Female Mouse Model Research Articles

Assembly of Genetically Engineered Ionizable Protein Nanocage-based Nanozymes for Intracellular Superoxide Scavenging

Nanozymes play a pivotal role in mitigating excessive oxidative stress, however, determining their specific enzyme-mimicking activities for intracellular free radical scavenging is challenging due to endo-lysosomal entrapment. In this study, we employ a genetic engineering strategy to generate ionizable ferritin nanocages (iFTn), enabling their escape from endo-lysosomes and entry into the cytoplasm. Specifically, ionizable repeated Histidine-Histidine-Glutamic acid (9H2E) sequences are genetically incorporated into the outer surface of human heavy chain FTn, followed by the assembly of various chain-like nanostructures via a two-armed polyethylene glycol (PEG). Utilizing endosome-escaping ability, we design iFTn-based tetrameric cascade nanozymes with high superoxide dismutase- and catalase-mimicking activities. The in vivo protective effects of these ionizable cascade nanozymes against cardiac oxidative injury are demonstrated in female mouse models of cardiac ischemia-reperfusion (IR). RNA-sequencing analysis highlight the crucial role of these nanozymes in modulating superoxide anions-, hydrogen peroxide- and mitochondrial functions-relevant genes in IR injured cardiac tissue. These genetically engineered ionizable protein nanocarriers provide opportunities for developing ionizable drug delivery systems.

Proline exacerbates hepatic gluconeogenesis via paraspeckle-dependent mRNA retention.

Type 2 diabetes (T2D) is a global health issue characterized by abnormal blood glucose levels and is often associated with excessive hepatic gluconeogenesis. Increased circulating non-essential amino acids (NEAAs) are consistently observed in individuals with T2D; however, the specific contribution of each amino acid to T2D pathogenesis remains less understood. Here, we report an unexpected role of the NEAA proline in coordinating hepatic glucose metabolism by modulating paraspeckle, a nuclear structure scaffolded by the long non-coding RNA Neat1. Mechanistically, proline diminished paraspeckles in hepatocytes, liberating the retained mRNA species into cytoplasm for translation, including the mRNAs of Ppargc1a and Foxo1, contributing to enhanced gluconeogenesis and hyperglycaemia. We further demonstrated that the proline-paraspeckle-mRNA retention axis existed in diabetic liver samples, and intervening in this axis via paraspeckle restoration substantially alleviated hyperglycaemia in both female and male diabetic mouse models. Collectively, our results not only delineated a previously unappreciated proline-instigated, paraspeckle-dependent mRNA-retention mechanism regulating gluconeogenesis, but also spotlighted proline and paraspeckle as potential targets for managing hyperglycaemia.

Non-Genotoxic and Environmentally Relevant Lower Molecular Weight Polycyclic Aromatic Hydrocarbons Significantly Increase Tumorigenicity of Benzo[a]pyrene in a Lung Two-Stage Mouse Model.

The World Health Organization has classified air pollution as a carcinogen, and polycyclic aromatic hydrocarbons (PAHs) are major components of air particulates of carcinogenic concern. Thus far, most studies focused on genotoxic high molecular weight PAHs; however, recent studies indicate potential carcinogenicity of the non-genotoxic lower molecular weight PAHs (LMW PAHs) that are found in indoor and outdoor air pollution as well as secondhand cigarette smoke. We hypothesize that LMW PAHs contribute to the promotion stage of cancer when combined with benzo[a]pyrene (B[a]P), a legacy PAH. We specifically determined the effects of an LMW PAH mixture containing 1-methylanthracene (1MeA), fluoranthene (Flthn), and phenanthrene (Phe) combined with B[a]P on lung tumor promotion. To test this hypothesis, we used a two-stage, initiation/promotion BALB/ByJ female lung tumor mouse model. The mice were initiated with 3-methylcholanthrene followed by exposures to B[a]P, the LMW PAH mixture, and the combination of the LMW PAH mixture plus B[a]P, all at 10 mg/kg. The LMW PAHs combined with B[a]P significantly increased the promotion and incidence of lung tumors over that of B[a]P alone. The LMW PAHs in the absence of B[a]P did not significantly promote tumors, indicating strong co-promotional activities. We further assessed the effects of these PAHs on other hallmarks of cancer, namely, bronchoalveolar lavage fluid inflammatory infiltrates, pro-inflammatory transcripts, KC protein content, and mRNA expression of the gap junction (Gja1) and epiregulin (Ereg) genes. The LMW PAHs increased the biomarkers of inflammation, decreased Gja1 expression, and increased Ereg expression, all consistent with tumor promotion. This study indicates that non-genotoxic LMW PAHs can contribute to the cancer process and warrants further studies to assess the carcinogenic risks of other LMW PAHs.

