24-h Time Point Research Articles

Full-Length Transcriptome Sequencing Analysis and Characterization of WRKY Transcription Factors Responsive to Cadmium Stress in Arabis paniculata.

Arabis paniculata is a newly discovered hyperaccumulator known for its ability to accumulate multiple metals. WRKY proteins play a significant role in plant responses to various stresses, including cadmium (Cd) stress. However, there is limited research on the molecular biology of Arabis paniculata, especially regarding the WRKY family. In this study, we conducted third-generation sequencing for functional annotation and structural analysis of Arabis paniculata. We obtained 41,196 high-quality isoforms from the full-length transcriptome, with an average length of 1043 bp. A total of 26,670 genes were predicted against NR, Swissprot, KOG, and KEGG databases. Functional comparison using the KOG database revealed excellent annotation in 25 functional categories, with general function prediction (1822 items) being the most predominant. MISA analysis identified 12,593 SSR loci, with single nucleotide repeats being the largest category (44.83% of the total). Moreover, our predictions provide insights into 20,022 coding sequences (CDS), 811 transcription factors, and 17,963 LncRNAs. In total, 34 WRKY gene sequences were identified in Arabis paniculata. Bioinformatics analysis revealed diverse numbers of amino acids in these WRKYs (113 to 545 aa), and a conserved WRKYGQK sequence within the N-terminus of the WRKY protein. Furthermore, all WRKYs were found to be localized in the nucleus. Phylogenetic analysis classified the WRKY genes into three categories: I (14 members), II (17 members), and III (3 members). Category II was subsequently divided into four sub-categories: II-a (8 members), II-b (1 member), II-c (1 member), and II-d (7 members). Our quantitative real-time polymerase chain reaction (qRT-PCR) experiments revealed that ApWRKY23 and ApWRKY34 exhibited the highest expression levels at the 24-h time point, suggesting their potential role as the candidate genes for Cd stress response. These findings contribute to our understanding of the genomic information of Arabis paniculata and provide a basis for the analysis of its genetic diversity. Additionally, this study paves the way for a comprehensive exploration of the molecular mechanisms underlying the WRKY genes in Arabis paniculata under Cd stress conditions.

Biodistribution of the saponin-based adjuvant Matrix-M™ following intramuscular injection in mice

Novel adjuvants are extensively utilized in the development of safe and effective vaccines against emerging pathogens. Matrix-M™ adjuvant is a saponin-based adjuvant used in several active clinical development programs and in widespread use in the COVID-19 vaccine NVX-CoV2373. Here, we conducted a biodistribution study to better understand the mechanism of action and safety profile for Matrix-M™ adjuvant. Radiolabeled saponins or cholesterol were incorporated into Matrix-A™ particles, which represent 85% of Matrix-M™. Labeled Matrix-M™ adjuvant was given to mice by intramuscular injection with or without SARS-CoV-2 Spike protein. Radioactivity of the adjuvant components was quantified in local and systemic tissues at seven timepoints over a period of 1–168 h. The highest saponin levels were found at the 1-h timepoint at the injection site, in the draining (iliac) lymph nodes, and in urine. Saponins were rapidly cleared from these tissues, reaching very low levels by 48–72 h. Systemically, saponins were found at low levels in the plasma, kidneys, liver, and bone marrow, and were barely detectable in other investigated tissues. Cholesterol was also found at high levels at the injection site and in the draining lymph nodes. These levels declined rapidly at first, then plateaued at 24–48 h. Radiolabeled cholesterol was found at very low levels in other tissues at the earliest timepoints, until increasing and stabilizing after the 24-h timepoint, indicating entry into the endogenous cholesterol recycling pool. This study demonstrates a rapid distribution of Matrix-M™ adjuvant from the injection site to the draining lymph nodes, thus excluding a depot effect as central to the mechanism of action for this adjuvant. The diverging clearance patterns for saponins and cholesterol are suggestive of at least partial disassembly of the Matrix-particles, which has implications for the downstream effects of Matrix-M™ adjuvant on adaptive immune responses. Systemic exposure to toxicologically relevant tissues is very low.

