A novel dual mechanism-of-action bispecific PD-1-IL-2v armed by a "ßγ-only" interleukin-2 variant.

Introduction: Interleukin-2 (IL-2) is one of the first cytokines to be discovered as an immune agonist for cancer immunotherapy. Biased IL-2 variants had been discovered to eliminate Treg activation or enhance the tumor specific T cell cytotoxicity. However, all the biased IL-2 variants pose the risk to overstimulate immune response at a low-dose range. Here, we introduce a novel dual-MOA bispecific PD-1-IL-2v molecule with great anti-tumor efficacy in a high dosed manner. Methods: The novel IL-2 variant was designed by structural truncation and shuffling. The single armed bispecific PD-1-IL-2v molecule and IL-2v were studied by immune cell activations in vitro and in vivo and anti-tumor efficacy in mouse model. Results and discussion: The IL-2 variant in this bispecific antibody only binds to IL-2Rßγ complex in a fast-on/off manner without α, ß or γ single receptor binding. This IL-2v mildly activates T and NK cells without over stimulation, meanwhile it diminishes Treg activation compared to the wild type IL-2. This unique bispecific molecule with "ßγ-only" IL-2v can not only "in-cis" stimulate and expand CD8 T and NK cells moderately without Treg activation, but also block the PD-1/L1 interaction at a similar dose range with monoclonal antibody.

IL-13 facilitates ferroptotic death in asthmatic epithelial cells via SOCS1-mediated ubiquitinated degradation of SLC7A11.

Th2-high asthma is characterized by elevated levels of type 2 cytokines, such as interleukin 13 (IL-13), and its prevalence has been increasing worldwide. Ferroptosis, a recently discovered type of programmed cell death, is involved in the pathological process of Th2-high asthma; however, the underlying mechanisms remain incompletely understood. In this study, we demonstrated that the serum level of malondialdehyde (MDA), an index of lipid peroxidation, positively correlated with IL-13 level and negatively correlated with the predicted forced expiratory volume in 1 s (FEV1%) in asthmatics. Furthermore, we showed that IL-13 facilitates ferroptosis by upregulating of suppressor of cytokine signaling 1 (SOCS1) through analyzing immortalized airway epithelial cells, human airway organoids, and the ovalbumin (OVA)-challenged asthma model. We identified that signal transducer and activator of transcription 6 (STAT6) promotes the transcription of SOCS1 upon IL-13 stimulation. Moreover, SOCS1, an E3 ubiquitin ligase, was found to bind to solute carrier family 7 member 11 (SLC7A11) and catalyze its ubiquitinated degradation, thereby promoting ferroptosis in airway epithelial cells. Last, we found that inhibiting SOCS1 can decrease ferroptosis in airway epithelial cells and alleviate airway hyperresponsiveness (AHR) in OVA-challenged wide-type mice, while SOCS1 overexpression exacerbated the above in OVA-challenged IL-13-knockout mice. Our findings reveal that the IL-13/STAT6/SOCS1/SLC7A11 pathway is a novel molecular mechanism for ferroptosis in Th2-high asthma, confirming that targeting ferroptosis in airway epithelial cells is a potential therapeutic strategy for Th2-high asthma.

Activation of locus coeruleus noradrenergic neurons rapidly drives homeostatic sleep pressure.

Homeostatic sleep regulation is essential for optimizing the amount and timing of sleep, but the underlying mechanism remains unclear. Optogenetic activation of locus coeruleus noradrenergic neurons immediately increased sleep propensity following transient wakefulness. Fiber photometry showed that repeated optogenetic or sensory stimulation caused rapid declines of locus coeruleus calcium activity and noradrenaline release. This suggests that functional fatigue of noradrenergic neurons, which reduces their wake-promoting capacity, contributes to sleep pressure.

Label-Free Optical Technologies to Enhance Noninvasive Endoscopic Imaging of Early-Stage Cancers.

