Position Matters: Pyridine Regioisomers Influence Secondary Structure and Micelle Morphology in Polymer-Homopolypeptide Micelles.

Polymer-homopolypeptide block copolymers are a class of bioinspired materials that combine the processability and stability of synthetic polymers with the biocompatibility and unique secondary structures of peptides, such as α-helices and ß-sheets. These properties make them ideal candidates for a wide variety of applications, for example, in the pharmaceutical field, where they are frequently explored as building blocks for polymeric micelle drug delivery systems. While homopolypeptide side chains can be furnished with an array of different moieties to impart the copolymers with desirable properties, such as stimulus responsivity, pyridine derivatives represent an underutilized functional group for this purpose. Additionally, the interplay between polypeptide side chain structure, secondary conformation, and micelle morphology is not yet well understood, particularly in the case of structural regioisomers. Therefore, in this work, a series of polymer-homopolypeptide copolymers were prepared from a poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-PGA) backbone, where the pendant carboxylic acid groups were covalently conjugated to a series of pyridine regioisomers by carbodiimide coupling. These pyridine regioisomers differed only in the position of the nitrogen heteroatom, ortho, meta or para, relative to the linking group, generating a series of PEG-b-poly(pyridinylmethyl glutamate) (PEG-b-PMG) copolymers. Following self-assembly of the copolymers in aqueous solutions, dynamic light scattering (DLS) revealed differences in micelle hydrodynamic diameter (Dh) (ranging from ∼60 to 120 nm), while transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) revealed distinctive morphologies ranging from ellipsoidal, to cylindrical, and disc-like, suggesting that subtle changes in positional isomers in the polypeptide block may influence the micelle structure. Analysis of the PEG-b-PMG copolymer micelles by circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that differences in the morphology were associated with changes in polypeptide secondary structure, which in turn was influenced by the position of the pyridine heteroatom. Overall, these findings contribute to the broader understanding of the relationship between polypeptide structure and micelle morphology and serve as useful insight for the rational design of polymer-polypeptide nanoparticles.

Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 µm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

Assessment of yeast physiology during industrial-scale brewing practices using the redox-sensitive dye resazurin.

Beer refermentation in bottles is an industrial process utilized by breweries where yeast and fermentable extract are added to green beer. The beer is refermented for a minimum of 2 weeks before distribution, with the physiological state of the yeast a critical factor for successful refermentation. Ideally, fresh yeast that is propagated from a dedicated propagation plant should be used for refermentation in bottles. Here, we explored the applicability of the fluorescent and redox-sensitive dye, resazurin, to assess cellular metabolism in yeast and its ability to differentiate between growth stages. We applied this assay, with other markers of yeast physiology, to evaluate yeast quality during a full-scale industrial propagation. Resazurin allowed the discrimination between the different growth phases in yeast and afforded a more in-depth understanding of yeast metabolism during propagation. This assay can be used to optimize the yeast propagation process and cropping time to improve beer quality.

Early molecular imaging response assessment based on determination of total viable tumor burden in [68Ga]Ga-PSMA-11 PET/CT independently predicts overall survival in [177Lu]Lu-PSMA-617 radioligand therapy.

