Induction Of Levels Research Articles

Introducing energy into marine environments: A lab-scale static magnetic field submarine cable simulation and its effects on sperm and larval development on a reef forming serpulid

Non-chemical sources of anthropogenic environmental stress, such as artificial lights, noise and magnetic fields, are still an underestimate factor that may affect the wildlife. Marine environments are constantly subjected to these kinds of stress, especially nearby to urbanized coastal areas. In the present work, the effect of static magnetic fields, associated with submerged electric cables, was evaluated in gametes and early life stages of a serpulid polychaete, namely Ficopomatus enigmaticus. Specifically, biochemical/physiological impairments of sperm, fertilization rate inhibition and incorrect larval development were assessed. We evaluated differences between two selected magnetic field induction values (0.5 and 1 mT) along a range of exposure times (30 min–48 h), for a sound evaluation on this species. We found that a magnetic induction of 1 mT, a typical value that can be found at distance of tens of cm from a submerged cable, may be considered a biologically and ecologically relevant for sessile organisms and for coastal environments more generally. This value exerted statistically significant effects on membranes, DNA integrity, kinetic parameters and mitochondrial activity of sperm cells. Moreover, a significant reduction in fertilization rate was observed in sperm exposed to the same magnetic induction level (1 mT) for 3 h, compared to controls. Regarding early larval stages, 48-h exposure did not affect the correct development. Our results represent a starting point for a future focus of research on magnetic field effects on early life stages of aquatic invertebrates, using model species as representative for reef-forming/encrusting organisms and ecological indicators of soft sediment quality.

The role of skin biopsy in the detection of exposure to endocrine disrupting chemicals in Mediterranean cetaceans

Use of skin biopsy is proposed as a sensitive non-lethal technique for the hazard assessment of Mediterranean cetaceans exposed to endocrine disrupting chemicals (EDCs). EDCs are a structurally diverse group of compounds that may adversely affect the health of humans and wildlife or their progeny, by interaction with the endocrine system. In the Mediterranean environment top predators accumulate high concentrations of polyhalogenated aromatic hydrocarbons (PHAHs) and toxic metals, incurring high toxicological hazard. In this paper, the hypothesis that Mediterranean cetaceans are potentially at risk due to PHAH-EDCs is investigated using skin biopsy samples. Benzo-a-pyrene monoxigenase (BPMO) activity in skin biopsies was used as a potential indicator of exposure to different organochlorines (OCs) known to have endocrine disrupting properties. The main objective of this paper was to use this non-destructive ecotoxicological tool to define the potential hazard to Mediterranean odontocete and mysicete species, comparing the present data with values detected in other cetaceans from heavily polluted areas, affected by pseudohermaphroditism and other reproductive dysfunction. Subcutaneous tissue consisting of skin and blubber was obtained from striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), common dolphins (Delphinus delphis) and fin whales (Balaenoptera physalus) in the Mediterranean basin. Sampling was performed in the western Ligurian Sea, between Corsica and the French-Italian coast, and in the Ionian Sea. High concentrations of DDT metabolites and PCB congeners (known as Endocrine Disruptors) were detected in the different species. Significant differences in BPMO induction and OC levels were found between odontocetes and mysticetes. Differences in organochlorine bioaccumulation and consequently potential risk due to endocrine disruptors were primarily related to different positions in the marine food web. A statistical correlation was found between BPMO activity and organochlorine (op’DDT, a potent estrogen and antiandrogen and pp’DDE, a potent antiandrogen) levels in skin biopsy specimens of the endangered Mediterranean population of common dolphin. Several conclusions on the potential risk to Mediterranean cetaceans can be drawn from comparison of the levels of OC-EDs detected in Mediterranean odontocetes with those in white whales (Delphinapterus leucas) of the St Lawrence estuary and bowhead whales (Balaena mysticetus) affected by pseudohermaphroditism and other reproductive dysfunction. Finally, these results suggest that BPMO induction may be an early sign of exposure to EDCs such as OCs and a warning of the possibility of transgenerational effects through exposure of future generations via the placenta and milk.

