Electrochemical Investigation of the Stability of Poly-Phosphocholinated Liposomes.

Poly[2-(methacryloyloxy)ethyl phosphorylcholine] liposomes (pMPC liposomes) gained attention during the last few years because of their potential use in treating osteoarthritis. pMPC liposomes that serve as boundary lubricants are intended to restore the natural lubrication properties of articular cartilage. For this purpose, it is important that the liposomes remain intact and do not fuse and spread as a lipid film on the cartilage surface. Here, we investigate the stability of the liposomes and their interaction with two types of solid surfaces, gold and carbon, by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). With the aid of a hydrophilic species used as an electroactive probe in the solution, the charge transfer characteristics of the electrode surfaces are obtained. Additionally, from EIS, the capacitance characteristics of the surfaces are derived. No decrease of the peak currents and no displacement of the peak potentials to greater overpotentials are observed in the CV experiments. No decrease in the apparent capacitance and increase in the charge transfer resistance is observed in the EIS experiments. On the contrary, all parameters in both CV and EIS do change in the opposite direction. The obtained results confirm that there is only physical adsorption without fusion and spreading of the pMPC liposomes and without the formation of lipid films on the surfaces of both gold and carbon electrodes.

Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis.

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species-C1P, S1P, or SPC-IL-1ß, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1ß, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

American Cutaneous Leishmaniasis: Imported cases in Berlin 2000-2023.

BACKGROUND: American Cutaneous Leishmaniasis (ACL) shows variable response to therapy, but data on species-specific treatment efficacy is scarce. We describe the clinical characteristics and outcome of patients with ACL imported to a tertiary centre in Germany and determine whether species-specific therapy according to the 2014 "LeishMan" group recommendations is associated with cure. METHODS: A retrospective chart review was conducted at the Charité Institute of International Health in Berlin. We analysed data on PCR-confirmed ACL cases collected between 2000 and 2023. Systemic therapy included liposomal amphotericin B, miltefosine, pentavalent antimony, ketoconazole or itraconazole. Localized therapy included perilesional pentavalent antimony or paromomycin ointment. Cure was defined as re-epithelialization of ulcers or disappearance of papular-nodular lesions after 3 months of treatment. Logistic regression models were used to quantify the effect of species-specific systemic therapy on the outcome. RESULTS: 75 cases were analysed. Most patients were male (62%), median age was 35 years, no patient had a history of immunosuppression. The most common reason for travel was tourism (60%), the most common destination was Costa Rica (28%), the median duration of illness was 8 weeks, and most patients presented with ulcers (87%). Lesions were complex in 43%. The most common Leishmania (L.) species was L. braziliensis (28%), followed by L. panamensis (21%). 51/73 (70%) patients were cured after initial therapy and 17/21 (81%) after secondary therapy. Cure after systemic therapy was more frequent when species-specific treatment recommendations were followed (33/45; 73%), compared to when not followed, (6/17; 35%, P = 0.008). This association was independent of age, sex, previous therapy, complex lesions, and Leishmania species (adjusted OR, 5.06; 95% CI, 1.22-24.16). CONCLUSIONS: ACL is a rare, imported disease in Germany. Complex lesions were common, challenging successful therapy. This study highlights the importance of identifying the parasite species and suggests that a species-specific approach to treatment leads to better outcomes.

Mass Spectrometry Imaging Combined with Sparse Autoencoder Method Reveals Altered Phosphorylcholine Distribution in Imipramine Treated Wild-Type Mice Brains.

Mass spectrometry imaging (MSI) is essential for visualizing drug distribution, metabolites, and significant biomolecules in pharmacokinetic studies. This study mainly focuses on imipramine, a tricyclic antidepressant that affects endogenous metabolite concentrations. The aim was to use atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI)-MSI combined with different dimensionality reduction methods to examine the distribution and impact of imipramine on endogenous metabolites in the brains of treated wild-type mice. Brain sections from both control and imipramine-treated mice underwent AP-MALDI-MSI. Dimensionality reduction methods, including principal component analysis, multivariate curve resolution, and sparse autoencoder (SAE), were employed to extract valuable information from the MSI data. Only the SAE method identified phosphorylcholine (ChoP) as a potential marker distinguishing between the control and treated mice brains. Additionally, a significant decrease in ChoP accumulation was observed in the cerebellum, hypothalamus, thalamus, midbrain, caudate putamen, and striatum ventral regions of the treated mice brains. The application of dimensionality reduction methods, particularly the SAE method, to the AP-MALDI-MSI data is a novel approach for peak selection in AP-MALDI-MSI data analysis. This study revealed a significant decrease in ChoP in imipramine-treated mice brains.