PURPOSE: In patients with metastatic castration-resistant prostate cancer (mCRPC) treated with prostate-specific membrane antigen-targeted radioligand therapy (PSMA-RLT), the predictive value of PSMA PET/CT-derived response is still under investigation. Early molecular imaging response based on total viable tumor burden and its association with overall survival (OS) was explored in this study. METHODS: Sixty-six mCRPC patients who received [177Lu]Lu-PSMA-617 RLT within a prospective patient registry (REALITY Study, NCT04833517) were analyzed. Patients received a [68Ga]Ga-PSMA-11 PET/CT scan before the first and after the second cycle of PSMA-RLT. Total lesion PSMA (TLP) was determined by semiautomatic whole-body tumor segmentation. Molecular imaging response was assessed by change in TLP and modified PERCIST criteria. Biochemical response was assessed using standard serum PSA and PCWG3 criteria. Both response assessment methods and additional baseline parameters were analyzed regarding their association with OS by univariate and multivariable analysis. RESULTS: By molecular imaging, 40/66 (60.6%) patients showed partial remission (PR), 19/66 (28.7%) stable disease (SD), and 7/66 (10.6%) progressive disease (PD). Biochemical response assessment revealed PR in 34/66 (51.5%) patients, SD in 20/66 (30.3%), and PD in 12/66 (18.2%). Response assessments were concordant in 49/66 (74.3%) cases. On univariate analysis, both molecular and biochemical response (p = 0.001 and 0.008, respectively) as well as two baseline characteristics (ALP and ECOG) were each significantly associated with OS. The median OS of patients showing molecular PR was 24.6 versus 10.7 months in the remaining patients (with SD or PD). On multivariable analysis molecular imaging response remained an independent predictor of OS (p = 0.002), eliminating biochemical response as insignificant (p = 0.515). CONCLUSION: The new whole-body molecular imaging-derived biomarker, early change of total lesion PSMA (TLP), independently predicts overall survival in [177Lu]Lu-PSMA-617 RLT in mCRPC, outperforming conventional PSA-based response assessment. TLP might be considered a more distinguished and advanced biomarker for monitoring PSMA-RLT over commonly used serum PSA.

Altered N-linked glycosylation in endometrial cancer.

It is well established that cell surface glycans play a vital role in biological processes and their altered form can lead to carcinogenesis. Mass spectrometry-based techniques have become prominent for analysing N-linked glycans, for example using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Additionally, MALDI MS can be used to spatially map N-linked glycans directly from cancer tissue using a technique termed MALDI MS imaging (MALDI MSI). This powerful technique combines mass spectrometry and histology to visualise the spatial distribution of N-linked glycans on a single tissue section. Here, we performed N-glycan MALDI MSI on six endometrial cancer (EC) formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) consisting of eight EC patients with lymph node metastasis (LNM) and twenty without LNM. By doing so, several putative N-linked glycan compositions were detected that could significantly distinguish normal from cancerous endometrium. Furthermore, a complex core-fucosylated N-linked glycan was detected that could discriminate a primary tumour with and without LNM. Structural identification of these putative N-linked glycans was performed using porous graphitized carbon liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS). Overall, we observed higher abundance of oligomannose glycans in tumour compared to normal regions with AUC ranging from 0.85-0.99, and lower abundance of complex N-linked glycans with AUC ranges from 0.03-0.28. A comparison of N-linked glycans between primary tumours with and without LNM indicated a reduced abundance of a complex core-fucosylated N-glycan (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2, in primary tumour with associated lymph node metastasis. In summary, N-linked glycan MALDI MSI can be used to differentiate cancerous endometrium from normal, and endometrial cancer with LNM from endometrial cancer without.

Uncovering Tumor-Stroma Inter-relationships Using MALDI Mass Spectrometry Imaging.