NETosis induction reflects COVID-19 severity and long COVID: insights from a 2-center patient cohort study in Israel

BackgroundCOVID-19 severity and its late complications continue to be poorly understood. Neutrophil extracellular traps (NETs) form in acute COVID-19, likely contributing to morbidity and mortality. ObjectivesThis study evaluated immunothrombosis markers in a comprehensive cohort of acute and recovered COVID-19 patients, including the association of NETs with long COVID. MethodsOne-hundred-seventy-seven patients were recruited from clinical cohorts at 2 Israeli centers: acute COVID-19 (mild/moderate, severe/critical), convalescent COVID-19 (recovered and long COVID), along with 54 non-COVID controls. Plasma was examined for markers of platelet activation, coagulation, and NETs. Ex vivo NETosis induction capability was evaluated after neutrophil incubation with patient plasma. ResultsSoluble P-selectin, factor VIII, von Willebrand factor, and platelet factor 4 were significantly elevated in patients with COVID-19 versus controls. Myeloperoxidase (MPO)-DNA complex levels were increased only in severe COVID-19 and did not differentiate between COVID-19 severities or correlate with thrombotic markers. NETosis induction levels strongly correlated with illness severity/duration, platelet activation markers, and coagulation factors, and were significantly reduced upon dexamethasone treatment and recovery. Patients with long COVID maintained higher NETosis induction, but not NET fragments, compared to recovered convalescent patients. ConclusionsIncreased NETosis induction can be detected in patients with long COVID. NETosis induction appears to be a more sensitive NET measurement than MPO-DNA levels in COVID-19, differentiating between disease severity and patients with long COVID. Ongoing NETosis induction capability in long COVID may provide insights into pathogenesis and serve as a surrogate marker for persistent pathology. This study emphasizes the need to explore neutrophil-targeted therapies in acute and chronic COVID-19.

De Novo Synthesis of Poly(3-hydroxybutyrate-co-3-hydroxypropionate) from Oil by Engineered Cupriavidus necator.

Poly(3-hydroxybutyrate-co-3-hydroxypropionate) [P(3HB-co-3HP)] is a biodegradable and biocompatible polyester with improved and expanded material properties compared with poly(3-hydroxybutyrate) (PHB). This study engineered a robust malonyl-CoA pathway in Cupriavidus necator for the efficient supply of a 3HP monomer, and could achieve the production of [P(3HB-co-3HP)] from variable oil substrates. Flask level experiments followed by product purification and characterization found the optimal fermentation condition (soybean oil as carbon source, 0.5 g/L arabinose as induction level) in general consideration of the PHA content, PHA titer and 3HP molar fraction. A 5 L fed-batch fermentation (72 h) further increased the dry cell weight (DCW) to 6.08 g/L, the titer of [P(3HB-co-3HP)] to 3.11 g/L and the 3HP molar fraction to 32.25%. Further improving the 3HP molar fraction by increasing arabinose induction failed as the engineered malonyl-CoA pathway was not properly expressed under the high-level induction condition. With several promising advantages (broader range of economic oil substrates, no need for expensive supplementations such as alanine and VB12), this study indicated a candidate route for the industrial level production of [P(3HB-co-3HP)]. For future prospects, further studies are needed to further improve the strain and the fermentation process and expand the range of relative products.

Abstract 5329: High throughput screening strategies in the development of logic gated cell therapies