White light endoscopic imaging allows for the examination of internal human organs and is essential in the detection and treatment of early-stage cancers. To facilitate diagnosis of precancerous changes and early-stage cancers, label-free optical technologies that provide enhanced malignancy-specific contrast and depth information have been extensively researched. The rapid development of technology in the past two decades has enabled integration of these optical technologies into clinical endoscopy. In recent years, the significant advantages of using these adjunct optical devices have been shown, suggesting readiness for clinical translation. In this review, we provide an overview of the working principles and miniaturization considerations and summarize the clinical and preclinical demonstrations of several such techniques for early-stage cancer detection. We also offer an outlook for the integration of multiple technologies and the use of computer-aided diagnosis in clinical endoscopy. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 17 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Adequate nutrient intake mitigate the toxic effects of bromate on the rotifer Brachionus calyciflorus.

Bromate is receiving increased attention as a typical disinfection by-product in aquatic environments, but bromate toxicity tests on invertebrate such as Brachionus calyciflorus rotifer are inadequate. In the present study, the long-term toxicity tests on B. calyciflorus were performed during 21 days under the exposure of different bromate concentrations and two algal density conditions. Furthermore, we evaluated the feeding behaviors of the rotifers under the impact of bromate. The maximum population density of rotifers was significantly reduced at 100 and 200 mg/L bromate exposure at the two algal density conditions. However, we observed that the maximum population density and population growth rate of rotifers were higher at 3.0 × 106 cells/mL algal density than those at 1.0 × 106 cells/mL under the same conditions of bromate exposure. These results suggest that higher food density may have alleviated the negative effects of bromate on rotifers. Meanwhile, the ingestion rate at an algal density of 3.0 × 106 cells/mL was higher than that at 1.0 × 106 cells/mL. The present study provides a basic reference to comprehensively evaluate the toxic effects of bromate on aquatic organisms.

Cyclophosphamide reduces gene transcriptional activity and embryo in vitro development by inhibiting NF-κB expression through decreasing AcH4K12.

Cyclophosphamide (CTX), a widely used chemotherapeutic agent for cancer treatment, has been associated with long-term toxicity and detrimental effects on oocytes and ovaries, resulting in female reproductive dysfunction. This study aimed to investigate the potential impact of CTX on in vitro maturation (IVM) injury of porcine oocytes and subsequent embryonic development, as well as its effects on epigenetic modification and gene activation during early embryonic development. The results demonstrated that CTX treatment caused aberrant spindle structure and mitochondrial dysfunction during oocyte maturation, inducing DNA damage and early apoptosis, which consequently disrupted meiotic maturation. Indeed, CTX significantly reduced the in vitro developmental capacity of porcine embryos, and induced DNA damage and apoptosis in in vitro fertilization (IVF) blastocysts. Importantly, CTX induced abnormal histone modification of AcH4K12 in early porcine embryos. Moreover, addition of LBH589 before zygotic genome activation (ZGA) effectively increased AcH4K12 levels and restored the protein expression of NF-κB, which can effectively enhance the in vitro developmental potential of IVF embryos. The DNA damage and apoptosis induced by CTX compromised the quality of the blastocysts, which were recovered by supplementation with LBH589. This restoration was accompanied by down-regulation of BAX mRNA expression and up-regulation of BCL2, POU5F1, SOX2 and SOD1 mRNA expression. These findings indicated that CTX caused abnormal histone modification of AcH4K12 in early porcine embryos and reduced the protein expression of NF-κB, a key regulator of early embryo development, which may block subsequent ZGA processes.

Enhancing the image quality of blue light cystoscopy through green-hue correction and fogginess removal.