Rationally designed multimeric nanovaccines using icosahedral DNA origami for display of SARS-CoV-2 receptor binding domain.

Multivalent antigen display on nanoparticles can enhance the immunogenicity of nanovaccines targeting viral moieties, such as the receptor binding domain (RBD) of SARS-CoV-2. However, particle morphology and size of current nanovaccines are significantly different from those of SARS-CoV-2. Additionally, surface antigen patterns are not controllable to enable the optimization of B cell activation. Herein, we employ an icosahedral DNA origami (ICO) as a display particle for RBD nanovaccines, achieving morphology and diameter like the virus (91 ± 11 nm). The surface addressability of DNA origami permits facile modification of the ICO surface with numerous RBD antigen clusters (ICO-RBD) to form various antigen patterns. Using an in vitro screening system, we demonstrate that the antigen spacing, antigen copies within clusters and cluster number parameters of the surface antigen pattern all impact the ability of the nanovaccines to activate B cells. Importantly, the optimized ICO-RBD nanovaccines evoke stronger and more enduring humoral and T cell immune responses in female mouse models compared to soluble RBD antigens, and the multivalent display broaden the protection range of B cell responses to more mutant strains. Our vaccines activate similar humoral immunity, observable stronger cellular immunity and more memory immune cells compared to trimeric mRNA vaccines.

Metabolic syndrome impairs endometrial functioning and early pregnancy: an in vivo study.

Metabolic syndrome (MS) is increasingly associated with impaired reproductive health. This study aimed to assess the endometrial characteristics and reproductive outcomes of a female MS mouse model and evaluate metformin's therapeutic effects. Twenty-one-day-old female C57BL/6 mice were randomly divided into a high-fat diet group (N = 50) and a control group (N = 30) that received standard chow. After 11 weeks, a subset of HF mice (N = 25) was given oral metformin at 300 mg/kg/day, while the other ones continued on HF diet. After 15 weeks, mice were either sacrificed during estrus or mated and euthanized on day 7.5 of pregnancy (N = 15 per group). The estrous cycle, progesterone and estradiol levels, uterine morphology, endometrial cell proliferation, reproductive performance, and metformin's treatment effects were assessed. Mice on a high-fat diet developed MS, which was characterized by moderate glycemic dysregulation, increased cholesterol, insulin resistance, and central obesity. Experimental MS caused estrous cycle disruptions and increased serum progesterone levels, which were normalized by metformin. MS also affected endometrial histology, producing hyperplasia and altering cell proliferation, while metformin restored normal endometrial architecture by inhibiting cell proliferation. Additionally, MS impaired reproductive success by delaying coitus and reducing the ratio of implantation sites to corpora lutea, both of which were rectified by metformin. In conclusion, MS adversely affects reproductive function, but metformin offers improvement. Our findings highlight the need for further research on the impact of MS on reproduction and the exploration of treatments to enhance reproductive health in women with MS.

Dynamin-1 is a potential mediator in cancer-related cognitive impairment

Dynamin-1 (DNM1) is crucial for synaptic activity, neurotransmission, and associative memory, positioning it as a potential biomarker of cancer-related cognitive impairment (CRCI), a neurological consequence of cancer treatment characterized by memory loss, poor concentration, and impaired executive function. Through a stepwise approach, this study investigated the role of DNM1 in CRCI pathogenesis, incorporating both human data and animal models. The human study recruited newly diagnosed, chemotherapy-naïve adolescent and young adult cancer and non-cancer controls to complete a cognitive instrument (FACT-Cog) and blood draws for up to three time points. Following that, a syngeneic young-adult WT (C57BL/6) female mouse model of breast cancer chemobrain was developed to study DNM1 expression in the hippocampus. Samples from eighty-six participants with 30 adolescent and young adult (AYA) cancer and 56 non-cancer participants were analyzed. DNM1 levels were 32 ​% lower (P ​= ​0.041) among cancer participants compared to non-cancer prior to treatment. After receiving cytotoxic treatment, cognitively impaired cancer patients were found to have 46 ​% lower DNM1 levels than those without impairment (P ​= ​0.049). In murine breast cancer-bearing mice receiving chemotherapy, we found a greater than 40 ​% decline (P ​< ​0.0001) in DNM1 immunoreactivity in the hippocampal CA1 and CA3 subregions concurrent with a deterioration in spatial recognition memory (P ​< ​0.02), compared to control mice without exposure to cancer and chemotherapy. Consistently observed in both human and animal studies, the downregulation of DNM1 is linked with the onset of CRCI. DNM1 might be a biomarker and therapeutic target for CRCI.