Stability Enhancement by Hydrophobic Anchoring and a Cross-Linked Structure of a Phospholipid Copolymer Film for Medical Devices.

Surface modification using zwitterionic 2-methacryloyloxyethylphosphorylcholine (MPC) polymers is one of the most reasonable ways to prepare medical devices that can suppress undesired biological reactions such as blood coagulation. Usable MPC polymers are hydrophilic and water soluble, and their surface modification strategy involves exploiting the copolymer structures by adding physical or chemical bonding moieties. In this study, we developed copolymers composed of MPC, hydrophobic anchoring moiety, and chemical cross-linking unit to clarify the role of hydrophobic interactions in achieving biocompatible and long-term stable coatings. The four kinds of MPC copolymers with cross-linking units, such as 3-methacryloxypropyl trimethoxysilane (MPTMSi), and four different hydrophobic anchoring moieties, such as 3-(methacryloyloxy)propyltris(trimethylsiloxy)silane (MPTSSi) named as PMMMSi, n-butyl methacrylate (BMA) as PMBSi, 2-ethylhexyl methacrylate (EHMA) as PMESi, and lauryl methacrylate as PMLSi, were synthesized and coated on polydimethylsiloxane, polypropylene (PP), and polymethyl pentene. These copolymers were uniformly coated on the substrate materials PP and poly(methyl pentene) (PMP), to achieve hydrophilic and electrically neutral coatings. The results of the antibiofouling test showed that PMBSi repelled the adsorption of fluorescence-labeled bovine serum albumin the most, whereas PMLSi repelled it the least. Notably, all four copolymers suppressed platelet adhesion similarly. The variations in protein adsorption quantities among the four copolymer coatings were attributed to their distinct swelling behaviors in aqueous environments. Further investigations, including 3D scanning force microscopy and neutron reflectivity measurements, revealed that the PMLSi coating exhibited a higher water intake under aqueous conditions in comparison to the other coatings. Consequently, all copolymer coatings effectively prevented the invasion of platelets but the proteins penetrated the PMLSi network. Subsequently, the dynamic stability required to induce shear stress was evaluated using a circulation system. The results demonstrated that the PMMMSi and PMLSi coatings on PMP and PP exhibited exceptional platelet repellency and maintained high stability during circulation. This study highlights the potential of hydrophobic moieties to improve hemocompatibility and stability, offering potential applications in medical devices.

Fluorinated Polyethylenimine and Fluorinated Choline Phosphate Lipids Complex System for Efficient mRNA Delivery to Deep-Seated Tumor Tissues.

Efficiently delivering mRNA to the deep-seated cells of diseased tissues for therapeutic purposes remains a significant challenge. To address this, we leveraged the dual hydrophobic properties of fluorine atoms to conjugate fluorinated polyethylenimine (FPEI) with fluorinated choline phosphate (FCP) lipids. When one adjusted the ratio of N/F atoms to 2/1 and a 15% FCP content, the mRNA@FPEI-FCP carrier was optimized, achieving significant circulation and accumulation in deep tumor regions. Compared to control carriers lacking FCP or FPEI, mRNA@FPEI-FCP exhibited a 3.94-fold increase in tumor targeting and a 3.0-fold increase in deep delivery. Delivery of IL-2 mRNA to 4T1 breast tumors resulted in a tumor inhibition rate of 91.9%, with IL-2 levels reaching 149.2 pg/mL and 12.1% of CD4+ cells throughout the tumor, with no abnormal blood indexes. This FPEI and FCP composite delivery system demonstrates potent targeting of mRNA delivery to deep tumor tissues.

Determining tissue distribution of the oral antileishmanial agent miltefosine: a physiologically-based pharmacokinetic modeling approach.

