Dolutegravir and Folic Acid Interaction during Neural System Development in Zebrafish Embryos.

Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 µM) and observed the disruption of the anterior-posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain-hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)+ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.

Spleen tyrosine kinase inhibitor R406 has both antiviral and anti-inflammatory effects on severe influenza A infection.

Death due to severe influenza is usually a fatal complication of a dysregulated immune response more than the acute virulence of an infectious agent. Although spleen tyrosine kinase (SYK) as a critical immune signaling molecule and therapeutic target plays roles in airway inflammation and acute lung injury, the role of SYK in influenza virus infection is not clear. Here, we investigated the antiviral and anti-inflammatory effects of SYK inhibitor R406 on influenza infection through a coculture model of human alveolar epithelial (A549) and macrophage (THP-1) cell lines and mouse model. The results showed that R406 treatment increased the viability of A549 and decreased the pathogenicity and mortality of lethal influenza virus in mice with influenza A infection, decreased levels of intracellular signaling molecules under the condition of inflammation during influenza virus infection. Combination therapy with oseltamivir further ameliorated histopathological damage in the lungs of mice and further delayed the initial time to death compared with R406 treatment alone. This study demonstrated that phosphorylation of SYK is involved in the pathogenesis of influenza, and R406 has antiviral and anti-inflammatory effects on the treatment of the disease, which may be realized through multiple pathways, including the already reported SYK/STAT/IFNs-mediated antiviral pathway, as well as TNF-α/SYK- and SYK/Akt-based immunomodulation pathway.

Discovery of Dolutegravir Derivative against Liver Cancer via Inducing Autophagy and DNA Damage.

We introduced a terminal alkyne into the core structure of dolutegravir, resulting in the synthesis of 34 novel dolutegravir-1,2,3-triazole compounds through click chemistry. These compounds exhibited remarkable inhibitory activities against two hepatocellular carcinoma cell lines, Huh7 and HepG2. Notably, compounds 5e and 5p demonstrated exceptional efficacy, particularly against Huh7 cells, with IC50 values of 2.64 and 5.42 µM. Additionally, both compounds induced apoptosis in Huh7 cells, suppressed tumor cell clone formation, and elevated reactive oxygen species (ROS) levels, further promoting tumor cell apoptosis. Furthermore, compounds 5e and 5p activated the LC3 signaling pathway, inducing autophagy, and triggered the γ-H2AX signaling pathway, resulting in DNA damage in tumor cells. Compound 5e exhibited low toxicity, highlighting its potential as a promising anti-tumor drug.

Arginine and sodium fluoride affect the microbial composition and reduce biofilm metabolism and enamel mineral loss in an oral microcosm model.

OBJECTIVE: To assess the effects of arginine, with or without sodium fluoride (NaF; 1,450 ppm), on saliva-derived microcosm biofilms and enamel demineralization. METHODS: Saliva-derived biofilms were grown on bovine enamel blocks in 0.2 % sucrose-containing modified McBain medium, according to six experimental groups: control (McBain 0.2 %); 2.5 % arginine; 8 % arginine; NaF; 2.5 % arginine with NaF; and 8 % arginine with NaF. After 5 days of growth, biofilm viability was assessed by colony-forming units counting, laser scanning confocal microscopy was used to determine biofilm vitality and extracellular polysaccharide (EPS) production, while biofilm metabolism was evaluated using the resazurin assay and lactic acid quantification. Demineralization was evaluated by measuring pH in the culture medium and calcium release. Data were analyzed by Kruskal-Wallis' and Dunn's tests (p < 0.05). RESULTS: 8 % arginine with NaF showed the strongest reduction in total streptococci and total microorganism counts, with no significant difference compared to arginine without NaF. Neither 2.5 % arginine alone nor NaF alone significantly reduced microbial counts compared to the control, although in combination, a reduction in all microbial groups was observed. Similar trends were found for biofilm vitality and EPS, and calcium released to the growth medium. CONCLUSIONS: 8 % Arginine, with or without NaF, exhibited the strongest antimicrobial activity and reduced enamel calcium loss. Also, NaF enhanced the effects of 2.5 % arginine, yielding similar results to 8 % arginine for most parameters analyzed. CLINICAL SIGNIFICANCE: The results provided further evidence on how arginine, with or without NaF, affects oral microcosm biofilms and enamel mineral loss.