H2S scavenger as a broad-spectrum strategy to deplete bacteria-derived H2S for antibacterial sensitization

Bacteria-derived H2S plays multifunctional protective roles against antibiotics insult, and the H2S biogenesis pathway is emerging as a viable target for the antibacterial adjuvant design. However, the development of a pan-inhibitor against H2S-synthesizing enzymes is challenging and underdeveloped. Herein, we propose an alternative strategy to downregulate the H2S levels in H2S-producing bacteria, which depletes the bacteria-derived H2S chemically by H2S scavengers without acting on the synthesizing enzymes. After the screening of chemically diversified scaffolds and a structural optimization campaign, a potent and specific H2S scavenger is successfully identified, which displays efficient H2S depletion in several H2S-producing bacteria, potentiates both bactericidal agents and photodynamic therapy, enhances the bacterial clearance of macrophages and polymorphonuclear neutrophils, disrupts the formation of bacterial biofilm and increases the sensitivity of bacterial persister cells to antibiotics. Most importantly, such an H2S scavenger exhibits sensitizing effects with gentamicin in Pseudomonas aeruginosa -infected pneumonia and skin wound female mouse models. In aggregate, our results not only provide an effective strategy to deplete bacteria-derived H2S and establish the H2S biogenesis pathway as a viable target for persisters and drug-resistant bacteria, but also deliver a promising antibacterial adjuvant for potential clinical translation.

BMSCs-EVs Alleviate Pelvic Floor Dysfunction in Mice by Reducing Inflammation and Promoting Tissue Regeneration.

Pelvic floor dysfunctions (PFDs), which encompass pelvic organ prolapse (POP), stress urinary incontinence (SUI), and anal incontinence (AI), are common degenerative diseases in women. Bone marrow mesenchymal stem cells (BMSCs) hold promise for the treatment of PFDs. Extracellular vesicles (EVs) derived from BMSCs, have displayed an extensive role in intercellular communication and tissue repair. However, efficacy of the treatment using EVs originated from BMSCs on mouse models of PFD remains unknown. This study investigated the therapeutic potential of BMSC-derived EVs in a female PFD mouse model induced by vaginal distension (VD). Flow cytometry analysis confirmed the positive expression of BMSC-related markers, and successful induction of multilineage differentiation further validated their characteristics. As expected, the EVs extracted from BMSCs exhibited typical cup-shaped and circular-shaped structures. In the PFD model, BMSC-derived EVs significantly reduced the levels of inflammatory cytokines (p<0.05), improved tissue repair, and mitigated neutrophil infiltration. Furthermore, EVs promoted cell proliferation, decreased expression of relaxin receptors, increased expression of elastin, and elevated collagen content in the anterior vaginal wall tissue (p<0.05), suggesting beneficial effects on tissue regeneration and connective tissue restoration in PFD. BMSC-derived EVs effectively reduce tissue inflammation, promote tissue regeneration and connective tissue reconstruction, and improve pelvic support deficiency, thereby alleviating PFD induced by vaginal distension (VD) in vivo.