Miltefosine (MTS) is the only approved oral drug for treating leishmaniasis caused by intracellular Leishmania parasites that localize in macrophages of the liver, spleen, skin, bone marrow, and lymph nodes. MTS is extensively distributed in tissues and has prolonged elimination half-lives due to its high plasma protein binding, slow metabolic clearance, and minimal urinary excretion. Thus, understanding and predicting the tissue distribution of MTS help assess therapeutic and toxicologic outcomes of MTS, especially in special populations, e.g., pediatrics. In this study, a whole-body physiologically-based pharmacokinetic (PBPK) model of MTS was built on mice and extrapolated to rats and humans. MTS plasma and tissue concentration data obtained by intravenous and oral administration to mice were fitted simultaneously to estimate model parameters. The resulting high tissue-to-plasma partition coefficient values corroborate extensive distribution in all major organs except the bone marrow. Sensitivity analysis suggests that plasma exposure is most susceptible to changes in fraction unbound in plasma. The murine oral-PBPK model was further validated by assessing overlay of simulations with plasma and tissue profiles obtained from an independent study. Subsequently, the murine PBPK model was extrapolated to rats and humans based on species-specific physiological and drug-related parameters, as well as allometrically scaled parameters. Fold errors for pharmacokinetic parameters were within acceptable range in both extrapolated models, except for a slight underprediction in the human plasma exposure. These animal and human PBPK models are expected to provide reliable estimates of MTS tissue distribution and assist dose regimen optimization in special populations.

Exploring hydrophilic 2,2-di(indol-3-yl)ethanamine derivatives against Leishmania infantum.

Herein we report the design and the synthesis of a library of new and more hydrophilic bisindole analogues based on our previously identified antileishmanial compound URB1483 that failed the preliminary in vivo test. The novel bisindoles were phenotypically screened for efficacy against Leishmania infantum promastigotes and simultaneously for toxicity on human macrophage-like THP-1 cells. Among the less toxic compounds, eight bisindoles showed IC50 below 10 µM. The most selective compound 1h (selectivity index = 10.1, comparable to miltefosine) and the most potent compound 2c (IC50 = 2.7 µM) were tested for their efficacy on L. infantum intracellular amastigotes. The compounds also demonstrated their efficacy in the in vitro infection model, showing IC50 of 11.1 and 6.8 µM for 1h and 2c, respectively. Moreover, 1h showed a better toxicity profile than the commercial drug miltefosine. For all these reasons, 1h could be a possible new starting point for hydrophilic antileishmanial agents with low cytotoxicity on human macrophage-like cells.

A phase II, non-comparative randomised trial of two treatments involving liposomal amphotericin B and miltefosine for post-kala-azar dermal leishmaniasis in India and Bangladesh.

BACKGROUND: In Southeast Asia, treatment is recommended for all patients with post-kala-azar dermal leishmaniasis (PKDL). Adherence to the first-line regimen, twelve weeks of miltefosine (MF), is low and ocular toxicity has been observed with this exposure period. We assessed the safety and efficacy of two shorter-course treatments: liposomal amphotericin B (LAmB) alone and combined with MF. METHODOLOGY/PRINCIPAL FINDINGS: An open-label, phase II, randomized, parallel-arm, non-comparative trial was conducted in patients with parasitologically confirmed PKDL, 6 to ≤60 years. Patients were assigned to 20 mg/kg LAmB (total dose, in five injections over 15 days) alone or combined with allometric MF (3 weeks). The primary endpoint was definitive cure at 12 months, defined as complete resolution of papular and nodular lesions and >80% re-pigmentation of macular lesions. Definitive cure at 24 months was a secondary efficacy endpoint. 118/126 patients completed the trial. Definitive cure at 12 months was observed in 29% (18/63) patients receiving LAmB and 30% (19/63) receiving LAmB/MF (mITT), increasing to 58% and 66%, respectively, at 24 months. Most lesions had resolved/improved at 12 and 24 months for patients receiving LAmB (90%, 83%) and LAmB/MF (85%, 88%) by qualitative assessment. One death, unrelated to study drugs, was reported; no study drug-related serious adverse events were observed. The most frequent adverse drug reactions were MF-related vomiting and nausea, and LAmB-related hypokalaemia and infusion reactions. Most adverse events were mild; no ocular adverse events occurred. CONCLUSIONS/SIGNIFICANCE: Both regimens are suitably safe and efficacious alternatives to long-course MF for PKDL in South Asia. TRIAL REGISTRATION: CTRI/2017/04/008421.

Choline Phosphate-Grafted Nanozymes as Universal Extracellular Vesicle Probes for Bladder Cancer Detection.

