Cell Fragments Research Articles

Anchorage of bacterial effector at plasma membrane via selective phosphatidic acid binding to modulate host cell signaling

Binding phospholipid is a simple, yet flexible, strategy for anchorage of bacterial effectors at cell membrane to manipulate host signaling responses. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are the only two phospholipid species known to direct bacterial effectors to establish inner leaflet localization at the plasma membrane. Here, selectivity of phosphatidic acid (PA) by bacterial effectors for the plasma membrane anchorage and its molecular entity was identified. C-terminal BID domain of Bartonella T4SS effectors (Beps) directed the plasma membrane localization of Beps in host cells through binding with PA. A hydrophobic segment of the ‘HOOK’ subdomain from BID is inserted into the bilayer to enhance the interaction of positively charged residues with the lipid headgroups. Mutations of a conserved arginine facilitating the electrostatic interaction, a conserved glycine maintaining the stability of the PA binding groove, and hydrophobic residues determining membrane insertion, prevented the anchorage of Beps at the plasma membrane. Disassociation from plasma membrane to cytosol attenuated the BepC capacity to induce stress fiber formation and cell fragmentation in host cells. The substitution of alanine with aspartic acid at the -1 position preceding the conserved arginine residue hindered BepD anchoring at the plasma membrane, a vital prerequisite for its ability to elicit IL-10 secretion in host macrophages. In conclusion, our findings reveal the PA-binding properties of bacterial effectors to establish plasma membrane localization and will shed light on the intricate mechanisms employed by bacterial effectors within host cells.

Comprehensive utilization of Aspergillus niger waste mycelium based on physically induced autolysis: Preparation of chitosan and alternative nitrogen source

In industrial fermentation of citric acid, vast quantities of mycelium from Aspergillus niger (MAN) are produced, with an annual output of 20 million tons in China alone. The disposal of waste MAN poses a significant challenge and signifies a missed opportunity for resource recovery. In this study, physically induced autolysis (PIA) was employed to utilize the MAN proteins and chitosan. The results indicated abundant soluble proteins and sugars were released into the supernatant, while the cell fragments can be used to prepare chitosan. Under optimized autolytic conditions, the rate of total protein utilization of MAN was 78.7 %, and the rate of reserved chitin in the cell fragment was 93.8 %, outperforming other techniques. These findings were consistently reproducible in larger-scale experiments. Chitosan was prepared from cell fragments that eliminated the need for acid treatment and reduced alkali utilization by 50–67 %, thanks to PIA's effective removal of proteins from cell fragments. The rate of total sugar utilization, including both the content of the soluble sugars from autolysate and prepared chitosan, was 75.42 % of the total sugar of MAN. Additionally, the autolysate was proved to be an alternative nitrogen source in the fermentation media for microbial production of 2, 3-butanediol and citric acid, yielding concentrations comparable or superior to traditional sources. Thus, our findings not only achieved high component utilization rates but also facilitated environmentally friendly chitosan preparation, indicating the potential for innovative approaches to convert waste mycelium into valuable resources and advance the industry towards more efficient practices.

Mathematical model of dissolved microbial products in sewage treatment system

Water is the source of life, but all kinds of water resources in the world are suffering from different degrees of pollution. Water pollution leads to a serious shortage of available fresh water resources, and sewage treatment is the main way to solve water pollution. For the sewage after biological treatment of activated sludge, the organic matter contained in the effluent is mainly the dissolved microbial products (SMPs) produced in the process of microbial metabolism. The composition of SMPs is complex, mainly including macromolecular substances such as protein, polysaccharide, humic acid and DNA and cell fragments. The mathematical model of activated sludge is a quantitative description of the mathematical relationship between substrate degradation parameters and microbial growth. Starting from the Monod equation representing the relationship between substrate consumption and microbial growth, it combines the reactor theory and microbiology theory in the chemical field. Based on the principle of conservation of materials and Monod equation, the mathematical expression of organic degradation model was determined according to the collected data and empirical values of parameters. The general idea of activated sludge model No.1 (ASM1) for activated sludge process simulation was introduced, and the influence of sludge concentration on SBR process water treatment was explored. It was found that the removal rate of COD, ammonia nitrogen, total nitrogen and total phosphorus increases with the increase of sludge concentration. When C/N=8, the removal rate of ammonia nitrogen increased from 62% to 81%, and the removal rate of total nitrogen increased from 64% to 82%, with the most obvious effect.