Abstract Chimeric antigen receptor T cell (CAR T) therapy has demonstrated unprecedented therapeutic activity in hematologic malignancies. However, generating potent clinical responses against solid tumors remains a challenge for CAR T therapy. As the field strives to improve the therapeutic efficacy of CAR T cells with novel target antigens and enhanced potency, the risks of on-target toxicity pose a major barrier to progress. To address these challenges, we have developed engineered CAR T cells to target solid tumors through AND logic gates, where CAR expression is conditionally induced by a transcription factor released from a priming receptor (PrimeRTM) upon binding to the PrimeR antigen. The AND gate limits off-tumor toxicity as it requires both CAR and PrimeR antigen expression in the tumor microenvironment. To ensure PrimeR expression and signal transduction upon antigen binding, while minimizing residual ‘‘leaky’’ CAR induction in the absence of PrimeR antigen, we screened hundreds of PrimeR binders using both arrayed and pooled strategies. In an arrayed strategy, we engineered T cells from four donors in multiwell plates using CRISPR-mediated, non-viral, site-specific integration of logic gates bearing a variable PrimeR binder and a fixed MSLN CAR. In addition, we employed a pooled screening strategy, where we engineered T cells from two independent donors with a pool containing a subset of >300 of the same logic gates. Engineered T cells from both strategies were co-cultured with cell lines bearing either both CAR and PrimeR antigens or a single antigen, in order to evaluate fidelity and on-target functionality. In the arrayed setting, on-target functionality was quantified based on the levels of CAR induction, cytokine secretion, T cell activation, and target cell killing in the presence of both antigens, while fidelity was assessed based on the absence of these activity signals in the presence of a single antigen. In the pooled setting, sorting based on functional markers was performed and sequencing was used to quantify the relative abundance of cells with each logic gate in different sorted populations. On-target activity and circuit fidelity were then quantified based on enrichments in different sorted populations. Results from the pooled and arrayed screens were highly concordant. We combined the screen readouts to nominate a small set of PrimeR binders that exhibited both high fidelity and on-target functionality. We confirmed the desired characteristics of these binders with targeted arrayed screens in additional conditions as well as in in-vivo models. We have applied both screen strategies to select a small set of leads from hundreds of candidate PrimeR binders in the context of a logic-gated MSLN CAR. As pooled and arrayed screens come with different sets of limitations and advantages, both serve as important tools for the effective selection of receptors in the development of novel cell therapies. Citation Format: Li Wang, Sofia Kyriazopoulou Panagiotopoulou, Rona Harari-Steinfeld, Dasmanthie De Silva, Michelle Tan, Laura Lim, Angela Boroughs, Cate Sue, Jon Chen, Jamie Thomas, Mary Chua, Ed Yashin, Christine Shieh, Ryan Fong, Sophie Xu, Grace Zheng, Brendan Galvin, Aaron Cooper, Tarjei Mikkelsen, Nicholas Haining. High throughput screening strategies in the development of logic gated cell therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5329.

Abstract 2592: Helicobacter pylori-induced FGFR4 mediates nuclear accumulation of NRF2 in gastric tumorigenesis

Abstract Background: Fibroblast growth factor receptor 4 (FGFR4) is a member of the transmembrane tyrosine kinase receptor family and has been linked to a variety of malignancies. NRF2 (Nuclear factor-erythroid 2-related factor) is a cytoprotective factor and a critical regulator in the antioxidant response pathway. In this study, we sought to uncover novel functions of FGFR4 and its role in regulating the antioxidant response in gastric carcinogenesis. Methods and Results: Using western blot and immunofluorescence, H. pylori infection in gastric cancer cell lines demonstrated high levels of reactive oxygen species and induction of both FGFR4 and NRF2. Using Flow cytometry, FGFR4 silencing resulted in decrease of NRF2 with a significant spike in ROS levels and an increase in DNA damage and cell death. FGFR4 knockdown showed a significant decrease in NRF2 transcriptional activity as measured by NRF2 ARE luciferase reporter assay and resulted in reduced mRNA levels of HO-1 (Heme Oxygenase-1), which is a classical target of NRF2. These results were confirmed by immunofluorescence showing a significant increase of nuclear accumulation of NRF2 after H. pylori infection which was abolished after FGFR4 knockdown. Similar results were found using recombinant protein FGF19, a FGFR4 ligand. C57/B6 wild-type mice were infected with the pylori strain (PMSS1). An increase in FGFR4, NRF2, and HO-1 was seen by immunofluorescence, Western blot, and quantitative real-time PCR in H. pylori-infected mice vs control mice. We observed a reduction in NRF2 in our in vitro and in vivo models using H3B-6527, a specific FGFR4 inhibitor. We also discovered an association between an increase in FGFR4 and P62 protein expressions and NRF2 protein stability. We detected a significant increase in FGFR4, NRF2, and HO1 in dysplastic and neoplastic gastric lesions using the TFF1-KO mouse model which was further aggravated by H. pylori infection. In terms of the mechanism, utilizing proximity ligation and immunoprecipitation assays, we found that FGFR4 binds to P62 to inhibit the interaction between NRF2 and KEAP1, allowing NRF2 to avoid degradation facilitating its translocation and accumulation in the nucleus. Conclusion: These findings revealed that FGFR4 has a unique functional role in promoting gastric carcinogenesis by mediating accumulation and activation of the NRF2 antioxidant response. The use of FGFR4 inhibitors is a viable treatment option that warrants further research in patients with gastric cancer. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Xing Zhang, Zheng Chen, Ahmed Gomaa, Marwah Al-Mathkour, Selma Maacha, Heng Lu, Dunfa Peng, Zekuan Xu, Wael El-Rifai. Helicobacter pylori-induced FGFR4 mediates nuclear accumulation of NRF2 in gastric tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2592.