Reversion of metabolic dysfunction-associated steatohepatitis by skeletal muscle-directed FGF21 gene therapy

The highly prevalent metabolic dysfunction-associated steatohepatitis (MASH) is associated with liver steatosis, inflammation, and hepatocyte injury, which can lead to fibrosis and may progress to hepatocellular carcinoma and death. New treatment modalities such as gene therapy may be transformative for MASH patients. Here, we describe that one-time intramuscular administration of adeno-associated viral vectors of serotype 1 (AAV1) encoding native fibroblast growth factor 21 (FGF21), a key metabolic regulator, resulted in sustained increased circulating levels of the factor, which mediated long-term (>1 year) MASH and hepatic fibrosis reversion and halted development of liver tumors in obese male and female mouse models. AAV1-FGF21 treatment also counteracted obesity, adiposity, and insulin resistance, which are significant drivers of MASH. Scale-up to large animals successfully resulted in safe skeletal muscle biodistribution and biological activity in key metabolic tissues. Moreover, as a step toward the clinic, circulating FGF21 levels were characterized in obese, insulin-resistant and MASH patients. Overall, these results underscore the potential of the muscle-directed AAV1-FGF21 gene therapy to treat MASH and support its clinical translation.

Lifespan of male and female APP/PS1 and APPNL-F/NL-F mouse models of Alzheimer's disease.

Alzheimer's disease (AD) disproportionately affects women, yet most preclinical research studies are male-centric. We performed lifespan analyses of male and female AD mouse models (APP/PS1 and APPNL-F/NL-F) and their shared genetic background control (C57BL/6). Survival curves support significant sex differences between within genotypes. Minimal longevity revealed increased age in male APP/PS1, and decreased age in APPNL-F/NL-F mice. Maximal longevity revealed an increased average age in males. Furthermore, median lifespan differed between sex and genotype. This study supports sexual dimorphic survival in two mouse models of AD, emphasizing the need to examine mechanisms and treatments in both sexes.

Sympathetic Innervation of Interscapular Brown Adipose Tissue Is Not a Predominant Mediator of Oxytocin-Induced Brown Adipose Tissue Thermogenesis in Female High Fat Diet-Fed Rats.

Recent studies have indicated that hindbrain [fourth ventricle (4V)] administration of the neurohypophyseal hormone, oxytocin (OT), reduces body weight, energy intake and stimulates interscapular brown adipose tissue temperature (TIBAT) in male diet-induced obese (DIO) rats. What remains unclear is whether chronic hindbrain (4V) OT can impact body weight in female high fat diet-fed (HFD) rodents and whether this involves activation of brown adipose tissue (BAT). We hypothesized that OT-elicited stimulation of sympathetic nervous system (SNS) activation of interscapular brown adipose tissue (IBAT) contributes to its ability to activate BAT and reduce body weight in female high HFD-fed rats. To test this hypothesis, we determined the effect of disrupting SNS activation of IBAT on OT-elicited stimulation of TIBAT and reduction of body weight in DIO rats. We first measured the impact of bilateral surgical SNS denervation to IBAT on the ability of acute 4V OT (0.5, 1, and 5 µg ≈ 0.5, 0.99, and 4.96 nmol) to stimulate TIBAT in female HFD-fed rats. We found that the high dose of 4V OT (5 µg ≈ 4.96 nmol) stimulated TIBAT similarly between sham rats and denervated rats (p = NS). We subsequently measured the effect of bilateral surgical denervation of IBAT on the effect of chronic 4V OT (16 nmol/day ≈ 16.1 μg/day) or vehicle infusion to reduce body weight, adiposity and energy intake in female HFD-fed rats (N = 7-8/group). Chronic 4V OT reduced body weight gain (sham: -18.0 ± 4.9 g; denervation: -15.9 ± 3.7 g) and adiposity (sham: -13.9 ± 3.7 g; denervation: -13.6 ± 2.4 g) relative to vehicle treatment (p < 0.05) and these effects were similar between groups (p = NS). These effects were attributed, in part, to reduced energy intake evident during weeks 2 (p < 0.05) and 3 (p < 0.05). To test whether these results translate to other female rodent species, we also examined the effect of chronic 4V infusion of OT on body weight and adiposity in two strains of female HFD-fed mice. Similar to what we found in the HFD-fed rat model, we also found that chronic 4V OT (16 nmol/day) infusion resulted in reduced body weight gain, adiposity and energy intake in female DIO C57BL/6J and DBA/2J mice (p < 0.05 vs. vehicle). Together, these findings suggest that (1) sympathetic innervation of IBAT is not necessary for OT-elicited increases in BAT thermogenesis and weight loss in female HFD-fed rats and (2) the effects of OT to reduce weight gain and adiposity translate to other female mouse models of diet-induced obesity (DIO).