Indoor air bacterial quality and associated factors in prison inmate cells of East Hararghe Zone and Harari Regional State, Eastern Ethiopia.

Bacterial indoor air load refers to the level of bacteria within and around dwellings and other structures. Pathogens, bacterial cell fragments, and bacterial organisms' byproducts can all pose major issues indoors, especially in prison inmate cells. However, there is lack of data on bacterial load and contributing factors in the East Hararghe zone and Harari regional state. The lack of studies on microbiological indoor air quality in prisons with contributing factors will therefore be filled by this investigation. The study aimed to assess bacterial indoor air load and contributing factors in prison inmate cells from October 1 to October 30, 2020. An institutional cross-sectional study was employed. All of the prisons in the East Hararghe zone and the Harari regional state served as the study's and source population. 62 prison cells were used in the investigation. Samples were obtained using the passively settling plate technique. The data were evaluated through the use of SPSS statistical software, Excel, and the statistical procedures of ANOVA, correlation, and chi-square test. The maximum and minimum bacterial loads, were recorded at 8:00 am (3027 CFU/m3) and 2:00 pm (1048 (CFU/m3) respectively. The correlation between the temperature and bacterial load was strongly positive (r = 0.680, p = 0.047), and the correlation of the moisture content and bacterial load was strongly negative (r = -0.671, p = 0.039). The levels of bacteria were higher than the guideline (2000 CFU/m3). While the relative humidity of indoor air was negatively correlated with bacterial load, temperature and bacterial load were significantly positively correlated. Harari regional state and East Hararghe zone prison commissions should be alarmed to alleviate these problems. The building standards need to be completely updated to the latest standards.

Investigating the effect of linker peptides on the fragmentation of Fc-fusion proteins in transient gene expression of mammalian cells

Protein fragmentation is a critical quality attribute for Fc-fusion protein production in mammalian cells. In the production of viral non-structural proteins as the form of Fc-fusion protein, fragmentation of Fc-fusion proteins occurred in two transient gene expression (TGE) systems with human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. The introduction of a flexible empirical linker reduced fragmentation in HEK293 cells, but not in CHO cells. Additionally, two rigid empirical linkers failed to restore impaired Fc-fusion proteins in CHO cells. In vitro incubation assay using conditioned culture medium and cultures supplemented with protease inhibitor cocktail suggest that fragmentation of Fc-fusion proteins in CHO cells may be due to various host cell proteins released into the culture medium. These findings suggest that the introduction of linker peptides can improve the fragmentation of Fc-fusion proteins in mammalian cells, but exhibit different fragment patterns depending on the cell type.

Ameliorative effects of osthole on acrylamide-induced neurotoxicity in PC12 cells: Role of oxidative stress, apoptosis and ERK pathways.

The possible protective effects of osthole on acrylamide-induced neurotoxicity in PC12 cells. Cells were pretreated with different concentrations of osthole (1- 25μM) for 24h and then the IC50 value of acrylamide (5mM) was added. After 24h, cell viability and intracellular ROS content were detected by MTT assay and DCF-DA methods, respectively. Also, DNA fragmentation in apoptotic cells was determined by propidium iodide assay, and apoptosis (Caspase-3, Bax, Bcl-2, ERK, and P-ERK) was measured by the western blot method. Exposing PC12 cells to acrylamide diminished cell viability, and enhanced the intracellular ROS generation and the percentage of apoptotic cells. Furthermore, acrylamide elevated the P-ERK/ERK and Bax/Bcl-2 ratio, and the level of cleaved caspase-3 protein in PC12 cells. Pretreating cells with osthole enhanced cell viability and reduced ROS generation. Also, osthole (10μM) significantly reduced P-ERK/ERK and Bax/Bcl-2 ratio, the level of cleaved caspase-3 protein, and the percentage of apoptotic cells in comparison to the acrylamide group. Osthole can exhibit a protective effect on the neurotoxicity of acrylamide through the inhibition of oxidative stress and apoptosis in PC12 cells.

Role of layilin in regulating mitochondria-mediated apoptosis: a study on B cell lymphoma (BCL)-2 family proteins

BackgroundMalignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells.ResultsWe compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-XL between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions.ConclusionThis study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis.

PFKFB3 ameliorates ischemia-induced neuronal damage by reducing reactive oxygen species and inhibiting nuclear translocation of Cdk5.

