Cell selective BCL-2 inhibition enabled by lipid nanoparticles alleviates lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a high mortality rate due to limited treatment options. Current therapies cannot effectively reverse the damage caused by IPF. Research suggests that promoting programmed cell death (apoptosis) in myofibroblasts, the key cells driving fibrosis, could be a promising strategy. However, inducing apoptosis in healthy cells like epithelial and endothelial cells can cause unwanted side effects. This project addresses this challenge by developing a targeted approach to induce apoptosis specifically in myofibroblasts. We designed liposomes (LPS) decorated with peptides that recognize VCAM-1, a protein highly expressed on myofibroblasts in fibrotic lungs. These VCAM1-targeted LPS encapsulate Venetoclax (VNT), a small molecule drug that inhibits BCL-2, an anti-apoptotic protein. By delivering VNT directly to myofibroblasts, we hypothesize that VCAM1-VNT-LPS can selectively induce apoptosis in these cells, leading to reduced fibrosis and improved lung function. We successfully characterized VCAM1-VNT-LPS for size, surface charge, and drug loading efficiency. Additionally, we evaluated their stability over three months at different temperatures. In vitro and in vivo studies using a bleomycin-induced mouse model of lung fibrosis demonstrated the therapeutic potential of VCAM1-VNT-LPS. These studies showed a reduction in fibrosis-associated proteins (collagen, α-SMA, VCAM1) and BCL-2, while simultaneously increasing apoptosis in myofibroblasts. These findings suggest that VCAM1-targeted delivery of BCL-2 inhibitors using liposomes presents a promising and potentially selective therapeutic approach for IPF.

Cadherin-11 targeted cell-specific liposomes enabled skin fibrosis treatment by inducing apoptosis.

Continuous and aberrant activation of myofibroblasts is the hallmark of pathological fibrosis (e.g., abnormal wound healing). The deposition of excessive extracellular matrix (ECM) components alters or increases the stiffness of tissue and primarily accounts for multiple organ dysfunctions. Among various proteins, Cadherin-11 (CDH11) has been reported to be overexpressed on myofibroblasts in fibrotic tissues. Anti-apoptotic proteins such as (B cell lymphoma-2) (BCL-2) are also upregulated on myofibroblasts. Therefore, we hypothesize that CDH11 could be a targeted domain for cell-specific drug delivery and targeted inhibition of BCL-2 to ameliorate the development of fibrosis in the skin. To prove our hypothesis, we have developed liposomes (LPS) conjugated with CDH11 neutralizing antibody (antiCDH11) to target cell surface CDH11 and loaded these LPS with a BCL-2 inhibitor, Navitoclax (NAVI), to induce apoptosis of CDH11 expressing fibroblasts. The developed LPS were evaluated for physicochemical characterization, stability, in vitro therapeutic efficacy using dermal fibroblasts, and in vivo therapeutic efficacy in bleomycin-induced skin fibrosis model in mice. The findings from in vitro and in vivo studies confirmed that selectivity of LPS was improved towards CDH11 expressing myofibroblasts, thereby improving therapeutic efficacy with no indication of adverse effects. Hence, this novel research work represents a versatile LPS strategy that exhibits promising potential for treating skin fibrosis.

Big is not better: Comparing two alpha-Gal-bearing glycotopes in neoglycoproteins as biomarkers for Leishmania (Viannia) braziliensis infection.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfß conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfß1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

Traversing the Cell Wall: The Chitinolytic Activity of Histoplasma capsulatum Extracellular Vesicles Facilitates Their Release.

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis.

Pancreatic cancer is a highly lethal disease with obesity as one of the risk factors. Oncogenic KRAS mutations are prevalent in pancreatic cancer and can rewire lipid metabolism by altering fatty acid (FA) uptake, FA oxidation (FAO), and lipogenesis. Identification of the underlying mechanisms could lead to improved therapeutic strategies for treating KRAS-mutant pancreatic cancer. Here, we observed that KRASG12D upregulated the expression of SLC25A1, a citrate transporter that is a key metabolic switch to mediate FAO, fatty acid synthesis, glycolysis, and gluconeogenesis. In genetically engineered mouse models and human pancreatic cancer cells, KRASG12D induced SLC25A1 upregulation via GLI1, which directly stimulated SLC25A1 transcription by binding its promoter. The enhanced expression of SLC25A1 increased levels of cytosolic citrate, FAs, and key enzymes in lipid metabolism. In addition, a high-fat diet (HFD) further stimulated the KRASG12D-GLI1-SLC25A1 axis and the associated increase in citrate and FAs. Pharmacologic inhibition of SLC25A1 and upstream GLI1 significantly suppressed pancreatic tumorigenesis in KrasG12D/+ mice on a HFD. These results reveal a KRASG12D-GLI1-SLC25A1 regulatory axis, with SLC25A1 as an important node that regulates lipid metabolism during pancreatic tumorigenesis, thus indicating an intervention strategy for oncogenic KRAS-driven pancreatic cancer. SIGNIFICANCE: Upregulation of SLC25A1 induced by KRASG12D-GLI1 signaling rewires lipid metabolism and is exacerbated by HFD to drive the development of pancreatic cancer, representing a targetable metabolic axis to suppress pancreatic tumorigenesis.