Improved influenza vaccine responses after expression of multiple viral glycoproteins from a single mRNA.

Influenza viruses cause substantial morbidity and mortality every year despite seasonal vaccination. mRNA-based vaccines have the potential to elicit more protective immune responses, but for maximal breadth and durability, it is desirable to deliver both the viral hemagglutinin and neuraminidase glycoproteins. Delivering multiple antigens individually, however, complicates manufacturingand increases cost, thus it would be beneficial to express both proteins from a single mRNA. Here, we develop an mRNA genetic configuration that allows the simultaneous expression of unmodified, full-length NA and HA proteins from a single open reading frame. We apply this approach to glycoproteins from contemporary influenza A and B viruses and, after vaccination, observe high levels of functional antibodies and protection from disease in female mouse and male ferret challenge models. This approach may further efforts to utilize mRNA technology to improve seasonal vaccine efficacy by efficiently delivering multiple viral antigens simultaneously and in their native state.

Exercise activates AMPK in mouse and human pancreatic islets to decrease senescence.

Beta (β)-cell senescence contributes to type 2 diabetes mellitus (T2DM). While exercise is vital for T2DM management and significantly affects cellular ageing markers, its effect on β-cell senescence remains unexplored. Here, we show that short-term endurance exercise training (treadmill running, 1 h per day for 10 days) in two male and female mouse models of insulin resistance decreases β-cell senescence. In vivo and in vitro experiments revealed that this effect is mediated, at least in part, by training-induced increases in serum glucagon, leading to activation of 5'-AMP-activated protein kinase (AMPK) signalling in β-cells. AMPK activation resulted in the nuclear translocation of NRF2 and decreased expression of senescence markers and effectors. Remarkably, human islets from male and female donors with T2DM treated with serum collected after a 10-week endurance exercise training programme showed a significant decrease in the levels of senescence markers. These findings indicate that exercise training decreases senescence in pancreatic islets, offering promising therapeutic implications for T2DM.

Clinical-grade intranasal NGF fuels neurological and metabolic functions of Mecp2-deficient mice

Abstract MECP2 deficiency causes a broad spectrum of neuropsychiatric disorders that can affect both genders. Rett syndrome is the most common and is characterized by an apparently normal growth period followed by a regression phase in which patients lose most of their previously acquired skills. After this dramatic period, various symptoms progressively appear, including severe intellectual disability, epilepsy, apraxia, breathing abnormalities and motor deterioration. MECP2 encodes for an epigenetic transcription factor that is particularly abundant in the brain; consequently, several transcriptional defects characterize the Rett syndrome brain. The well-known deficiency of several neurotrophins and growth factors, together with the positive effects exerted by trofinetide, a synthetic analogue of insulin-like growth factor 1, in Rett patients and in mouse models of Mecp2 deficiency, prompted us to investigate the therapeutic potential of nerve growth factor. Initial in vitro studies demonstrated a healing effect of recombinant human GMP-grade NGF (rhNGF) on neuronal maturation and activity in cultured Mecp2-null neurons. Subsequently, we designed in vivo studies with clear translational potential using intranasally administered rhNGF already used in the clinic. The efficacy of rhNGF in vivo in Mecp2-null hemizygous male mice and heterozygous female mice was assessed. General well-being was evaluated by a conventional phenotypic score and motor performance through the Pole and Beam Walking tests, while cognitive function and interaction with the environment were measured by the Novel Object Recognition test and the Marble Burying test, respectively. At the end of the treatment, mouse cortices were dissected and bulk RNA sequencing was performed to identify the molecular pathways involved in the protective effects of rhNGF. In both male and female mouse models of Rett syndrome, rhNGF exerted positive effects on cognitive and motor functions. In male hemizygous mice, which suffer from significantly more severe and rapidly advancing symptoms, the drug’s ability to slow the disease’s progression was more pronounced. The unbiased research for the molecular mechanisms triggering the observed benefits revealed a strong positive effect on gene sets related to oxidative phosphorylation, mitochondrial structure and function. These results were validated by demonstrating the drug’s ability to improve mitochondrial structure and respiration in Mecp2-null cerebral cortices. Furthermore, Gene Ontology analyses indicated that NGF exerted the expected improvement in neuronal maturation. We conclude that intranasal administration of rhNGF is a non-invasive and effective route of administration for the treatment of Rett syndrome and possibly for other neurometabolic disorders with overt mitochondrial dysfunction.