Abstract 2640: An innovative site-specific dual-payload antibody drug conjugate (dpADC) combining a novel Topo1 inhibitor and an immune agonist delivers a strong immunogenic cell death (ICD) and antitumor response in vitro and in vivo

Abstract Background: Antibody-drug conjugate, also known as a targeted chemotherapy, leverages the targeting feature of mAb and the efficient cell-killing ability of cytotoxin to deliver cytotoxic drugs to tumor specifically. Among the FDA approved 15 ADCs and over 100 in clinical trial, the majority contains a single type cytotoxin payload. Combination of chemotherapies or multiple agents are widely therapeutic approach to enhance treatment efficiency, although frequently combo therapies could lead to higher toxicity, long term chemotherapies could result in drug resistance and tumor relapse. To solve above challenge, here we report an innovative dual-payload antibody-drug conjugate (dpADC) with 2 type of different drug payloads with different MoA, the conjugate is constructed based on unique enzymatic site-specific conjugation technology iLDC/iGDC. The dpADC leads to an enhanced immunogenic cell death (ICD) and antitumor response in vitro and in vivo via a synergistic MoA. Results: A novel Topo1 inhibitor TopoIx and an immune agonist were stably conjugated to an anti-Trop2 mAb in a site-specific manner, conjugate homogeneity and precise DAR were characterized. The dpADC showed a strong cytotoxicity on the proliferation of Trop2-expressing cells, meanwhile bystander killing activity of TopoIx also effectively inhibits growth ofTrop2-negative cells. This dpADC also triggers IP10/IL6 secretion when co-cultured with PBMC/monocyte and Trop2-positive cancer cells, with induction level correlated with Trop 2 expression level. No free payload was released after 96hr incubation at 37 oC in human plasma, demonstrating high linker stability. The dpADC demonstrated a robust antitumor response against diverse Trop2+ tumors in vivo. Its’ combination with anti-mPD-1 resulted in synergistic anti-tumor response. In a tumor rechallenge model, the mice that had cleared of Trop2+ tumors after dpADC treatment were protected against the re-implantation of tumor cells with same or different neoantigen, suggesting the presence of a robust immunological memory. Conclusion: In summary, this novel dpADC demonstrates potent antitumor activities in vitro and in vivo and leads to an enhanced tumor cell killing ability and a prolonged immunological memory via a synergistic MoA. Based on this site-specific, dual-enzyme orthogonal catalytic conjugation platform, we can easily integrate MoA-varied drugs in one targeting modality to realize a synergistic, durable anti-tumor therapy. Citation Format: Meijun Xiong, Beibei Fan, Xinju Gao, Yajun Sun, Cao Lv, Yu Si, Xiao Liu, Lili Shi, Paul H. Song, Gang Qin. An innovative site-specific dual-payload antibody drug conjugate (dpADC) combining a novel Topo1 inhibitor and an immune agonist delivers a strong immunogenic cell death (ICD) and antitumor response in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2640.

Identification of a Far-Red Light-Inducible Promoter that Exhibits Light Intensity Dependency and Reversibility in a Cyanobacterium.

As the demand for sustainable energy has increased, photoautotrophic cyanobacteria have become a popular platform for developing tools in synthetic biology. Although genetic tools are generally available for several model cyanobacteria, such tools have not yet been developed for many other strains potentially suitable for industrial applications. Additionally, most inducible promoters in cyanobacteria are controlled by chemical compounds, but adding chemicals into growth media on an industrial scale is neither cost-effective nor environmentally friendly. Although using light-controlled promoters is an alternative approach, only a cyanobacterial expression system inducible by green light has so far been described and employed for such applications. In this study, we have established a conjugation-based technique to express a reporter gene (eyfp) in the nonmodel cyanobacterium, Chlorogloeopsis fritschii PCC 9212. We also identified a promoter specifically activated by far-red light from the Far-Red Light Photoacclimation gene cluster of Leptolyngbya sp. JSC-1. This promoter, PchlFJSC1, was successfully used to drive eyfp expression. PchlFJSC1 is tightly regulated by light quality (i.e., wavelength) and leads to an approximately 30-fold increase in EYFP production when cells were exposed to far-red light. The induction level was controlled by the far-red light intensity, and induction stopped when cells were returned to visible light. This system has the potential for further applications in cyanobacteria by providing an additional choice of light wavelength to control gene expression. Collectively, this study developed a functional gene-expression system for C. fritschii PCC 9212 that can be regulated by exposing cells to far-red light.

