full article

In the field of genetic code expansion (GCE), improvements in the efficiency of noncanonical amino acid (ncAA) incorporation have received continuous attention. By analyzing the reported gene sequences of giant virus species, we noticed some sequence differences at the tRNA binding interface. On the basis of the structural and activity differences between Methanococcus jannaschii Tyrosyl-tRNA Synthetase (MjTyrRS) and mimivirus Tyrosyl-tRNA Synthetase (MVTyrRS), we found that the size of the anticodon-recognized loop of MjTyrRS influences its suppression activity regarding triplet and specific quadruplet codons. Therefore, three MjTyrRS mutants with loop minimization were designed. The suppression of wild-type MjTyrRS loop-minimized mutants increased by 1.8–4.3-fold, and the MjTyrRS variants enhanced the activity of the incorporation of ncAAs by 15–150% through loop minimization. In addition, for specific quadruplet codons, the loop minimization of MjTyrRS also improves the suppression efficiency. These results suggest that loop minimization of MjTyrRS may provide a general strategy for the efficient synthesis of ncAAs-containing proteins. Full article

Growth factors are a class of proteins that play a role in the proliferation (the increase in the number of cells resulting from cell division) and differentiation (when a cell undergoes changes in gene expression becoming a more specific type of cell) of cells. They can have both positive (accelerating the normal healing process) and negative effects (causing cancer) on disease progression and have potential applications in gene therapy and wound healing. However, their short half-life, low stability, and susceptibility to degradation by enzymes at body temperature make them easily degradable in vivo. To improve their effectiveness and stability, growth factors require carriers for delivery that protect them from heat, pH changes, and proteolysis. These carriers should also be able to deliver the growth factors to their intended destination. This review focuses on the current scientific literature concerning the physicochemical properties (such as biocompatibility, high affinity for binding growth factors, improved bioactivity and stability of the growth factors, protection from heat, pH changes or appropriate electric charge for growth factor attachment via electrostatic interactions) of macroions, growth factors, and macroion-growth factor assemblies, as well as their potential uses in medicine (e.g., diabetic wound healing, tissue regeneration, and cancer therapy). Specific attention is given to three types of growth factors: vascular endothelial growth factors, human fibroblast growth factors, and neurotrophins, as well as selected biocompatible synthetic macroions (obtained through standard polymerization techniques) and polysaccharides (natural macroions composed of repeating monomeric units of monosaccharides). Understanding the mechanisms by which growth factors bind to potential carriers could lead to more effective delivery methods for these proteins, which are of significant interest in the diagnosis and treatment of neurodegenerative and civilization diseases, as well as in the healing of chronic wounds. Full article

Profilins are ubiquitous allergens with conserved structural elements. Exposure to profilins from different sources leads to IgE-cross-reactivity and the pollen–latex–food syndrome. Monoclonal antibodies (mAbs) that cross-react with plant profilins and block IgE-profilin interactions are relevant for diagnosis, epitope mapping, and specific immunotherapy. We generated IgGs mAbs, 1B4, and 2D10, against latex profilin (anti-rHev b 8) that inhibit the interaction of IgE and IgG4 antibodies from sera of latex- and maize-allergic patients by 90% and 40%, respectively. In this study, we evaluated 1B4 and 2D10 recognition towards different plant profilins, and mAbs recognition of rZea m 12 mutants by ELISAs. Interestingly, 2D10 highly recognized rArt v 4.0101 and rAmb a 8.0101, and to a lesser extent rBet v 2.0101, and rFra e 2.2, while 1B4 showed recognition for rPhl p 12.0101 and rAmb a 8.0101. We demonstrated that residue D130 at the α-helix 3 in profilins, which is part of the Hev b 8 IgE epitope, is essential for the 2D10 recognition. The structural analysis suggests that the profilins containing E130 (rPhl p 12.0101, rFra e 2.2, and rZea m 12.0105) show less binding with 2D10. The distribution of negative charges on the profilins’ surfaces at the α-helices 1 and 3 is relevant for the 2D10 recognition, and that may be relevant to explain profilins’ IgE cross-reactivity. Full article