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) plays an essential role in glycolysis and in the antioxidant pathway associated with glutathione. Therefore, we investigated the effects of PFKFB3 on oxidative and ischemic damage. We synthesized a fusion protein of transactivator of transcription (Tat)-PFKFB3 to facilitate its passage into the intracellular space and examine its effects against oxidative stress induced by hydrogen peroxide (H2O2) treatment and ischemic damage caused by occlusion of the common carotid arteries for 5min in gerbils. The Tat-PFKFB3 protein was efficiently delivered into HT22 cells in a concentration- and time-dependent manner, with higher levels observed 18h after treatment. Furthermore, treatment with 6 µM Tat-PFKFB3 demonstrated intracellular delivery into HT22 cells, as analyzed through immunocytochemical staining. Moreover, it significantly ameliorated the reduction of cell viability induced by 200 µM H2O2 treatment. Tat-PFKFB3 treatment also alleviated H2O2-induced DNA fragmentation and reactive oxygen species formation in HT22 cells. In gerbils, the intraperitoneal administration of 2mg/kg Tat-PFKFB3 efficiently delivered the substance to all hippocampal areas, including the hippocampal CA1 region. This administration significantly mitigated ischemia-induced hyperlocomotion, long-term memory deficits, and ischemic neuronal death in the hippocampal CA1 region after ischemia. Additionally, treatment with 2mg/kg Tat-PFKFB3 significantly ameliorated the translocation of Cdk5 from the cytosol to the nucleus in the hippocampal CA1 region 24h after ischemia, but not in other regions. The treatment also significantly reduced reactive oxygen species formation in the CA1 region. These findings suggest that Tat-PFKFB3 reduces neuronal damage in the hippocampal CA1 region after ischemia through the reduction of Cdk5 signaling and reactive oxygen species formation. Therefore, Tat-PFKFB3 may have potential applications in reducing ischemic damage.

Targeting Hypoxia and Autophagy Inhibition via Delivering Sonodynamic Nanoparticles With HIF-2α Inhibitor for Enhancing Immunotherapy in Renal Cell Carcinoma.

Immune checkpoint blockers (ICBs) therapy stands as the first-line treatment option for advanced renal cell carcinoma (RCC). However, its effectiveness is hindered by the immunosuppressive tumor microenvironment (TME). Sonodynamic therapy (SDT) generates tumor cell fragments that can prime the host's antitumor immunity. Nevertheless, the hypoxic microenvironment and upregulated autophagy following SDT often lead to cancer cell resistance. In response to these challenges, a hypoxia-responsive polymer (Poly(4,4'-azobisbenzenemethanol-PMDA)-mPEG5k, P-APm) encapsulating both a HIF-2α inhibitor (belzutifan) and the ultrasonic sensitize (Chlorin e6, Ce6) is designed, to create the nanoparticle APm/Ce6/HIF. APm/Ce6/HIF combined with ultrasound (US) significantly suppresses tumor growth and activates antitumor immunity in vivo. Moreover, this treatment effectively transforms the immunosuppressive microenvironment from "immune-cold" to "immune-hot", thereby enhancing the response to ICBs therapy. The findings indicate that APm/Ce6/HIF offers a synergistic approach combining targeted therapy with immunotherapy, providing new possibilities for treating RCC.

Investigation of optimal sperm storage conditions for short-term storage

Objective The quality of sperm cells is important role in the success rates of assisted reproduction technology (ART) treatments. The quality of the sperm cells shows variations depending on the temperature of short-term semen storage as well as the methods of semen preparation. Thus, this study aimed to investigate the sperm viability, motility and DNA fragmentation following different sperm preparation methods and short-term storage conditions, respectively. Materials and Methods A total of 25 semen samples were evaluated. In the first part of this study, different incubation temperatures were investigated in two groups, in such the first group involved the semen samples and the second group involved the sperm cells separated by density gradient centrifugation method, respectively. The samples in each group were incubated at 4°C, room temperature (21°C) and 37°C for 24 hours, respectively. The sperm cell qualities were evaluated by mobility analysis, DNA fragmentation by acridine orange staining and sperm cell viability by propidium iodide staining. Results and Discussion The analysis outcome demonstrated that the mobility, DNA fragmentation and viability of the sperm cells were statistically different when incubated at RT (21°C) in both groups. Furthermore, samples prepared by the density gradient centrifugation method were shown to have better quality. The optimum short-term storage temperature was detected to be the room temperature. Conclusion The conclusion of this investigation is crucial to assess storage conditions in ART clinics. This study provides essential data for short-term sperm storage and preparation methods to improve the success rates of ART clinics. Bangladesh Journal of Medical Science Vol. 23 No. 04 October’24 Page : 1137-1141