Pharmacological inhibition of the CB1 cannabinoid receptor restores abnormal brain mitochondrial CB1 receptor expression and rescues bioenergetic and cognitive defects in a female mouse model of Rett syndrome

BackgroundDefective mitochondria and aberrant brain mitochondrial bioenergetics are consistent features in syndromic intellectual disability disorders, such as Rett syndrome (RTT), a rare neurologic disorder that severely affects mainly females carrying mutations in the X-linked MECP2 gene. A pool of CB1 cannabinoid receptors (CB1R), the primary receptor subtype of the endocannabinoid system in the brain, is located on brain mitochondrial membranes (mtCB1R), where it can locally regulate energy production, synaptic transmission and memory abilities through the inhibition of the intra-mitochondrial protein kinase A (mtPKA). In the present study, we asked whether an overactive mtCB1R-mtPKA signaling might underlie the brain mitochondrial alterations in RTT and whether its modulation by systemic administration of the CB1R inverse agonist rimonabant might improve bioenergetics and cognitive defects in mice modeling RTT.MethodsRimonabant (0.3 mg/kg/day, intraperitoneal injections) was administered daily to symptomatic female mice carrying a truncating mutation of the Mecp2 gene and its effects on brain mitochondria functionality, systemic oxidative status, and memory function were assessed.ResultsmtCB1R is overexpressed in the RTT mouse brain. Subchronic treatment with rimonabant normalizes mtCB1R expression in RTT mouse brains, boosts mtPKA signaling, and restores the defective brain mitochondrial bioenergetics, abnormal peripheral redox homeostasis, and impaired cognitive abilities in RTT mice.LimitationsThe lack of selectivity of the rimonabant treatment towards mtCB1R does not allow us to exclude that the beneficial effects exerted by the treatment in the RTT mouse model may be ascribed more broadly to the modulation of CB1R activity and distribution among intracellular compartments, rather than to a selective effect on mtCB1R-mediated signaling. The low sample size of few experiments is a further limitation that has been addressed replicating the main findings under different experimental conditions.ConclusionsThe present data identify mtCB1R overexpression as a novel molecular alteration in the RTT mouse brain that may underlie defective brain mitochondrial bioenergetics and cognitive dysfunction.

A Foundation Model Identifies Broad-Spectrum Antimicrobial Peptides against Drug-Resistant Bacterial Infection

Development of potent and broad-spectrum antimicrobial peptides (AMPs) could help overcome the antimicrobial resistance crisis. We develop a peptide language-based deep generative framework (deepAMP) for identifying potent, broad-spectrum AMPs. Using deepAMP to reduce antimicrobial resistance and enhance the membrane-disrupting abilities of AMPs, we identify, synthesize, and experimentally test 18 T1-AMP (Tier 1) and 11 T2-AMP (Tier 2) candidates in a two-round design and by employing cross-optimization-validation. More than 90% of the designed AMPs show a better inhibition than penetratin in both Gram-positive (i.e., S. aureus) and Gram-negative bacteria (i.e., K. pneumoniae and P. aeruginosa). T2-9 shows the strongest antibacterial activity, comparable to FDA-approved antibiotics. We show that three AMPs (T1-2, T1-5 and T2-10) significantly reduce resistance to S. aureus compared to ciprofloxacin and are effective against skin wound infection in a female wound mouse model infected with P. aeruginosa. In summary, deepAMP expedites discovery of effective, broad-spectrum AMPs against drug-resistant bacteria.