Withdrawal from repeated nicotine vapor exposure increases somatic signs of physical dependence, anxiety-like behavior, and brain reward thresholds in adult male rats.

Nicotine vapor consumption via electronic nicotine delivery systems has increased over the last decade. While prior work has shed light on the health effects of nicotine vapor inhalation, its unique effects on the brain and behavior have not been thoroughly explored. In this study we assessed markers of withdrawal following 14 days of nicotine vapor exposure. For Experiment 1, 21 adult male rats were exposed to ambient air or 6, 12, or 24 mg/mL nicotine vapor for 14 consecutive days. Following exposure on day 14, rats were injected with the nicotinic receptor antagonist mecamylamine (3.0 mg/mL) and assessed for somatic withdrawal signs and anxiety-like behavior in the elevated plus maze. For Experiment 2, 12 adult male rats were tested for intracranial self-stimulation (ICSS) immediately following exposure to vehicle vapor (50%/50%, vegetable glycerin/propylene glycol) or 24 mg/mL nicotine vapor, for 14 consecutive days. ICSS behavior was assessed for an additional 14 days, following cessation of repeated vapor exposure. Results reveal that rats with repeated nicotine vapor exposure display an increase in behavioral indicators of withdrawal following injection of mecamylamine (precipitated withdrawal). Additionally, increases in ICSS stimulation thresholds, indicative of reduced brain reward sensitivity, persist following cessation of repeated nicotine vapor exposure (spontaneous withdrawal). These data suggest that repeated e-cigarette use leads to nicotine dependence and withdrawal that affects behavior and brain reward function. Further characterization of the health effects of nicotine vapor is necessary to improve treatment strategies for nicotine use disorder and public health policies related to novel nicotine delivery systems.

Fatty acid elongases 1-3 have distinct roles in mitochondrial function, growth, and lipid homeostasis in Trypanosoma cruzi.

Trypanosomatids are a diverse group of uniflagellate protozoan parasites that include globally relevant pathogens such as Trypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase system typically used for de novo fatty acid (FA) synthesis in other eukaryotes. Instead, these microbes have evolved a modular FA elongase (ELO) system comprised of individual ELO enzymes (ELO1-4) that can operate processively to generate long chain- and very long chain-FAs. The importance of ELO's for maintaining lipid homeostasis in trypanosomatids is currently unclear, given their ability to take up and utilize exogenous FAs for lipid synthesis. To assess ELO function in T. cruzi, we generated individual KO lines, Δelo1, Δelo2, and Δelo3, in which the genes encoding ELO1-3 were functionally disrupted in the parasite insect stage (epimastigote). Using unbiased lipidomic and metabolomic analyses, in combination with metabolic tracing and biochemical approaches, we demonstrate that ELO2 and ELO3 are required for global lipid homeostasis, whereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth in T. cruzi epimastigotes. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that, we propose, merits further examination of the trypanosomatid ELO pathway as critical for central metabolism.

Synthesis and Photoreactivity of 7-Nitroindoline-S-thiocarbamates.

The photolytic properties of N-acyl-7-nitroindolines make these compounds attractive as photocleavable protecting groups and "caged" compounds for the light-induced release ("uncaging") of biologically active compounds and as acylating reagents under neutral conditions. However, the synthesis of N-acyl-7-nitroindolines usually requires multiple steps, and the direct acylation of 7-nitroindolines can be quite challenging. 7-Nitroindolines with other types of N-carbonyl-containing groups may also be photoreactive and could potentially be better accessible. Here we demonstrate the short and efficient synthesis of 5-bromo-7-nitroindoline-S-thiocarbamates, a new class of photoreactive compounds, and the study of some of their photochemical and photophysical properties. Using 5-bromo-7-nitroindoline-S-ethylthiocarbamate as a model compound, we show that it can undergo one-photon and two-photon photolysis at 350 and 710 nm, respectively. Our experimental data and quantum chemistry calculations support a photolysis pathway that differs from photolysis pathways previously reported for N-acyl-7-nitroindolines. The photolysis with 350 nm light results in 5-bromo-7-nitrosoindoline, which is in equilibrium with its dimeric form(s), as supported by experiment and theory. This study expands the scope of photoreactive 7-nitroindoline derivatives and informs the development of novel photocleavable compounds.