Exploring the Roles of Vitamins C and D and Etifoxine in Combination with Citalopram in Depression/Anxiety Model: A Focus on ICAM-1, SIRT1 and Nitric Oxide.

The study of intercellular adhesion molecule-1 (ICAM-1) and SIRT1, a member of the sirtuin family with nitric oxide (NO), is emerging in depression and anxiety. As with all antidepressants, the efficacy is delayed and inconsistent. Ascorbic acid (AA) and vitamin D (D) showed antidepressant properties, while etifoxine (Etx), a GABAA agonist, alleviates anxiety symptoms. The present study aimed to investigate the potential augmentation of citalopram using AA, D and Etx and related the antidepressant effect to brain and serum ICAM-1, SIRT1 and NO in an animal model. BALB/c mice were divided into naive, control, citalopram, citalopram + etx, citalopram + AA, citalopram + D and citalopram + etx + AA + D for 7 days. On the 8th day, the mice were restrained for 8 h, followed by a forced swim test and marble burying test before scarification. Whole-brain and serum expression of ICAM-1, Sirt1 and NO were determined. Citalopram's antidepressant and sedative effects were potentiated by ascorbic acid, vitamin D and etifoxine alone and in combination (p < 0.05), as shown by the decreased floating time and rearing frequency. Brain NO increased significantly (p < 0.05) in depression and anxiety and was associated with an ICAM-1 increase versus naive (p < 0.05) and a Sirt1 decrease (p < 0.05) versus naive. Both ICAM-1 and Sirt1 were modulated by antidepressants through a non-NO-dependent pathway. Serum NO expression was unrelated to serum ICAM-1 and Sirt1. Brain ICAM-1, Sirt1 and NO are implicated in depression and are modulated by antidepressants.

Discovery of a Macrocyclic Influenza Cap-Dependent Endonuclease Inhibitor.

Influenza viruses (IFVs) have caused several pandemics and have claimed numerous lives since their first record in the early 20th century. While the outbreak of COVID-19 seemed to expel influenza from the sight of people for a short period of time, it is not surprising that it will recirculate around the globe after the coronavirus has mutated into a less fatal variant. Baloxavir marboxil (1), the prodrug of baloxavir (2) and a cap-dependent endonuclease (CEN) inhibitor, were approved by the FDA for the first treatment in almost 20 years. Despite their high antiviral potency, drug-resistant variants have been observed in clinical trials. Herein, we report a novel CEN inhibitor 8 with a delicately designed macrocyclic scaffold that exhibits a significantly smaller shift of inhibitory activity toward baloxavir-resistant variants.

An orally active entry inhibitor of influenza A viruses protects mice and synergizes with oseltamivir and baloxavir marboxil.

Seasonal or pandemic illness caused by influenza A viruses (IAVs) is a major public health concern due to the high morbidity and notable mortality. Although there are several approved drugs targeting different mechanisms, the emergence of drug resistance calls for new drug candidates that can be used alone or in combinations. Small-molecule IAV entry inhibitor, ING-1466, binds to hemagglutinin (HA) and blocks HA-mediated viral infection. Here, we show that this inhibitor demonstrates preventive and therapeutic effects in a mouse model of IAV with substantial improvement in the survival rate. When administered orally it elicits a therapeutic effect in mice, even after the well-established infection. Moreover, the combination of ING-1466 with oseltamivir phosphate or baloxavir marboxil enhances the therapeutic effect in a synergistic manner. Overall, ING-1466 has excellent oral bioavailability and in vitro absorption, distribution, metabolism, excretion, and toxicity profile, suggesting that it can be developed for monotherapy or combination therapy for the treatment of IAV infections.

Characterization of Spodoptera littoralis (Lepidoptera: Noctuidae) resistance to indoxacarb: inheritance mode, realized heritability, and fitness costs.

Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is a major economic pest attacking a variety of crops in Egypt and other Mediterranean countries. S. littoralis has developed resistance to both traditional and novel insecticides. The current study investigated S. littoralis resistance to indoxacarb regarding inheritance mode, realized heritability (h2), and fitness costs. An indoxacarb-resistant strain (Indoxa-SEL) was obtained by selecting a field strain with indoxacarb. Indoxa-SEL strain outperformed the susceptible one (Indoxa-S) by 29.77-fold after 16 consecutive generations of selection. Based on the LC50 values of the progenies of reciprocal crosses F1 (R♂ × S♀) and F1' (R♀ × S♂), S. littoralis resistance to indoxacarb was found to be autosomal and partially recessive. Chi-square tests for goodness-of-fit between observed and expected mortalities of self-bred F1 and resistant strain reciprocal crosses revealed that the resistance was controlled by multiple genes. The resistant strain had a relative fitness of 0.80, with significantly increased total preovipositional period of females, egg, larvae, pupae, preadult, adult, and total longevity period. The estimated realized heritability value in the Indoxa-SEL strain was 0.21. The current study will contribute to sustaining indoxacarb efficacy and designing effective resistance management programs against S. littoralis.

Effects of thiamethoxam on physiological and molecular responses to potato plant (Solanum tuberosum), green peach aphid (Myzus persicae), and parasitoid (Aphidius gifuensis).

BACKGROUND: To improve integrated pest management (IPM) performance it is essential to assess pesticide side effects on host plants, insect pests, and natural enemies. The green peach aphid (Myzus persicae Sulzer) is a major insect pest that attacks various crops. Aphidius gifuensis is an essential natural enemy of M. persicae that has been applied effectively in controlling M. persicae. Thiamethoxam is a neonicotinoid pesticide widely used against insect pests. RESULTS: The current study showed the effect of thiamethoxam against Solanum tuberosum, M. persicae, and A. gefiuensis and the physiological and molecular response of the plants, aphids, and parasitoids after thiamethoxam application. Thiamethoxam affected the physical parameters of S. tuberosum and generated a variety of sublethal effects on M. persicae and A. gefiuensis, including nymph development time, adult longevity, and fertility. Our results showed that different thiamethoxam concentrations [0.1, 0.5, and 0.9 µm active ingredient (a.i.)/L] on different time durations (2, 6, and 10 days) increased the antioxidant enzyme activities SOD, POD, and CAT of S. tuberosum, M. persicae, and A. gefiuensis significantly compared with the control. Our results also showed that different thiamethoxam concentrations (0.1, 0.5, and 0.9 µm a.i./L) on different time durations (2, 6, and 10 days) increased the expression of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione-S-transferase (GST) genes of S. tuberosum, M. persicae, and A. gefiuensis compared with the control. CONCLUSION: Our findings reveal that using thiamethoxam at suitable concentrations and time durations for host plants and natural enemies may enhance natural control through the conservation of natural enemies by overcoming any fitness disadvantages. © 2024 Society of Chemical Industry.

The impact of PA/I38 substitutions and PA polymorphisms on the susceptibility of zoonotic influenza A viruses to baloxavir.

Genetic reassortment of avian, swine, and human influenza A viruses (IAVs) poses potential pandemic risks. Surveillance is important for influenza pandemic preparedness, but the susceptibility of zoonotic IAVs to the cap-dependent endonuclease inhibitor baloxavir acid (BXA) has not been thoroughly researched. Although an amino acid substitution at position 38 in the polymerase acidic protein (PA/I38) in seasonal IAVs reduces BXA susceptibility, PA polymorphisms at position 38 are rarely seen in zoonotic IAVs. Here, we examined the impact of PA/I38 substitutions on the BXA susceptibility of recombinant A(H5N1) viruses. PA mutants that harbored I38T, F, and M were 48.2-, 24.0-, and 15.5-fold less susceptible, respectively, to BXA than wild-type A(H5N1) but were susceptible to the neuraminidase inhibitor oseltamivir acid and the RNA polymerase inhibitor favipiravir. PA mutants exhibited significantly impaired replicative fitness in Madin-Darby canine kidney cells at 24 h postinfection. In addition, in order to investigate new genetic markers for BXA susceptibility, we screened geographically and temporally distinct IAVs isolated worldwide from birds and pigs. The results showed that BXA exhibited antiviral activity against avian and swine viruses with similar levels to seasonal isolates. All viruses tested in the study lacked the PA/I38 substitution and were susceptible to BXA. Isolates harboring amino acid polymorphisms at positions 20, 24, and 37, which have been implicated in the binding of BXA to the PA endonuclease domain, were also susceptible to BXA. These results suggest that monitoring of the PA/I38 substitution in animal-derived influenza viruses is important for preparedness against zoonotic influenza virus outbreaks.

