Dependent Manner Research Articles

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2.

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.

C-C chemokine ligand 2 signaling promotes skeletal muscle wasting in non-tumor and breast tumor mouse models.

Despite advancements in treatment, approximately 25% of breast cancer patients experience long-term skeletal muscle wasting (SMW), which limits mobility, reduces drug tolerance and adversely impacts survival. By understanding the underlying molecular mechanisms of SMW, we may develop new strategies to alleviate this condition and improve the lives of breast cancer patients. Chemokines are small soluble factors that regulate homing of immune cells to tissues during inflammation. In breast cancers, overexpression of the C-C chemokine ligand 2 (CCL2) correlates with unfavorable prognosis. Elevated levels of CCL2 in peripheral blood indicate possible systemic effects of this chemokine in breast cancer patients. Here, we investigated the role of CCL2 signaling on SMW in a tumor and non-tumor context. In vitro, increasing concentrations of CCL2 inhibits myoblast and myotube function through C-C chemokine receptor 2 (CCR2) dependent mechanisms involving JNK, SMAD3 and AMPK signaling. In healthy mice, delivery of recombinant CCL2 protein promotes SMW in a dose dependent manner. In vivo knockdown of breast tumor derived CCL2 partially protects against SMW. Overall, chronic, upregulated CCL2/CCR2 signaling positively regulates SMW, with implications on therapeutic targeting.

The effect of low-dose photodynamic therapy usingthe photosensitizer chloroaluminum phthalocyanine on a scratch wound model in skin fibroblasts.

Different approaches on wound healing have been developed over the years but they suffer from high costs and adverse effects for the patients. The current paper was designed to study low dose PDT, a novel healing approach, in an in vitro fibroblasts wound healing model. Chloroaluminum phthalocyanine (AlClPc) was used as photosensitizer and was activated by a red diode laser at 661 nm. After PDT optimization, wound closure rate and reactive oxygen species were quantified by image processing and analysis. Our results revealed that wound healing rates were significantly higher in PDT treated groups than in the control. Additionally, the study revealed that a prolonged ROS increase did not promote wound closure, while a small increase acted as a trigger, resulting in faster wound closure. Concluding, low dose PDT using AlClPc enhances wound healing in vitro in a ROS dependent manner, allowing the assumption of similar positive effects in vivo.

GNA13 suppresses proliferation of ER+ breast cancer cells via ERα dependent upregulation of the MYC oncogene

GNA13 (Gα13) is one of two alpha subunit members of the G12/13 family of heterotrimeric G-proteins which mediate signaling downstream of GPCRs. It is known to be essential for embryonic development and vasculogenesis and has been increasingly shown to be involved in mediating several steps of cancer progression. Recent studies found that Gα13 can function as an oncogene and contributes to progression and metastasis of multiple tumor types, including ovarian, head and neck and prostate cancers. In most cases, Gα12 and Gα13, as closely related α-subunits in the subfamily, have similar cellular roles. However, in recent years their differences in signaling and function have started to emerge. We previously identified that Gα13 drives invasion of Triple Negative Breast Cancer (TNBC) cells in vitro. As a highly heterogenous disease with various well-defined molecular subtypes (ER+ /Her2−, ER+ /Her2+, Her2+, TNBC) and subtype associated outcomes, the function(s) of Gα13 beyond TNBC should be explored. Here, we report the finding that low expression of GNA13 is predictive of poorer survival in breast cancer, which challenges the conventional idea of Gα12/13 being universal oncogenes in solid tumors. Consistently, we found that Gα13 suppresses the proliferation in multiple ER+ breast cancer cell lines (MCF-7, ZR-75-1 and T47D). Loss of GNA13 expression drives cell proliferation, soft-agar colony formation and in vivo tumor formation in an orthotopic xenograft model. To evaluate the mechanism of Gα13 action, we performed RNA-sequencing analysis on these cell lines and found that loss of GNA13 results in the upregulation of MYC signaling pathways in ER+ breast cancer cells. Simultaneous silencing of MYC reversed the proliferative effect from the loss of GNA13, validating the role of MYC in Gα13 regulation of proliferation. Further, we found Gα13 regulates the expression of MYC, at both the transcript and protein level in an ERα dependent manner. Taken together, our study provides the first evidence for a tumor suppressive role for Gα13 in breast cancer cells and demonstrates for the first time the direct involvement of Gα13 in ER-dependent regulation of MYC signaling. With a few exceptions, elevated Gα13 levels are generally considered to be oncogenic, similar to Gα12. This study demonstrates an unexpected tumor suppressive role for Gα13 in ER+ breast cancer via regulation of MYC, suggesting that Gα13 can have subtype-dependent tumor suppressive roles in breast cancer.