Helicobacter pylori VacA-induced mitochondrial damage in the gastric pit cells of the antrum and therapeutic rescue

Exploring host cell specificity, pathogenicity, and molecular mechanisms of the vacuolating cytotoxin A (VacA), secreted by Helicobacter pylori (Hp) is crucial for developing novel treatment strategies. VacA affects subcellular events, particularly mitochondria, at a cell-type-specific level. However, the lack of reliable models that mimic VacA-induced subcellular damages and enable novel drug screening linked to the human stomach clinically limits our understanding of the mitochondrial networks in vivo. Here, human antrum gastric organoids (hAGOs) and tissue samples from Hp-infected patients were used to show the toxic effects of VacA-induced mitochondrial damage mainly in mucus-producing gastric pit cells by employing transcriptional, translational, and functional analyses. In VacA-intoxicated or Hp-infected hAGOs, robust mitochondrial fragmentation in gastric pit cells reduced ATP production during respiration, and loss of mucosal barrier integrity was first demonstrated experimentally. Using hAGOs, clinically relevant small molecules were screened for efficacy, and MLN8054, an Aurora kinase A inhibitor, reversed VacA-induced mitochondrial damage and loss of gastric epithelium integrity. MLN8054 was effective in VacA-treated and Hp-infected hAGOs and mice, highlighting hAGOs as a promising drug-screening model. These findings suggest that mitochondrial quality control may serve as a promising therapeutic target for Hp VacA-mediated toxicity and disease progression.

DMF-ChIP-seq for Highly Sensitive and Integrated Epigenomic Profiling of Low-Input Cells.

Mapping genome-wide DNA-protein interactions (DPIs) provides insights into the epigenetic landscape of complex biological systems and elucidates the mechanisms of epigenetic regulation in biological progress. However, current technologies in DPI profiling still suffer from high cell demands, low detection sensitivity, and large reagent consumption. To address these problems, we developed DMF-ChIP-seq that builds on digital microfluidic (DMF) technology to profile genome-wide DPIs in a highly efficient, cost-effective, and user-friendly way. The entire workflow including cell pretreatment, antibody recognition, pA-Tn5 tagmentation, fragment enrichment, and PCR amplification is programmatically manipulated on a single chip. Leveraging closed submicroliter reaction volumes and a superhydrophobic interface, DMF-ChIP-seq presented higher sensitivity in peak enrichment than other current methods, with high accuracy (Pearson Correlation Coefficient (PCC) > 0.86) and high repeatability (PCC > 0.92). Furthermore, DMF-ChIP-seq was capable of processing the samples with as few as 8 cells while maintaining a high signal-to-noise ratio. By applying DMF-ChIP-seq, H3K27ac histone modification of early embryonic cells during differentiation was profiled for the investigation of epigenomic landscape dynamics. With the benefits of high efficiency and sensitivity in DPI analysis, the system provides great promise in studying epigenetic regulation during various biological processes.

How Protein Depletion Balances Thrombosis and Bleeding Risk in the Context of Platelet's Activatory and Negative Signaling.

Platelets are small cell fragments that play a crucial role in hemostasis, requiring fast response times and fine signaling pathway regulation. For this regulation, platelets require a balance between two pathway types: the activatory and negative signaling pathways. Activatory signaling mediators are positive responses that enhance stimuli initiated by a receptor in the platelet membrane. Negative signaling regulates and controls the responses downstream of the same receptors to roll back or even avoid spontaneous thrombotic events. Several blood-related pathologies can be observed when these processes are unregulated, such as massive bleeding in activatory signaling inhibition or thrombotic events for negative signaling inhibition. The study of each protein and metabolite in isolation does not help to understand the role of the protein or how it can be contrasted; however, understanding the balance between active and negative signaling could help develop effective therapies to prevent thrombotic events and bleeding disorders.

Death in the taste bud: Morphological features of dying taste cells and engulfment by Type I cells.

