Insight into Mantle Cell Lymphoma Pathobiology, Diagnosis, and Treatment Using Network-Based and Drug-Repurposing Approaches.

Mantle cell lymphoma (MCL) is a rare, incurable, and aggressive B-cell non-Hodgkin lymphoma (NHL). Early MCL diagnosis and treatment is critical and puzzling due to inter/intra-tumoral heterogeneity and limited understanding of the underlying molecular mechanisms. We developed and applied a multifaceted analysis of selected publicly available transcriptomic data of well-defined MCL stages, integrating network-based methods for pathway enrichment analysis, co-expression module alignment, drug repurposing, and prediction of effective drug combinations. We demonstrate the "butterfly effect" emerging from a small set of initially differentially expressed genes, rapidly expanding into numerous deregulated cellular processes, signaling pathways, and core machineries as MCL becomes aggressive. We explore pathogenicity-related signaling circuits by detecting common co-expression modules in MCL stages, pointing out, among others, the role of VEGFA and SPARC proteins in MCL progression and recommend further study of precise drug combinations. Our findings highlight the benefit that can be leveraged by such an approach for better understanding pathobiology and identifying high-priority novel diagnostic and prognostic biomarkers, drug targets, and efficacious combination therapies against MCL that should be further validated for their clinical impact.

A spatiotemporal atlas of the lepidopteran pest Helicoverpa armigera midgut provides insights into nutrient processing and pH regulation.

BACKGROUND: Caterpillars from the insect order Lepidoptera are some of the most widespread and destructive agricultural pests. Most of their impact is at the larval stage, where the midgut epithelium mediates the digestion and absorption of an astonishing amount of food. Although this tissue has been the subject of frequent investigation in Lepidoptera, a comprehensive expression atlas has yet to be generated. RESULTS: Here, we perform RNA-sequencing and proteomics on the gut of the polyphagous pest Helicoverpa armigera across, life stages, diet types, and compartments of the anterior-posterior axis. A striking relationship between the structural homology and expression pattern of a group of sugar transporters was observed in the early larval stages. Further comparisons were made among the spatial compartments of the midgut, which suggested a putative role for vATPases and SLC9 transporters in the generation of alkaline conditions in the H. armigera midgut. CONCLUSIONS: This comprehensive resource will aid the scientific community in understanding lepidopteran gut physiology in unprecedented resolution. It is hoped that this study advances the understanding of the lepidopteran midgut and also facilitates functional work in this field.

Towards analyzing the potential of exosomes to deliver microRNA therapeutics.

Exosome selectivity mechanisms underlying exosome-target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome-based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC-5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo-transmission electron microscopy; (c) cellular uptake studies of rhodamine-labeled exosomes in normal and cancer cells, providing to exosomes either "autologous" or "heterologous" cellular delivery environments; and (d) loading exogenous Alexa Fluor 488-labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC-5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising "bio-shuttles," being pharmacologically exploitable in the context of theranostic applications.

Mosquitoes cloak their legs to resist insecticides.

Malaria incidence has halved since the year 2000, with 80% of the reduction attributable to the use of insecticides. However, insecticide resistance is now widespread, is rapidly increasing in spectrum and intensity across Africa, and may be contributing to the increase of malaria incidence in 2018. The role of detoxification enzymes and target site mutations has been documented in the major malaria vector Anopheles gambiae; however, the emergence of striking resistant phenotypes suggests the occurrence of additional mechanisms. By comparing legs, the most relevant insect tissue for insecticide uptake, we show that resistant mosquitoes largely remodel their leg cuticles via enhanced deposition of cuticular proteins and chitin, corroborating a leg-thickening phenotype. Moreover, we show that resistant female mosquitoes seal their leg cuticles with higher total and different relative amounts of cuticular hydrocarbons, compared with susceptible ones. The structural and functional alterations in Anopheles female mosquito legs are associated with a reduced uptake of insecticides, substantially contributing to the resistance phenotype.

ProteoSign: an end-user online differential proteomics statistical analysis platform.