Synovial fibroblasts from children with oligoarticular juvenile idiopathic arthritis induce migration and prolong viability of neutrophils

IntroductionLittle is known of the processes that trigger neutrophil activation in the joint of patients with oligoarticular juvenile idiopathic arthritis (oJIA), and if synovial fibroblasts (S-Fib) play an important role in the activation. Therefore, we aimed to investigate whether S-Fib derived from oJIA patients drive neutrophil activation.MethodsSynovial fluid (SF) was collected from patients with oJIA. S-Fib were isolated from the SF of n = 7 patients through passaging. Subsequently, the S-Fib were primed or not with 20% of pooled SF. Supernatants were used to study migration of neutrophils in a transwell system. Additionally, the influence of S-Fib on neutrophils were studied in co-cultures. Phenotype and viability were assessed by flow cytometry. Neutrophil function was tested through the production of reactive oxygen species (ROS), and supernatants were tested for myeloperoxidase (MPO) release and elastase activity.ResultsSupernatants of S-Fib induced neutrophil migration (n = 5, p = 0.0491), which was further pronounced using supernatants from SF-primed S-Fib (p = 0.0063). Additionally, co-culture between SF-primed S-Fib and neutrophils resulted in prolonged viability (n = 5, p = 0.0094), with little effect on activation markers, e.g., CD11b. Conversely, co-culture did not induce functional alterations (n = 4), such as production of ROS (p > 0.1570), release of MPO (p > 0.4934) or elastase activity (p > 0.0904). Finally, supernatant stimulation did not replicate the results of prolonged viability (p = 0.9102), suggesting a role of cell-contact.ConclusionS-Fib from patients with oJIA induce migration of neutrophils via soluble mediators and, in addition, S-Fib prolong neutrophil viability in a cell-contact dependent manner. These mechanisms could be important for accumulation of neutrophils during arthritis.

Identification of Early Protein Damage-Associated Molecular Pattern (DAMPs) Candidates from Cardiac Cells Subjected to an In Vitro Myocardial Ischemic Injury

Damage-associated molecular patterns (DAMPs) are intracellular molecules that are released from cells that undergo injury. Protein-DAMPs are abundant biomolecules that could potentially be a novel biomarker for pathology. Myocardial ischemia injury is a causative process for ischemic heart disease, which is now the leading cause of global deaths. Early diagnosis is necessary to save patient lives, so the discovery of new early cardiac biomarkers is crucial. Here, we examined the protein-DAMPs expression in ischemic buffer collected from adult ventricular myocytes cells subjected to various timepoints of simulated ischemia (sI). Adult ventricular myocytes cells (AC-16) were subjected to sublethal simulated ischemia (sI) by treatment with ischemic buffer for 5, 10, 20, and 30 minutes (I5, I10, I20, and I30, respectively). After simulated ischemia was achieved, the ischemic buffer was collected and determined for proteins profiling by Shotgun proteomics approach. Cell viability was reduced in a time dependent manner in ischemia. Targeted proteomics reveals 21 proteins found to highly increase in early ischemic injury and can still be detected until 30 minutes of ischemia. Lactotransferrin (LTF) was determined to be the protein with the greatest impact, exhibiting both fast and sustained release throughout the whole study time. The present study identified the protein damage associated molecular pattern (DAMPs) released specifically from ischemia-induced cardiomyocytes injury, which could potentially be candidates to be early biomarkers for myocardial ischemic injury. Further studies should be focused on in vivo validation of potential protein markers that could be used in practical clinical applications.

