Viability-PCR for the selective detection of and in live bacteria-containing products.

To exert their beneficial effects, microorganisms used in live bacteria-containing products must be viable and present in certain amounts. In this study, we developed a viability assay based on quantitative PCR coupled with propidium monoazide for the identification and enumeration of viable and . In order to optimize the protocol, the thermal inactivation conditions for the two target microorganisms and the PMA concentration inhibiting DNA amplification from the dead cells while allowing it from the live cells were first determined.

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-μm diameter limit for 3D migration through gaps in the extracellular matrix.

Mapping cardiac remodeling in chronic kidney disease.

Patients with advanced chronic kidney disease (CKD) mostly die from sudden cardiac death and recurrent heart failure. The mechanisms of cardiac remodeling are largely unclear. To dissect molecular and cellular mechanisms of cardiac remodeling in CKD in an unbiased fashion, we performed left ventricular single-nuclear RNA sequencing in two mouse models of CKD.

Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.

Matrix Gla Protein drives stemness and tumor initiation in ovarian cancer.

Ovarian cancer (OC) displays the highest mortality among gynecological tumors, mainly due to early peritoneal dissemination, the high frequency of tumor relapse following primary debulking, and the development of chemoresistance. All these events are thought to be initiated and sustained by a subpopulation of neoplastic cells, termed ovarian cancer stem cells (OCSC), that are endowed with self-renewing and tumor-initiating properties. This implies that interfering with OCSC function should offer novel therapeutic perspectives to defeat OC progression.

Live biotherapeutic products and their regulatory framework in Italy and Europe.

In Italy and Europe, live microorganisms-containing products meant to be used by vulnerable or sick people for preventing or curing a disease are defined as live biotherapeutic products and are regulated as biological drugs. As such, they must undergo extensive quality, safety and efficacy testing and evaluation before receiving a marketing authorization. This review describes the regulatory framework of live biotherapeutic products with special focus on the European Pharmacopoeia monograph 3053 that set mandatory requirements for this kind of medicines, including verification of the number of live microorganisms and absence of certain contamination indicator microorganisms.

Recent advances in kinase signaling network profiling by mass spectrometry.

Mass spectrometry-based phosphoproteomics is currently the leading methodology for the study of global kinase signaling. The scientific community is continuously releasing technological improvements for sensitive and fast identification of phosphopeptides, and their accurate quantification. To interpret large-scale phosphoproteomics data, numerous bioinformatic resources are available that help understanding kinase network functional role in biological systems upon perturbation.

Proteomics to study cancer immunity and improve treatment.

Cancer survival and progression depend on the ability of tumor cells to avoid immune recognition. Advances in the understanding of cancer immunity and tumor immune escape mechanisms enabled the development of immunotherapeutic approaches. In patients with otherwise incurable metastatic cancers, immunotherapy resulted in unprecedented response rates with the potential for durable complete responses.

Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing.

In large-scale quantitative mass spectrometry (MS)-based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high-pH reversed-phase peptide chromatographic fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affect sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT-based fractionation strategies against single-shot label-free quantification with spectral library-free data independent acquisition (LFQ-DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT-fractionation approaches in a phosphoproteomics workflow, we compared two different high-pH reversed-phase fractionation strategies, microflow (MF) and stage-tip fractionation (STF), while scaling the peptide input amount down from 12.

Phosphorylation of SHP2 at Tyr62 Enables Acquired Resistance to SHP2 Allosteric Inhibitors in FLT3-ITD-Driven AML.

Unlabelled: The protein tyrosine phosphatase SHP2 is crucial for oncogenic transformation of acute myeloid leukemia (AML) cells expressing mutated receptor tyrosine kinases. SHP2 is required for full RAS-ERK activation to promote cell proliferation and survival programs. Allosteric SHP2 inhibitors act by stabilizing SHP2 in its autoinhibited conformation and are currently being tested in clinical trials for tumors with overactivation of the RAS/ERK pathway, alone and in various drug combinations.

Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Type E Strains from China.

Previously, a whole-genome comparison of three type E strains from Italy and the United States with different type E strains indicated that the / gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the / gene. Herein, we report the whole genome sequences for four neurotoxigenic type E strains that originated in China.

Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures.

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models.

Notch3 contributes to T-cell leukemia growth via regulation of the unfolded protein response.

Unfolded protein response (UPR) is a conserved adaptive response that tries to restore protein homeostasis after endoplasmic reticulum (ER) stress. Recent studies highlighted the role of UPR in acute leukemias and UPR targeting has been suggested as a therapeutic approach. Aberrant Notch signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), as downregulation of Notch activity negatively affects T-ALL cell survival, leading to the employment of Notch inhibitors in T-ALL therapy.

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping.

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus that cleaves primarily on the C-terminal side of proline and alanine residues.

Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites.

Type E Botulinum Neurotoxin-Producing Clostridium butyricum Strains Are Aerotolerant during Vegetative Growth.

Clostridium butyricum, the type species of the genus , is considered an obligate anaerobe, yet it has been shown to grow in the presence of oxygen. strains atypically producing the botulinum neurotoxin type E are the leading cause of type E human botulism in Italy. Here, we show that type E botulinum neurotoxin-producing strains growing exponentially were able to keep growing and producing toxin upon exposure to air, although less efficiently than under ideal oxygen-depleted conditions.

Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress.

The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress.

Galleria mellonella as an in vivo model for assessing the protective activity of probiotics against gastrointestinal bacterial pathogens.

The antagonistic activity against gastrointestinal bacterial pathogens is an important property of probiotic bacteria and a desirable feature for pre-selection of novel strains with probiotic potential. Pre-screening of candidate probiotics for antibacterial activity should be based on in vitro and in vivo tests. This study investigated whether the protective activity of probiotic bacteria against gastrointestinal bacterial pathogens can be evaluated using Galleria mellonella larvae as an in vivo model.

Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains.

Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression.

Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties.

Numb-dependent integration of pre-TCR and p53 function in T-cell precursor development.

Numb asymmetrically segregates at mitosis to control cell fate choices during development. Numb inheritance specifies progenitor over differentiated cell fates, and, paradoxically, also promotes neuronal differentiation, thus indicating that the role of Numb may change during development. Here we report that Numb nuclear localization is restricted to early thymocyte precursors, whereas timed appearance of pre-T-cell receptor (pre-TCR) and activation of protein kinase Cθ promote phosphorylation-dependent Numb nuclear exclusion.