Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory decline and cognitive impairment. Research on biomarkers can aid in early diagnosis, monitoring disease progression, evaluating treatment efficacy, and advancing fundamental research. We conducted a cross-sectional longitudinal study to see if there is an association between AD patients and age-matched healthy controls for their physiologic skin characteristics, such as pH, hydration, transepidermal water loss (TEWL), elasticity, microcirculation, and ApoE genotyping. The study used the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating-Sum of the Boxes (CDR-SB) scales as references to quantify the presence of disease, if any. Our findings demonstrate that AD patients have a dominantly neutral pH, greater skin hydration, and less elasticity compared to the control subjects. At baseline, the tortuous capillary percentage negatively correlated with MMSE scores in AD patients. However, AD patients who carry the ApoE E4 allele and exhibit a high percentage of tortuous capillaries and capillary tortuous numbers have shown better treatment outcomes at six months. Therefore, we believe that physiologic skin testing is a rapid and effective way to screen, monitor progression, and ultimately guide the most appropriate treatment for AD patients. Full article

Rhodesain is the main cysteine protease of Trypanosoma brucei rhodesiense, the parasite causing the acute lethal form of Human African Trypanosomiasis. Starting from the dipeptide nitrile CD24, the further introduction of a fluorine atom in the meta position of the phenyl ring spanning in the P3 site and the switch of the P2 leucine with a phenylalanine led to CD34, a synthetic inhibitor that shows a nanomolar binding affinity towards rhodesain (K_i = 27 nM) and an improved target selectivity with respect to the parent dipeptide nitrile CD24. In the present work, following the Chou and Talalay method, we carried out a combination study of CD34 with curcumin, a nutraceutical obtained from Curcuma longa L. Starting from an affected fraction (f_a) of rhodesain inhibition of 0.5 (i.e., the IC₅₀), we observed an initial moderate synergistic action, which became a synergism for f_a values ranging from 0.6 to 0.7 (i.e., 60–70% inhibition of the trypanosomal protease). Interestingly, at 80–90% inhibition of rhodesain proteolytic activity, we observed a strong synergism, resulting in 100% enzyme inhibition. Overall, in addition to the improved target selectivity of CD34 with respect to CD24, the combination of CD34 + curcumin resulted in an increased synergistic action with respect to CD24 + curcumin, thus suggesting that it is desirable to use CD34 and curcumin in combination. Full article

Atherosclerotic cardiovascular disease (ACVD) is the leading cause of death worldwide. Although current therapies, such as statins, have led to a marked reduction in morbidity and mortality from ACVD, they are associated with considerable residual risk for the disease together with various adverse side effects. Natural compounds are generally well-tolerated; a major recent goal has been to harness their full potential in the prevention and treatment of ACVD, either alone or together with existing pharmacotherapies. Punicalagin (PC) is the main polyphenol present in pomegranates and pomegranate juice and demonstrates many beneficial actions, including anti-inflammatory, antioxidant, and anti-atherogenic properties. The objective of this review is to inform on our current understanding of the pathogenesis of ACVD and the potential mechanisms underlying the beneficial actions of PC and its metabolites in the disease, including the attenuation of dyslipidemia, oxidative stress, endothelial cell dysfunction, foam cell formation, and inflammation mediated by cytokines and immune cells together with the regulation of proliferation and migration of vascular smooth muscle cells. Some of the anti-inflammatory and antioxidant properties of PC and its metabolites are due to their strong radical-scavenging activities. PC and its metabolites also inhibit the risk factors of atherosclerosis, including hyperlipidemia, diabetes mellitus, inflammation, hypertension, obesity, and non-alcoholic fatty liver disease. Despite the promising findings that have emerged from numerous in vitro, in vivo, and clinical studies, deeper mechanistic insights and large clinical trials are required to harness the full potential of PC and its metabolites in the prevention and treatment of ACVD. Full article

In the last decades, it has been shown that biofilm-associated infections in most cases are caused by rather two or even more pathogens than by single microorganisms. Due to intermicrobial interactions in mixed communities, bacteria change their gene expression profile, in turn leading to alterations in the biofilm structure and properties, as well as susceptibility to antimicrobials. Here, we report the alterations of antimicrobials efficiency in mixed biofilms of Staphylococcus aureus–Klebsiella pneumoniae in comparison with mono-species biofilms of each counterpart and discuss possible mechanisms of these alterations. In cell clumps detached from dual-species biofilms, S. aureus became insensitive to vancomycin, ampicillin, and ceftazidime compared to solely S. aureus cell clumps. In turn, the increased efficiency of amikacin and ciprofloxacin against both bacteria could be observed, compared to mono-species biofilms of each counterpart. Scanning electron microscopy and confocal microscopy indicate the porous structure of the dual-species biofilm, and differential fluorescent staining revealed an increased number of polysaccharides in the matrix, in turn leading to more loose structure and thus apparently providing increased permeability of the dual-species biofilm to antimicrobials. The qRT-PCR showed that ica operon in S. aureus became repressed in mixed communities, and polysaccharides are produced mainly by K. pneumoniae. While the molecular trigger of these changes remains undiscovered, detailed knowledge of the alterations in antibiotic susceptibility to given drugs opens doors for treatment correction options for S. aureus–K. pneumoniae biofilm-associated infections. Full article