Spatial Response to Pivekimab Sunirine (IMGN632) In Vivo in a BPDCN Model

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematologic malignancy, which often manifests with skin lesions in addition to lymph node, blood, and bone marrow involvement. Standard of care treatments for BPDCN patients are intense chemotherapy or tagraxofusp (Elzonris®). Although many patients initially respond to standard-of-care therapies, most will eventually relapse. Overexpression of CD123 (IL-3Rα receptor) is a hallmark of BPDCN thus making this antigen an attractive target. Pivekimab sunirine is an antibody-drug-conjugate (ADC) comprised of a humanized anti-CD123 monoclonal antibody conjugated to a potent DNA-damaging agent, FGN849 via a cleavable linker. Pivekimab sunirine was granted orphan drug and Breakthrough Therapy designation and is currently being tested for the treatment of BPDCN patients as monotherapy and, as a triplet therapy in combination with azacitidine and venetoclax for the treatment of AML patients. Preclinically, pivekimab sunirine has demonstrated potent activity against BPDCN cells (Blood 2018 132:3956) and importantly, showed a favorable safety profile and promising efficacy in BPDCN patients (Hemasphere. 2022 Jun; 6 (Suppl): 1708-1709; ASH 2021, Abstract #1284). The goal of this study was to investigate the response to pivekimab sunirine in a pre-clinical model of BPDCN and to monitor spatial response in vivo. Pivekimab sunirine efficacy was evaluated in vitro on GEN2.2, a patient-derived BPDCN cell line, for 24h, 48h, or 72h at different concentrations. We observed a concentration and time-dependent efficacy at 48h or 72h of treatment with EC50s between 4pM and 12pM and between 1.3pM and 3pM respectively. Limited cytotoxicity was observed at the 24h time point. The efficacy of pivekimab sunirine was further confirmed in vivo in the GEN2.2 BPDCN model. Pivekimab sunirine treatment with either 0.024mg/kg or 0.08mg/kg doses at QWx3 (once weekly for 3 weeks) dosing regimen was able to restrain the tumor burden and significantly enhance survival compared to the vehicle group To assess spatial response to pivekimab sunirine, we utilized a bioluminescent (BLI) GEN2.2-luc model. Briefly, NSG mice were injected with 1x10 6 GEN2.2-luc cells followed by Fc receptor blockade on Day 2 for 24h. On Day 3, mice were treated with pivekimab sunirine (0.024mg/kg; QWx3) or vehicle intravenously. Organs were harvested on day 16 for the vehicle-treated group and day 30 for the pivekimab sunirine-treated group and tumor burden was assessed using luciferin. The vehicle group showed the presence of tumor cells in the bone marrow, spleen, and lungs. These results were confirmed by HES staining. Pivekimab sunirine treated mice showed no sign of tumor cells in any of the organs in BLI evaluation. HES staining confirmed the elimination of tumor cells in the lungs and the liver while few remaining tumor cells were observed in the spleen and the bone marrow. Overall, this work enabled us to validate the efficacy of pivekimab sunirine in a BPDCN model and to monitor its spatial effect. Data showed that pivekimab sunirine treatment significantly reduced tumor burden and increased survival compared to the vehicle group. Pivekimab sunirine treatment eradicated tumor cells in the lungs and the liver, while reducing the number of tumor cells in the spleen and bone marrow. Pivekimab sunirine is a potent ADC targeting CD123 and is highly efficacious against an aggressive BPDCN cell line model. This finding reinforces the importance of its use for the treatment of BPDCN patients.

Oxycodone withdrawal is associated with increased cocaine self-administration and aberrant accumbens glutamate plasticity in rats

Individuals with opioid use disorder (OUD) frequently use other substances, including cocaine. Opioid withdrawal is associated with increased likelihood of cocaine use, which may represent an attempt to ameliorate opioid withdrawal effects. Clinically, 30% of co-using individuals take opioids and cocaine exclusively in a sequential manner. Preclinical studies evaluating mechanisms of drug use typically study drugs in isolation. However, polysubstance use is a highly prevalent clinical issue and thus, we established a novel preclinical model of sequential oxycodone and cocaine self-administration (SA) whereby rats acquired oxycodone and cocaine SA in an A-B-A-B design. Somatic signs of withdrawal were evaluated at 0, 22, and 24h following oxycodone SA, with the 24h timepoint representing somatic signs immediately following cocaine SA. Preclinically, aberrant glutamate signaling within the nucleus accumbens core (NAcore) occurs following use of cocaine or opioids, whereby medium spiny neurons (MSNs) rest in a potentiated or depotentiated state, respectively. Further, NAcore glial glutamate transport via GLT-1 is downregulated following SA of either drug alone. However, it is not clear if cocaine can exacerbate opioid-induced changes in glutamate signaling. In this study, NAcore GLT-1 protein and glutamate plasticity were measured (via AMPA/NMDA ratio) following SA. Rats acquired SA of both oxycodone and cocaine regardless of sex, and the acute oxycodone-induced increase in somatic signs at 22h was positively correlated with cocaine consumption during the cocaine testing phase. Cocaine use following oxycodone SA downregulated GLT-1 and reduced AMPA/NMDA ratios compared to cocaine use following food SA. Further, oxycodone SA alone was associated with reduced AMPA/NMDA ratio. Together, behavioral signs of oxycodone withdrawal may drive cocaine use and further dysregulate NAcore glutamate signaling.