Blue light cystoscopy (BLC) is a guideline-recommended endoscopic tool to detect bladder cancer with high sensitivity. Having clear, high-quality images during cystoscopy is crucial to the sensitive, efficient detection of bladder tumors; yet, important diagnostic information is often missed or poorly visualized in images containing illumination artifacts or impacted by impurities in the bladder. In this study, we introduce computational methods to remove two common artifacts in images from BLC videos: green hue and fogginess. We also evaluate the effect of artifact removal on the perceptual quality of the BLC images through a survey study and computation of Blind/Referenceless Image Spatial Quality Evaluator scores on the original and enhanced images. We show that corrections and enhancements made to cystoscopy images resulted in a better viewing experience for clinicians during BLC imaging and reliably restored lost tissue features that were important for diagnostics. Incorporating these enhancements during clinical and OR procedures may lead to more comprehensive tumor detection, fewer missed tumors during TURBT procedures, more complete tumor resection and shorter procedure time. When used in off-line review of cystoscopy videos, it may also better guide surgical planning and allow more accurate assessment and diagnosis.

Multiparameter interferometric polarization-enhanced imaging differentiates carcinoma in situ from inflammation of the bladder: an ex vivo study.

Significance: Successful differentiation of carcinoma in situ (CIS) from inflammation in the bladder is key to preventing unnecessary biopsies and enabling accurate therapeutic decisions. Current standard-of-care diagnostic imaging techniques lack the specificity needed to differentiate these states, leading to false positives. Aim: We introduce multiparameter interferometric polarization-enhanced (MultiPIPE) imaging as a promising technology to improve the specificity of detection for better biopsy guidance and clinical outcomes. Approach: In this ex vivo study, we extract tissue attenuation-coefficient-based and birefringence-based parameters from MultiPIPE imaging data, collected with a bench-top system, to develop a classifier for the differentiation of benign and CIS tissues. We also analyze morphological features from second harmonic generation imaging and histology slides and perform imaging-to-morphology correlation analysis. Results: MultiPIPE enhances specificity to differentiate CIS from benign tissues by nearly 20% and reduces the false-positive rate by more than four-fold over clinical standards. We also show that the MultiPIPE measurements correlate well with changes in morphological features in histological assessments. Conclusions: The results of our study show the promise of MultiPIPE imaging to be used for better differentiation of bladder inflammation from flat tumors, leading to a fewer number of unnecessary procedures and shorter operating room (OR) time.

A parabrachial-hypothalamic parallel circuit governs cold defense in mice.

Thermal homeostasis is vital for mammals and is controlled by brain neurocircuits. Yet, the neural pathways responsible for cold defense regulation are still unclear. Here, we found that a pathway from the lateral parabrachial nucleus (LPB) to the dorsomedial hypothalamus (DMH), which runs parallel to the canonical LPB to preoptic area (POA) pathway, is also crucial for cold defense. Together, these pathways make an equivalent and cumulative contribution, forming a parallel circuit. Specifically, activation of the LPB → DMH pathway induced strong cold-defense responses, including increases in thermogenesis of brown adipose tissue (BAT), muscle shivering, heart rate, and locomotion. Further, we identified somatostatin neurons in the LPB that target DMH to promote BAT thermogenesis. Therefore, we reveal a parallel circuit governing cold defense in mice, which enables resilience to hypothermia and provides a scalable and robust network in heat production, reshaping our understanding of neural circuit regulation of homeostatic behaviors.

Ginsenoside Rg1 activates brown adipose tissue to counteract obesity in high-fat diet-fed mice by regulating gut microbes and bile acid composition.

Obesity is a global health problem strongly linked to gut microbes and their metabolites. In this study, ginsenoside Rg1 (Rg1) reduced lipid droplet size and hepatic lipid accumulation by activating uncoupling protein 1 expression in brown adipose tissue (BAT), which in turn inhibited high-fat diet (HFD)-induced weight gain in mice. Furthermore, the intestinal flora of mice was altered, the abundance of Lachnoclostridium, Streptococcus, Lactococcus, Enterococcus and Erysipelatoclostridium was upregulated, and the concentrations of fecal bile acids were altered, with cholic acid and taurocholic acid concentrations being significantly increased. In addition, the beneficial effects of Rg1 were eliminated in mice treated with a combination of antibiotics. In conclusion, these results suggest that Rg1 activates BAT to counteract obesity by regulating gut microbes and bile acid composition in HFD-fed mice.

Inflammatory response triggered by avian hepatitis E virus in vivo and in vitro.