Profiling of proteome dynamics is crucial for understanding cellular behavior in response to intrinsic and extrinsic stimuli and maintenance of homeostasis. Over the last 20 years, mass spectrometry (MS) has emerged as the most powerful tool for large-scale identification and characterization of proteins. Bottom-up proteomics, the most common MS-based proteomics approach, has always been challenging in terms of data management, processing, analysis and visualization, with modern instruments capable of producing several gigabytes of data out of a single experiment. Here, we present ProteoSign, a freely available web application, dedicated in allowing users to perform proteomics differential expression/abundance analysis in a user-friendly and self-explanatory way. Although several non-commercial standalone tools have been developed for post-quantification statistical analysis of proteomics data, most of them are not end-user appealing as they often require very stringent installation of programming environments, third-party software packages and sometimes further scripting or computer programming. To avoid this bottleneck, we have developed a user-friendly software platform accessible via a web interface in order to enable proteomics laboratories and core facilities to statistically analyse quantitative proteomics data sets in a resource-efficient manner. ProteoSign is available at http://bioinformatics.med.uoc.gr/ProteoSign and the source code at https://github.com/yorgodillo/ProteoSign.

Spatial proximity of homologous alleles and long noncoding RNAs regulate a switch in allelic gene expression.

Physiological processes rely on the regulation of total mRNA levels in a cell. In diploid organisms, the transcriptional activation of one or both alleles of a gene may involve trans-allelic interactions that provide a tight spatial and temporal level of gene expression regulation. The mechanisms underlying such interactions still remain poorly understood. Here, we demonstrate that lipopolysaccharide stimulation of murine macrophages rapidly resulted in the actin-mediated and transient homologous spatial proximity of Tnfα alleles, which was necessary for the mono- to biallelic switch in gene expression. We identified two new complementary long noncoding RNAs transcribed from the TNFα locus and showed that their knockdown had opposite effects in Tnfα spatial proximity and allelic expression. Moreover, the observed spatial proximity of Tnfα alleles depended on pyruvate kinase muscle isoform 2 (PKM2) and T-helper-inducing POZ-Krüppel-like factor (ThPOK). This study suggests a role for lncRNAs in the regulation of somatic homologous spatial proximity and allelic expression control necessary for fine-tuning mammalian immune responses.

Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation.

The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the Cα atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.

Rapid label-free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome.

Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.

Focal adhesion kinase phosphorylates the phosphatase and tensin homolog deleted on chromosome 10 under the control of p110δ phosphoinositide-3 kinase.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is regulated by various mechanisms that are not fully understood. This includes regulation by Tyr phosphorylation by a mechanism that remains elusive. Here, we show that focal adhesion kinase (FAK) phosphorylates PTEN in vitro, in cell-free systems and in cells. Furthermore, by mass spectrometry, we identified Tyr336 on PTEN as being phosphorylated by FAK. Tyr336 phosphorylation increased phosphatase activity, protein-lipid interaction, and protein stability of PTEN. In cells, including primary mouse macrophages and human cancer cell lines, FAK was found to be negatively regulated by p110δ phosphoinositide-3 kinase (PI3K), whereas the activation of FAK was positively regulated by RhoA-associated kinase (ROCK). Indeed, the phosphorylation of FAK was unexpectedly increased in macrophages derived from mice expressing kinase-dead p110δ. Pharmacologic inactivation of RhoA/ROCK reduced the phosphorylation of FAK to normal levels in cells with genetically inactivated p110δ. Likewise, pharmacologic inactivation of FAK reduced the phosphorylation of PTEN in cells expressing kinase-dead p110δ and restored the functional defects of p110δ inactivation, including Akt phosphorylation and cell proliferation. This work identifies FAK as a target of p110δ PI3K that links RhoA with PTEN and establishes for the first time that PTEN is a substrate of FAK-mediated Tyr phosphorylation.

Soluble MHC-II proteins promote suppressive activity in CD4+ T cells.

Soluble MHCII (sMHCII) molecules are present in body fluids of healthy individuals and are considered to be involved in the maintenance of self tolerance, and are also related to various diseases. Their concentration increases during in vivo antigen-specific tolerogenic stimulation and it was recently shown that exosome-mediated tolerance is MHCII dependent. At the cellular level, sMHCII proteins compete with membrane MHCII for T-cell receptor binding on CD4(+) T cells. Immunoaffinity purification techniques isolated sMHCII antigens from the serum of human serum albumin (HSA) -tolerant mice as a single highly glycosylated protein of ~ 60,000 molecular weight, specifically interacting with anti-class II antibodies in Western blotting and ELISA. Mass spectroscopy showed that these sMHCII proteins were loaded with the tolerogenic peptide as well as multiple self peptides. At the cellular level, sMHCII suppressed antigen-specific, and to a lesser degree antigen-non-specific, spleen cell proliferation and induced CD25 in naive T cells. In T cells activated by antigen-seeded macrophages, sMHCII decreased CD28 and increased CTLA-4 protein expression, while decreasing interleukin-2 and increasing interleukin-10 production. In this case, sMHCII proteins were shown to decrease ZAP-70 and LAT phosphorylation. The results presented here for the first time provide evidence for the role of sMHCII proteins in immune response suppression and maintenance of tolerance, revealing novel regulatory mechanisms for immune system manipulation.