Characterization of the zebrafish gabra1sa43718/sa43718 germline loss of function allele confirms a function for Gabra1 in motility and nervous system development.

Mutation of the GABRA1 gene is associated with neurodevelopmental defects and epilepsy. GABRA1 encodes for the α1 subunit of the gamma-aminobutyric acid type A receptor (GABAAR), which regulates the fast inhibitory impulses of the nervous system. Multiple model systems have previously been developed to understand the function of GABRA1 during development, but these models have produced complex and at times incongruent data. Thus, additional model systems are required to validate and substantiate previously published results. We investigated the behavioral swim patterns associated with a nonsense mutation of the zebrafish gabra1 (sa43718 allele) gene. The sa43718 allele causes a decrease in gabra1 mRNA expression, which is associated with light induced hypermotility, one phenotype associated with seizure like behavior in zebrafish. Mutation of gabra1 was accompanied by decreased mRNA expression of gabra2, gabra3, and gabra5, indicating a reduction in the expression of additional alpha sub-units of the GABAAR. Although multiple sub-units were decreased in total expression, larvae continued to respond to pentylenetetrazole (PTZ) indicating that a residual GABAAR exists in the sa43718 allele. Proteomics analysis demonstrated that nonsense mutation of gabra1 is associated with abnormal expression of proteins that regulate proton transport, ion homeostasis, vesicle transport, and mitochondrial protein complexes. These data support previous studies performed in a zebrafish nonsense allele created by CRISPR/Cas9 and validate that loss of function mutations in the gabra1 gene result in seizure like phenotypes with abnormal function of inhibitory synapses.

Missense and nonsense mutations of the zebrafish hcfc1a gene result in contrasting mTor and radial glial phenotypes.

Mutations in the HCFC1 transcriptional co-factor protein are the cause of cblX syndrome and X-linked intellectual disability (XLID). cblX is the more severe disorder associated with intractable epilepsy, abnormal cobalamin metabolism, facial dysmorphia, cortical gyral malformations, and intellectual disability. In vitro, murine Hcfc1 regulates neural precursor (NPCs) proliferation and number, which has been validated in zebrafish. However, conditional deletion of mouse Hcfc1 in Nkx2.1 + cells increased cell death, reduced Gfap expression, and reduced numbers of GABAergic neurons. Thus, the role of this gene in brain development is not completely understood. Recently, knock-in of both a cblX (HCFC1) and cblX-like (THAP11) allele were created in mice. Knock-in of the cblX-like allele was associated with increased expression of proteins required for ribosome biogenesis. However, the brain phenotypes were not comprehensively studied due to sub-viability. Therefore, a mechanism underlying increased ribosome biogenesis was not described. We used a missense, a nonsense, and two conditional zebrafish alleles to further elucidate this mechanism during brain development. We observed contrasting phenotypes at the level of Akt/mTor activation, the number of radial glial cells, and the expression of two downstream target genes of HCFC1, asxl1 and ywhab. Despite these divergent phenotypes, each allele studied demonstrates with a high degree of face validity when compared to the phenotypes reported in the literature. Collectively, these data suggest that individual mutations in the HCFC1 protein result in differential mTOR activity which may be associated with contrasting cellular phenotypes.

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6-7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galß. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

Serodiagnosis and therapeutic monitoring of New-World tegumentary leishmaniasis using synthetic type-2 glycoinositolphospholipid-based neoglycoproteins.