Assessment of Micronuclei Frequency in the Peripheral Blood of Adult and Pediatric Patients Receiving Fractionated Total Body Irradiation

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = −0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

Neutrophil extracellular trap formation and gene programs distinguish TST/IGRA sensitization outcomes among Mycobacterium tuberculosis exposed persons living with HIV.

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and show no evidence of immune sensitization to Mycobacterium tuberculosis (Mtb) as defined by persistently negative tuberculin skin tests (TST) and interferon gamma release assays (IGRA). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophil Mtb infection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected with Mtb H37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. Since there was no significant differential transcriptional response at 1h between infected PMNHITTIN and PMNHIT, we focused on the 6h timepoint. When compared to uninfected PMN, PMNHITTIN displayed 3106 significantly upregulated and 3548 significantly downregulated differentially expressed genes (DEGs) (absolute cutoff of a log2FC of 0.2, FDR < 0.05) whereas PMNHIT demonstrated 3816 significantly upregulated and 3794 significantly downregulated DEGs following 6h Mtb infection. Contrasting the log2FC 6h infection response to Mtb from PMNHITTIN against PMNHIT, 2285 genes showed significant differential response between the two groups. Overall PMNHITTIN had a lower fold change response to Mtb infection compared to PMNHIT. According to pathway enrichment, Apoptosis and NETosis were differentially regulated between HITTIN and HIT PMN responses after 6h Mtb infection. To corroborate the blunted NETosis transcriptional response measured among HITTIN, fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTIN compared to PMNHIT, showing that PMNHITTIN have a distinct response to Mtb.

UDP-glucuronosyltransferase is involved in susceptibility of Chironomus kiiensis Tokunaga, 1936 (Diptera: Chironomidae) to insecticides

UDP-glucuronosyltransferases (UGTs) could transform various exogenous and endogenous compounds, which help detoxification of pesticides in insects. To investigate the role of UGTs in the detoxification metabolism of insecticides in Chironomus kiiensis, CkUGT302M1, CkUGT302N1, CkUGT308N1 and CkUGT36J1 genes were identified with 1449–1599 bp encoding 482–532 amino acids. Four UGT genes shared 40.86∼53.36% identity with other homologous insect species, and expressed in all developmental stages, notably in the larval and adult stages. Expression of CkUGTs was higher in the gastric caecum, midgut and head. Moreover, CkUGTs expression and activity were significantly increased in C. kiiensis larvae in exposure to sublethal concentrations of carbaryl, deltamethrin and phoxim, respectively. To further explore the functions of UGT genes, the CkUGT308N1 was effectively silenced in 4th instar C. kiiensis larvae by RNA interference, which resulted in the mortality of dsCkUGT308N1 treated larvae increased by 71.43%, 111.11% and 62.50% under sublethal doses of carbaryl, deltamethrin and phoxim at the 24-h time point, respectively. The study revealed that the CkUGT308N1 gene in C. kiiensis could contribute to the metabolism of pesticides and provide a scientific basis for evaluating the water pollution of pesticides.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

Due to antibiotic tolerance of microbes within a biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for short-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid (T2DA), which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. Titanium coupons were drop coated with 0.75mg of DSF in ethanol and dried. Surface characteristics and presence of DSF were confirmed with Fourier Transform infrared spectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS), and water contact angle. Antimicrobial assays on 12-well plates analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-hour incubation, but only sustained through 72 hours for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-hour time point, but not through 72 hours for E. coli. Although approximately 60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. In this study we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

Cloning and Functional Analysis of the VfRR17 Gene from tung tree (Vernicia fordii).

