Institute of Medical Biology

Lipidomic studies demand analytical platforms capable of capturing the structural diversity and complexity of lipid species with accuracy, precision, and speed. The Echo® MS+ system, an advanced acoustic ejection (AE) system coupled with a mass spectrometer (MS), offers a genuinely high-throughput, contactless workflow for comprehensive lipid profiling. In this study, we developed an automated high-throughput workflow based on a single-phase lipid extraction using 1-octanol, and methanol with 10mM ammonium formate as a carrier solvent, to measure lipid profiles covering 10 lipid classes, namely, phosphatidylcholine (PC), lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, triacylglycerol, diacylglycerol, sphingomyelin, cholesteryl ester and cholesterol from plasma samples. We optimized sample preparation protocols, solvent systems, and droplet ejection parameters to maintain sensitivity and minimize variability of lipid signals. The extraction recoveries for lipid class representatives, typically between 89% and 95%, were found to be comparable to those obtained with the traditional butanol/methanol extraction method. We also tested the extract integrity; 1-octanol extracts did not show any degradation of lipids after the storage for several days. We assessed the fast analysis AE-MS mode, operating at a speed of 4 seconds per sample, for the quantitation of targeted lipid panels. The signal reproducibility for the lipid class representatives was better than 6% and the linear response was confirmed across 2 orders of magnitude. The semi-quantitative profile from AE-MS analysis of a target panel of 14 ceramides in NIST SRM 1950 was consistent with the published data. Additionally, we investigated a 9-second-wide peak AE-MS mode, operating at a speed of 12 seconds per sample, for its utility in untargeted qualitative lipid analysis. We employed the data dependent high-resolution (HR) MS acquisition. Data analysis of plasma extracts involved a search for the 14 most common plasma lipid classes, and it resulted in a comprehensive lipid profile, consistently identifying 200 most abundant lipid molecular ions per polarity in all samples in 384-well plate; a total of 338 lipid were reported as sum compositions. The MS information was supplemented with lipid structure details and tentative molecular species assignments based on the corresponding MS/MS data. For example, 42 major PC lipids measured in HR MS were found to correspond to 69 PC molecular species. This new technique, combining the single-phase 1-octanol plasma extraction with AE-MS analysis offers a carryover-free workflow, enabling ultra-fast comprehensive lipid measurements using small nanolitre extracts and minimal solvent volumes.

Current antibiotics and chemotherapeutics are becoming ineffective because pathogenic bacteria and tumor cells have developed multiple drug resistance. Therefore, it is necessary to find new substances that can be used in treatment, either alone or as sensitizing molecules in combination with existing drugs. Peptaibols are bioactive, membrane-active peptides of non-ribosomal origin, mainly produced by filamentous fungi such as Trichoderma spp. This study focused on producing peptaibol-rich extracts from Trichoderma atroviride O1, cultivated on malt extract agar (MA) under circadian and constant darkness conditions for 13 days. Peptaibol production was detected by MALDI-TOF mass spectrometry after six days of cultivation. The extracts demonstrated antibacterial activity against Staphylococcus aureus strains, particularly the methicillin-resistant variant, but not against the Gram-negative Pseudomonas aeruginosa. Quorum sensing interference revealed that a peptaibol-rich extract suppressed Vibrio campbellii BAA-1119’s AI-2 signaling system to a degree comparable with gentamycin. Beyond antibacterial properties, the extracts exhibited notable antiproliferative activity against human ovarian cancer cells and their adriamycin-resistant subline in both 2D and 3D models. Specifically, MA-derived extracts reduced ovarian cancer cell viability by 70% at 50 μg/mL, especially under light/dark regime of cultivation. Compared to previously published results for PDA-based extracts, MA cultivation shifted the biological effects of peptaibol-containing extracts toward anticancer potential. These findings support the idea that modifying fungal cultivation parameters, the bioactivity of secondary metabolite mixtures can be tailored for specific therapeutic applications.

ABSTRACTBackgroundInnovative use of a point‐of‐care ultrasound (POCUS) device facilitates the diagnosis and management of potential challenges associated with Ellanse treatment. Our case series of 6 patients illustrates the effectiveness of POCUS in managing challenges associated with Ellanse treatment. Ultrasound analysis is performed in all 6 patients, and management decisions are guided by ultrasound findings.MethodOur case series of 6 patients (A, B, C, D, E & F) consists of Ellanse‐treated patients. They presented problems associated with (1) facial overfilling, (2) diagnostic challenges, (3) underfilling requiring POCUS‐guided treatment, (4) confirmation of nodule dissolution, and (5) pretreatment vascular assessment.ResultsPatients A and B presented with complaints of facial overfilling, and ultrasound analysis attributed the overfilling to previous autologous fat grafting and hyaluronic acid filler treatments respectively. Patient C complained of facial cheekbone prominence after receiving 26 mL of Ellanse in her entire face over 3 years. The patient was reassured that her cheek prominence was secondary to neocollagenesis following ultrasound analysis and refused further intervention. Patient D had successful dissolution of a nodule associated with Ellanse, and this was confirmed with photographs and POCUS imaging. Patient E had underdevelopment of collagen in the left midcheek following initial Ellanse treatment 3 months ago. 0.2 mL of additional Ellanse was injected with a 25G cannula under POCUS guidance to the left cheek with satisfactory correction. Patient F had three previous sessions of Ellanse treatments. She underwent vascular anatomy analysis with an ultrasound Doppler mode prior to the fourth Ellanse treatment. The presence of dense neocollagenesis following Ellanse can alter vascular patterns. A preprocedural analysis is recommended in planning subsequent treatment.ConclusionOur limited case series of 6 patients provides useful insights into the effectiveness of POCUS as the initial imaging modality of choice when managing Ellanse‐treated patients. POCUS imaging provides valuable diagnostic information (Patient A, B, C and D) and therapeutic purposes such as POCUS guided injection and pretreatment assessment (Patient D and E). POCUS can be the initial imaging modality of choice as it is convenient and allows clinical management to be made based on imaging findings.