P-442 Genome edition in endometrial stroma cells leading to decreased expression of UDP-glucuronic acid decarboxylase I (UXS1) alters the molecular interactions of the implantation area

Abstract Study question Does UDP-glucuronic acid decarboxylase 1 (UXS1) expression in endometrial stroma cells have a direct effect on the early stages of implantation? Summary answer Decreased expression of UXS1 was found to alter adhesion, invasion, development and function related genes of implantation in 3D cultures in a time dependent manner. What is known already The implantation process is governed by a complex interaction of genes and molecular pathways. Some of those genes are well-defined, however, as fertility problems increase with escalating rates of recurrent implantation failure or abortions, with an effort to reveal those genes that may be more significant than known for implantation RNA-seq analyses are used. UDP-glucuronic acid decarboxylase 1 (UXS1) is an enzyme involved in the detoxification and conversion of UDP-glucuronic acid to UDP-xylose. Loss of UXS1 led to defective glycosylation, increased protein degradation, and decreased overall protein levels, but no direct relation to has been stated before. Study design, size, duration Two different Matrigel based 3D culture systems were constructed with labeled mouse endometrium epithelial cells, stroma cells and blastocysts for 24-72 hours (n = 24). Sorted cells were sequenced for RNA for determination of differentially expressed genes. Of over 4000 genes, a highly expressed one was UXS1 in the endometrial stroma cells, The gene was knocked out via CRISPR/Cas9. The impact of decline of UXS1 in stroma cells on implantation parameters has been evaluated. Participants/materials, setting, methods CB6F1 female mice were superovulated. Blastocysts were obtained, endometrium was used for isolation of epithelial and stroma cells. 3D cultures were constructed for RNA-seq analysis. Three gRNAs were designed for CRISPR/Cas9 experiments. Cultures were constructed with knock out stroma and wild epithelial cells, wild blastocysts. On hours 24 and 72 of culture, gene and protein expression analyses for e-cadherin, MMP9, entactin, LIF; Hoxa10, Wnt4, ESR1, PR, fibronectin have been accomplished. Supported by TUBITAK 118S676. Main results and the role of chance All adherence molecules tested (e-cadherin, entactin, fibronectin) have shown significant increase compared to non targeted (the cell has Cas9, but no gRNA) groups (p < 0.00001). As e-cadherin has decreased detrimentally from 24 to 72 hours, fibronectin has continued to increase. LIF, an important gene for embryo-blastocyst interphase, has increased steeply and then decreased in the long term culture (p < 0.00001). ESR1 and PR (estrogen and progesterone receptors) increased compared to non-targeted groups but they were lower than the control groups with no genome edition (p < 0.01). The invasion gene MMP9 has increased steeply in the first 24 hours, then declined suddenly at hour 72 (p < 0.0001). The developmental genes Wnt4 and Hoxa10 were also affected significantly, as Wnt4 has decreased and Hoxa10 has increased compared to non-targeted groups (p < 0.001, p < 0.0001 respectively), however the expressions did not change from 24 to 72 hours. These findings were mostly supported by the protein expressions depicted by IF staining. The data can be interpreted as UXS1 has some fundamental effects during the implantation process as its decreased expression changes the adhesion and invasion patterns with unexpectedly high and low pattern, and it deteriorates differential expression of LIF during the critical interaction of endometrial cells and the blastocyst. Limitations, reasons for caution - This is a 3D in vitro implantation model constructed with mouse cells, so it cannot reflect in vivo process perfectly. - The genome edition of primary cells can show lower yield than cell lines. - There are numerous parameters of implantation, more tests can always be added. Wider implications of the findings - Data arising from various RNAseq analyses for implantation is accumulating and it is possible to find new genes and their connections and contribution can be revealed. - The new genes tested in vitro can further be re-examined in knock out mouse models or can be scanned in the abortus materials. Trial registration number not applicable

P-272 Cannabis negatively impacts female fertility: an in vitro and clinical investigation