Hepatitis E virus (HEV) is relevant to public health worldwide, and it affects a variety of animals. Big liver and spleen disease (BLS) and hepatitis-splenomegaly syndrome (HSS) associated with avian HEV (aHEV) were first reported in 1988 and in 1991, respectively. Here, cell culture-adapted aHEV genotype 3 strain, YT-aHEV (YT strain), a typical genotype isolated in China, was used for basic and applied research. We evaluated liver injury during the early stages of infection caused by the YT strain in vivo. Both in vivo and in vitro experimental data demonstrated that viral infection induces innate immunity, with mRNA expression levels of two key inflammatory factors, interleukin-1ß (IL-1ß) and IL-18, significantly upregulated. The YT strain infection was associated with the activation of Toll-like receptors (TLRs), nuclear factor kappa B (NF-κB), caspase-1, and NOD-like receptors (NLRs) in the liver and primary hepatocellular carcinoma epithelial cells (LMH). Moreover, inhibiting c-Jun N-terminal kinase, extracellular signal-regulated kinase (ERK1 or 2), P38, NF-κB, or caspase-1 activity has different effects on NLRs, and there is a mutual regulatory relationship between these signaling pathways. The results show that SB 203580, U0126, and VX-765 inhibited IL-1ß and IL-18 induced by the YT strain, whereas Pyrrolidinedithiocarbamate (PDTC) had no significant effect on the activity of IL-1ß and IL-18. Pretreatment of cells with SP600125 had an inhibitory effect on IL-18 but not on IL-1ß. The analysis of inhibition results suggests that there is a connection between Mitogen-activated protein kinase (MAPK), NF-κB, and the NLRs signaling pathways. This study explains the relationship between signaling pathway activation (TLRs, NF-κB, MAPK, and NLR-caspase-1) and viral-associated inflammation caused by YT strain infection, which will help to dynamic interaction between aHEV and host innate immunity.

Combined inhibitory effect of interferon and antiserum on avian hepatitis E virus.

Epidemiologic investigations in recent years have shown that the detection rate of avian hepatitis E virus (HEV) in chicken flocks is increasing in China. Nevertheless, effective prevention and control measures are still lacking. In this study, specific pathogen-free (SPF) chicken serum against HEV was prepared using recombinant HEV open reading frames (ORF2 and ORF3) proteins as immunogens. An SPF chicken infection model was established by intravenous inoculation of chick embryos. Swab samples were collected at 7, 14, 21, and 28 d of age and used to detect avian HEV load, along with other indicators, by fluorescence quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay. The therapeutic effects on blocking vertical HEV transmission were observed, by using the methods of antibody application alone, mixed, or combined application of each of the 2 antibodies with type I interferon. The results showed that type I interferon alone or in combination with antiserum reduced the positive rate of HEV from 100 to 62.5% and 25%, respectively. However, the avian HEV-positivity rate was reduced to 75, 50, and 37.5% after type I interferon was used alone or in combination with antisera against ORF2 and ORF3, respectively. The inhibitory effect of type I interferon alone or in combination with an antiserum, on HEV replication was more significant in cells than in vivo. In this study, the inhibitory effect of type I interferon alone or in combination with an antiserum on avian HEV replication was observed in vitro and in vivo, providing the necessary technical reserve for disease prevention and control.

A review of advanced oxidation process towards organic pollutants and its potential application in fracturing flowback fluid.

Fracturing flowback fluid (FFF) including various kinds of organic pollutants that do harms to people and new treatments are urgently needed. Advanced oxidation processes (AOPs) are suitable methods in consideration with molecular weight, removal cost and efficiency. Here, we summarize the recent studies about AOP treatments towards organic pollutants and discuss the application prospects in treatment of FFF. Immobilization and loading methods of catalysts, evaluation method of degradation of FFF, and continuous treatment process flow are discussed in this review. In conclusion, further studies are urgently needed in aspects of catalyst loading methods, macromolecule organic evaluation methods, industrial process, and pathways of macromolecule organics' decomposition.