To explore the effects and mechanism of Modified Liu-an Decoction (MLAD) on Th1/Th2 function and the Notch signaling pathway in guinea pigs with cough variant asthma (CVA). 60 SPF Hartley guinea pigs were randomized into six groups (n = 10): the blank control group, model group, montelukast sodium group (MS group), as well as MLAD group of low-dose, mid-dose and high-dose. Intraperitoneal injection of ovalbumin, aluminum hydroxide sensitization, and nebulized inhalation of ovalbumin were performed in all guinea pigs of non-blank control groupto induce CVA. Cough frequency and airway resistance were recorded. The chronic inflammatory infiltration in the airways was detected by using HE and Masson staining smears. The IL-2, IL-4, IL-12, and IL-13 levels were measured by enzyme-linked immunoassay (ELISA). The expression of Delta, Jagged1, Notch1, and NICD proteins in the lung tissues were analyzed by Western blot. In terms of airway sensitivity, coughing time and airway resistance were reduced in the MLAD and MS groups compared to the model group (P < 0.05). MLAD reduced the inflammatory infiltration in the lungs of CVA guinea pigs compared with the model group (P < 0.05).For cytokines in BALF, the contents of IL-2 and IL-12 were increased in the MS group and each dose group of MLAD compared with the model group, and the contents of IL-4 and IL-13 were decreased compared with them.The contents of IL-12 were significantly different in each dose group of MLAD compared with the model group; the contents of IL-2 and IL-4 differed with increasing concentrations of MLAD. 4 content with increasing MLAD concentration, the more significant difference. IL-13 content decreased significantly in the MLAD low dose group compared with the model group (P < 0.05). In the serum cytokine model group IL-2, IL-12, IL-13 content were significantly higher than the blank group, IL-4 content was lower than the blank group, and the difference between each intervention group and the model group was not regular. The expression levels of Jagged1, Notch, and NICD proteins in the CVA guinea pigs in the MS group and the low-dose MLAD group were decreased compared with those in the model group, and the expression of Delta1 protein was increased compared with that in the model group (P < 0.05). The addition of Liuan Decoction to the lungs can clear lungs and remove heat and strengthen the spleen to resolve phlegm, congestion, edema degree, reduce airway inflammation, and at the same time can reduce peri-tracheal collagen deposition, effectively improve airway remodeling, by regulating the dynamic balance of Th1/Th2. The specific downstream regulatory mechanisms of Notch signaling pathway need to be explored in further studies.

Diabetes mellitus (DM) is a chronic metabolic non-communicable disease with the ability to cause serious microvascular and macrovascular complications throughout the body, including in the eye. Diabetic retinopathy (DR), present in one-third of patients with diabetes, is a vision-threatening complication caused by uncontrolled diabetes, which greatly affects the retinal blood vessels and the light-sensitive inner retina, eventually leading to blindness. Several epidemiological studies elucidate that DR can vary by age of onset, duration, types of diabetes, and ethnicity. Recent studies show that the pathogenesis of diabetic retinopathy has spread its roots beyond merely being the result of hyperglycemia. The complexity of its etiopathology and diagnosis makes therapeutic intervention challenging. This review throws light on the pathological processes behind DR, the cascade of events that follow it, as well as the available and emerging treatment options.

BCV and tecovirimat are the only two chemical drugs that have been approved to treat smallpox and can be requested for Mpox treatment through a single-patient Emergency Investigational New Drug (EIND) application. Disappointedly, the efficacy of tecovirimat manifested in a recent clinical trial is far from being satisfactory, while the clinical efficacy of BCV is still inconclusive. Given that MPXV, variola and other emerging orthopoxviruses are posing serious threats to global health, it is urgent to develop better therapeutics. In this study, we tested the antiviral effects of three novel prodrugs, which were designed based on previously reported parent drugs, either (S)-HPMPC (cidofovir) or (S)-HPMPA. We found that one of the (S)-HPMPA-based prodrugs, ODE-(S)-HPMPA formate, exhibited significantly better anti-orthopoxvirus activity than BCV both in vitro and in vivo, which also inhibited human adenovirus type 2 and type 21 more efficiently than BCV. Most strikingly, the EC50 and EC90 of ODE-(S)-HPMPA formate against MPXV were more than 40-fold lower than those of BCV. In contrast, we observed that the anti-HSV-1 activities of the (S)-HPMPA-based prodrugs were less effective than those of the cidofovir-based prodrugs (BCV and BCV formate), especially in vivo. Moreover, we showed for the first time that cytidine and adenine analog combined therapies could provide mice with complete protection against lethal challenges of both vaccinia and HSV-1. Collectively, we propose that both the ODE-(S)-HPMPA formate and the BCV/ODE-(S)-HPMPA formate combination are worth further investigations for their potential clinical applications.