Tung tree (Vernicia fordii) is one of the four major woody oilseed species in China. However, its fruit yield is severely hampered by the low number of female flowers and the imbalanced male-to-female flower ratio, which is a problem that restricts the development of the oilseed industry. Previous research has demonstrated that the exogenous application of cytokinins can significantly augment the number of female flowers, although the underlying regulatory mechanism remains elusive. To elucidate the involvement of VfRR17, a member of the A-type ARRs family, in the exogenous cytokinin regulation of flower sexual differentiation in tung tree, this study conducted a comprehensive bioinformatic analysis of the physicochemical properties, structural characteristics, and evolutionary relationships of the protein encoded by VfRR17. Additionally, gene function analysis was performed using subcellular localization, qRT-PCR, and genetic transformation techniques. The findings revealed that the VfRR17 gene's coding region spanned 471 bp, encoding an unstable protein of 156 amino acids with a relative molecular mass of 17.4 kDa. Phylogenetic analysis demonstrated a higher similarity between VfRR17 of the tung tree and the RR17 gene of Jatropha curcas, Ricinus communis, Hevea brasiliensis, and other species within the Euphorbiaceae family compared to other species, with the greatest similarity of 86% observed with the RR17 gene of Jatropha curcas. The qRT-PCR analysis indicated that VfRR17 exhibited high expression levels during the early stage of tung tree inflorescence buds following 6-BA treatment, peaking at 24 h and displaying a 3.47-fold increase compared to that at 0 h. In female and male flowers of the tung tree, the expression in female flowers during the 1 DBF period was significantly higher than in male flowers, exhibiting a difference of approximately 47.91-fold. Furthermore, notable differential expression was observed in the root, leaf, and petiole segments of the tung tree under low-temperature stress at the 12-h time point. In transgenic Arabidopsis, the VfRR17 lines and wild-type lines exhibited significantly different flowering times under an exogenous 6-BA treatment at a concentration of 2 mg/L, with the VfRR17 lines experiencing an 11-day delay compared to the wild-type lines. Additionally, the number of fruit pods in VfRR17 transgenic Arabidopsis lines was significantly reduced by 28 compared to the wild-type lines at a 6-BA concentration of 3 mg/L. These findings suggest that VfRR17 likely plays a critical role in regulating flower development in response to exogenous 6-BA, providing valuable insights into the mechanisms underlying exogenous 6-BA-mediated regulation of female flower development in the tung tree.

Characteristics and response cutoff of octreotide suppression test in thyrotrophin (TSH)-secreting pituitary adenomas.

Somatostatin analogs are recommended for preoperative therapy in thyrotrophin secreting pituitary adenomas (TSHomas). Octreotide suppression test (OST) was designed to differentiate TSHomas with resistance to thyroid hormones, while its ability to test the sensitivity of SSA has not been fully studied. To test the sensitivity of SSA in TSHomas with OST. We collected 48 pathologically confirmed TSHoma patients with complete 72 h' data of OST into analysis. Octreotide suppression test. Sensitivity timepoint and cutoff of OST. During the entire OST, the TSH descended maximally 89.07% (73.85%, 96.77%), while the FT3 and FT4 declined slowly [43.40% (37.80%, 54.44%) and 26.59% (19.01%, 33.13%), respectively]. The 24th hour was the timepoint wherein the stability occurs for TSH, and the 48th hour for FT3 and FT4 during OST. In the patients who received both short- and long-acting somatostatin analogs (SSA), the 24-h timepoint was the most predictive timepoint for the percentage of TSH decline (Spearman's rank correlation analysis, r = .571, p < .001), while the 72-h timepoint was optimal for predicting the magnitude of TSH decline (Spearman's rank correlation analysis, r = .438, p = .005). In the 24th timepoint, a positive correlation was also observed between TSH suppression rate and the percentage decrease and absolute value decrease of FT3 and FT4. Furthermore, in patients treated with long-acting SSA, the 72-h timepoint was optimal for predicting both the percentage (Spearman's rank correlation analysis, r = .587, p = .01) and magnitude (Spearman's rank correlation analysis, r = .474, p = .047) of TSH decline. The 24th hour was the optimal timepoint with 44.54% (50% of median value of TSH in 72-hOST) decrease of TSH being the observing cutoff. The adverse effect of OST was predominantly occurred in the gastrointestinal system and no severe event occurred during OST. Paradoxical response could occur in OST and it did not influence the effect of SSA as long as sensitivity was confirmed. A high level of hormonal control was achieved in the SSA-sensitive patients. OST can be used as an efficient tool to guide the adequate use of SSA.