Abstract Study question How does tetrahydrocannabinol (THC) impacts human oocyte maturation and female fertility? Summary answer THC alters the oocyte’s RNA profile and affects meiotic spindle formation resulting in an increased rate of aneuploidy in the oocyte and in the blastocyst. What is known already In the past decades, cannabis consumption and legalization has been increasing globally. It is the third most used substance by reproductive aged people and our group demonstrated that tetrahydrocannabinol (THC) reaches the ovarian follicle. THC is known to impact male fertility, but due to the challenges associated with investigating the female gamete in humans, there is no study about the human oocyte. Animal studies have shown that THC impacts oocyte maturation in a concentration dependent manner, but between models, the results are conflicting. Study design, size, duration For the in vitro exposure assessment, GV oocytes naïve to THC were collected from consenting patients undergoing IVF-ICSI from July 2022 to June 2024 (IRB: Veritas #16518). Per patient, oocytes were split into three groups: Control, THC1 (physiological concentration of THC, based on previous human studies) and THC2 (supra-physiological concentration, based on animal studies), and cultured for 24h in our standard clinical in vitro maturation media. Participants/materials, setting, methods Single-cell RNAseq and pathway analysis were conducted to identify key pathways altered by THC treatments (n = 86 from 24 patients). Spindle morphology was assessed using confocal microscopy. Polar body biopsy was performed and ploidy assessed using our clinical low pass whole genome sequencing pipeline. Also, we conducted a multi-year retrospective study (n = 1059 patients) by measuring the concentration of THC in their follicular fluid by mass spectrometry and collecting and comparing IVF outcomes between cases and controls. Main results and the role of chance In vitro, THC treatment marginally increased the maturation rate (46% for Ctrl (n = 103), 51% for THC1 (n = 103) and 58% for THC2 (n = 102)). Using time-lapse imaging, no difference was observed between key developmental stages of oocyte maturation including GVBD and polar body extrusion. Single-cell RNAseq revealed significant alterations to the transcriptome profile of THC treated oocytes. When treated with THC1 (physiological concentration), 89 upregulated and 227 downregulated genes were identified, this was exaggerated when treated with THC2 (supraphysiologic concentration) 402 upregulated and 62 downregulated genes were identified. Pathway analysis showed that differentially expressed genes in THC1 group were associated with immune system and synaptic transmission, while THC2 group showed genes enriched in meiotic spindle formation and apoptosis. Moreover, preliminary results demonstrated abnormal spindle configuration and misaligned chromosomes in THC-treated groups and a concurrent decrease in the number of euploid oocytes. Furthermore, retrospective analysis of clinical IVF outcomes revealed that patients whose FF was positive for THC (62 patients, 6%) also had a slight increased maturation rate (77% vs 73%, p=ns), but a significant reduction of their embryo euploid rate when compared to control patients (59% vs 70%, p < 0.01). Limitations, reasons for caution The in vitro exposure to THC utilized GV oocytes that did not mature after ovarian stimulation, so they might have a reduced capacity of becoming MII. The THC measurement was assessed only on the day of retrieval and we do not know the route, dose, or duration of THC consumption. Wider implications of the findings This is the first study investigating the impact of cannabis on human oocytes. Our data suggests that cannabis consumption may reduce the number of euploid embryos available for transfer. With cannabis legalization increasing worldwide, further investigation into its consequences is critical for clinical consultation and legalization guidelines. Trial registration number Not applicable

Collagen concentration regulates neutrophil extravasation and migration in response to infection in an endothelium dependent manner

IntroductionAs the body’s first line of defense against disease and infection, neutrophils must efficiently navigate to sites of inflammation; however, neutrophil dysregulation contributes to the pathogenesis of numerous diseases that leave people susceptible to infections. Many of these diseases are also associated with changes to the protein composition of the extracellular matrix. While it is known that neutrophils and endothelial cells, which play a key role in neutrophil activation, are sensitive to the mechanical and structural properties of the extracellular matrix, our understanding of how protein composition in the matrix affects the neutrophil response to infection is incomplete.MethodsTo investigate the effects of extracellular matrix composition on the neutrophil response to infection, we used an infection-on-a-chip microfluidic device that replicates a portion of a blood vessel endothelium surrounded by a model extracellular matrix. Model blood vessels were fabricated by seeding human umbilical vein endothelial cells on 2, 4, or 6 mg/mL type I collagen hydrogels. Primary human neutrophils were loaded into the endothelial lumens and stimulated by adding the bacterial pathogen Pseudomonas aeruginosa to the surrounding matrix.ResultsCollagen concentration did not affect the cell density or barrier function of the endothelial lumens. Upon infectious challenge, we found greater neutrophil extravasation into the 4 mg/mL collagen gels compared to the 6 mg/mL collagen gels. We further found that extravasated neutrophils had the highest migration speed and distance in 2mg/mL gels and that these values decreased with increasing collagen concentration. However, these phenomena were not observed in the absence of an endothelial lumen. Lastly, no differences in the percent of extravasated neutrophils producing reactive oxygen species were observed across the various collagen concentrations.DiscussionOur study suggests that neutrophil extravasation and migration in response to an infectious challenge are regulated by collagen concentration in an endothelial cell-dependent manner. The results demonstrate how the mechanical and structural aspects of the tissue microenvironment affect the neutrophil response to infection. Additionally, these findings underscore the importance of developing and using microphysiological systems for studying the regulatory factors that govern the neutrophil response.