The crystal structure of Z-Gly-Aib-Gly-Aib-OtBu.

The synthetic peptide Z-Gly-Aib-Gly-Aib-OtBu was dissolved in methanol and crystallized in a mixture of ethyl acetate and petroleum ether. The crystals belong to the centrosymmetric space group P4/n that is observed less than 0.3% in the Cambridge Structural Database. The first Gly residue assumes a semi-extended conformation (φ ±62°, ψ ∓131°). The right-handed peptide folds in two consecutive ß-turns of type II' and type I or an incipient 310 -helix, and the left-handed counterpart folds accordingly in the opposite configuration. In the crystal lattice, one molecule is linked to four neighbors in the ab-plane via hydrogen bonds. These bonds form a continuous network of left- and right-handed molecules. The successive ab-planes stack via apolar contacts in the c-direction. An ethyl acetate molecule is situated on and close to the fourfold axis.

The Escherichia coli peripheral inner membrane proteome.

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Amine-substituted heterocyclic thioamide Cu(I) and Ag(I) complexes as effective anticancer and antibacterial agents targeting the periplasm of E. coli bacteria.

Metal complexes showing dual activity against cancer and bacterial infections are currently the focus of significant interest for their potential in treating life-threatening diseases. Aiming to investigate the impact of ligand substituents on these bioactivity properties of Group 11 d10 metal complexes, we herein present a series of mononuclear Cu(I) and Ag(I) complexes featuring the bis-NH2-substituted heterocyclic thioamide dap2SH (=4,6-diaminopyrimidine-2-thione), namely [AgCl(dap2SH)(PPh3)2] (1), [CuBr(dap2SH)(PPh3)2] (2), [CuBr(dap2SH)(xantphos)] (3), [Ag(dap2S)(xantphos)] (4), and [Cu(dap2S)(xantphos)] (5) (xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene). Complexes were characterized by means of different physicochemical methods (i.e., single crystal X-ray diffraction as well as FTIR, NMR, UV-Vis and fluorescence spectroscopy), and studied in-vitro for their antibacterial and anticancer activity against a variety of bacterial strains and cancer cell lines. Complexes 1-3 effectively inhibited both Gram (+) and Gram (-) bacterial growth, while cellular uptake studies for the most potent complex 1 against E. coli bacteria revealed the accumulation of Ag(I) ions in the periplasm of the bacteria. A high anti-proliferative effect was observed for 1 and 5 against A549, MCF7 and PC3 cancer cell lines, with 1 being capable of inducing apoptosis in A549 cells, as suggested by flow cytometry analysis. DNA interaction studies revealed the capacity of 1 to intercalate between base-pairs of CT DNA. All complexes had a moderate-to-high capacity to scavenge free radicals preventing oxidative stress. Molecular docking calculations, in combination with the experimentally obtained data, provided insights for potential mechanisms of the bioactivity of the complexes.

Fingerprinting Breast Milk; insights into Milk Exosomics.

Breast milk, often referred to as "liquid gold," is a complex biofluid that provides essential nutrients, immune factors, and developmental cues for newborns. Recent advancements in the field of exosome research have shed light on the critical role of exosomes in breast milk. Exosomes are nanosized vesicles that carry bioactive molecules, including proteins, lipids, nucleic acids, and miRNAs. These tiny messengers play a vital role in intercellular communication and are now being recognized as key players in infant health and development. This paper explores the emerging field of milk exosomics, emphasizing the potential of exosome fingerprinting to uncover valuable insights into the composition and function of breast milk. By deciphering the exosomal cargo, we can gain a deeper understanding of how breast milk influences neonatal health and may even pave the way for personalized nutrition strategies.

Proteomics in Childhood Acute Lymphoblastic Leukemia: Challenges and Opportunities.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and one of the success stories in cancer therapeutics. Risk-directed therapy based on clinical, biologic and genetic features has played a significant role in this accomplishment. Despite the observed improvement in survival rates, leukemia remains one of the leading causes of cancer-related deaths. Implementation of next-generation genomic and transcriptomic sequencing tools has illustrated the genomic landscape of ALL. However, the underlying dynamic changes at protein level still remain a challenge. Proteomics is a cutting-edge technology aimed at deciphering the mechanisms, pathways, and the degree to which the proteome impacts leukemia subtypes. Advances in mass spectrometry enable high-throughput collection of global proteomic profiles, representing an opportunity to unveil new biological markers and druggable targets. The purpose of this narrative review article is to provide a comprehensive overview of studies that have utilized applications of proteomics in an attempt to gain insight into the pathogenesis and identification of biomarkers in childhood ALL.