Arctiin alleviates functional constipation by enhancing intestinal motility in mice.

Functional constipation (FC), a common symptom that is primarily associated with intestinal motility dysfunction, is a common problem worldwide. Arctiin (Arc) is a lignan glycoside isolated from the Chinese herbal medicine Arctium lappa L., which is a health food in China. The present study aimed to evaluate the laxative effects of Arc against FC in mice. A model of FC induced by loperamide (5 mg/kg) was established in male Institute of Cancer Research (ICR) mice. Arc was administered at a dose of 100 mg/kg as a protective agent. The faecal status, intestinal motility and histological analyses were evaluated. Furthermore, the levels of gastrointestinal motility-associated neurotransmitters, such as motilin (MTL), nitric oxide (NO), and brain-derived neurotrophic factor (BDNF) and the protective effect of Arc on interstitial cells of Cajal (ICC) were assessed. Arc treatment reversed the loperamide-induced reduction in faecal number and water content and the intestinal transit ratio in ICR mice. Histological analysis confirmed that Arc administration mitigated colonic injury. Moreover, Arc treatment increased levels of motilin and brain-derived neurotrophic factor while decreasing nitric oxide levels and ICC injury in the colon of FC mice. Arc decreased inflammation induction and aquaporin expression levels. Owing to its pro-intestinal motility property, Arc was shown to have a protective effect against FC and may thus serve as a promising therapeutic strategy for the management of FC.

Repurposing Verapamil to Enhance Killing of T-ALL Cells by the mTOR Inhibitor Everolimus.

New therapies are needed for patients with T-cell lymphoblastic leukemia (T-ALL) who do not respond to standard chemotherapy. Our previous studies showed that the mTORC1 inhibitor everolimus increases reactive oxygen species (ROS) levels, decreases the levels of NADPH and glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), and induces apoptosis in T-ALL cells. Studies in T-ALL-xenografted NOD/SCID mice demonstrated that everolimus improved their response to the glucocorticoid (GC) dexamethasone. Here we show that verapamil, a calcium antagonist used in the treatment of supraventricular tachyarrhythmias, enhanced the effects of everolimus on ROS and cell death in T-ALL cell lines. The death-enhancing effect was synergistic and was confirmed in assays on a panel of therapy-resistant patient-derived xenografts (PDX) and primary samples from T-ALL patients. The verapamil-everolimus combination produced a dramatic reduction in the levels of G6PD and induction of p38 MAPK phosphorylation. Studies of NOD/SCID mice inoculated with refractory T-ALL PDX cells demonstrated that the addition of verapamil to everolimus plus dexamethasone significantly reduced tumor growth in vivo. Taken together, our results provide a rationale for repurposing verapamil in association with mTORC inhibitors and GC to treat refractory T-ALL.

Mini-αA-Crystallin Stifled Melittin-Induced Haemolysis and Lymphocyte Lysis

Melittin, the most potent pharmacological ingredient of honey bee venom, induces haemolysis, lymphocyte lysis, long-term pain, localised inflammation, and hyperalgesia. In this study, efforts were made to subdue the melittin’s ill effects using a chaperone peptide called ‘mini-αA-crystallin’ (MAC) derived from eye lens αA-crystallin. Haemolytic test on human red blood cells, percentage viability, and DNA diffusion assay on Human peripheral blood lymphocytes (HPBLs) were performed with melittin in the presence or absence of MAC. Propidium iodide and Annexin V-FITC dual staining were performed to analyse quantitative levels of necrotic and apoptotic induction by melittin in the presence or absence of MAC on HPBLs using a flow cytometer. A computational study to find out the interactions between MAC and melittin was undertaken by modelling the structure of MAC using a PEP-FOLD server. The result showed that MAC inhibited melittin-induced lysis in nucleated (lymphocytes) and enucleated (RBC) cells. Flow cytometric analysis revealed a substantial increase in the necrotic and late apoptotic cells after treating HPBLs with melittin (4 µg/ml) for 24 h. Treatment with MAC at a 2:1 molar ratio prevented HPBLs from developing melittin-induced necrosis and late apoptosis. In the docking study, hydrogen, van der Waals, π-π stacking, and salt bridges were observed between the MAC and melittin complex, confirming a strong interaction between them. The MAC-melittin complex was stable during molecular dynamics simulation. These findings may be beneficial in developing a medication for treating severe cases of honeybee stings.