Synchrotron small-angle X-ray diffraction is the method of choice for nm-scale structural studies of striated muscle under physiological conditions and on millisecond time scales. The lack of generally applicable computational tools for modeling X-ray diffraction patterns from intact muscles has been a significant barrier to exploiting the full potential of this technique. Here, we report a novel “forward problem” approach using the spatially explicit computational simulation platform MUSICO to predict equatorial small-angle X-ray diffraction patterns and the force output simultaneously from resting and isometrically contracting rat skeletal muscle that can be compared to experimental data. The simulation generates families of thick–thin filament repeating units, each with their individually predicted occupancies of different populations of active and inactive myosin heads that can be used to generate 2D-projected electron density models based on known Protein Data Bank structures. We show how, by adjusting only a few selected parameters, we can achieve a good correspondence between experimental and predicted X-ray intensities. The developments presented here demonstrate the feasibility of combining X-ray diffraction and spatially explicit modeling to form a powerful hypothesis-generating tool that can be used to motivate experiments that can reveal emergent properties of muscle. Full article

Trichomes are attractive cells for terpenoid biosynthesis and accumulation in Artemisia annua. However, the molecular process underlying the trichome of A. annua is not yet fully elucidated. In this study, an analysis of multi-tissue transcriptome data was performed to examine trichome-specific expression patterns. A total of 6646 genes were screened and highly expressed in trichomes, including artemisinin biosynthetic genes such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1). Mapman and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that trichome-specific genes were mainly enriched in lipid metabolism and terpenoid metabolism. These trichome-specific genes were analyzed by a weighted gene co-expression network analysis (WGCNA), and the blue module linked to terpenoid backbone biosynthesis was determined. Hub genes correlated with the artemisinin biosynthetic genes were selected based on TOM value. ORA, Benzoate carboxyl methyltransferase (BAMT), Lysine histidine transporter-like 8 (AATL1), Ubiquitin-like protease 1 (Ulp1) and TUBBY were revealed as key hub genes induced by methyl jasmonate (MeJA) for regulating artemisinin biosynthesis. In summary, the identified trichome-specific genes, modules, pathways and hub genes provide clues and shed light on the potential regulatory mechanisms of artemisinin biosynthesis in trichomes in A. annua. Full article

Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N–glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs—clindamycin, diltiazem, lidocaine, and warfarin—was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10⁻⁵–10⁻⁶ M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected. Full article

The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid–liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes. Full article

One of the most frequent solid tumors in children is neuroblastoma, which has a variety of clinical behaviors that are mostly influenced by the biology of the tumor. Unique characteristics of neuroblastoma includes its early age of onset, its propensity for spontaneous tumor regression in newborns, and its high prevalence of metastatic disease at diagnosis in individuals older than 1 year of age. Immunotherapeutic techniques have been added to the previously enlisted chemotherapeutic treatments as therapeutic choices. A groundbreaking new treatment for hematological malignancies is adoptive cell therapy, specifically chimeric antigen receptor (CAR) T cell therapy. However, due to the immunosuppressive nature of the tumor microenvironment (TME) of neuroblastoma tumor, this treatment approach faces difficulties. Numerous tumor-associated genes and antigens, including the MYCN proto-oncogene (MYCN) and disialoganglioside (GD2) surface antigen, have been found by the molecular analysis of neuroblastoma cells. The MYCN gene and GD2 are two of the most useful immunotherapy findings for neuroblastoma. The tumor cells devise numerous methods to evade immune identification or modify the activity of immune cells. In addition to addressing the difficulties and potential advancements of immunotherapies for neuroblastoma, this review attempts to identify important immunological actors and biological pathways involved in the dynamic interaction between the TME and immune system. Full article

The development of desorption/ionization (DI) mass spectrometric (MS) assays for drug quantification in tissue sections and their validation according to regulatory guidelines would enable their universalization for applications in (clinical) pharmacology. Recently, new enhancements in desorption electrospray ionization (DESI) have highlighted the reliability of this ion source for the development of targeted quantification methods that meet requirements for method validation. However, it is necessary to consider subtle parameters leading to the success of such method developments, such as the morphology of desorption spots, the analytical time, and sample surface, to cite but a few. Here, we provide additional experimental data highlighting an additional important parameter, based on the unique advantage of DESI-MS on continuous extraction during analysis. We demonstrate that considering desorption kinetics during DESI analyses would largely help (i) reducing analytical time during profiling analyses, (ii) verifying solvent-based drug extraction using the selected sample preparation method for profiling and imaging modes, and (iii) predicting the feasibility of imaging assays using samples in a given expected concentration range of the targeted drug. These observations will likely serve as precious guidance for the development of validated DESI-profiling and imaging methods in the future. Full article

Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production. SAMs are comprised of a “dead” Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications. Full article

Radicinin is a phytotoxic dihydropyranopyran-4,5-dione isolated from the culture filtrates of Cochliobolus australiensis, a phytopathogenic fungus of the invasive weed buffelgrass (Cenchrus ciliaris). Radicinin proved to have interesting potential as a natural herbicide. Being interested in elucidating the mechanism of action and considering radicinin is produced in small quantities by C. australiensis, we opted to use (±)-3-deoxyradicinin, a synthetic analogue of radicinin that is available in larger quantities and shows radicinin-like phytotoxic activities. To obtain information about subcellular targets and mechanism(s) of action of the toxin, the study was carried out by using tomato (Solanum lycopersicum L.), which, apart from its economic relevance, has become a model plant species for physiological and molecular studies. Results of biochemical assays showed that (±)-3-deoxyradicinin administration to leaves induced chlorosis, ion leakage, hydrogen peroxide production, and membrane lipid peroxidation. Remarkably, the compound determined the uncontrolled opening of stomata, which, in turn, resulted in plant wilting. Confocal microscopy analysis of protoplasts treated with (±)-3-deoxyradicinin ascertained that the toxin targeted chloroplasts, eliciting an overproduction of reactive singlet oxygen species. This oxidative stress status was related by qRT-PCR experiments to the activation of transcription of genes of a chloroplast-specific pathway of programmed cell death. Full article

The exposure of ionizing radiation during early gestation often leads to deleterious and even lethal effects; however, few extensive studies have been conducted on late gestational exposures. This research examined the behavior al effects of C57Bl/6J mouse offspring exposed to low dose ionizing gamma irradiation during the equivalent third trimester. Pregnant dams were randomly assigned to sham or exposed groups to either low dose or sublethal dose radiation (50, 300, or 1000 mGy) at gestational day 15. Adult offspring underwent a behavioral and genetic analysis after being raised under normal murine housing conditions. Our results indicate very little change in the behavioral tasks measuring general anxiety, social anxiety, and stress-management in animals exposed prenatally across the low dose radiation conditions. Quantitative real-time polymerase chain reactions were conducted on the cerebral cortex, hippocampus, and cerebellum of each animal; results indicate some dysregulation in markers of DNA damage, synaptic activity, reactive oxygen species (ROS) regulation, and methylation pathways in the offspring. Together, our results provide evidence in the C57Bl/6J strain, that exposure to sublethal dose radiation (<1000 mGy) during the last period of gestation leads to no observable changes in behaviour when assessed as adults, although some changes in gene expression were observed for specific brain regions. These results indicate that the level of oxidative stress occurring during late gestation for this mouse strain is not sufficient for a change in the assessed behavioral phenotype, but results in some modest dysregulation of the genetic profile of the brain. Full article

The ultimate objective of this review is to encourage a multi-disciplinary and integrated methodological approach that, starting from the recognition of some current uncertainties, helps to deepen the molecular bases of ozone treatment effects on human and animal well-being and to optimize their performance in terms of reproducibility of results, quality, and safety. In fact, the common therapeutic treatments are normally documented by healthcare professionals’ prescriptions. The same applies to medicinal gases (whose uses are based on their pharmacological effects) that are intended for patients for treatment, diagnostic, or preventive purposes and that have been produced and inspected in accordance with good manufacturing practices and pharmacopoeia monographs. On the contrary, it is the responsibility of healthcare professionals, who thoughtfully choose to use ozone as a medicinal product, to achieve the following objectives: (i) to understand the molecular basis of the mechanism of action; (ii) to adjust the treatment according to the clinical responses obtained in accordance with the principles of precision medicine and personalized therapy; (iii) to ensure all quality standards. Full article

McCune–Albright syndrome (MAS) is a rare sporadic condition defined by the classic triad of fibrous dysplasia of bone, café au lait skin macules, and hyperfunctioning endocrinopathies. The molecular basis of MAS has been ascribed to the post-zygotic somatic gain-of-function mutations in the GNAS gene, which encodes the alpha subunit of G proteins, leading to constitutive activation of several G Protein-Coupled Receptors (GPCRs). The co-occurrence of two of the above-mentioned cardinal clinical manifestations sets the diagnosis at the clinical level. In this case report, we describe a 27-month-old girl who presented with gonadotropin-independent precocious puberty secondary to an estrogen-secreting ovarian cyst, a café au lait skin macule and growth hormone, and prolactin excess, and we provide an updated review of the scientific literature on the clinical features, diagnostic work-up, and therapeutic management of MAS. Full article