Abnormal HCK/glutamine/autophagy axis promotes endometriosis development by impairing macrophage phagocytosis.

The presence of extensive infiltrated macrophages with impaired phagocytosis is widely recognised as a significant regulator for the development of endometriosis (EMs). Nevertheless, the metabolic characteristics and the fundamental mechanism of impaired macrophage phagocytosis are yet to be clarified. Here, we observe that there is the decreased expression of haematopoietic cellular kinase (HCK) in macrophage of peritoneal fluid from EMs patients, which might be attributed to high oestrogen and hypoxia condition. Of note, HCK deficiency resulted in impaired macrophage phagocytosis, and increased number and weight of ectopic lesions in vitro and in vivo. Mechanistically, this process was mediated via regulation of glutamine metabolism, and further upregulation of macrophage autophagy in a c-FOS/c-JUN dependent manner. Additionally, macrophages of EMs patients displayed insufficient HCK, excessive autophagy and phagocytosis dysfunction. In therapeutic studies, supplementation with glutamine-pre-treated macrophage or Bafilomycin A1 (an autophagy inhibitor)-pre-treated macrophage leads to the induction of macrophage phagocytosis and suppression of EMs development. This observation reveals that the aberrant HCK-glutamine-autophagy axis results in phagocytosis obstacle of macrophage and further increase the development risk of Ems. Additionally, it offers potential therapeutic approaches to prevent EMs, especially patients with insufficient HCK and macrophage phagocytosis dysfunction.

Evaluation of antimicrobial and anticancer efficacy of silver nanoparticles phytofabricated by Nyctanthes arbor-tristis L. leaf extract

Nyctanthes arbor-tristis L. (Oleaceae), commonly known as harsingar, is a plant with potential medicinal properties. The plant was used in traditional folk medicine and as a pharmacological activity. The present study reports a rapid and eco-accommodating technique to synthesize silver nanoparticles AgNPs with low cost and with no need to heat, using aqueous extracts of N. arbor-tristis L. Phytochemical analysis was conducted to detect the existence of alkaloids, flavonoids, saturated sugar, saponins, glycosides, steroids, tannins, terpenoids, and proteins in the plant. A color change observed in the test confirmed the synthesis of AgNPs. SEM, XRD, EDX, FTIR, and UV – Visible spectrophotometer (DLS) were used to characterize the synthesized AgNPs. The antibacterial potential of AgNPs was further tested against different bacterial strains such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and Klebsiella pneumonia displayed by finding the maximum zone of inhibition observed against S. typhi of 24.6 mm, S. aureus 21.6±0.57 mm, P. aeruginosa 19 mm, K. pneumonia 17 mm and E. coli 15.3±0.57 mm. Furthermore, the synthesized AgNPs were also exhibited as anticancer properties against MDA-231 cell line Human Breast cancer cell line which was determined dose dependent manner and their concentration of 2.5 to 30 μg/mL respectively, and noted the inhibitory range at 30 μg/mL of its concentration, which was further tested in high inhibitory effect on their leaf extract high concentration in cytotoxic, bactericidal experiments.

Arnicolide D Inhibits Oxidative Stress-Induced Breast Cancer Cell Growth And Invasion Through Apoptosis, Ferroptosis, And Parthanatos.