Natural course of AIDS following HIV-2 infection.

In a 21-year-old female, AIDS following infection with HIV-2 was diagnosed alongside an HIV-associated high-grade B cell lymphoma. Treatment of HIV-2 with dolutegravir, emtricitabine, and tenofovir resulted in viral suppression and slow recovery of CD4 cell counts. Treatment of lymphoma caused significant adverse effects but led to complete remission. The patient denied sexual activity and intravenous drug abuse. The patient had been born to an HIV-2-positive mother but appropriate perinatal testing based on national guidelines had remained negative. This case recapitulates the natural course of HIV-2 infection.

Genomic Analysis of Influenza A and B Viruses Carrying Baloxavir Resistance-Associated Substitutions Serially Passaged in Human Epithelial Cells.

Baloxavir marboxil (baloxavir) is an FDA-approved inhibitor of the influenza virus polymerase acidic (PA) protein. Here, we used next-generation sequencing to compare the genomic mutational profiles of IAV H1N1 and H3N2, and IBV wild type (WT) and mutants (MUT) viruses carrying baloxavir resistance-associated substitutions (H1N1-PA I38L, I38T, and E199D; H3N2-PA I38T; and IBV-PA I38T) during passaging in normal human bronchial epithelial (NHBE) cells. We determined the ratio of nonsynonymous to synonymous nucleotide mutations (dN/dS) and identified the location and type of amino acid (AA) substitutions that occurred at a frequency of ≥30%. We observed that IAV H1N1 WT and MUT viruses remained relatively stable during passaging. While the mutational profiles for IAV H1N1 I38L, I38T, and E199D, and IBV I38T MUTs were relatively similar after each passage compared to the respective WTs, the mutational profile of the IAV H3N2 I38T MUT was significantly different for most genes compared to H3N2 WT. Our work provides insight into how baloxavir resistance-associated substitutions may impact influenza virus evolution in natural settings. Further characterization of the potentially adaptive mutations identified in this study is needed.

Frequency and diversity of indoxacarb resistance in Australian Helicoverpa armigera (Lepidoptera: Noctuidae).

Annual indoxacarb resistance in Helicoverpa armigera (Hübner) populations collected from various crops in Australia was monitored between 2013 and 2023. Resistance frequency determined by F2 screening using a predetermined discriminating dose of indoxacarb, was lowest in the 2013-2014 and 2015-2016 seasons at 0.0164 and 0.0246, respectively. Resistance then increased significantly to a ten-year high of 0.0869 in 2018-2019 but declined to 0.0557 in 2019-2020 during a severe drought, remaining relatively stable thereafter to 2023. Indoxacarb resistance was first detected in H. armigera collected from maize in the Gwydir valley, New South Wales, in 2013 (strain GY7-39). In 2017, a second indoxacarb resistant H. armigera strain (UN1U3-10) was isolated from a population collected in chickpeas in the Liverpool Plains, New South Wales. Indoxacarb resistance of this strain was characterized to evaluate its potential to compromise the ongoing effectiveness of insecticide resistance management strategies in Australian farming systems. Survival at the discriminating dose of indoxacarb in UN1U3-10 was 28.9, 52.6, 86.7, and 92.9% in the F2, F3, F4, and F5, respectively. Following introgression with a susceptible strain and reselection with the discriminating dose of indoxacarb, the resistance ratio of UN1U3-10 was approximately 800-fold. Resistance was autosomal, incompletely dominant and conferred by more than 1 locus. While indoxacarb resistance in UN1U3-10 did not confer to emamectin benzoate or spinetoram and there was no evidence of major cross-resistance to the Bt toxins Cry1A, Cry2A or Vip3A, there was 5-fold reduced sensitivity to chlorantraniliprole. Indoxacarb resistance was suppressed by approximately 10-fold by PBO with no synergism by TPP or DEM, suggesting the involvement of cytochrome P450 enzymes. A stability analysis indicated a fitness cost may be associated with the genes that confer resistance in the UN1U3-10 strain. The potential risk for diverse indoxacarb resistance in the Australian H. armigera population is discussed.

