Bombus terrestris are efficient pollinators in forestry and agriculture, with higher cold tolerance than other bees. Yet, their cold tolerance mechanism remains unclear. Aquaporins (AQPs) function as cell membrane proteins facilitating rapid water flow, aiding in osmoregulation. Recent studies highlight the importance of insect AQPs in dehydration and cold stress. Comparative transcriptome analysis of B. terrestris under cold stress revealed up-regulation of four AQPs, indicating their potential role in cold tolerance. Seven AQPs-Eglp1, Eglp2, Eglp3, DRIP, PRIP, Bib, and AQP12L-have been identified in B. terrestris. These are widely expressed in various tissues, particularly in the alimentary canal and Malpighian tubules. Functional analysis of BterAQPs in the Xenopus laevis oocytes expressing system showed distinct water and glycerol selectivity, with BterDrip exhibiting the highest water permeability. Molecular modeling of BterDrip revealed six transmembrane domains, two NPA motifs, and an ar/R constriction region (Phe131, His256, Ser265, and Arg271), likely contributing to its water selectivity. Silencing BterDRIP accelerated mortality in B. terrestris under cold stress, highlighting the crucial role of BterDRIP in their cold tolerance and providing a molecular mechanism for their cold adaptation.

Exudates of non-healing wounds contain drivers of pathogenicity. We utilized >800 exudates from non-healing and healing wounds of diverse etiologies, collected by three different methods, to develop a wound-specific, cell-based functional biomarker assay. Human dermal fibroblast proliferation served as readout to a) to differentiate between healing and non-healing wounds, b) follow the healing process of individual patients, and c) assess the effects of therapeutics for chronic wounds ex vivo. We observed a strong correlation between wound chronicity and inhibitory effects of individual exudates on fibroblast proliferation, with good diagnostic sensitivity (76-90%, depending on the sample collection method). Transition of a clinically non-healing to a healing phenotype restored fibroblast proliferation and extracellular matrix formation while reducing inflammatory cytokine production. Transcriptional analysis of fibroblasts exposed to ex vivo non-healing wound exudates revealed an induction of inflammatory cytokine- and chemokine pathways and the unfolded protein response, indicating that these changes may contribute to the pathology of non-healing wounds. Testing the wound therapeutics platelet derived growth factor and silver sulfadiazine yielded responses in line with clinical experience and indicate the usefulness of the assay to search for and profile new therapeutics.

Autoantibodies against type I interferon (IFN) are associated with a worse outcome in COVID-19. The measurement of cytokine-neutralizing autoantibodies has been limited, hindering understanding of their role in clinical practice. We showed that an easy and reliable assay can be reproduced and validated to measure the neutralizing potency of autoantibodies directed to type I or type II IFN. Identifying of anti-cytokine autoantibodies might reflect on early treatments for subsequent infections, such as with antivirals or virus-neutralizing monoclonal antibodies.

UNASSIGNED: Shigella is the etiologic agent of a bacillary dysentery known as shigellosis, which causes millions of infections and thousands of deaths worldwide each year due to Shigella\'s unique lifestyle within intestinal epithelial cells. Cell adhesion/invasion assays have been extensively used not only to identify targets mediating host-pathogen interaction, but also to evaluate the ability of Shigella-specific antibodies to reduce virulence. However, these assays are time-consuming and labor-intensive and fail to assess differences at the single-cell level.
UNASSIGNED: Here, we developed a simple, fast and high-content method named visual Adhesion/Invasion Inhibition Assay (vAIA) to measure the ability of anti-Shigellaantibodies to inhibit bacterial adhesion to and invasion of epithelial cells by using the confocal microscope Opera Phenix.
UNASSIGNED: We showed that vAIA performed well with a pooled human serum from subjects challenged with S. sonnei and that a specific anti-IpaD monoclonal antibody effectively reduced bacterial virulence in a dose-dependent manner.
UNASSIGNED: vAIA can therefore inform on the functionality of polyclonal and monoclonal responses thereby supporting the discovery of pathogenicity mechanisms and the development of candidate vaccines and immunotherapies. Lastly, this assay is very versatile and may be easily applied to other Shigella species or serotypes and to different pathogens.

Most investigations on the immune cell-activating potency of IgA used purified total IgA and/or specific isolated cell populations. As IgA2 has been reported to be more pro-inflammatory than IgA1, we aimed to employ a fast and convenient whole blood-based assay to individually probe the capacity of the two IgA subclasses to activate immune cells in close physiological conditions. To this end, whole blood from healthy donors (n = 10) was stimulated with immobilized IgA1, IgA2m1 or IgA2m2 (the two main allotypic variants of IgA2). Activation of major leukocyte subsets was measured using a 10-color flow cytometry panel providing access to the expression of 5 activation markers on 6 different immune cell subsets. While capturing some heterogeneity of responses among donors, IgA2m1 and IgA2m2 systematically showed a stronger activation profile compared to IgA1 in a variety of dimensions. For example, both IgA2 allotypes led to stronger modulations of CD54, CD11b, CD62L, CD66b or CD69, on both or either monocytes or neutrophils, indicating a more pronounced pro-inflammatory effect for this subclass than IgA1. By taking into account donor-specific soluble and cellular components this whole blood-based functional approach provides new perspectives to further investigate IgA effector functions in mechanistic studies and/or translational research.