Methionine, cysteine, and butylated hydroxytoluene enhance cryosurvival of ram semen on post-thaw and post-incubation time points.

Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders is yet to be found. This study was designed to investigate the different doses of methionine (2.5 and 5mM), cysteine (1 and 2mM), and butylated hydroxytoluene (BHT) (1 and 2mM) for ram semen cryopreservation on post-thaw and post-incubation (6h) time points over spermatological parameters. Semen samples were collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5mM methionine, 5mM methionine, 1mM cysteine, 2mM cysteine, 1mM BHT, and 2mM BHT). Semen samples were put into French straws (0.25mL), and freezing procedure (two-step) was conducted via a programmable gamete freezer. At both time points, motility, HOST, PSA-FITC, and TUNEL assays were made to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results compared to the control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6h of time. The study demonstrated that supplementing sperm freezing extenders with previous antioxidants may create new approaches to cryopreservation procedures, and through increasing success rate of freezing, fertility results may increase to better results in near future.

In Vitro Study of the Biological and Physical Properties of Dual-Cure Resin-Modified Calcium Silicate-Based Cement

The aim of the present study was to compare the biological and mechanical properties of a novel dual-cure, resin-modified calcium silicate material, Theracal PT® (TP), with those of Theracal LC® (TL) and BiodentineTM (BD). The cell counting kit-8 was used on human dental pulp cells to test cell the viability of the three materials. Antibacterial activity of TP, TL, and BD against Enterococcus faecalis was investigated under anaerobic conditions. The ability of the materials to support odontogenic differentiation was studied by examining the relative gene expression of osteocalcin (OCN), osteopontin (OPN), and Collagen I (ColI) using real-time polymerase chain reaction. For mechanical property tests, microhardness was evaluated using the Vickers microhardness (VHN) test, and the bond strength to the resin was evaluated using a shear bond test machine. There was no significant difference in cell viability between TL and TP after 48 h, and BD showed the highest cell viability, while TP showed the highest antibacterial effect. At the 12-h time point, there was no significant difference in ColI and OCN expression between BD and TP, but TP showed a higher expression of OPN than BD. However, at the 48-h time point, ColI and OCN showed higher levels of expression for BD than for TP and TL. At the same time point, only OPN had a higher diffusion for TP than for BD. TP demonstrated a VHN of approximately 30-35. This value was higher than that of TL and lower than that of BD. In contrast to VHN, the shear bond strength to resin was significantly higher for TL and TP than for BD. TP showed lower biocompatibility than BD but higher OPN expression and antibacterial effects than BD and TL. TP showed higher shear bond strength than BD and higher VHN than TL and BD at the 24-h time point.

Deletion of Cyclic GMP-AMP Synthase Aggravates Concanavalin A-Induced Acute Hepatic Injury by Facilitating Leukocyte Chemotaxis

Growing evidence demonstrates that cyclic GMP-AMP synthase (cGAS), as a cytosolic DNA sensor, is essential for activating innate immunity and regulating inflammatory response against cellular damage. However, its role in immune-mediated hepatitis remains unclear. Here by challenging the cGAS knockout (KO) and their littermate wide-type (WT) mice with intravenous ConA injection to induce acute immune-mediated liver injury, we found that lack of cGAS drastically aggravated liver damage post ConA treatment for 24h, reflected by increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and amplified hepatic necrosis. The number of apoptotic hepatocytes was also significantly increased in the KO mice. RNA-sequencing analysis revealed that leukocyte chemotaxis and migration-related genes were remarkably upregulated in the KO livers. Consistently, immunofluorescence assays illustrated that the infiltrating F4/80-positive macrophages, Ly6G-positive neutrophils, and CD3-positive T cells were all significantly increased in the KO liver sections. The hepatic expression of the pro-inflammatory genes was elevated as well. Supporting the in vivo findings, the knockdown of cGAS in cultured macrophages showed promoted migration potential and enhanced pro-inflammatory gene expression. These results collectively demonstrated that deletion of cGAS could aggravate ConA-induced acute liver injury, at least at the 24-h time point, and its mechanism might be related to facilitating leukocyte chemotaxis and promoting liver inflammatory response.

Photobiomodulation at 660nm promotes collagen synthesis via downregulation of HIF-1α expression without photodamage in human scleral fibroblasts in vitro in a hypoxic environment.