Tumorigenesis involves a complex interplay between genetically modified cancer cells and their adjacent normal tissue, the stroma. We used an established breast cancer mouse model to investigate this inter-relationship. Conditional activation of Rho-associated protein kinase (ROCK) in a model of mammary tumorigenesis enhances tumor growth and progression by educating the stroma and enhancing the production and remodeling of the extracellular matrix. We used peptide matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify the proteomic changes occurring within tumors and their stroma in their regular spatial context. Peptides were ranked according to their ability to discriminate between the two groups, using a receiver operating characteristic tool. Peptides were identified by liquid chromatography tandem mass spectrometry, and protein expression was validated by quantitative immunofluorescence using an independent set of tumor samples. We have identified and validated four key proteins upregulated in ROCK-activated mammary tumors relative to those expressing kinase-dead ROCK, namely, collagen I, α-SMA, Rab14, and tubulin-ß4. Rab14 and tubulin-ß4 are expressed within tumor cells, whereas collagen I is localized within the stroma. α-SMA is predominantly localized within the stroma but is also expressed at higher levels in the epithelia of ROCK-activated tumors. High expression of COL1A, the gene encoding the pro-α 1 chain of collagen, correlates with cancer progression in two human breast cancer genomic data sets, and high expression of COL1A and ACTA2 (the gene encoding α-SMA) are associated with a low survival probability (COLIA, p = 0.00013; ACTA2, p = 0.0076) in estrogen receptor-negative breast cancer patients. To investigate whether ROCK-activated tumor cells cause stromal cancer-associated fibroblasts (CAFs) to upregulate expression of collagen I and α-SMA, we treated CAFs with medium conditioned by primary mammary tumor cells in which ROCK had been activated. This led to abundant production of both proteins in CAFs, clearly highlighting the inter-relationship between tumor cells and CAFs and identifying CAFs as the potential source of high levels of collagen 1 and α-SMA and associated enhancement of tissue stiffness. Our research emphasizes the capacity of MALDI-MSI to quantitatively assess tumor-stroma inter-relationships and to identify potential prognostic factors for cancer progression in human patients, using sophisticated mouse cancer models.

[Hip-spine syndrome-current developments and state of the evidence]. / Hip-Spine-Syndrom ­ Aktuelle Entwicklungen und Evidenzlage.

The movements between the spine, pelvis and hip joints are coordinated to allow for a physiological balance of the upper body and the pelvis during sitting and standing. Degenerative changes or spondylodesis of the spine result in decreased pelvic mobility and can lead to increased rates of instability in the presence of total hip arthroplasty (THA). To reduce the risk of THA dislocation several authors have recently recommended functional radiographs of the spine and pelvis, as well as individual safe zones for THA components. The aim of this article is to summarize the current body of knowledge regarding the influence of spinopelvic alignment on THA and provide recommendations for everyday clinical practice.

The Emerging Role of Cytoskeletal Proteins as Reliable Biomarkers.

Cytoskeletal proteins are essential building blocks of cells. More than 100 cytoskeletal and cytoskeleton-associated proteins are known and for some, their function and regulation are understood in great detail. Apart from cell shape and support, they facilitate many processes such as intracellular signaling and transport, and cancer related processes such as proliferation, migration, and invasion. During the last decade, comparative proteomic studies have identified cytoskeletal proteins as in vitro markers for tumor progression and metastasis. Here, these results are summarized and a number of unrelated studies are highlighted, identifying the same cytoskeletal proteins as potential biomarkers. These findings might indicate that the abundance of these potential markers of tumor progression is associated with the biological outcome and are independent of the cancer origin. This correlates well with recently published results from the Cancer Genome Atlas, indicating that cancers show remarkable similarities in their analyzed molecular information, independent of their organ of origin. It is postulated that the quantification of cytoskeletal proteins in healthy tissues, tumors, in adjacent tissues, and in stroma, is a great source of molecular information, which might not only be used to classify tumors, but more importantly to predict patients' outcome or even best treatment choices.

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) for Monitoring of Drug Response in Primary Cancer Spheroids.

Malignant ascites is a fluid, which builds up in the abdomen and contains cancer cells in the form of single cells or multicellular clusters called spheroids. Malignant ascites has been observed in patients suffering from ovarian, cervical, gastric, colorectal, pancreatic, endometrial, or primary liver cancer. The spheroids are believed to play a major role in chemo resistance and metastasis of the cancer. To ease the discomfort of patients, malignant ascites (MA) is often drained from the abdomen using a procedure called paracentesis. MA retrieved via this minimal invasive procedure is a great source for cancer spheroids, which can be used for testing chemotherapeutic drugs and drug combinations. Herein, the existing workflow is adapted to make concurrent monitoring of drug accumulation, drug response, and drug metabolites feasible using primary spheroids or spheroids grown without a scaffolding matrix. To achieve this, those spheroids are embedded in matrigel, before fixing them with formalin. This makes it possible to process, store, and ship samples at room temperature. This new approach might be used to choose the best targeted therapy for each patient and thereby facilitate personalized medicine.