Stamnagathi (Cichorium spinosum L.) is an indigenous plant species well-known for its health-promoting properties. Salinity is a long-term issue with devastating consequences on land and farmers. Nitrogen (N) constitutes a crucial element for plant growth and development (chlorophyll, primary metabolites, etc.). Thus, it is of paramount importance to investigate the impact of salinity and N supply on plants’ metabolism. Within this context, a study was conducted aiming to assess the impact of salinity and N stress on the primary metabolism of two contrasting ecotypes of stamnagathi (montane and seaside). Both ecotypes were exposed to three different salinity levels (0.3 mM—non-saline treatment, 20 mM—medium, and 40 mM—high salinity level) combined with two different total-N supply levels: a low-N at 4 mM and a high-N at 16 mM, respectively. The differences between the two ecotypes revealed the variable responses of the plant under the applied treatments. Fluctuations were observed at the level of TCA cycle intermediates (fumarate, malate, and succinate) of the montane ecotype, while the seaside ecotype was not affected. In addition, the results showed that proline (Pro) levels increased in both ecotypes grown under a low N-supply and high salt stress, while other osmoprotectant metabolites such as γ-aminobutyric acid (GABA) exhibited variable responses under the different N supply levels. Fatty acids such as α-linolenate and linoleate also displayed variable fluctuations following plant treatments. The carbohydrate content of the plants, as indicated by the levels of glucose, fructose, α,α-trehalose, and myo-inositol, was significantly affected by the applied treatments. These findings suggest that the different adaptation mechanisms among the two contrasting ecotypes could be strongly correlated with the observed changes in their primary metabolism. This study also suggests that the seaside ecotype may have developed unique adaptation mechanisms to cope with high N supply and salinity stress, making it a promising candidate for future breeding programs aimed at developing stress tolerant varieties of C. spinosum L. Full article

Rett syndrome (RTT, online MIM 312750) is a devastating neurodevelopmental disorder characterized by motor and cognitive disabilities. It is mainly caused by pathogenetic variants in the X-linked MECP2 gene, encoding an epigenetic factor crucial for brain functioning. Despite intensive studies, the RTT pathogenetic mechanism remains to be fully elucidated. Impaired vascular function has been previously reported in RTT mouse models; however, whether an altered brain vascular homeostasis and the subsequent blood–brain barrier (BBB) breakdown occur in RTT and contribute to the disease-related cognitive impairment is still unknown. Interestingly, in symptomatic Mecp2-null (Mecp2^-/y, Mecp2^tm1.1Bird) mice, we found enhanced BBB permeability associated with an aberrant expression of the tight junction proteins Ocln and Cldn-5 in different brain areas, in terms of both transcript and protein levels. Additionally, Mecp2-null mice showed an altered expression of different genes encoding factors with a role in the BBB structure and function, such as Cldn3, Cldn12, Mpdz, Jam2, and Aqp4. With this study, we provide the first evidence of impaired BBB integrity in RTT and highlight a potential new molecular hallmark of the disease that might open new perspectives for the setting-up of novel therapeutic strategies. Full article

Atrial fibrillation is a disease with a complex pathophysiology, whose occurrence and persistence are caused not only by aberrant electrical signaling in the heart, but by the development of a susceptible heart substrate. These changes, such as the accumulation of adipose tissue and interstitial fibrosis, are characterized by the presence of inflammation. N-glycans have shown great promise as biomarkers in different diseases, specifically those involving inflammatory changes. To assess the changes in the N-glycosylation of the plasma proteins and IgG in atrial fibrillation, we analyzed the N-glycosylation of 172 patients with atrial fibrillation, before and six months after a pulmonary vein isolation procedure, with 54 cardiovascularly healthy controls. An analysis was performed using ultra-high-performance liquid chromatography. We found one oligomannose N-glycan structure from the plasma N-glycome and six IgG N-glycans, mainly revolving around the presence of bisecting N-acetylglucosamine, that were significantly different between the case and control groups. In addition, four plasma N-glycans, mostly oligomannose structures and a derived trait that was related to them, were found to be different in the patients who experienced an atrial fibrillation recurrence during the six-month follow-up. IgG N-glycosylation was extensively associated with the CHA₂DS₂-VASc score, confirming its previously reported associations with the conditions that make up the score. This is the first study looking at the N-glycosylation patterns in atrial fibrillation and warrants further investigation into the prospect of glycans as biomarkers for atrial fibrillation. Full article

The search for molecules to be targeted that are involved in apoptosis resistance/increased survival and pathogenesis of onco-hematological malignancies is ongoing since these diseases are still not completely understood. Over the years, a good candidate has been identified in the Heat Shock Protein of 70kDa (HSP70), a molecule defined as “the most cytoprotective protein ever been described”. HSP70 is induced in response to a wide variety of physiological and environmental insults, allowing cells to survive lethal conditions. This molecular chaperone has been detected and studied in almost all the onco-hematological diseases and is also correlated to poor prognosis and resistance to therapy. In this review, we give an overview of the discoveries that have led us to consider HSP70 as a therapeutic target for mono- or combination-therapies in acute and chronic leukemias, multiple myeloma and different types of lymphomas. In this excursus, we will also consider HSP70 partners, such as its transcription factor HSF1 or its co-chaperones whose druggability could indirectly affect HSP70. Finally, we will try to answer the question asked in the title of this review considering that, despite the effort made by research in this field, HSP70 inhibitors never reached the clinic. Full article