Small RNA deep sequencing revealed microRNAs' involvement in modulating cellular senescence and immortalization state.

Unlike rodent cells, spontaneous immortalization of avian cells and human cells is a very rare event. According to patent publications and current literature, there are no more than 4 spontaneously immortalized chicken embryo fibroblast (CEF) cell lines established up to date. One of those cell lines is ADOL (Avian Disease and Oncology Laboratory) ZS-1 cell line, which was established by continuous passaging of the CEFs derived from the specific pathogen free (SPF) 0.TVB*S1 (commonly known as rapid feathering susceptible or RFS) genetic line of chickens. The RFS genetic line of chickens was developed and has been maintained on the SPF chicken farm of USDA-ARS facility, ADOL, in East Lansing, Michigan, which is known as one of a few lines of chickens that are free of any known avian endogenous virus genes. To explore potential roles that epigenetic factors may play in modulating cellular senescence processes and spontaneous immortalization state, total RNAs extracted from samples of the RFS primary CEFs, RFS CEFs reached the 21st passage, and the ZS-1 cells were subjected to small RNA sequencing. Collectively, a total of 531 miRNAs was identified in the 3 types of samples. In contrast to the primary CEF samples, 50 miRNAs were identified with significantly differential expression only in the 21st passage samples; a different subset of 63 differentially expressed miRNAs was identified only in the ZS-1 samples; the majority of differentially expressed miRNAs identified in both the 21st passage CEF and the ZS-1 samples were more or less directionally consistent. Gene Ontology analysis results suggested that the epigenetic factor, miRNAs, plays a role in modulating the cellular senescence and spontaneous immortalization processes through various bioprocesses and key pathways including ErbB and MAPK signaling pathways. These findings provided the experimental and bioinformatic evidence for a better understanding on the epigenetic factor of miRNAs in association with cellular senescence and spontaneous immortalization process in avian cells.

Dynamic changes in intestinal microbiota and metabolite composition of pre-weaned beef calves.

Gut microbes and their metabolites are essential for maintaining host health and production. The intestinal microflora of pre-weaned calves gradually tends to mature with growth and development and has high plasticity, but few studies have explored the dynamic changes of intestinal microbiota and metabolites in pre-weaned beef calves. In this study, we tracked the dynamics of faecal microbiota in 13 new-born calves by 16S rRNA gene sequencing and analysed changes in faecal amino acid levels using metabolomics. Calves were divided into the relatively high average daily gain group (HA) and the relatively low average daily gain group (LA) for comparison. The results demonstrated that the alpha diversity of the faecal microbiota increased with calf growth and development. The abundance of Porphyromonadaceae bacterium DJF B175 increased in the HA group, while that of Lactobacillus reuteri decreased. The results of the LEfSe analysis showed that the microbiota of faeces of HA calves at eight weeks of age was enriched with P. bacterium DJF B175, while Escherichia coli and L. reuteri were enriched in the microbiota of faeces of LA calves. Besides, the total amino acid concentration decreased significantly in the eighth week compared with that in the first week (P < 0.05). Overall, even under the same management conditions, microorganisms and their metabolites interact to play different dynamic regulatory roles. Our results provide new insights into changes in the gut microbiota and metabolites of pre-weaned calves.

Effects of myostatin gene knockout on porcine extraocular muscles.

Myostatin (MSTN), a negative regulator of skeletal muscle mass, is not well known in extraocular muscles (EOMs). EOMs are specialized skeletal muscles. Hence, in this study, the effect of MSTN on the superior rectus (SR) and superior oblique (SO) of 2-month-old MSTN knockout (MSTN-/-) and wild-type (WT) pigs of the same genotype was investigated. SR (P < 0.01) and SO (P < 0.001) fiber cross-sectional areas of MSTN-/- pigs were significantly larger than those of WT pigs. Compared with WT pigs, MSTN-/- SO displayed a decrease in type I fibers (WT: 27.24%, MSTN-/-: 10.32%, P < 0.001). Type IIb fibers were higher in MSTN-/- pigs than in WT pigs (WT: 30.38%, MSTN-/-: 62.24%, P < 0.001). The trend in SR was the same as that in SO, although the trend in SO was greater than that in SR. The expression of myogenic differentiation factor (MyoD) and myogenic (MyoG) showed a significant increase in MSTN-/- SO (about 2.5-fold and 2-fold, respectively at the gene expression level, about 1.5-fold at the protein level) compared with WT pigs. MSTN plays an important role in the development of EOMs and regulates the muscle fiber type by modulating the gene expression of MyoD and MyoG in pigs.