Egg White as a Quality Control in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI).

The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during sample preparation can be used to improve the mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for the quality check of sample preparation and data acquisition, which is particularly relevant when either many spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).

18F-FDG PET/CT: an unexpected case of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a rare, genetic neurodegenerative disorder often presenting with emotional, cognitive and behavioral abnormalities before manifestation of disease defining motor symptoms. Cognitive impairment is a frequent clinical feature caused by different dementia subtypes. Imaging cortical and subcortical glucose metabolism via 18F-FDG PET/CT can help to discriminate the underlying disease. CASE PRESENTATION: The patient is a 54-year old man presenting with progressive cognitive impairment and mild orofacial dyskinesia. 18F-FDG PET/CT of the brain revealed a severe bilateral hypometabolism in the striatum. Following imaging Huntington's disease was suspected and a molecular genetic testing confirmed the diagnosis. CONCLUSIONS: Huntington's disease is a rare but important differential diagnosis of cognitive impairment, especially before motor symptoms are manifest. 18F-FDG PET is capable to show early striatal dysfunction in HD even when structural imaging is normal. We conclude that, in cases with negative family history the HD characteristic metabolic pattern can lead to the diagnosis when no other dementia-suspected changes are present.

Annexin A2 and alpha actinin 4 expression correlates with metastatic potential of primary endometrial cancer.

The prediction of lymph node metastasis using clinic-pathological data and molecular information from endometrial cancers lacks accuracy and is therefore currently not routinely used in patient management. Consequently, although only a small percentage of patients with endometrial cancers suffer from metastasis, the majority undergo radical surgery including removal of pelvic lymph nodes. Upon analysis of publically available data and published research, we compiled a list of 60 proteins having the potential to display differential abundance between primary endometrial cancers with versus those without lymph node metastasis. Using data dependent acquisition LC-ESI-MS/MS we were able to detect 23 of these proteins in endometrial cancers, and using data independent LC-ESI-MS/MS the differential abundance of five of those proteins was observed. The localization of the differentially expressed proteins, was visualized using peptide MALDI MSI in whole tissue sections as well as tissue microarrays of 43 patients. The proteins identified were further validated by immunohistochemistry. Our data indicate that annexin A2 protein level is upregulated, whereas annexin A1 and α actinin 4 expression are downregulated in tumours with lymph node metastasis compared to those without lymphatic spread. Moreover, our analysis confirmed the potential of these markers, to be included in a statistical model for prediction of lymph node metastasis. The predictive model using highly ranked m/z values identified by MALDI MSI showed significantly higher predictive accuracy than the model using immunohistochemistry data. In summary, using publicly available data and complementary proteomics approaches, we were able to improve the prediction model for lymph node metastasis in EC.

Lymph node metastasis of primary endometrial cancers: Associated proteins revealed by MALDI imaging.

Metastasis is a crucial step of malignant progression and is the primary cause of death from endometrial cancer. However, clinicians presently face the challenge that conventional surgical-pathological variables, such as tumour size, depth of myometrial invasion, histological grade, lymphovascular space invasion or radiological imaging are unable to predict with accuracy if the primary tumour has metastasized. In the current retrospective study, we have used primary tumour samples of endometrial cancer patients diagnosed with (n = 16) and without (n = 27) lymph node metastasis to identify potential discriminators. Using peptide matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), we have identified m/z values which can classify 88% of all tumours correctly. The top discriminative m/z values were identified using a combination of in situ sequencing and LC-MS/MS from digested tumour samples. Two of the proteins identified, plectin and α-Actin-2, were used for validation studies using LC-MS/MS data independent analysis (DIA) and immunohistochemistry. In summary, MALDI-MSI has the potential to identify discriminators of metastasis using primary tumour samples.

Ectrodactyly and Lethal Pulmonary Acinar Dysplasia Associated with Homozygous FGFR2 Mutations Identified by Exome Sequencing.