Taste buds comprise 50-100 epithelial derived cells, which are renewed throughout the life of an organism. Immature cells enter the bud at its base, maturing into one of three distinct cell types. How taste cells die and/or exit the bud, however, remains unclear. Here we present morphological data obtained through Serial Blockface Scanning Electron Microscopy of murine circumvallate taste buds, revealing several taste cells at the end of their life (4-6 per bud). Cells we identify as dying share certain morphological features typical of apoptosis: swollen endoplasmic reticulum, large lysosomes, degrading organelles, distended outer nuclear membranes, heterochromatin reorganization, cell shrinkage, and cell and/or nuclear fragmentation. Based on these features, we divide the cells into "early" and "late" stage dying cells. Most early stage dying cells have Type II cell morphologies, while a few display Type III cell features. Many dying cells maintain contacts with nerve fibers, but those fibers often appear detached from the main trunk of an afferent nerve fiber. Dying cells, like mature Type II and Type III taste cells, are surrounded by Type I taste cells, the glial-like cells of the bud. In many instances Type I cells appear to be engulfing their dying neighbors, suggesting a novel, phagocytic role for Type I cells. Surprisingly, virtually no Type I cells, which have the shortest residence time in taste buds, display features of apoptosis. The ultimate fate of Type I cells therefore remains enigmatic.

Manufacturing processes, additional nutritional value and versatile food applications of fresh microalgae Spirulina.

Spirulina is capable of using light energy and fixing carbon dioxide to synthesize a spectrum of organic substances, including proteins, polysaccharides, and unsaturated fatty acids, making it one of the most coveted food resources for humanity. Conventionally, Spirulina products are formulated into algal powder tablets or capsules. However, the processing and preparation of these products, involving screw pump feeding, extrusion, high-speed automation, and high-temperature dewatering, often result in the rupture of cell filaments, cell fragmentation, and the unfortunate loss of vital nutrients. In contrast, fresh Spirulina, cultivated within a closed photobioreactor and transformed into an edible delight through harvesting, washing, filtering, and sterilizing, presents a refreshing taste and odor. It is gradually earning acceptance as a novel health food among the general public. This review delves into the manufacturing processes of fresh Spirulina, analyzes its nutritional advantages over conventional algal powder, and ultimately prospects the avenues for fresh Spirulina's application in modern food processing. The aim is to provide valuable references for the research and development of new microalgal products and to propel the food applications of microalgae forward.

Abstract MP13: Hemolysis-mediated Prolyl Endo-Peptidase plasma release – a natural protective mechanism against extreme blood pressure elevations?

During hypertensive crises (BP >180/120 mm Hg), turbulent passage of red blood cells through severely constricted blood vessels can cause blood cell fragmentation and intravascular hemolysis. Consistent with this, in mice we found that a rapid and large increase in BP (by ~60-70 mm Hg) induced by an Ang II-infusion (0.2 ug/g BW) resulted in hemolysis. Prolyl Endo-Peptidase (PEP) is an intracellular enzyme that converts Ang II to Ang-(1-7), thereby shifting from a powerful vasopressor peptide to a counter regulatory peptide. The Ang II-induced acute BP increase that caused a visible hemolysis was associated with a ~4-fold increase in PEP activity (Figure, Panel A). A massive hemolysis induced by freeze-thawing of blood (cryo-hemolysis), moreover, was associated with a ~20x increase in PEP activity as compared to native plasma (Panel B). Of note, mouse cryo-hemolyzed plasma (HP) ex vivo was able to produce substantially more Ang-(1-7) peptide from spiked Ang II than equivalent amount of non-hemolyzed plasma (P). In addition, when PEP inhibitor, S-17092, was added to cryo-hemolyzed plasma (HP inh), Ang-(1-7) formation from Ang II was greatly diminished (Panel C). This suggests that blood corpuscles acting via intracellular PEP are endowed with a larger capacity than native plasma to catabolize the vasoconstrictor peptide Ang II to the vasodilator peptide Ang-(1-7). In conclusion, a large and rapid increase in BP after Ang II infusion causes hemolysis which is associated with the release of intracellular PEP enzyme from blood cells. We propose that the release of PEP confers a protective mechanism against severe BP elevations during hypertensive crisis by granting higher plasma Ang II-degrading capability thereby lowering Ang II and BP. This occurs at the expense of temporary hemolysis in the setting of hypertensive crisis as seen with malignant hypertension.

Embryo multinucleation: detection, possible origins, and implications for treatment.