The increasing prevalence of myopia is a global public health issue. Because of the complexity of myopia pathogenesis, current control methods for myopia have great limitations. The aim of this study was to explore the effect of photobiomodulation (PBM) on human sclera fibroblasts (HSFs) under hypoxia, in the hope of providing new ideas for myopia prevention and control. Hypoxic cell model was established at 0, 6, 12, and 24h time points to simulate myopia microenvironment and explore the optimal time point. Control, hypoxia, hypoxia plus light, and normal plus light cell models were set up for the experiments, and cells were incubated for 24 or 48h after PBM (660nm, 5J/cm2), followed by evaluation of hypoxia-inducible factor 1α (HIF-1α) and collagen I a1 (COL1A1) proteins using Western blotting and immunofluorescence, and photo damage was detected by CCK-8, scratch test, and flow cytometry assays. We also used transfection technology to further elucidate the regulatory mechanism. The change of target proteins is most obvious when hypoxia lasts for 24h (p < 0.01). PBM at 660nm increased extracellular collagen content (p < 0.001) and downregulated expression of HIF-1α (p < 0.05). This treatment did not affect the migration and proliferation of cells (p > 0.05), and effectively inhibited apoptosis under hypoxia (p < 0.0001). After overexpression of HIF-1α, the effect of PBM was attenuated (p > 0.05). Photobiomodulation at 660nm promotes collagen synthesis via downregulation of HIF-1α expression without photodamage.

Comparative evaluation of the effect of calcium silicate and epoxy resinbased root canal sealers on postoperative pain in patients with primary endodontic lesion: A randomized clinical study.

This study's objective was to assess and compare postoperative pain response between bioceramic sealer (Nishika BG) and epoxy resin based (AH Plus) in patients with primary endodontic lesion at time interval of 24 h, 48 h, and 7 days using the Visual Analog Scale (VAS). The study included 40 individuals with necrotic pulp and apical periodontitis. Calcium hydroxide was administered as the intracanal medication during the two-visit endodontic therapy. They were subsequently allocated randomly to either the AH Plus root canal sealer or the Nishika Canal Sealer BG, with a total of 20 participants in each group. Patients were told to use a VAS to rate their postoperative pain severity as none, minimal, moderate, or severe after 24 h, 48 h, and 7 days following obturation using the appropriate sealers. Nishika Canal Sealer BG (CS-BG) has a lower pain score as compared to the AH Plus group at 24-h time point. VAS ratings for both the groups decreased over time. According to the intergroup analysis, there was a significant difference in postoperative pain at the 24 h (P = 0.022), but not at the 48 h or 7 days (P > 0.05). Although bioceramic sealer (Nishika Canal Sealer BG) resulted in significantly lower levels of pain as compared to epoxy resin-based sealer (AH Plus) at 24-h interval, there was no significant difference of postoperative pain at 48-h interval and 7-day period.

Abstract WP230: Human Peripheral Blood Gene Expression Associated With 90day Ischemic Stroke Outcomes

Objective: Identifying the molecular underpinnings associated with long-term functional outcome after ischemic stroke (IS) could guide search for treatments for improved outcome. Thus, we identified early peripheral immune gene expression (GE) responses associated with 90-day IS outcome, as measured by the Barthel Index (BI). Methods: RNA from 108 samples from three peripheral blood draws (≤3h – before thrombolytic treatment; 5h and 24h - post-treatment) from 36 CLEAR-trial subjects was analyzed on Affymetrix arrays. We performed Analysis of Covariance accounting for treatment (tPA or tPA+eptifibatide), hypercholesterolemia, hypertension, diabetes, age, sex and BI. Genes whose partial correlation with BI was significant ( P &lt;0.005) were identified. Results: There were 206, 865, and 209 genes that significantly correlated with 90-day BI at ≤3h, 5h and 24h post ictus , respectively. The gene lists at all three time-points were enriched in B-cell specific genes (hypergeometric probability p&lt;0.05), while the 5h gene list was also enriched in neutrophil-specific genes, and the 24h gene list was also enriched in monocyte-specific genes. B Cell Receptor Signaling pathway was predicted activated in subjects with better 90-day outcome at the ≤3h time-point, while ICOS-ICOSL Signaling in T Helper Cells and Role of NFAT in Regulation of the Immune Response were predicted activated at the 24h time-point.Immune-related pathways were enriched at three time-points including B cell-related, IL-7, and T cell signaling pathways. Apoptosis was suppressed in patients with better 90-day outcome at 3h and 24h post ictus . Among the genes associated with the 90-day BI were genes important for stroke and repair after stroke, such as KCNG1 , Potassium voltage-gated channel subfamily G member 1, which like other potassium channels could modulate stroke outcome. KCNG1 expression was positively correlated with 90-day BI at ≤3h and 5h. TERML4 , implicated in inflammatory response and human coronary arterial calcification had expression that positively correlated with BI at all three time-points. Conclusion: The findings expand our understanding of the early molecular biology associated with long-term stroke outcome and may serve as potential targets to improve outcome.