Ectrodactyly/split hand-foot malformation is genetically heterogeneous with more than 100 syndromic associations. Acinar dysplasia is a rare congenital lung lesion of unknown etiology, which is frequently lethal postnatally. To date, there have been no reports of combinations of these two phenotypes. Here, we present an infant from a consanguineous union with both ectrodactyly and autopsy confirmed acinar dysplasia. SNP array and whole-exome sequencing analyses of the affected infant identified a novel homozygous Fibroblast Growth Factor Receptor 2 (FGFR2) missense mutation (p.R255Q) in the IgIII domain (D3). Expression studies of Fgfr2 in development show localization to the affected limbs and organs. Molecular modeling and genetic and functional assays support that this mutation is at least a partial loss-of-function mutation, and contributes to ectrodactyly and acinar dysplasia only in homozygosity, unlike previously reported heterozygous activating FGFR2 mutations that cause Crouzon, Apert, and Pfeiffer syndromes. This is the first report of mutations in a human disease with ectrodactyly with pulmonary acinar dysplasia and, as such, homozygous loss-of-function FGFR2 mutations represent a unique syndrome.

Novel IEF Peptide Fractionation Method Reveals a Detailed Profile of N-Terminal Acetylation in Chemotherapy-Responsive and -Resistant Ovarian Cancer Cells.

Although acetylation is regarded as a common protein modification, a detailed proteome-wide profile of this post-translational modification may reveal important biological insight regarding differential acetylation of individual proteins. Here we optimized a novel peptide IEF fractionation method for use prior to LC-MS/MS analysis to obtain a more in depth coverage of N-terminally acetylated proteins from complex samples. Application of the method to the analysis of the serous ovarian cancer cell line OVCAR-5 identified 344 N-terminally acetylated proteins, 12 of which are previously unreported. The protein peptidyl-prolyl cis-trans isomerase A (PPIA) was detected in both the N-terminally acetylated and unmodified forms and was further analyzed by data-independent acquisition in carboplatin-responsive parental OVCAR-5 cells and carboplatin-resistant OVCAR-5 cells. This revealed a higher ratio of unacetylated to acetylated N-terminal PPIA in the parental compared with the carboplatin-resistant OVCAR-5 cells and a 4.1-fold increase in PPIA abundance overall in the parental cells relative to carboplatin-resistant OVCAR-5 cells (P = 0.015). In summary, the novel IEF peptide fractionation method presented here is robust, reproducible, and can be applied to the profiling of N-terminally acetylated proteins. All mass spectrometry data is available as a ProteomeXchange repository (PXD003547).

MALDI Mass Spectrometry Imaging Reveals Decreased CK5 Levels in Vulvar Squamous Cell Carcinomas Compared to the Precursor Lesion Differentiated Vulvar Intraepithelial Neoplasia.

Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC.

Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer.

The timely detection of gastric cancer will contribute significantly towards effective treatment and is aided by the availability and reliability of appropriate biomarkers. A combination of several biomarkers can improve the sensitivity and specificity of cancer detection and this work reports results from a panel of 4 proteins. By combining a validated preclinical mouse model with a proteomic approach we have recently discovered novel biomarkers for the detection of gastric cancer. Here, we investigate the specificity of four of those biomarkers (afamin, clusterin, VDBP and haptoglobin) for the detection of gastric cancer using two independent methods of validation: ELISA, and a non antibody based method: Multiple Reaction Monitoring with High Resolution Mass Spectrometry (MRM-HR). All four biomarkers reliably differentiated GC from benign patient serum, and also in a small cohort of 11 early stage cases. We also present a novel isoform specific biomarker alpha-1-antitrypsin (A1AT) that was identified using a mouse model for gastric cancer. This isoform is distinct in charge and mobility in a pH gradient and was validated using human samples by isoelectric focussing and Western-blot (IEF-WB). This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

p84 forms a negative regulatory complex with p110γ to control PI3Kγ signalling during cell migration.