Breast cancer is the most common malignant tumor in women, and its pathogenesis is very complicated. More and more studies have found that Traditional Chinese Medicine plays an important role in tumor prevention. To investigate the mechanism of arnicolide D isolated from Centipeda minima in breast cancer. Cell Counting Kit-8 (CCK-8), western blot, RT-qPCR, ELISA, flow cytometry, and Transwell were used to detect the effect of arnicolide D on the biological function of breast cancer cells. Arnicolide D promoted reactive oxygen species (ROS) production and induced a decrease in mitochondrial membrane potential in breast cancer cells, thereby inhibiting cell viability and increasing lactate dehydrogenase (LDH) release. Arnicolide D activated the classical apoptosis pathway to induce cell apoptosis; it significantly promoted PARP-1 expression, enhanced the nuclear translocation of apoptosis-inducing factor (AIF), and reduced the expression of AIF in mitochondria, indicating that it can induce the occurrence of parthanatos in a ROS dependent manner. In addition, arnicolide D down-regulated glutathione peroxidase 4 (GPX4) expression and increased the accumulation of Fe2+ and malondialdehyde (MDA), thereby activating ferroptosis. Apoptosis inhibitor, ferroptosis inhibitor, PARP inhibitor, PARP-1 siRNA, AIF siRNA and GPX4 overexpression vector significantly attenuated the inhibitory effect of arnicolide D on cell viability and reduced LDH release, which indicates that arnicolide D inhibits breast cancer cell growth by inducing apoptosis, parthanatos and ferroptosis. Arnicolide D also reduced breast cancer cell invasion and inhibited the expression of matrix metallopeptidase (MMP)-2 and MMP-9. Arnicolide D can activate a variety of cell death modes by inducing oxidative stress, thereby inhibiting the growth and invasion of breast cancer cells, indicating that arnicolide D has a good anti-tumor effect.

Antipyretic and Anti-inflammatory Effects of Sanchen Pediatric Antipyretic Paste

Objective:: This study aimed to investigate the antipyretic and anti-inflammatory effects and mechanisms of Sanchen Pediatric antipyretic Paste (SPAP). Methods:: Rats were randomly divided into 7 groups: negative control group, positive control group, positive treatment group, SPAP high-dose, medium-dose and low-dose groups, and blank substrate group. Except normal control group, rats in other groups were subcutaneously injected with 10% dry yeast suspension (10mL/kg) on the back to establish a fever model, and the normal group was given the same volume of saline. At the 4 h and 7 h after modeling, each treatment group was subjected to corresponding SPAP intervention. Anal temperature was measured every one hour after modeling for 10 consecutive times. The average temperature-rise curve, maximum temperature and body temperature response index (TRI) were calculated to evaluate the efficacy of SPAP. After the last temperature measurement, abdominal aortic blood was collected, and serum was separated from blood. The levels of IL-6, TNF-α and CRP in serum were determined by enzyme-linked immunosorbent assay. Results:: The body temperature of the positive control group was significantly increased (p < 0.01) 4 hours after model establishment, and that of the SPAP groups significantly decreased (p < 0.01) after 5 hours of the model establishment. The levels of Serum IL-6, TNF-α and CRP were significantly decreased (p < 0.01) in a dose-dependent manner. Conclusion:: SPAP has an obvious antipyretic effect on fever caused by dry yeast in rats. One of its antipyretic mechanisms may be associated with inhibiting inflammatory reactions by reducing the production or accelerating the degradation of endogenous thermogenic cytokines, such as IL-6, TNF- α, and CRP.