Mediterranean Diet, Ketogenic Diet or MIND Diet for Aging Populations with Cognitive Decline: A Systematic Review.

(1) Background: Compelling evidence shows that dietary patterns can slow the rate of cognitive decline, suggesting diet is a promising preventive measure against dementia. (2) Objective: This systematic review summarizes the evidence of three dietary patterns, the Mediterranean diet, the ketogenic diet and the MIND diet, for the prevention of cognitive decline. (3) Methods: A systematic search was conducted in major electronic databases (PubMed, ScienceDirect and Web of Science) up until 31 January 2022, using the key search terms "Mediterranean diet", "ketogenic diet", "MIND diet", "dementia", "cognition" and "aging". A statistical analysis was performed using RoB 2 and the Jadad scale to assess the risk of bias and methodological quality in randomized controlled trials. (4) Results: Only RCTs were included in this study; there were eleven studies (n = 2609 participants) of the Mediterranean diet, seven studies (n = 313) of the ketogenic diet and one study (n = 37) of the MIND diet. The participants' cognitive statuses were normal in seven studies, ten studies included patients with mild cognitive impairments and two studies included Alzheimer's disease patients. (5) Conclusion: All three dietary interventions have been shown to slow the rate of cognitive decline in the included studies. The Mediterranean diet was shown to be beneficial for global cognition after 10 weeks of adherence, the ketogenic diet had a beneficial effect for patients with diabetes mellitus and improved verbal recognition, while the MIND diet showed benefits in obese patients, improving working memory, verbal recognition, memory and attention.

Prevalence of SOD1 allele associated with degenerative myelopathy in canine population in Greece.

Canine degenerative myelopathy (CDM) is a late-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (c.118G > A). The purpose of this study was to determine the genotype and allele frequencies of this mutation in 108 dogs, mainly in Belgian Malinois and German Shepherd dogs with (CDM-affected group) and without CDM clinical symptoms (control group) in Greece. Genotyping of the c.118G > A mutation was possible by Sanger sequencing and PCR-RFLP. The observed genotype frequencies for the control group were 89.4% for the homozygous (G/G), 9.6% for the heterozygous (A/G), and 0.96% for the homozygous mutant (A/A) allele. The mutant allele was not common in the Belgian Malinois dogs (allele frequency = 0.029), but quite common in the German Shepherd dogs (allele frequency = 0.138). In the CDM affected group, all 4 dogs were homozygous for the mutant allele. These frequencies were close to those expected, indicating no significant departure from Hardy-Weinberg equilibrium. A strong but not statistically significant association between the mutant allele and CDM was observed. A previously identified deletion upstream of the mutation of interest was found at a high frequency (0.361) in the population.

Interruption of p53-MDM2 Interaction by Nutlin-3a in Human Lymphoma Cell Models Initiates a Cell-Dependent Global Effect on Transcriptome and Proteome Level.

In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.

Analysis of the urine proteome via a combination of multi-dimensional approaches.

Urine is a biological fluid that is non-invasively and easily harvested, and exhibits high stability from the proteomics point of view. At the downside, the overall low protein content of urine as well as the presence of low- and high-abundance proteins underscores the need for protein enrichment. As a continuation of previous efforts towards the comprehensive characterization of the urine proteome, the current study targeted the mining of urine proteins through the combined application of different protein separation methodologies, specifically, liquid chromatography and preparative electrophoresis along with 1D gel electrophoresis and protein identification by mass spectrometry. In order to enhance comparison and integration of different experimental data sets, the "standard" urine sample developed within the European Kidney and Urine Proteomics (EuroKUP) COST Action, was employed. As a contribution to the existing knowledge, we focused on maintaining and providing information about experimental mass of the identified proteins as well as information pertaining to their relative abundance--as allowed by technical limitations--thus providing an initial view of different isoforms representation and facilitating their future characterization. The difficulties in comparing proteome mining data sets become once more evident, underscoring the need for adopting standardized ways for data reporting as well as for potential new approaches for data analysis involving a thorough investigation of received information at the peptide level.