Policosanol protects against Alzheimer's disease-associated spatial cognitive decline in male rats: possible involved mechanisms.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by cognitive decline and synaptic failure. The present study was designed to explore the possible protective effects of policosanol (PCO) on spatial cognitive capacity, long-term potentiation (LTP) induction, oxidant/antioxidant status, and Aβ plaques formation in an AD rat model induced by intracerebroventricular (ICV) injection of Aβ1-40. Healthy adult male Wistar rats were randomly divided into control, sham (ICV injection of 5µl phosphate-buffered saline), AG (50mg/kg; P.O., as PCO vehicle), PCO (50mg/kg; P.O.), AD model (ICV injection of 5µl Aβ), AD + AG (50mg/kg; P.O.), and AD + PCO (50mg/kg; P.O.). Treatments were performed for eight consecutive weeks. At the end of the treatment course, spatial learning and memory functions, hippocampal long-term potentiation (LTP) induction, malondialdehyde (MDA), and total thiol group (TTG) levels, as well as the formation of Aβ plaques, were examined. The results showed that injection of Aβ reduced spatial learning and memory abilities in the Barnes maze test, which was accompanied by decreases in field excitatory postsynaptic potential (fEPSP) slope, population spike (PS) amplitude, and TTG level and increases in Aβ plaque accumulation and MDA content. In contrast, PCO treatment improved all the above-mentioned changes in the Aβ-infused rats. The results suggest that amelioration of hippocampal synaptic plasticity impairment, modulation of oxidant/antioxidant status, and inhibition of Aβ plaque formation by PCO may be the mechanisms behind its protective effect against AD-associated spatial cognitive decline.

P663 Post-ustekinumab induction IL12, IL23, and ustekinumab levels are associated with clinical response in a multi-centre prospective cohort study of Crohn’s disease patients: results from the AURORA Study including ANZIBDC Cohort

Abstract Background Ustekinumab (UST) is a monoclonal antibody targeting IL-12 and IL-23 through their shared p40 subunit. This study aimed to determine the clinical outcomes after UST treatment in Crohn’s disease (CD) patients in a real-world setting, and to investigate the association of clinical outcomes with IL-12, IL-23 and UST levels. Methods A multi-centre prospective observational cohort study of UST for moderate to severe CD was conducted. Patients were recruited from 19 Australian centres between Sep 2019 and Apr 2022. Clinical assessments were performed at baseline study visit (SV) 1 and post-induction (SV2). Patients were assessed every 6 months during maintenance therapy to 18 months (SV4). Clinical response and remission rates were determined using PRO2 definitions (STRIDE II guidelines). Logistic regression analyses were performed to identify predictors of clinical response and remission. UST levels were measured post induction and interleukin levels including IL12p40 and IL-23p19 were measured at SV1 and SV2 using ProQuantum ELISA assays. Results A total of 198 patients were recruited: median age 40.7 years, 54.3% male, median duration of disease 90.5 months, 12.8% active smokers, and 49.7% on concomitant immunomodulatory therapy. The majority (58.4%) were biologic-naïve and 82 patients were previously exposed to biologics (51.9%; IFX n=42, ADA n=50, VDZ n=9). Clinical response was achieved in 137 patients (75.7%), and remission in 84 (46.4%) with higher rates of response (85.2% vs 61.6%, p=0.001) and remission (54.6% vs 34.2%, p=0.006) in biologic naïve patients compared to biologic-exposed. For the 114 patients with 18 months (SV4) of follow-up, durable clinical response was maintained in 66.8% and remission in 50.0%. Dose escalation (90mg 4 weekly) was administered to 13 patients (6.6%) during induction, 48 (42.1%) in maintenance, and 15 patients (13.2%) received IV re-induction during maintenance. There was a significant reduction in IL-12 and IL-23 levels from SV1 to SV2 in responders (p=0.0001), but no significant reduction in non-responders (Figure 1). Clinical response at SV4 (n=101) was associated with higher post-induction UST levels (p=0.03) (Table 1). The combination of IL-12. IL-23 and UST level at SV2 predicted long-term response at SV4 (Sn 73%, Sp 71%, AUROC 0.765), but not at SV2 (Figure 2). Conclusion This large real-world study confirms that UST is most effective in bio-naïve CD patients with significantly higher response and remission rates than bio-experienced patients. The combination of post-induction IL-12, IL-23, and UST levels was associated with response at 18 months and could represent a novel predictor of long-term clinical outcomes.