Urinary extracellular vesicles (uEVs) are regarded as highly promising liquid-biopsy biomarkers for the early diagnosis and prognosis of bladder cancer (BC). However, detection of uEVs remains technically challenging owing to their huge heterogeneity and ultralow abundance in real samples. We herein present a choline phosphate-grafted platinum nanozyme (Pt@CP) that acts as a universal EV probe for the construction of a high-throughput and high-sensitivity immunoassay, which allowed multiplex profiling of uEV protein markers for BC detection. With the Pt@CP-based immunoassays, three uEV protein markers (MUC-1, CCDC25, and GLUT1) were identified for BC, by which the BC cases (n = 48), cystitis patients (n = 27), and healthy donors (n = 24) were discriminated with high clinical sensitivity and specificity (area under curve = 98.3%). For the BC cases (n = 9) after surgery, the Pt@CP-based immunoassay could report the postoperative residual tumor that cannot be observed by cystoscopy, which is clinically significant for assessing BC recurrence. This work provides generally high sensitivity for EV detection, facilitating the discovery and clinical use of EV-based biomarkers.

Investigation of miltefosine-model membranes interactions at the molecular level for two different PS levels modeling cancer cells.

Miltefosine (MLT) is a broad-spectrum drug included in the alkylphospholipids (APL) used against leishmania and various types of cancer. The most crucial feature of APLs is that they are thought to only kill cancerous cells without harming normal cells. However, the molecular mechanism of action of APLs is not completely understood. The increase in the phosphatidylserine (PS) ratio is a marker showing the stage of cancer and even metastasis. The goal of this research was to investigate the molecular effects of miltefosine at the molecular level in different PS ratios. The effects of MLT on membrane phase transition, membrane orders, and dynamics were studied using DPPC/DPPS (3:1) and DPPC/DPPS (1:1) multilayer (MLV) vesicles mimicking DPPS ratio variation, Differential Scanning Calorimetry (DSC), and Fourier Transform Infrared spectroscopy (FTIR). Our findings indicate that miltefosine is evidence at the molecular level that it is directed towards the tumor cell and that the drug's effect increases with the increase of anionic lipids in the membrane depending on the stage of cancer.

Case Report: Management of Acanthamoeba Rhinosinusitis in a Patient with Chronic Lymphocytic Leukemia.

Infections caused by free-living amoebae pose a significant public health threat owing to growing populations of immunocompromised hosts combined with diagnostic delays, treatment difficulties, and high case fatality rates. Nasopharyngeal infections caused by Acanthamoeba are rare and the optimal treatment is not well established. We report a case of Acanthamoeba rhinosinusitis in a patient with chronic lymphocytic leukemia who presented with headaches and chronic rhinosinusitis refractory to multiple courses of antibiotics. A diagnosis of Acanthamoeba rhinosinusitis was established through broad-range polymerase chain reaction testing on sinus tissue. The patient had a favorable response to treatment, which included surgical debridement, cessation of immunosuppressants, and a three-drug regimen consisting of miltefosine, fluconazole, and sulfadiazine.

Effect of barium silicate on mechanical properties, transmittance, and protein adsorption of resin for additive manufacturing containing zwitterionic polymer.

STATEMENT OF PROBLEM: Studies on the effect of barium silicate on the material properties of additively manufactured (AM) resins containing 2-methacryloyloxyethyl phosphorylcholine (MPC) for dental applications are lacking. PURPOSE: The purpose of this in vitro study was to evaluate the mechanical properties, transmittance, and protein adsorption of MPC-containing AM resin incorporated with different barium silicate contents and to compare these findings with those of a commercially available unfilled AM resin marketed for definitive restorations. MATERIAL AND METHODS: Resins incorporating 6 wt% MPC and 4 different concentrations of barium silicate (10 wt%, MB10; 20 wt%, MB20; 30 wt%, MB30; and 40 wt%, MB40) were prepared. An MPC-containing resin with no filler was also prepared (0 wt%, MBN). Surface roughness (n=15), Vickers hardness (n=15), flexural strength and modulus (n=15), fracture toughness (n=15), transmittance (n=15), and protein adsorption (n=3) of the filled resin specimens were measured and compared with those of commercially available unfilled resin specimens. All data were analyzed using the Kruskal-Wallis and Dunn tests (α=.05). RESULTS: All experimental resins had higher surface roughness than the unfilled resin (P≤.048). MB40 had higher hardness, flexural strength, flexural modulus, and fracture toughness than most other groups (P≤.047). MB10 had higher transmittance than most other groups (P≤.012). All experimental resins had lower protein adsorption than the unfilled resin, regardless of the barium silicate content (P≤.023). CONCLUSIONS: The experimental resin containing 6 wt% MPC and 40 wt% barium silicate showed better mechanical properties and lower protein adsorption than the resin with no MPC or ceramic fillers. Transmittance decreased with the increase of barium silicate in the resins.