A community cluster of influenza A(H3N2) virus infection with reduced susceptibility to baloxavir due to a PA E199G substitution in Japan, February to March 2023.

A community cluster of influenza A(H3N2) caused by viruses with an E199G substitution in PA was detected in Nara, Japan, between February and March 2023. The three patients with these mutant viruses had not received antiviral treatment before specimen collection but patients in the same hospital had. The sequences of the mutant viruses were closely related, suggesting clonal spread in Nara. They showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear.

Role of ERK1/2 Signaling in Cinnabarinic Acid-Driven Stanniocalcin 2-Mediated Protection against Alcohol-Induced Apoptosis.

We have previously shown that a bona fide aryl hydrocarbon receptor (AhR) agonist, cinnabarinic acid (CA), protects against alcohol-induced hepatocyte apoptosis via activation of a novel AhR target gene, stanniocalcin 2 (Stc2). Stc2 translates to a secreted disulfide-linked hormone, STC2, known to function in cell development, calcium and phosphate regulation, angiogenesis, and antiapoptosis-albeit the comprehensive mechanism by which the CA-AhR-STC2 axis confers antiapoptosis is yet to be characterized. In this study, using RNA interference library screening, downstream antiapoptotic molecular signaling components involved in CA-induced STC2-mediated protection against ethanol-induced apoptosis were investigated. RNA interference library screening of kinases and phosphatases in Hepa1 cells and subsequent pathway analysis identified mitogen-activated protein kinase (MAPK) signaling as a critical molecular pathway involved in CA-mediated protection. Specifically, phosphorylation of ERK1/2 was induced in response to CA treatment without alterations in p38 and JNK signaling pathways. Silencing Stc2 in Hepa1 cells and in vivo experiments performed in Stc2-/- (Stc2 knockout) mice, which failed to confer CA-mediated protection against ethanol-induced apoptosis, showed abrogation of ERK1/2 activation, underlining the significance of ERK1/2 signaling in CA-STC2-mediated protection. In conclusion, activation of ERK1/2 signaling in CA-driven AhR-dependent Stc2-mediated protection represents a novel mechanism of protection against acute alcohol-induced apoptosis. SIGNIFICANCE STATEMENT: Previous studies have shown the role of stanniocalcin 2 (Stc2) in cinnabarinic acid (CA)-mediated protection against alcohol-induced apoptosis. Here, using RNA interference library screening and subsequent in vivo studies, the functional significance of ERK1/2 activation in CA-induced Stc2-mediated protection against acute ethanol-induced apoptosis was identified. This study is thus significant as it illustrates a comprehensive downstream mechanism by which CA-induced Stc2 protects against alcoholic liver disease.

Blood telomere length gain in people living with HIV switching to dolutegravir plus lamivudine versus continuing triple regimen: a longitudinal, prospective, matched, controlled study.