Phosphoinositide 3-kinase γ (PI3Kγ) consists of the catalytic subunit p110γ that forms a mutually exclusive heterodimer with one of the two adaptor subunits, p101 or p84. Although activation of PI3Kγ is necessary for cell migration downstream of G-protein-coupled receptor engagement, particularly within the immune system, aberrant PI3Kγ signalling has been associated with transformation, increased migration and the progression of multiple cancer types. Regulation of PI3Kγ signal activation and duration is critical to controlling and maintaining coordinated cellular migration; however, the mechanistic basis for this is not well understood. We have recently demonstrated that, in contrast to the tumour-promoting potential of p110γ and p101, p84 possesses tumour-suppressor activity, suggesting a negative regulatory role within PI3Kγ signalling. The present study investigated the role of p84 phosphorylation in the context of PI3Kγ signalling, cell migration and p84-mediated tumour suppression. Two putative phosphorylation sites were characterised within p84, Ser358 and Thr607. Expression of wild-type p84 reduced the oncogenic potential of MDA.MB.231 cells and inhibited metastatic lung colonisation in vivo, effects that were dependent on Thr607. Furthermore, loss of Thr607 enhanced migration of MDA.MB.231 cells in vitro and prevented the induction of p84/p110γ dimers. The dimerisation of wild-type p84 with p110γ was not detected at the plasma membrane, indicating an inhibitory interaction preventing PI3Kγ lipid-kinase activity. In contrast, Ser358 phosphorylation was not determined to be critical for p84 activity in the context of migration. Our findings suggest that p84 binding to p110γ may represent a novel negative feedback signal that terminates PI3Kγ activity.

Prevalence of obesity and associated health risks in soldiers of the German Armed Forces.

BACKGROUND: Obesity rates are rising in the armed forces of Western democratic countries, impacting military readiness and health. This highlights the need for preventive health risk assessments and countermeasures. METHODS: Using mandatory health examination data from 2018 to 2022, we analyzed the prevalence of obesity, health risks, and associated specific military risk factors (rank and unit) in 43,214 soldiers of the German Armed Forces. Statistical methods included χ2 contingencies and binary logistic regressions. RESULTS: The prevalence of obesity (BMI ≥ 30) was 18.0%. Male soldiers (OR = 3.776) and those with an officer's rank (OR = 1.244) had an increased chance for obesity. Serving in a combat unit reduced the chance of being obese (OR = .886). Considering BMI and waist circumference, 2.4% of the total sample faced extremely high cardiovascular and metabolic health risks, while 11.0% and 11.6% had very high or high health risks, respectively. CONCLUSIONS: Our data underscore the importance of targeting obesity-related health risk factors in soldiers to ensure their well-being and deployment readiness.

A systematic review and in silico analysis of studies investigating the ischaemic penumbra proteome in animal models of experimental stroke.

Ischaemic stroke results in the formation of a cerebral infarction bordered by an ischaemic penumbra. Characterising the proteins within the ischaemic penumbra may identify neuro-protective targets and novel circulating markers to improve patient care. This review assessed data from studies using proteomic platforms to compare ischaemic penumbra tissues to controls following experimental stroke in animal models. Proteins reported to differ significantly between penumbra and control tissues were analysed in silico to identify protein-protein interactions and over-represented pathways. Sixteen studies using rat (n = 12), mouse (n = 2) or primate (n = 2) models were included. Heterogeneity in the design of the studies and definition of the penumbra were observed. Analyses showed high abundance of p53 in the penumbra within 24 hours of permanent ischaemic stroke and was implicated in driving apoptosis, cell cycle progression, and ATM- MAPK- and p53- signalling. Between 1 and 7 days after stroke there were changes in the abundance of proteins involved in the complement and coagulation pathways. Favourable recovery 1 month after stroke was associated with an increase in the abundance of proteins involved in wound healing. Poor recovery was associated with increases in prostaglandin signalling. Findings suggest that p53 may be a target for novel therapeutics for ischaemic stroke.