Single-cell RNA sequencing reveals the effects of high-fat diet on oocyte and early embryo development in female mice

BackgroundObesity is a global health issue with detrimental effects on various human organs, including the reproductive system. Observational human data and several lines of animal experimental data suggest that maternal obesity impairs ovarian function and early embryo development, but the precise pathogenesis remains unclear.MethodsWe established a high-fat diet (HFD)-induced obese female mouse model to assess systemic metabolism, ovarian morphology, and oocyte function in mice. For the first time, this study employed single-cell RNA sequencing to explore the altered transcriptomic landscape of preimplantation embryos at different stages in HFD-induced obese mice. Differential gene expression analysis, enrichment analysis and protein-protein interactions network analysis were performed.ResultsHFD-induced obese female mice exhibited impaired glucolipid metabolism and insulin resistance. The ovaries of HFD mice had a reduced total follicle number, an increased proportion of atretic follicles, and irregular granulosa cell arrangement. Furthermore, the maturation rate of embryonic development by in vitro fertilization of oocytes was significantly decreased in HFD mice. Additionally, the transcriptional landscapes of preimplantation embryos at different stages in mice induced by different diets were significantly distinguished. The maternal-to-zygotic transition was also affected by the failure to remove maternal RNAs and to turn off zygotic genome expression.ConclusionsHFD-induced obesity impaired ovarian morphology and oocyte function in female mice and further led to alterations in the transcriptional landscape of preimplantation embryos at different stages of HFD mice.

Plasma lipidomics in early APP/PS1 female mouse model and its relationship with brain: Is it affected by the estrous cycle?

BackgroundAlzheimer's disease (AD) is the most prevalent dementia, showing higher incidence in women. Besides, lipids play an essential role in brain, and they could be dysregulated in neurodegeneration. Specifically, impaired plasma lipid levels could predict early AD diagnosis. This work aims to identify the main plasma lipids altered in early AD female mouse model and evaluate their relationship with brain lipidome. Also, the possible involvement of the estrous cycle in lipid metabolism has been evaluated.MethodsPlasma samples of wild-type (n = 10) and APP/PS1 (n = 10) female mice of 5 months of age were collected, processed, and analysed using a lipidomic mass spectrometry-based method. A statistical analysis involving univariate and multivariate approaches was performed to identify significant lipid differences related to AD between groups. Also, cytology tests were conducted to confirm estrous cycle phases.ResultsThree hundred thirty lipids were detected in plasma, 18 of them showed significant differences between groups; specifically, some triacylglycerols, cholesteryl esters, lysophosphatidylcholines, phosphatidylcholines, and ether-linked phosphatidylcholines, increased in early AD; while other phosphatidylcholines, phosphatidylethanolamines, ceramides, and ether-linked phosphatidylethanolamines decreased in early AD. A multivariate approach was developed from some lipid variables, showing high diagnostic indexes (70% sensitivity, 90% specificity, 80% accuracy). From brain and plasma lipidome, some significant correlations were observed, mainly in the glycerophospholipid family. Also, some differences were found in both plasma and brain lipids, according to the estrous cycle phase.ConclusionsTherefore, lipid alterations can be identified in plasma at early AD stages in mice females, with a relationship with brain lipid metabolism for most of the lipid subfamilies, suggesting some lipids as potential AD biomarkers. In addition, the estrous cycle monitoring could be relevant in female studies.

Highly potent and broadly neutralizing anti-CD4 trimeric nanobodies inhibit HIV-1 infection by inducing CD4 conformational alteration

Despite advancements in antiretroviral therapy (ART) suppressing HIV-1 replication, existing antiviral drugs pose limitations, including lifelong medication, frequent administration, side effects and viral resistance, necessitating novel HIV-1 treatment approaches. CD4, pivotal for HIV-1 entry, poses challenges for drug development due to neutralization and cytotoxicity concerns. Nevertheless, Ibalizumab, the sole approved CD4-specific antibody for HIV-1 treatment, reignites interest in exploring alternative anti-HIV targets, emphasizing CD4’s potential value for effective drug development. Here, we explore anti-CD4 nanobodies, particularly Nb457 from a CD4-immunized alpaca. Nb457 displays high potency and broad-spectrum activity against HIV-1, surpassing Ibalizumab’s efficacy. Strikingly, engineered trimeric Nb457 nanobodies achieve complete inhibition against live HIV-1, outperforming Ibalizumab and parental Nb457. Structural analysis unveils Nb457-induced CD4 conformational changes impeding viral entry. Notably, Nb457 demonstrates therapeutic efficacy in humanized female mouse models. Our findings highlight anti-CD4 nanobodies as promising HIV-1 therapeutics, with potential implications for advancing clinical treatment against this global health challenge.

Cyclization increases bactericidal activity of arginine-rich cationic cell-penetrating peptide for Neisseria gonorrhoeae.

We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.