BACKGROUND: Pathogenic (P)/likely pathogenic (LP) SMAD3 variants cause Loeys-Dietz syndrome type 3 (LDS3), which is characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with osteoarthritis.
OBJECTIVE: Investigate the impact of P/LP SMAD3 variants with functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs), to optimize interpretation of SMAD3 variants.
METHODS: A retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and functional analyses on SMAD3 patient-derived VSMCs and SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts.
RESULTS: Individuals with dominant negative (DN) SMAD3 variant in the MH2 domain exhibited more major events (66.7% vs. 44.0%, P = 0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. The age at first major event was 35.0 years [IQR 29.0-47.0] in individuals with DN variants in MH2, compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (P = 0.065). Fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential in HI SMAD3 variant fibroblasts. HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix formation compared to control cell lines.
CONCLUSIONS: Distinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and SMA and MYH11 expression. The differences between DN and HI SMAD3 variant fibroblasts and VSMCs potentially contribute to the differences in disease manifestation. Notably, myofibroblast differentiation seems a suitable alternative in vitro test system compared to VSMCs.

UNASSIGNED: Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown.
UNASSIGNED: To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1β and IL-8 in the human monocyte cell line THP-1 upon bMV treatment.
UNASSIGNED: Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1β and IL-8 expressions.
UNASSIGNED: Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

UNASSIGNED: The ITGB6 gene encoding a protein that can regulate the integrin αvβ6 heterodimer protein expression in different status was shown to play an important role in multiple human cancers, such as brain cancer, colon cancer and oral cancer, and is related to clinical progression. This study aims to explore the function and the mechanism of the ITGB6 gene or protein in pancreatic cancer.
UNASSIGNED: We examined the expression of ITGB6 in pancreatic cancer using immunohistochemistry and analyzed the relationship between the expression of ITGB6 and the clinicopathologic features in pancreatic cancer patients. In addition, a bioinformatic method was used to analyze the ITGB6 mRNA level in pancreatic tumor tissues compared with normal pancreatic tissues and to analyze the correlation between high KIF23 expression and prognosis in pancreatic cancer patients. Moreover, colony formation assay, MTT assay, cell scratch, cell invasion and western blot assays in vitro and a xenograft mouse model in vivo were performed to analyze the effect of KIF23 on proliferation and invasion of pancreatic cancer cells.
UNASSIGNED: Increased expression of ITGB6 was significantly correlated with poor clinical outcome in both our clinical data and TCGA data of pancreatic cancer. Furthermore, functional assays revealed that ITGB6 knockdown in vivo and in vitro might inhibit cancer cell proliferation and the ability of invasion or migration.
UNASSIGNED: Our data suggest that ITGB6 is associated with pancreatic cancer malignant progression. Hence, ITGB6 may serve as a potential target of pancreatic cancer for future research, and further study is needed.

Alzheimer\'s disease (AD) is a neurodegenerative disease and the most frequent cause of dementia. It is characterized by the accumulation in the brain of two pathological protein aggregates: amyloid-β peptides (Aβ) and abnormally phosphorylated tau. The progressive cognitive decline observed in patients strongly correlates with the synaptic loss. Many lines of evidence suggest that soluble forms of Aβ accumulate into the brain where they cause synapse degeneration. Stopping their spreading and/or targeting the pathophysiological mechanisms leading to synaptic loss would logically be beneficial for the patients. However, we are still far from understanding these processes. Our objective was therefore to develop a versatile model to assay and study Aβ-induced synaptotoxicity. We integrated a microfluidic device that physically isolates synapses from presynaptic and postsynaptic neurons with a microelectrode array. We seeded mouse primary cortical cells in the presynaptic and postsynaptic chambers. After functional synapses have formed in the synaptic chamber, we exposed them to concentrated conditioned media from cell lines overexpressing the wild-type or mutated amyloid precursor protein and thus secreting different levels of Aβ. We recorded the neuronal activity before and after exposition to Aβ and quantified Aβ\'s effects on the connectivity between presynaptic and postsynaptic neurons. We observed that the application of Aβ on the synapses for 48 h strongly decreased the interchamber connectivity without significantly affecting the neuronal activity in the presynaptic or postsynaptic chambers. Thus, through this model, we are able to functionally assay the impact of Aβ peptides (or other molecules) on synaptic connectivity and to use the latter as a proxy to study Aβ-induced synaptotoxicity. Moreover, since the presynaptic, postsynaptic, and synaptic chambers can be individually targeted, our assay provides a powerful tool to evaluate the involvement of candidate genes in synaptic vulnerability and/or test therapeutic strategies for AD.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as μ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and β-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and β-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting β-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and β-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.