In situ one-pot synthesis of chitosan@ P,N-CPDs as a ratiometric scatter up-conversion sensor for α-lipoic acid

The rapid sensitive determination of α-lipoic acid (ALA) is essential for monitoring biological functions. Nevertheless, the lack of chromophore and its occurrence in complex biological matrices renders its determination challenging and requires additional treatment steps which lengthen assay procedure and affects method sensitivity. Herein, through the coordination of frequency doubling scatter (FDS) of polysaccharide strands with up-conversion of the embedded in situ formed P, N- carbonized polymer dots (CPDs), a novel ratiometric sensing platform was constructed. One-pot synthesis of chitosan decorated P, N-CPDs (chitosan@ P,N-CPDs) was attempted through microwave-assisted crosslinking-accelerated carbonization, using phosphoric acid as crosslinker and dopant. The constructed platform responded to the presence of α-lipoic acid (ALA) in concentration dependent manner (R2 = 0.995) between 1.65–100 μg/mL, with detection limit of 0.55 μg/mL, good accuracy (%E = 0.52 %) and precision (%RSD < 2 %). Consistent results with comparison method confirmed sensor reliability for determination of ALA in urine, Thiotacid® and Thiotacid compound® tablets, with no interference from the coexisting highly absorbing water-soluble vitamins, thiamine, and cyanocobalamin, or other potential interferents. Thus, the developed high performance sensing platform could be adopted for ALA determination as it is much less prone to spectral interferences and hence more discerning than those operated in single wavelength mode.

Fazirsiran for Adults with Alpha-1 Antitrypsin Deficiency Liver Disease: A Phase 2 Placebo Controlled Trial (SEQUOIA)

Homozygous ZZ alpha-1 antitrypsin (AAT) deficiency produces mutant AAT (Z-AAT) proteins in hepatocytes, leading to progressive liver fibrosis. We evaluated the safety and efficacy of an investigational RNA interference therapeutic, fazirsiran, that degrades Z-AAT mRNA, reducing deleterious protein synthesis. This ongoing, phase 2 study randomized 40 patients to subcutaneous placebo or fazirsiran 25/100/200 mg. The primary endpoint was percentage change in serum Z-AAT concentration from baseline to Week 16. Patients with fibrosis on baseline liver biopsy received treatment on Day 1, Week 4, and then every 12 weeks, and had a second liver biopsy at or after Weeks 48, 72, or 96. Patients without fibrosis received two doses on Day 1 and Week 4. At Week 16, least-squares mean percent declines in serum Z-AAT concentration were -61%, -83% and -94% with fazirsiran 25/100/200 mg, respectively, versus placebo (all P< .0001). Efficacy was sustained through Week 52. At post-dose liver biopsy, fazirsiran reduced median liver Z-AAT concentration by 93% compared with an increase of 26% with placebo. All fazirsiran-treated patients had histological reduction from baseline in hepatic globule burden. Portal inflammation improved in 5/12 and 0/8 patients with baseline score >0 in the fazirsiran and placebo groups, respectively. Histological METAVIR score improved by >1 point in 7/14 and 3/8 patients with fibrosis >F0 at baseline in the fazirsiran and placebo groups, respectively. No adverse events led to discontinuation and pulmonary function tests remained stable. Fazirsiran reduced serum and liver concentrations of Z-AAT in a dose dependent manner and reduced hepatic globule burden (NCT03945292).

Monoclonal antibodies for equine IL-1β enable the quantification of mature IL-1β in horses

Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1β in a fluorescent bead-based assay and for staining of IL-1β-producing immune cells by flow cytometry. The bead-based assay for equine IL-1β had a linear quantification range between 60pg/ml to 960ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1β after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1β bead-based assay. A comparison of two commercial equine IL-1β ELISA kits with the new IL-1β fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1β in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1β recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1β in equine PBMC after LPS stimulation were CD14+ monocytes. IL-1β secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1β within the cells even when proteolytic cleavage for IL-1β activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1β in horses.

Restoring hippocampal glucose metabolism rescues cognition across Alzheimer's disease pathologies.