Alzheimer’s disease (AD) is a major contributor to dementia and the most common neurodegenerative disorder. In AD pathophysiology, matrix metalloproteinases (MMPs)—proteolytic enzymes, best known to be responsible for remodeling and degradation of the extracellular matrix—were suggested to play an important role. Due to the diverse nature of the published data and frequent inconsistent results presented in available papers, it was considered essential to analyze all aspects of MMP literature with respect to AD pathophysiology and attempt to outline a unifying concept for understanding their role in AD. Thus, the main contribution of this review article is to summarize the most recent research on the participation of MMP in AD pathophysiology obtained using the cell cultures to understand the molecular principles of their action. Furthermore, an updated comprehensive view regarding this topic based exclusively on papers from human studies is provided as well. It can be concluded that determining the exact role of any particular MMPs in the AD pathophysiology holds promise for establishing their role as potential biomarkers reflecting the severity or progression of this disease or for developing new therapeutic agents targeting the processes that lead to AD.

This post hoc analysis evaluated the efficacy and safety of intravenous belimumab 10 mg/kg in the South Korean subgroup of patients with systemic lupus erythematosus (SLE) enrolled in the North East Asia (NEA) study (GSK Study BEL113750; NCT01345253). NEA was a double-blind, placebo-controlled, randomized Phase 3 trial. Patients with active, autoantibody-positive SLE were randomized 2:1 to belimumab or placebo plus standard therapy administered on Days 0, 14, and 28, and then every 28 days up to Week 48. The primary efficacy endpoint in this analysis was SLE Responder Index 4 (SRI-4) response rate at Week 52, defined as the proportion of patients achieving a ≥4-point reduction in Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index (SELENA-SLEDAI) score, no worsening (<0.3 increase from baseline) in Physician Global Assessment, no new British Isles Lupus Assessment Group (BILAG) A domain and <2 new BILAG B domain scores. Among 100 South Korean patients enrolled in NEA, 54/66 (81.8%) belimumab- and 24/34 (70.6%) placebo-treated patients completed the double-blind phase. Significantly more belimumab- than placebo-treated patients achieved SRI-4 response at Week 52 (n = 35/66, 53.0% vs. n = 8/34, 23.5%; odds ratio [OR; 95% confidence interval (CI)]: 3.67 [1.45, 9.28]; p = .0061). The proportion of patients experiencing ≥1 adverse event was similar between groups (belimumab: n = 60/66, 90.9% vs. placebo: n = 31/34, 91.2%). No new safety signals emerged in this subgroup analysis. Belimumab was efficacious for the treatment of SLE and well tolerated among the South Korean subgroup of patients from the NEA study.

Congenital anomalies, diseases, and injuries may result in osteochondral damage. Recently, a big hope has been given to somatic stem cells (SSCs) which are characterized as undifferentiated cells with an ability of long-term self-renewing and plasticity. They are adherent with a fibroblast-like morphology in vitro and express various surface markers (e.g. CD29, CD73, CD90, and CD105), but they are negative for CD31, CD34, CD45, and HLA-DR. SSCs secrete various bioactive molecules, which are involved in processes of regeneration. The main goal of the present study was the characterization and comparison of biological properties of SSCs obtained from adipose tissue, dental pulp, and urine concerning osteochondral regeneration. SSCs were maintained in an appropriate growth medium up to the third passage and were analyzed by light and electron microscope. The immunophenotype was analyzed by flow cytometry. The kinetics of proliferation was measured by MTT assay. Human Cytokine/Chemokine Multiplex Assay was used, and SSCs secretory profile was measured by Luminex MAGPIX® Instrument. Pellet cultures and a chondrogenic medium were used to induce chondrogenic differentiation. Osteogenic differentiation was induced by the osteogenic medium. Chondrogenic and osteogenic differentiation was analyzed by real-time PCR. SSCs had similar fibroblast-like morphology. They have similar kinetics of proliferation. SSCs shared the expression CD29, CD44, CD73, CD90, and CD105. They lack expression of CD29 and CD34. SSCs secerned similar levels of IL10 and IL18 while differing in IFN-gamma, IL6, IL8, MCP-1, and RANTES production. SSCs possess a similar capacity for chondrogenic differentiation but slightly differ in osteogenic differentiation. In conclusion, it can be emphasized that SSCs from adipose tissue, dental pulp, and urine share the majority of cellular characteristics typical for SSCs and have great potential to be used in osteochondral tissue regeneration.