Different concentrations of fetal bovine serum affect cytokine modulation in Lipopolysaccharide-activated apical papilla cells in vitro.

Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-β1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-β1, OPG, IL-6, and CCL2 by APCs in vitro.

Efficacy and safety of Felpreva®, a spot-on formulation for cats containing emodepside, praziquantel and tigolaner against experimental infestation with the Australian paralysis tick Ixodes holocyclus

The Australian paralysis tick Ixodes holocyclus continues to be a serious threat to companion animals along Australia’s east coast. The tick produces a potent neurotoxin which causes a rapidly ascending flaccid paralysis, which if left untreated, can result in the death of the animal. There is currently only a limited number of products registered in Australia for the treatment and control of paralysis ticks in cats. Felpreva® is an effective spot-on combination containing emodepside, praziquantel and tigolaner. To investigate the therapeutic and long-term persistent efficacy of Felpreva® (2.04% w/v emodepside, 8.14% w/v praziquantel and 9.79% w/v tigolaner) against experimental infestation with I. holocyclus in cats, two studies were undertaken. Fifty cats were included in the studies on study Day -17. These cats were immunized against paralysis tick holocyclotoxin prior to the study commencing. Immunity to holocyclotoxin was confirmed with a tick carrying capacity (TCC) test conducted prior to treatment. Cats were treated once on Day 0. Group 1 cats were treated with the placebo formulation and Group 2 cats were treated with Felpreva®. Cats were infested on Days -14 (tick carrying capacity test), 0, 28, 56, 70, 84 and 91 (weeks 4, 8, 10, 12 and 13). Ticks were counted on cats 24 h, 48 h and 72 ​h post-treatment and infestation, except during the tick carrying capacity test when they were counted approximately 72 ​h post-infestation only. The 24-h and 48-h assessments were conducted without removing the ticks. The ticks were assessed, removed and discarded at the 72-h assessment time-points. Significant differences in total live tick counts at ∼24 h, ∼48 h and ∼72 ​h post-infestation were observed between the treatment and control group. Differences were significant (P ​< ​0.05 to ​< ​0.001) in all instances. Treatment efficacies of 98.1–100% were observed ∼72 ​h post-infestation through to 13 weeks (94 days) post-treatment. These results show that a single application of Felpreva® provides effective treatment and control against induced infestation with paralysis ticks for 13 weeks.

The Comparison of Cerebral Oxygenation among Mechanically Ventilated Children Receiving Protocolized Sedation and Analgesia versus Clinician's Decision in Pediatric Intensive Care Unit.

Adequate sedation and analgesia are two crucial factors affecting recovery of intensive care patients. Improper use of sedation and analgesia in intensive care patients may adversely lead to brain oxygen desaturation. This study aims to determine cerebral oxygenation as measured by near-infrared spectroscopy (NIRS) and inotropic interventions received among mechanically ventilated children in the pediatric intensive care unit (PICU). This study is a nested case - control study in the PICU of Indonesian tertiary hospital. Children aged 1 month to 17 years on mechanical ventilation and were given sedation and analgesia were included in the study. Subjects were divided into two groups according to the protocol of the main study (Clinical Trial ID NCT04788589). Cerebral oxygenation was measured by NIRS at five time points (before sedation, 5-min, 1, 6, and 12 h after sedation). Thirty-nine of the 69 subjects were categorized into the protocol group and the rest were in the control group. A decrease of >20% NIRS values was found among subjects in the protocol group at 5-min (6.7%), 1-h (11.1%), 6-h (26.3%), and 12-h (23.8%) time-point. The mean NIRS value was lower and the inotropic intervention was more common in the control group (without protocol), although not statistically significant. This study found that mechanically ventilated children who received sedation and analgesia based on the protocol had a greater decrease of >20% NIRS values compared to the other group. The use of sedation and analgesia protocols must be applied in selected patients after careful consideration.