Gastrodia elata Blume extract improves high-fat diet-induced type 2 diabetes by regulating gut microbiota and bile acid profile.

In this study, we aimed to characterize the anti-type 2 diabetes (T2D) effects of Gastrodia elata Blume extract (GEBE) and determine whether these are mediated through modification of the gut microbiota and bile acids. Mice were fed a high-fat diet (HFD), with or without GEBE, and we found that GEBE significantly ameliorated the HFD-induced hyperglycemia, insulin resistance, and inflammation by upregulating glucose transporter 4 (GLUT4) and inhibiting the toll-like receptor 4-nuclear factor kappa-B signaling pathway in white adipose tissue (WAT). In addition, we found that GEBE increased the abundance of Faecalibaculum and Lactobacillus, and altered the serum bile acid concentrations, with a significant increase in deoxycholic acid. The administration of combined antibiotics to mice to eliminate their intestinal microbiota caused a loss of the protective effects of GEBE. Taken together, these findings suggest that GEBE ameliorates T2D by increasing GLUT4 expression in WAT, remodeling the gut microbiota, and modifying serum bile acid concentrations.

Application of ORF3 Subunit Vaccine for Avian Hepatitis E Virus.

Avian hepatitis E virus (HEV) is the main etiologic pathogen of chicken big liver and spleen disease which is widely prevalent in China in recent years. However, due to the lack of a highly effective culture system in vitro, a genetically engineered subunit vaccine is the main direction of vaccine development. In this study, ORF3 genes of VaHEV strain from laying hens and YT-aHEV strain from broilers were amplified, respectively, and ORF3 protein was successfully expressed by Escherichia coli prokaryotic expression system. The serum samples were collected periodically to detect avian HEV antibodies by indirect immunofluorescence after specific pathogen free chickens immunized with the two proteins and their mixed proteins, the results showed that all serum samples were positive for antibodies to avian HEV. The antibody-positive chickens were artificially challenged with the cell-adapted strain YT-aHEV strain. The chickens from the immunized control group were infected successfully; no fecal detoxification was detected in the immunized group. In this study, two representative strains of ORF3 subunit vaccines of laying hens and broilers were prepared by prokaryotic expression, the immune effects of different proteins of these were evaluated through immunization and challenge studies in vivo, which provided a new technical possibility for prevention and control of avian HEV.

Theoretical insight into the acidity and cooperativity effect of the LLM-105∙(HNO3)2 system.

In order to reveal the nature of the acidity in explosive product, the acidities and cooperativity effects from the intermolecular H-bonding interactions in the LLM-105∙∙∙(HNO3)2 ternary systems were investigated at the B3LYP/6-311 + + G** and M062X/6-311 + + G** levels, with the integral equation formalism polarized continuum model (IEFPCM) based on the self-consistent-reaction-field. The results show that for the ternary systems, the intermolecular H-bonding interactions are stronger than those in the binary complexes, resulting in the lower [H+] concentrations and larger pKa1 values upon the ternary-complex formations. However, there is no obvious correlation between the acidities and cooperativity effects or APT charges of the H atoms involving the H-bonds. Surface electrostatic potential (ESP) and reduced density gradient are used to reveal the nature of the H-bond and acidity. Interestingly, the acidity is closely related to the ESPs of the H atom (VS,H) involving the intermolecular H-bond, but not to the statistical quantities. This is mainly because both acidity and VS,H are the local properties of system, whereas the cooperativity and statistical quantity of ESP are the global property.