Background: Abdominal aortic aneurysms (AAAs) are permanent dilations of the abdominal aorta with 4–5 times greater prevalence in males than in females. The aim of this study is to define whether Celastrol, a pentacyclic triterpene from the root extracts of Tripterygium wilfordii, supplementation influences angiotensin II (AngII)-induced AAAs in hypercholesterolemic mice. Methods: Age-matched (8–12 weeks old) male and female low-density lipoprotein (Ldl) receptor-deficient mice were fed a fat-enriched diet supplemented with or without Celastrol (10 mg/kg/day) for five weeks. After one week of diet feeding, mice were infused with either saline (n = 5 per group) or AngII (500 or 1000 ng/kg/min, n = 12–15 per group) for 28 days. Results: Dietary supplementation of Celastrol profoundly increased AngII-induced abdominal aortic luminal dilation and external aortic width in male mice as measured by ultrasonography and ex vivo measurement, with a significant increase in incidence compared to the control group. Celastrol supplementation in female mice resulted in significantly increased AngII-induced AAA formation and incidence. In addition, Celastrol supplementation significantly increased AngII-induced aortic medial elastin degradation accompanied by significant aortic MMP9 activation in both male and female mice compared to saline and AngII controls. Conclusions: Celastrol supplementation to Ldl receptor-deficient mice ablates sexual dimorphism and promotes AngII-induced AAA formation, which is associated with increased MMP9 activation and aortic medial destruction. Full article

Microarrays are one of the trailblazing technologies of the last two decades and have displayed their importance in all the associated fields of biology. They are widely explored to screen, identify, and gain insights on the characteristics traits of biomolecules (individually or in complex solutions). A wide variety of biomolecule-based microarrays (DNA microarrays, protein microarrays, glycan microarrays, antibody microarrays, peptide microarrays, and aptamer microarrays) are either commercially available or fabricated in-house by researchers to explore diverse substrates, surface coating, immobilization techniques, and detection strategies. The aim of this review is to explore the development of biomolecule-based microarray applications since 2018 onwards. Here, we have covered a different array of printing strategies, substrate surface modification, biomolecule immobilization strategies, detection techniques, and biomolecule-based microarray applications. The period of 2018–2022 focused on using biomolecule-based microarrays for the identification of biomarkers, detection of viruses, differentiation of multiple pathogens, etc. A few potential future applications of microarrays could be for personalized medicine, vaccine candidate screening, toxin screening, pathogen identification, and posttranslational modifications. Full article

The 70 kDa heat shock proteins (HSP70s) are a group of highly conserved and inducible heat shock proteins. One of the main functions of HSP70s is to act as molecular chaperones that are involved in a large variety of cellular protein folding and remodeling processes. HSP70s are found to be over-expressed and may serve as prognostic markers in many types of cancers. HSP70s are also involved in most of the molecular processes of cancer hallmarks as well as the growth and survival of cancer cells. In fact, many effects of HSP70s on cancer cells are not only related to their chaperone activities but rather to their roles in regulating cancer cell signaling. Therefore, a number of drugs directly or indirectly targeting HSP70s, and their co-chaperones have been developed aiming to treat cancer. In this review, we summarized HSP70-related cancer signaling pathways and corresponding key proteins regulated by the family of HSP70s. In addition, we also summarized various treatment approaches and progress of anti-tumor therapy based on targeting HSP70 family proteins. Full article

Alzheimer’s disease (AD) is a typical progressive neurodegenerative disorder, and with multiple possible pathogenesis. Among them, coumarin derivatives could be used as potential drugs as monoamine oxidase-B (MAO-B) inhibitors. Our lab has designed and synthesized coumarin derivatives based on MAO-B. In this study, we used nuclear magnetic resonance (NMR)-based metabolomics to accelerate the pharmacodynamic evaluation of candidate drugs for coumarin derivative research and development. We detailed alterations in the metabolic profiles of nerve cells with various coumarin derivatives. In total, we identified 58 metabolites and calculated their relative concentrations in U251 cells. In the meantime, the outcomes of multivariate statistical analysis showed that when twelve coumarin compounds were treated with U251cells, the metabolic phenotypes were distinct. In the treatment of different coumarin derivatives, there several metabolic pathways changed, including aminoacyl-tRNA biosynthesis, D-glutamine and D-glutamate metabolism, glycine, serine and threonine metabolism, taurine and hypotaurine metabolism, arginine biosynthesis, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, glutathione metabolism and valine, leucine and isoleucine biosynthesis. Our work documented how our coumarin derivatives affected the metabolic phenotype of nerve cells in vitro. We believe that these NMR-based metabolomics might accelerate the process of drug research in vitro and in vivo. Full article