Cell cycle regulation is crucial to assure expansion of a cell population, while preserving genome integrity. This notion is especially relevant to fertilization and early embryo development, a time when the cell cycle transforms from meiotic into mitotic cycles. Zygote-to-embryo transition is acutely error-prone, causing major developmental perturbations, including cleavage delays, tri- and multi-chotomous cleavages, and cell fragmentation. Another such alteration is bi- and multinucleation, consisting of the simultaneous formation of two or more nuclei at interphase. Indeed, multinucleation affects a large proportion of early human embryos, typically at the two-cell stage. Mechanistically, several factors, including spindle dysfunction, failed cleavage, and cell fusion, may generate this cell anomaly. In assisted reproduction treatment, multinucleation is associated with reduced developmental rates and lower implantation rates in Days 2-3 embryo transfers. However, many multinucleated embryos can develop to the blastocyst stage. In blastocyst transfers, the current evidence does not suggest a major impact of a previous history of multinucleation on the odds of euploidy or successful treatment outcomes. Human embryo multinucleation remains a not-fully-understood but developmentally relevant and intriguing phenomenon which requires further research of its generative mechanisms and clinical implications.

Sublethal Effects Induced by a Cyflumetofen Formulation on Honeybee Apis mellifera L. Workers: Assessment of Midgut, Hypopharyngeal Glands, and Fat Body Integrity.

Worldwide, both cultivated and wild plants are pollinated by the honey bee, Apis mellifera. Bee numbers are declining as a result of a variety of factors, including increased pesticide use. Cyflumetofen controls pest mites in some plantations pollinated by bees, which may be contaminated with residual sublethal concentrations of this pesticide, in nectar and pollen. We evaluated the effects of a sublethal concentration of a cyflumetofen formulation on the midgut, hypopharyngeal gland, and fat body of A. mellifera workers orally exposed for 72 h or 10 days. The midgut epithelium of treated bees presented digestive cells with cytoplasm vacuoles and some cell fragmentation, indicating autophagy and cell death. After being exposed to the cyflumetofen formulation for 72 h, the midgut showed a higher injury rate than the control bees, but after 10 days, the organs had recovered. In the hypopharyngeal gland of treated bees, the end apparatus was filled with secretion, suggesting that the acaricide interferes with the secretory regulation of this gland. Histochemical tests revealed differences in the treated bees in both exposure periods in the midgut and hypopharyngeal glands. The acaricide caused cytotoxic effects on the midgut digestive cells, with apical protrusions, plasma membrane rupture, and several vacuoles in the cytoplasm, features of cell degeneration. In the hypopharyngeal glands of the treated bees, the secretory cells presented small electron-dense and large electron-lucent secretory granules. The fat body cells had no changes in comparison with the control bees. In conclusion, the cyflumetofen formulation at sublethal concentrations causes damage to the midgut and the hypopharyngeal glands of honey bee, which may compromise the functions of these organs and colony fitness. Environ Toxicol Chem 2024;43:2455-2465. © 2024 SETAC.

Platelets isolation and ectonucleotidase assay: Revealing functional aspects of the communication between the vasculature and the immune system

Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5′-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems.

Sequential delivery of photosensitizers and checkpoint inhibitors by engineered bacteria for enhanced cancer photodynamic immunotherapy.

Engineered bacteria-based cancer therapy has increasingly been considered to be a promising therapeutic strategy due to the development of synthetic biology. Wherein, engineering bacteria-mediated photodynamic therapy (PDT)-immunotherapyshows greater advantages and potential in treatment efficiency than monotherapy. However, the unsustainable regeneration of photosensitizers (PSs) and weak immune responses limit the therapeutic efficiency. Herein, we developed an engineered bacteria-based delivery system for sequential delivery of PSs and checkpoint inhibitors in cancer PDT-immunotherapy. The biosynthetic pathway of 5-aminolevulinic acid (5-ALA) was introduced into Escherichia coli, yielding a supernatant concentration of 172.19 mg/L after 10 h of growth. And another strain was endowed with the light-controllable releasement of anti-programmed cell death-ligand 1 nanobodies (anti-PD-L1). This system exhibited a collaborative effect, where PDT initiated tumor cell death and the released tumor cell fragments stimulated immunity, followed by the elimination of residual tumor cells. The tumor inhibition rate reached 74.97%, and the portion of activated T cells and inflammatory cytokines were reinforced. The results demonstrated that the engineered bacteria-based collaborative system could sequentially deliver therapeutic substance and checkpoint inhibitors, and achieve good therapeutic therapy. This paper will provide a new perspective for the cancer PDT-immunotherapy.