A Real-World Population Pharmacokinetic (PK) Study of Two Recombinant Factor VIII (rFVIII) Concentrates in Boys with Severe Hemophilia a without Inhibitors

Introduction To personalize long-term prophylaxis in boys with hemophilia A with a severe bleeding phenotype, the individual's pharmacokinetic (PK) profile must be established. Traditional FVIII PK assessment has required a 2-3-day washout, which can place boys at risk of bleeding, and up to 11 post-infusion blood samples collected over a 48h period (Lee et al., ISTH guidelines, 2001). Population PK models with Bayesian forecasting are currently available that permit the use of a no-washout PK protocol with sparse sampling, which is clinically practical and less demanding for patients and their families. Our previous experience with a no-washout, single-clinic visit, reverse 2-sampling time point (24 and 3h) PK protocol established that this practical, patient-friendly PK protocol was sufficiently accurate for generating clinically useful individual PK profiles in patients receiving a standard half-life (SHL) FVIII concentrate [antihemophilic factor (recombinant) plasma/albumin-free method (Advate®)] for long-term prophylaxis (Blanchette et al., Thromb Haemost, 2021). Aim To determine the accuracy of a clinically practical single-clinic visit, reverse 2-sampling time point population PK protocol for generating individual PK profiles [clearance (Cl) and terminal half-life (t1/2)] in boys with severe hemophilia A without inhibitors receiving either an SHL [antihemophilic factor (recombinant) BAY 81-8973 (Kovaltry®)] or an extended half-life (EHL) FVIII concentrate [antihemophilic factor (recombinant) PEGylated BAY 94-9027 (Jivi®)] for long-term prophylaxis. Methods In this multicenter observational study, participants had their first infusion given at home. Of a total of 14 boys with hemophilia A without inhibitors receiving long-term prophylaxis, 5 receiving a SHL completed a single clinic visit, reverse 2-point (24 and 3h) and a multiple clinic visit 6-point (baseline, 1, 3, 8, 24 and 48h) PK sampling protocol; and 4 boys completed a SHL single clinic visit reverse 2-point (48 and 3h) and a 2 clinic visit reverse 3-point (48, 3 and 24h) PK sampling protocol. 5 boys receiving EHL completed single clinic visit, reverse 2-point (72 and 3h) and 2-clinic visit 3-point (72, 3 and 48h) PK sampling protocols and they had multiple spot samplings (n=12) at various post-infusion times to evaluate the precision of their predicted PK profiles based on their 2-point PK sampling protocol. One-stage (OS) and chromogenic (CHR) FVIII:C and von Willebrand factor antigen (VWF:Ag) assays were performed in a central reference coagulation laboratory in Kingston, Ontario. PK parameters (Cl and t1/2) were estimated using the WAPPS-Hemo population PK program. The median of the % difference between the 2-sampling time point PK protocol compared to the 6- and 3- sampling time point PK protocols is reported. Results 14 boys with hemophilia A (FVIII:C <2%; median age: 14.5 yrs, range: 5-19 yrs) participated in the study. The 2-point PK protocol for both SHL and EHL showed a higher Cl and lower t1/2 when compared to either a 6-sampling time point (for SHL) or a 3-sampling time point (for either SHL or EHL) PK assessment (Table 1). The difference was greatest for the 2-point (24 and 3h) vs the 6-point (Table 1). In the case of the 2-point (48 and 3h for SHL and 72 and 3h for EHL) vs 3-point (an additional 24h time point for SHL and 48h for EHL) PK parameter estimates, the 2-point protocol gave values that were all within 6% of those obtained with the 3-sampling time point PK (Table 1). The OS and CHR assays gave similar estimations for Cl and t1/2 with the exception of t1/2 when comparing the 6-point to the 2-point PK protocol (Table 1). Evaluation of the 2-sampling time point PK protocol through spot sampling showed approximately 66% and 81% of FVIII:C values falling within the 95% prediction limits for the OS and CHR assays, respectively (n=12 for EHL OS; n=11 for EHL CHR). There was a strong association between the observed FVIII:C levels from the spot sampling and their predicted values from the 2-point PK for EHL (R2=94% and 71% for the OS and CHR assays, respectively) (Fig. 1). Conclusion The reverse 2-point, single clinic visit, PK protocol (24/48 or 72h and 3h) is a practical approach to generate PK parameters that provide clinically useful information for guiding personalized prophylaxis regimens for boys receiving SHL or EHL for long-term prophylaxis. Funding Supported by Bayer (Investigator-Initiated Research grant) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal