Owner-Perception of the Effects of Two Long-Lasting Dog-Appeasing Pheromone Analog Devices on Situational Stress in Dogs

Devices that release a synthetic analog of the canine-appeasing pheromone can help to relax dogs during stressful situations, but they usually last for only one month. Two new devices with this analog were tested by owners of dogs showing signs of stress in a range of everyday situations: Zenidog™ collar, lasting three months, and Zenidog™ diffusing gel, lasting two months (Virbac, Carros, France). They were compared against reference products that last for one month. In the three-month study with collars, one group received Zenidog™ collar, one received the reference collar, and one group of dogs wore an antiparasitic collar alongside a Zenidog™ collar. In the two-month study with diffusers, groups received either the unpowered Zenidog™ gel diffuser or the reference electric diffuser. Owners regularly completed a questionnaire that assessed seventeen general behaviors and sources of fear and eleven specific signs of stress. Global scores for these two main scales were calculated, and the evolution of scores was compared between groups. Non-parametric tests with a Bonferroni correction were used for statistical analysis. An improvement of all global scores was observed in all groups (p < 0.001), including in puppies, and there was no difference between groups. Zenidog™ devices were as effective as the reference devices and lasted longer.

The Potential Antimicrobial Action of Human Mucin 7 15-Mer Peptide and Its Metal Complexes

Mucin 7 (encoded byMUC7) is a human salivary protein that has a role in the natural immune system. Fragments of mucin 7 exhibit antimicrobial activity against bacteria and yeast. Although the antimicrobial properties of peptides have been known and studied for decades, the exact mechanism of action of antimicrobial peptides (AMPs) is still unclear. It is known that some AMPs require divalent metal ions to activate their activity. Herein, we investigated three 15-mer MUC7 peptides, one of which (mother peptide, sequence, L3) is a synthetic analog of a fragment naturally excised from MUC7 (with His3, His8, and His 14) and its two structural analogs, containing only two histidine residues, His3, His13 and His8, His13 (L2 and L1, respectively). Since there is a correlation between lipophilicity, the presence of metal ions (such as Cu(II) and Zn(II)) and antimicrobial activity of AMP, antimicrobial properties of the studied peptides, as well as their complexes with Cu(II) and Zn(II) ions, were tested for activity against Gram-positive (Enterococcus faecalis, Staphylococcus epidermidis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria and fungi (Candida albicans). The results were correlated with their lipophilicity. Coordination and thermodynamic studies (potentiometry, UV-Vis, CD) revealed the formation of mainly mononuclear complexes in solution for all studied systems with different stability in the physiological pH range.

1045. Safety of the Synthetic Saponin Adjuvant TQL1055: Preliminary Results from a First-in-humans Trial

Background Saponin adjuvants reliably enhance immune response to a variety of antigens, but their use is hindered by dose-limiting toxicities and supply constraints. TQL1055 is a semi-synthetic analog of the natural saponin adjuvant QS-21, rationally modified to improve tolerability and enable large-scale manufacturing. We previously showed that the combination of acellular pertussis vaccine (aP) and TQL1055 was well-tolerated and increased anti-pertussis toxin (PT) antibody responses in mice and rabbits, with a no observed adverse effect level (NOAEL) &gt; 2000 mcg/dose. Methods Here we report interim results from a Phase 1 first-in-humans dose-escalation study of TQL1055. Healthy adults 18 to 50 years of age were sequentially enrolled into 6 groups (n=12/group) and randomized 10:2 to receive one intramuscular dose of aP + TQL1055 or aP alone on Day 1. TQL1055 dose increased by group from 25 to 800 mcg (Figure 1). Local adverse events (AEs) (injection site pain, redness, swelling) and systemic AEs (fever, chills, headache, fatigue, myalgia, arthralgia, nausea, vomiting, diarrhea) were solicited through Day 8. Clinical laboratory panels (chemistry, hematology, coagulation) were performed on Days 1 (pre-dose), 8, and 29. Serious AEs were collected through Day 365. Antibodies to PT were assessed at all visits. Figure 1. Study Design Results Blinded safety data from the first four groups (n=48) through Day 8 were analyzed, including 2 subjects/group receiving aP alone. All solicited AEs were mild or moderate (Figure 2). Local AEs, mainly injection site pain, occurred in 75% of subjects (mild 65%, moderate 10%). The incidence of total local AEs increased with TQL1055 dose, from 50% at 25 mcg to 92% at 200 mcg. The mean duration of local AEs was 1.8 days and also increased with TQL1055 dose, from 1.3 days at 25 mcg to 2.1 days at 200 mcg. Systemic AEs, mostly fatigue, headache, and nausea, occurred in 63% of subjects (mild 40%, moderate 23%), with no fevers. The mean duration of systemic AEs was 1.4 days, with no association with TQL1055 dose. No severe or serious adverse events were reported. Figure 2. Solicited Adverse Events by Severity and TQL1055 Dose Conclusion In this early analysis, the safety profile of aP + TQL1055 appears similar to that of licensed aP vaccines, without severe or prolonged injection site pain. These data support further dose escalation and assessment of immunogenicity. Disclosures Sean R. Bennett, MD PhD, Adjuvance Technologies (Employee) Tyler Martin, MD, Adjuvance Technologies (Employee, Shareholder)

Effects of His-Phe-Arg-Trp-Pro-Gly-Pro peptide on free-radical oxidation processes in conditions of chronic restraint stress

Studying the effects of regulatory peptides on the stress-induced shifts in the bodily processes is of great fundamental and applied significance. Currently, a wide range of peptide neurotropic drugs, affecting the stress response development, are used in medicine, and new promising molecules are being studied. The study was aimed to assess the effects of the adrenocorticotropic hormone (ACTH) synthetic analog, ACTH(6-9)-Pro-Gly-Pro, administered at a dose of 5, 50 and 500 μg/kg, on the free-radical oxidation processes in Wistar rats, subjected to chronic restraint stress (CRS) during two weeks. Serum levels of 8-oxo-2'-deoxyguanosine (8-OHdG) and superoxide dismutase 3 (SOD3) were assessed by enzyme immunoassay, and the levels of thiobarbituric acid reactive substances (TBARS) were assessed by fluorimetric method. CRS lead to the significant increase in the 8-OHdG levels by 18.4% (p = 0.01) and the decrease in the SOD3 levels by 14.3% (p = 0.01), however, it had no effect on the levels of TBARS. ACTH(6-9)-Pro-Gly-Pro, administered at a dose of 5 and 50 μg/kg, significantly decreased the levels of 8-OHdG by 19.8% (p = 0.03) and 30% (p = 0.001), respectively. Thus, it was found that CRS resulted in oxidative stress in animals. ACTH(6-9)-Pro-Gly-Pro administration at a dose of 5 and 50 μg/kg inhibits the stress-induced free-radical oxidation processes.

FLLL32 Triggers Caspase-Mediated Apoptotic Cell Death in Human Oral Cancer Cells by Regulating the p38 Pathway

Oral cancer is the most common oral malignant tumor in Taiwan. Although there exist several methods for treatment, oral cancer still has a poor prognosis and high recurrence. FLLL32, a synthetic analog of curcumin with antitumor activity, is currently known to induce melanoma apoptosis and inhibit tumor growth in various cancers. However, few studies have examined the mechanisms of FLLL32 in oral cancer. In this study, we explore whether FLLL32 induces apoptosis in oral cancer. We determined that FLLL32 can inhibit the cell viability of oral cancer. Next, we analyzed the effect of FLLL32 on the cell cycle of oral cancer cells and observed that the proportion of cells in the G2/M phase was increased. Additionally, annexin-V/PI double staining revealed that FLLL32 induced apoptosis in oral cancer cells. Data from the Human Apoptosis Array revealed that FLLL32 increases the expression of cleaved caspase-3 and heme oxygenase-1 (HO-1). FLLL32 activates proteins such as caspase-8, caspase-9, caspase-3, PARP, and mitogen-activated protein kinases (MAPKs) in apoptosis-related molecular mechanisms. Moreover, by using MAPK inhibitors, we suggest that FLLL32 induces the apoptosis of oral cancer cells through the p38 MAPK signaling pathway. In conclusion, our findings suggest that FLLL32 is a potential therapeutic agent for oral cancer by inducing caspase-dependent apoptosis and HO-1 activation through the p38 pathway. We believe that the activation of HO-1 and the p38 pathway by FLLL32 represent potential targets for further research in oral cancer.

Effect of Ivermectin and Atorvastatin on Nuclear Localization of Importin Alpha and Drug Target Expression Profiling in Host Cells from Nasopharyngeal Swabs of SARS-CoV-2- Positive Patients

Nuclear transport and vesicle trafficking are key cellular functions involved in the pathogenesis of RNA viruses. Among other pleiotropic effects on virus-infected host cells, ivermectin (IVM) inhibits nuclear transport mechanisms mediated by importins and atorvastatin (ATV) affects actin cytoskeleton-dependent trafficking controlled by Rho GTPases signaling. In this work, we first analyzed the response to infection in nasopharyngeal swabs from SARS-CoV-2-positive and -negative patients by assessing the gene expression of the respective host cell drug targets importins and Rho GTPases. COVID-19 patients showed alterations in KPNA3, KPNA5, KPNA7, KPNB1, RHOA, and CDC42 expression compared with non-COVID-19 patients. An in vitro model of infection with Poly(I:C), a synthetic analog of viral double-stranded RNA, triggered NF-κB activation, an effect that was halted by IVM and ATV treatment. Importin and Rho GTPases gene expression was also impaired by these drugs. Furthermore, through confocal microscopy, we analyzed the effects of IVM and ATV on nuclear to cytoplasmic importin α distribution, alone or in combination. Results showed a significant inhibition of importin α nuclear accumulation under IVM and ATV treatments. These findings confirm transcriptional alterations in importins and Rho GTPases upon SARS-CoV-2 infection and point to IVM and ATV as valid drugs to impair nuclear localization of importin α when used at clinically-relevant concentrations.

Strategies for the De Novo Synthesis of Highly Substituted Pyridine Scaffolds: Unified Total Synthesis of the Limonoid Alkaloids

Highly substituted pyridine scaffolds are found in many biologically active natural products and therapeutics. Accordingly, numerous complementary de novo approaches to obtain differentially substituted pyridines have been disclosed. This article delineates the evolution of the synthetic strategies designed to assemble the demanding tetrasubstituted pyridine core present in the limonoid alkaloids isolated from Xylocarpus granatum, including xylogranatopyridine B, granatumine A and related congeners. The most efficient and convergent construction of the core framework present in xylogranatopyridine B involved a Liebeskind pyridine synthesis and late-stage benzylic oxidation. By contrast, the synthesis of the bislactone limonoid alkaloids, such as granatumine A which exhibited moderate PTP1B-inhibitory activities, necessitated the development of a novel pyran-to-pyridine conversion. In addition, NMR calculations suggested structural misassignment of several limonoid alkaloids, and predicted their C3-epimers as the correct structures, which was further validated unequivocally through chemical synthesis. While preliminary results of the pNPP assays showed that these bislactone limonoid alkaloids were only weakly inhibitory against PTP1B, C3-deoxy-xylogranatin F, an unnatural synthetic analog, was demonstrated to be more potent than the other congeners.

Total Syntheses of Pladienolide-Derived Spliceosome Modulators

Pladienolides, an emerging class of naturally occurring spliceosome modulators, exhibit interesting structural features, such as highly substituted 12-membered macrocycles and epoxide-containing diene side chains. The potential of pladienolides as anti-cancer agents is confirmed by H3B-8800, a synthetic analog of this natural product class, which is currently under Phase I clinical trials. Since its isolation in 2004 and the first total synthesis in 2007, a dozen total syntheses and synthetic approaches toward the pladienolide class have been reported to date. This review focuses on the eight completed total syntheses of naturally occurring pladienolides or their synthetic analogs, in addition to a synthetic approach to the main framework of the natural product.

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

ABSTRACTBillions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to β-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to “calm down” its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Life’s gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo.SIGNIFICANCE STATEMENTThe (apparent) rigidity of the genetic code, as well as its universality, have long since ushered explorations into expanding the code with synthetic, new-to-nature building blocks and testing its boundaries. While nowadays even proteome-wide incorporation of synthetic amino acids has been reported on several occasions1–3, little is known about the underlying mechanisms.We here report ALE with auxotrophic E. coli that yielded successful proteome-wide replacement of Trp by its synthetic analog [3,2]Tpa accompanied with the selection for loss of RpoS4 function. Such laboratory domestication of bacteria by the acquisition of rpoS mitigation mutations is beneficial not only to overcome the stress of nutrient (Trp) starvation but also to evolve the paths to use environmental xenobiotics (e.g. [3,2]Tp) as essential nutrients for growth.We pose that regulatory constraints rather than a rigid and conserved protein translation apparatus are Life’s gatekeepers of the canonical amino acid repertoire (at least where close structural analogs are concerned). Our findings contribute a step towards understanding possible environmental causes of genetic changes and their relationship to evolution.Our evolved strain affords a platform for homogenous protein labeling with [3,2]Tpa as well as for the production of biomolecules5, which are challenging to synthesize chemically. Top-down synthetic biology will also benefit greatly from breaking through the boundaries of the frozen bacterial genetic code, as this will enable us to begin creating synthetic cells capable to utilize an expanded range of substrates essential for life.