BACKGROUND: Blood telomere length (BTL) is a validated biomarker of aging. ART reduces immunosenescence and has benefits in terms of BTL in people living with HIV (PLWH). However, it has also been observed that ART containing NRTIs, such as tenofovir or abacavir, which are potent inhibitors of human telomerase activity in vitro, might negatively affect BTL. Here we investigated the effects on BTL 1 year after switching to a dual therapy (DT) with dolutegravir + lamivudine versus maintaining a standard triple therapy (TT) with a two-NRTI backbone and an anchor drug. METHODS: This was a longitudinal, prospective, matched, controlled study that included virologically suppressed adults on stable three-drug ART who either switched at baseline (BL) to DT or maintained TT. The DT and TT groups were 1:1 matched for age, sex, years since HIV diagnosis, years on ART and anchor drug. BTL was assessed by a monochrome multiplex qPCR at BL and after 48 weeks (W48). RESULTS: We enrolled 120 PLWH, i.e. 60 participants in each group. At BL, the BTL means were comparable between the two groups (P = 0.973). At W48, viro-immunological status was stable and an overall increase in the mean BTL was observed, i.e., +0.161 (95%CI, 0.054-0.268) (P = 0.004). However, the within-group analysis showed a significant mean BTL gain in the DT group (P = 0.003) but not in the TT group (P = 0.656). CONCLUSIONS: In this setting of virologically suppressed PLWH, simplifying to dolutegravir + lamivudine was associated with a higher gain in BTL than maintaining triple therapy after the 1 year follow-up. These findings suggest that as a simplification strategy dolutegravir + lamivudine might have a positive effect on BTL.

Natural products with 1,2-oxazine scaffold: occurrence, chemical diversity, bioactivity, synthesis, and biosynthesis.

Covering: up to the end of July, 20231,2-Oxazine is a heterocyclic scaffold rarely found in natural products and is characterized by a directly connected N-O bond in a six-membered ring. Since the discovery of geneserine, the first 1,2-oxazine-containing natural product (1,2-oxazine NP) being isolated from Calabar bean (Physostigma venenosum) in 1925, a total of 76 naturally occurring 1,2-oxazine NPs have been isolated and identified from various sources, which have attracted the attention of researchers in the field of natural product chemistry, organic synthesis, biosynthesis, and pharmacology. This review summarizes the chemical family of 1,2-oxazine NPs, focusing on their source organisms, structural diversities, chemical synthesis, and biosynthesis.

Selection of Rattus norvegicus cumulus-oocyte complex for vitrification by brilliant cresyl blue.

The influence of the method of evaluating developmentally competent oocytes on their viability after cryopreservation still needs to be better understood. The objective of this study was to determine the cleavage and embryo developmental rates after parthenogenetic activation of cumulus-oocyte complexes (COCs) selected by different concentrations of brilliant cresyl blue (BCB) and cryopreservation. In the first experiment, COCs were separated into groups and incubated for 1 h in medium containing BCB (13 µM, 16 µM, or 20 µM). The control group was not exposed to BCB staining. In the second experiment, COCs were divided into four groups: 13 µM BCB(+), 13 µM BCB(-), fresh control (selected by morphologic observation and immediately in vitro matured) and vitrified control (selected by morphologic evaluation, vitrified, and in vitro matured). In the first experiment, the 13 µM BCB group displayed greater development rates at the morula stage (65.45%, 36/55) when compared with the other groups. In the second experiment, cleavage (47.05%, 72/153) and morula development (33.55%, 51/153) of the control group of fresh COCs were increased compared with the other groups. However, when comparing morula rates between vitrified COC control and BCB(+) groups, the BCB(+) group had better results (19.23%, 5/26 and 64.7%, 11/17, respectively). Our best result in rat COC selection by BCB staining was obtained using a concentration of 13 µM. This selection could be a valuable tool to improve vitrification outcomes, as observed by the BCB(+) group that demonstrated better results compared with the vitrified COC control.

Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

Ampakine CX614 increases respiratory rate in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra compacta (SNpc). In a mouse model of PD induced by the injection of 6-hydroxydopamine (6-OHDA) into the caudate putamen (CPu) dyspnea events are very common. Neuroanatomical and functional studies show that the number of glutamatergic neurons in the pre-Bötzinger Complex (preBötC) are reduced. We hypothesize that the neuronal loss, and consequently loss of glutamatergic connections in the respiratory network previously investigated, are responsible for the breathing impairment in PD. Here, we tested whether ampakines (CX614), a subgroup of AMPA receptor positive allosteric modulators, could stimulate the respiratory activity in PD-induced animals. CX614 (50 µM) injected intraperitoneally or directly into the preBötC region reduced the irregularity pattern and increased the respiratory rate by 37% or 82%, respectively, in PD-induced animals. CX614 also increased the respiratory frequency in healthy animals. These data suggest that ampakine CX614 could become a tool to restore breathing in PD.