American tegumentary leishmaniasis (TL) caused by Leishmania braziliensis is characterized by a spectrum of clinical presentations, ranging from localized cutaneous ulcers (CL), mucosal (ML), or disseminated (DL) disease, to a subclinical (SC) asymptomatic form. Current diagnosis based on parasite culture and/or microscopy lacks sensitivity and specificity. Previous studies showed that patients with CL and ML have very high levels of Leishmania-specific anti-α-Gal antibodies. However, the native parasite α-Gal glycotope(s) is(are) still elusive, thus they have not yet been explored for a more accurate TL diagnosis. Using a chemiluminescent immunoassay, we evaluated the seroreactivity of TL patients across its clinical spectrum, and of endemic (EC) and nonendemic healthy controls (NEC) against three synthetic neoglycoproteins (NGP29b, NGP30b, and NGP28b), respectively comprising the L. major-derived type-2 glycoinositolphospholipid (GIPL)-1 (Galfß1,3Manα), GIPL-2 (Galα1,3Galfß1,3Manα), and GIPL-3 (Galα1,6Galα1,3Galfß) glycotopes. Contrary to NGP29b and NGP30b, NGP28b exhibited high sensitivity and specificity to a CL serum pool. More importantly, NGP28b reacted strongly and specifically with individual sera from distinct clinical forms of TL, especially with SC sera, with 94% sensitivity and 97% specificity, by post-two-graph receiver-operating characteristic curve analysis. Contrary to NGP29b, NGP28b showed low cross-reactivity with Chagas disease and control (NEC/EC) sera. Additionally, seroreactivity of CL patients against NGP28b was significantly decreased after successful chemotherapy, indicating that L. braziliensis-specific anti-α-Gal antibodies may serve as an early biomarker of cure in CL. Our data also points towards the applicability of L. major type-2 GIPL-3-derived Galα1,6Galα1,3Galfß glycotope for the serological diagnosis of American TL, particularly of the subclinical form.

A comparative proteomics study of Arabidopsis thaliana responding to the coexistence of BPA and TiO2-NPs at environmentally relevant concentrations.

Through the applications of recycling sewage sludge to soils as nutrients, bisphenol A (BPA) and titanium dioxide nanoparticles (TiO2-NPs) are commonly found in the agricultural environment. Previous studies have reported that BPA and nanoparticles are harmful to the environment. However, the combined toxicity of both compounds is not yet understood. This work presented an in-depth proteomic analysis of Arabidopsis thaliana exposed to BPA and TiO2-NPs concurrently at environmentally relevant levels. Seeds were simultaneously treated with varying concentrations of BPA (0, 10, 100, and 1000 µg·kg-1) and TiO2-NPs (0, 1, 10 and 100 mg·kg-1). In treatment of 1000 µg·kg-1 BPA and 100 mg·kg-1 TiO2-NPs, highest seed germination rate (87.97%, p < 0.05) was observed. Shorter primary roots but more branched roots were obtained in treatments of high BPA and NPs concentrations (100, 1000 µg·kg-1 BPA and 10, 100 mg·kg-1 TiO2-NPs) while no significant effects on plant height and biomass were found. In the comparative analysis, both concentration related positive and negative effects were observed, such as regulation of cell proliferation (positive), root hair elongation (positive), cellular response to oxidative stress (negative), and cell wall organization (negative). In response to the stress caused by BPA and TiO2-NPs, some proteins related to plant root development, such as CD48E, DNAJ2 and GL24, were up-regulated explaining the shorter primary root length and more branched roots. Moreover, Arabidopsis may have stimulated its ability of resource transportation and energy metabolism to overcome the stress and maintain or somehow enhance their growth by up-regulating proteins like TBB6, CALM1, RAA2A, G3PP2 and KASC1. Our comparative proteomics analysis also highlighted multiple biological processes that consequently lead to the stability of plant growth and its stress adaptation. The results demonstrated that applying biosolids to soil as a fertilizer may be considered as a sustainable practice.

Specific Recognition of ß-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients.

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients' blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method's limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal ß-galactofuranosyl (ß-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients' sera react specifically with synthetic ß-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfß1,3Manpα-(CH2)3SH (glycan G29SH) and Galfß1,3Manpα1,2-[Galfß1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfß1,3Manpα1,2-[Galfß1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These N-glycans are predicted to modulate spike binding to the host receptor by stabilizing its open conformation and host immunity evasion. Here, we investigated the essentiality of both the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Ablation of host N-glycosylation using RNA interference or inhibitors, including FDA-approved drugs, reduced the spread of the infection, including that of variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Under these conditions, cells produced fewer virions and some completely lost their infectivity. Furthermore, partial enzymatic deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, we propose protein N-glycosylation as a targetable pathway with clinical potential for treatment of COVID-19. IMPORTANCE The coronavirus SARS-CoV-2 uses its spike surface proteins to infect human cells. Spike proteins are heavily modified with several N-glycans, which are predicted to modulate their function. In this work, we show that interfering with either the synthesis or attachment of spike N-glycans significantly reduces the spread of SARS-CoV-2 infection in vitro, including that of several variants. As new SARS-CoV-2 variants, with various degrees of resistance against current vaccines, are likely to continue appearing, halting virus glycosylation using repurposed human drugs could result in a complementary strategy to reducing the spread of COVID-19 worldwide.