Asymmetric Ruthenium-Catalyzed Carbonyl Allylations by Gaseous Allene via Hydrogen Auto-Transfer: 1° vs 2° Alcohol Dehydrogenation for Streamlined Polyketide Construction.

Iodide-bound ruthenium-JOSIPHOS complexes catalyze the redox-neutral C-C coupling of primary alcohols 2a-2r with the gaseous allene (propadiene) 1a to form enantiomerically enriched homoallylic alcohols 3a-3r with complete atom-efficiency. Using formic acid as reductant, aldehydes dehydro-2a and dehydro-2c participate in reductive C-C coupling with allene to deliver adducts 3a and 3c with comparable levels of asymmetric induction. Deuterium labeling studies corroborate a mechanism in which alcohol dehydrogenation triggers allene hydroruthenation to form transient allylruthenium-aldehyde pairs that participate in carbonyl addition. Notably, due to a kinetic preference for primary alcohol dehydrogenation, chemoselective C-C coupling of 1°,2°-1,3-diols occurs in the absence of protecting groups. As illustrated by the synthesis of C7-C15 of spirastrellolide B and F (7 vs 17 steps), C3-C10 of cryptocarya diacetate (3 vs 7 or 9 steps), and a fragment common to C8'-C14' of mycolactone F (1 vs 4 steps) and C22-C28 marinomycin A (1 vs 9 steps), this capability streamlines type I polyketide construction.

CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells.

Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CYP3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G decreased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1G GA HepG2 cells. In summary, we successfully constructed CYP3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and enzyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells. SIGNIFICANCE STATEMENT: Cytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5. This study successfully established CYP3A4*1G (G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1G regulates the basal and RIF-induced expression of CYP3A4 and CYP3A5.

Specified Parameters in Designing Porous Materials Using Magnetic Nanotechnologies

The research is devoted to solving the problem of regulating the porosity parameters during the manufacturing process under magnetic field conditions. The process of synthesizing magnetic nanocomponents based on a mixture of divalent and trivalent iron oxides was given. The use of nanocomponents allowed for improving the conditions for creating porous materials. A device with adjustable magnetic induction was developed to produce porous materials in a magnetic field. The study of the porous material’s structure with the nanopowder content in the magnetic field conditions showed a clear dependence of the structure on the magnetic parameters. When the content of nanocomponents increased to 0.3 %, and the magnetic field induction increased to 2.5 mT, the dispersion of pore sizes decreased by 8–10 times, the density of pores – increased by 15–20 times, and the average diameter of pores – decreased by 12–15 times. Mathematical dependencies that determine the porosity parameters for different values of the magnetic nanopowder content and the level of magnetic induction in the ring electromagnet were proposed. The obtained dependencies allowed for assigning the level of magnetic technological parameters to ensure the given porosity parameters. The developed methods of magnetic technology for creating porous materials can increase the quality and ensure the required porosity level.

Anion-accelerated asymmetric Nazarov cyclization: access to vicinal all-carbon quaternary stereocenters.

We have developed a catalytic asymmetric Nazarov cyclization that results in the formation of two contiguous all-carbon quaternary stereocenters in high yield with excellent levels of asymmetric induction. This method requires no catalyst recognition elements in the starting materials that are simple diketoesters. Geometrically pure E or Z isomers of the starting material lead to diastereomerically pure products with high enantioselectivity because the species that undergoes cyclization is a rhodium enolate that is configurationally stable.