An injectable and antifouling hydrogel prevents the development of abdominal adhesions by inhibiting the CCL2/CCR2 interaction.

Abdominal adhesion, a serious complication of abdominal surgery, often resists mitigation by current drug administration and physical barriers. To address this issue, we developed an injectable, antifouling hydrogel through the free-radical polymerization of methacrylate chondroitin sulfate (CS-GMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) monomers, dubbed the CGM hydrogel. We systematically analyzed its physicochemical properties, including rheological strength, biocompatibility, and antifouling capabilities. A rat abdominal cecum adhesion model was constructed to assess the effectiveness of CGM hydrogel in preventing postoperative adhesion and recurrent adhesion. In addition, multi-omics analyses identified the relationship between adhesion development and CCL2/CCR2 interaction. Notably, CGM hydrogel can thwart the recruitment and aggregation of fibroblasts and macrophages by inhibiting the CCL2/CCR2 interaction. Moreover, CGM hydrogel significantly dampens the activity of fibrosis-linked cytokines (TGF-ßR1) and recalibrates extracellular matrix deposition-related cytokines (t-PA and PAI-1, Col Ⅰ and MMP-9). Cumulatively, the dual action of CGM hydrogel-as a physical barrier and cytokine regulator-highlights its promising potential in clinical application for abdominal adhesion prevention.

Photodynamic therapy offers a novel approach to managing miltefosine-resistant cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a neglected disease caused by Leishmania parasites. The oral drug miltefosine is effective, but there is a growing problem of drug resistance, which has led to increasing treatment failure rates and relapse of infections. Photodynamic therapy (PDT) combines a light source and a photoactive drug to promote cell death by oxidative stress. Although PDT is effective against several pathogens, its use against drug-resistant Leishmania parasites remains unexplored. Herein, we investigated the potential of organic light-emitting diodes (OLEDs) as wearable light sources, which would enable at-home use or ambulatory treatment of CL. We also assessed its impact on combating miltefosine resistance in Leishmania amazonensis-induced CL in mice. The in vitro activity of OLEDs combined with 1,9-dimethyl-methylene blue (DMMB) (OLED-PDT) was evaluated against wild-type and miltefosine-resistant L. amazonensis strains in promastigote (EC50 = 0.034 µM for both strains) and amastigote forms (EC50 = 0.052 µM and 0.077 µM, respectively). Cytotoxicity in macrophages and fibroblasts was also evaluated. In vivo, we investigated the potential of OLED-PDT in combination with miltefosine using different protocols. Our results demonstrate that OLED-PDT is effective in killing both strains of L. amazonensis by increasing reactive oxygen species and stimulating nitric oxide production. Moreover, OLED-PDT showed great antileishmanial activity in vivo, allowing the reduction of miltefosine dose by half in infected mice using a light dose of 7.8 J/cm2 and 15 µM DMMB concentration. In conclusion, OLED-PDT emerges as a new avenue for at-home care and allows a combination therapy to overcome drug resistance in cutaneous leishmaniasis.

Mechanical properties, cytotoxicity, and protein adsorption of three-dimensionally printable hybrid resin containing zwitterionic polymer and silicate-based composites for dental restorations.

OBJECTIVE: To evaluate the mechanical and biological properties of three-dimensionally (3D) printable resins filled with 2-methacryloyloxyethyl phosphorylcholine (MPC) and silicate-based composites and compare with those of a commercially available 3D-printable resin for definitive restorations. METHODS: A group of 3D-printable hybrid resins (HRs) filled with 6 wt% MPC and three different compositions of silicate-based composites (barium silicate to zirconium silicate ratios: 1.50:1 for HR1, 0.67:1 for HR2, and 0.25:1 for HR3) were prepared. The HR groups were compared with the commercially available unfilled 3D-printable resin (CR) marketed for definitive restorations in terms of flexural strength and modulus, fracture toughness, surface roughness, Vickers hardness, light transmittance (all, n = 15), cytotoxicity, and protein adsorption (both, n = 3). All data were analyzed by using non-parametric Kruskal-Wallis and Dunn's tests (α=0.05). RESULTS: The HR groups had significantly higher flexural strength, modulus, fracture toughness, and hardness values than the CR (P < 0.001). HR3 had the highest surface roughness and light transmittance among the groups (P ≤ 0.006). None of tested resins showed cytotoxicity. Both HR2 and HR3 showed significantly lower protein adsorption than the CR, with a difference of approximately 60% (P ≤ 0.026). CONCLUSION: Both HR2 and HR3 exhibited superior mechanical properties (flexural strength, flexural modulus, fracture toughness, and Vickers hardness), light transmittance, and protein-repellent activity than the CR, with no impact on cytotoxicity. CLINICAL SIGNIFICANCE: The MPC/silicate-based composite-filled resins may be a suitable alternative for definitive restorations, given their higher mechanical properties and promising biological properties to prevent microbial adhesion and subsequent biofilm formation, as well as their non-cytotoxic properties.

Broad specificity of monoclonal IgA (TEPC15-IgA) for enteric bacteria via phosphorylcholine-mediated interaction.

Immunoglobulin A (IgA) is notable for its broad specificity toward multiple bacteria. Phosphorylcholine (PC) plays a role in the infection of pathogenic bacteria carrying PC and in the induction of IgA responses in the host immune system. The commercially available mouse monoclonal IgA, TEPC15-IgA, is a distinctive antibody with specificity for PC, warranting further exploration of its response to PC-bearing enteric bacteria. In this study, using 17 different enteric bacteria, including 3 aerobic and 14 anerobic bacteria that could be cultured in vitro, we confirmed that TEPC15-IgA recognizes 4 bacterial species: Lactobacillus taiwanensis, Limosilactobacillus frumenti, Streptococcus infantis, and Escherichia coli, although reactivity varied. Interestingly, TEPC15-IgA did not react with four of six Lactobacillus species used. Moreover, distinct target molecules associated with PC in L. taiwanensis and L. frumenti were evident, differing in molecular weight. These findings suggest that the natural generation of PC-specific IgA could prevent PC-mediated infections and potentially facilitate the formation of a microflora rich in indigenous bacteria with PC, particularly in the gastrointestinal tract.

CRISPR-Cas9 high-throughput screening to study drug resistance in Leishmania infantum.

Parasites of the genus Leishmania pose a global health threat with limited treatment options. New drugs are urgently needed, and genomic screens have the potential to accelerate target discovery, mode of action, and resistance mechanisms against these new drugs. We describe here our effort in developing a genome-wide CRISPR-Cas9 screen in Leishmania, an organism lacking a functional nonhomologous end joining system that must rely on microhomology-mediated end joining, single-strand annealing, or homologous recombination for repairing Cas9-induced double-stranded DNA breaks. A new vector for cloning and expressing single guide RNAs (sgRNAs) was designed and proven to be effective in a small pilot project while enriching specific sgRNAs during drug selection. We then developed a whole-genome library of 49,754 sgRNAs, targeting all the genes of Leishmania infantum. This library was transfected in L. infantum expressing Cas9, and these cells were selected for resistance to two antileishmanials, miltefosine and amphotericin B. The sgRNAs the most enriched in the miltefosine screen targeted the miltefosine transporter gene, but sgRNAs targeting genes coding for a RING-variant protein and a transmembrane protein were also enriched. The sgRNAs the most enriched by amphotericin B targeted the sterol 24 C methyltransferase genes and a hypothetical gene. Through gene disruption experiments, we proved that loss of function of these genes was associated with resistance. This study describes the feasibility of carrying out whole-genome CRISPR-Cas9 screens in Leishmania provided that a strong selective pressure is applied. Such a screen can be used for accelerating the development of urgently needed antileishmanial drugs.IMPORTANCELeishmaniasis, a global health threat, lacks adequate treatment options and drug resistance exacerbates the challenge. This study introduces a CRISPR-Cas9 screening approach in Leishmania infantum, unraveling mechanisms of drug resistance at a genome-wide scale. Our screen was applied against two main antileishmanial drugs, and guides were enriched upon drug selection. These guides targeted known and new targets, hence validating the use of this screen against Leishmania. This strategy provides a powerful tool to expedite drug discovery as well as potential therapeutic targets against this neglected tropical disease.