Impaired cerebral glucose metabolism is a pathologic feature of Alzheimer Disease (AD), and recent proteomic studies highlight a disruption of glial carbohydrate metabolism with disease progression. Here, we report that inhibition of indoleamine-2,3-dioxygenase 1 (IDO1), which metabolizes tryptophan to kynurenine (KYN) in the first step of the kynurenine pathway, rescues hippocampal memory function and plasticity in preclinical models of amyloid and tau pathology by restoring astrocytic metabolic support of neurons. Activation of IDO1 in astrocytes by amyloid-beta 42 and tau oligomers, two major pathological effectors in AD, increases KYN and suppresses glycolysis in an AhR-dependent manner. Conversely, pharmacological IDO1 inhibition restores glycolysis and lactate production. In amyloid-producing APP Swe -PS1 ΔE9 and 5XFAD mice and in tau-producing P301S mice, IDO1 inhibition restores spatial memory and improves hippocampal glucose metabolism by metabolomic and MALDI-MS analyses. IDO1 blockade also rescues hippocampal long-term potentiation (LTP) in a monocarboxylate transporter (MCT)-dependent manner, suggesting that IDO1 activity disrupts astrocytic metabolic support of neurons. Indeed, in vitro mass-labeling of human astrocytes demonstrates that IDO1 regulates astrocyte generation of lactate that is then taken up by human neurons. In co-cultures of astrocytes and neurons derived from AD subjects, deficient astrocyte lactate transfer to neurons was corrected by IDO1 inhibition, resulting in improved neuronal glucose metabolism. Thus, IDO1 activity disrupts astrocytic metabolic support of neurons across both amyloid and tau pathologies and in a model of AD iPSC-derived neurons. These findings also suggest that IDO1 inhibitors developed for adjunctive therapy in cancer could be repurposed for treatment of amyloid- and tau-mediated neurodegenerative diseases.

Long-term NMN treatment increases lifespan and healthspan in mice in a sex dependent manner.

Nicotinamide adenine dinucleotide (NAD) is essential for many enzymatic reactions, including those involved in energy metabolism, DNA repair and the activity of sirtuins, a family of defensive deacylases. During aging, levels of NAD + can decrease by up to 50% in some tissues, the repletion of which provides a range of health benefits in both mice and humans. Whether or not the NAD + precursor nicotinamide mononucleotide (NMN) extends lifespan in mammals is not known. Here we investigate the effect of long-term administration of NMN on the health, cancer burden, frailty and lifespan of male and female mice. Without increasing tumor counts or severity in any tissue, NMN treatment of males and females increased activity, maintained more youthful gene expression patterns, and reduced overall frailty. Reduced frailty with NMN treatment was associated with increases in levels of Anerotruncus colihominis, a gut bacterium associated with lower inflammation in mice and increased longevity in humans. NMN slowed the accumulation of adipose tissue later in life and improved metabolic health in male but not female mice, while in females but not males, NMN increased median lifespan by 8.5%, possible due to sex-specific effects of NMN on NAD + metabolism. Together, these data show that chronic NMN treatment delays frailty, alters the microbiome, improves male metabolic health, and increases female mouse lifespan, without increasing cancer burden. These results highlight the potential of NAD + boosters for treating age-related conditions and the importance of using both sexes for interventional lifespan studies.

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2.

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found Tg WIP, a protein secreted from Toxoplasma , played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that Tg WIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant Tg WIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a Tg WIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which Tg WIP modulates the migration dynamics of infected DCs in vitro .

GVHD targets organoid-forming bile duct stem cells via a TGF-β–dependent manner

AbstractGraft-versus-host disease (GVHD) is a major life-threatening complication that occurs after allogeneic hematopoietic cell transplantation (HCT). Although adult tissue stem cells have been identified as targets of GVHD in the skin and gut, their role in hepatic GVHD is yet to be clarified. In the current study, we explored the fate of bile duct stem cells (BDSCs), capable of generating liver organoids in vitro, during hepatic GVHD after allogeneic HCT. We observed a significant expansion of biliary epithelial cells (BECs) on injury early after allogeneic HCT. Organoid-forming efficiency from the bile duct was also significantly increased early after allogeneic HCT. Subsequently, the organoid-forming efficiency from bile ducts was markedly decreased in association with the reduction of BECs and the elevation of plasma concentrations of bilirubin, suggesting that GVHD targets BDSCs and impairs the resilience of BECs. The growth of liver organoids in the presence of liver-infiltrating mononuclear cells from allogeneic recipients, but not from syngeneic recipients, was significantly reduced in a transforming growth factor-β (TGF-β)–dependent manner. Administration of SB-431542, an inhibitor of TGF-β signaling, from day 14 to day 28, protected organoid-forming BDSCs against GVHD and mitigated biliary dysfunction after allogeneic HCT, suggesting that BDSCs are a promising therapeutic target for hepatic GVHD.