Regulation of Cytotoxic Immune Effector Function by AJ3 Probiotic Bacteria in Amyotrophic Lateral Sclerosis (ALS).

Our recent studies indicated that amyotrophic lateral sclerosis (ALS) patients suffer from significantly elevated levels of interferon-gamma (IFN-γ) secretion by natural killer (NK) and CD8+ T cells, which may be responsible for the immune-pathologies seen in central nervous system and in peripheral organs of the patients. In order to counter such elevated induction of IFN-γ in patients we designed a treatment strategy to increase anti-inflammatory cytokine interleukin-10 (IL-10) by the use of probiotic strains which significantly increase the levels of IL-10. Therefore, in this paper we demonstrate disease specific functions of Al-Pro (AJ3) formulated for the adjunct treatment of auto-immune diseases including ALS, and compared the function with CA/I-Pro (AJ4) for the treatment of cancer and viral diseases, and NK-CLK (AJ2) for maintenance of immune balance and promotion of disease prevention. The three different formulations of probiotic bacteria have distinct profiles of activation of peripheral blood mononuclear cells (PBMCs), NK, and CD8+ T cells, and their induced activation is different from those mediated by either IL-2 or IL-2 + anti-CD16 monoclonal antibodies (mAbs) or IL-2 + anti-CD3/CD28 mAbs. IL-2 + anti-CD16 mAb activation of PBMCs and NK cells had the highest IFN-γ/IL-10 ratio, whereas IL-2 combination with sAJ4 had the next highest followed by IL-2 + sAJ2 and the lowest was seen with IL-2 + sAJ3. Accordingly, the highest secretion of IFN-γ was seen when the PBMCs and NK cells were treated with IL-2 + sAJ4, intermediate for IL-2 + sAJ2 and the lowest with IL-2 + sAJ3. The levels of IFN-γ induction and the ratio of IFN-γ to IL-10 induced by different probiotic bacteria formulation in the absence of IL-2 treatment remained much lower when compared to those treated in the presence of IL-2. Of note is the difference between NK cells and CD8+ T cells in which synergistic induction of IFN-y by IL-2 + sAJ4 was significantly higher in NK cells than those seen by CD8+ T cells. Based on these results, sAJ3 should be effective in alleviating auto-immunity seen in ALS since it will greatly regulate the levels and function of IFN-γ negatively, decreasing overactivation of cytotoxic immune effectors and prevention of death in motor neurons.

A Shielding System Proposal for the Cabling of Electric Glass Melters

This paper analysesthe impact on workers of the electromagnetic field produced by large furnaces for glass production. The paper also propose a solution for the reduction and the containment of magnetic induction levels below the limits imposed by national and international directives. Using a three-dimensional numerical calculation code based on the hypothesis of filiform conductors in the air (Biot-savart law). The choice of the solution is based on the technique named highly coupled magnetically passive loop technique (HMCPL). The solution was then implemented and a comparison between the magnetic induction values before and after the installation of the shielding solution is presented.

Redox-sensitive small GTPase H-Ras in murine astrocytes, an in vitro study

ABSTRACT Background Although the protooncogenes small GTPases Ras are redox-sensitive proteins, how they are regulated by redox signaling in the central nervous system (CNS) is still poorly understood. Alteration in redox-sensitive targets by redox signaling may have myriad effects on Ras stability, activity and localization. Redox-mediated changes in astrocytic RAS may contribute to the control of redox homeostasis in the CNS that is connected to the pathogenesis of many diseases. Results and Methods Here, we investigated the transient physiological induction, at both transcriptional and translational levels, of small GTPases Ras in response to redox stimulation. Cultured astrocytes were treated with hydrogen peroxide as in bolus addition and relative mRNA levels of murine hras and kras genes were detected by qRT-PCR. We found that de novo transcription of hras mRNA in reactive astrocytes is redox-sensitive and mimics the prototypical redox-sensitive gene iNOS. Protein abundance in combination with protein turnover measurements by cycloheximide-chase experiments revealed distinct translation efficiency, GTP-bound enrichment, and protein turnover rates between the two isoforms H-Ras and K-Ras. Conclusion Reports from recent years support a significant role of H-Ras in driving redox processes. Beyond its canonical functions, Ras may impact on the core astrocytic cellular machinery that operates during redox stimulation.