Ectoine, a compatible solute and valuable cosmetic additive, is synthesized from L-aspartate-β-semialdehyde (ASA) via a three-step pathway catalyzed by EctABC. To investigate the metabolic basis of ectoine overproduction in an engineered E. coli strain E2, we employed an integrated analysis of metabolomics and quantitative PCR (qPCR). The results suggest that E2 exhibits a coordinated upregulation of central metabolism: metabolomic profiles showed elevated levels of PPP intermediates and key precursors (aspartate and oxaloacetate), consistent with increased precursor and NADPH supply. qPCR analysis confirmed the upregulated expression of genes involved in the TCA cycle, aspartate biosynthesis, and the Ectabc operon. Based on this multi-omics insight, the competing pathway of ASA toward methionine biosynthesis was selected as a potential target for metabolic intervention via knockout of the metA gene. Based on these findings, a precise metabolic intervention was implemented: knockout of the metA gene to block this diversion, which, supported by an optimized feeding strategy, resulted in a final ectoine titer of 115.52 g/L in the derived strain E2-1 after 72 hours of fermentation. This study provides insights into the metabolic basis of high-yield ectoine production and presents a paradigm of systems-level analysis for targeted engineering, providing a robust framework and a clear theoretical foundation for the rational design of advanced microbial cell factories.
Quorum sensing inhibitors (QSIs) can inhibit the quorum sensing regulated physiological functions of bacteria and might be useful in drug-resistant bacterial infection therapy. The culture medium of a mangrove derived actinobacterium Streptomyces chumphonensis HN2-56, which exhibiting higher anti-quorum sensing activity, was optimized in this study. The results showed that the GS medium was the most favourable medium for the bioactivity of HN2-56 and that the content of soluble starch in the GS medium had a significant impact on the yield of metabolites. By culturing under the optimized condition (soluble starch 40 g/L, KNO3 4 g/L, salinity of 0 and pH 6.5), both metabolites production and anti-quorum sensing activity of HN2-56 against the purple pigment (violacein) production of Chromobacterium violaceum 12472 were significantly increased (41.8% and 177.9%, respectively). Five pure compounds were obtained from the extract of HN2-56 and identified as 2,2-[bis-4-(2,3-dihydroxypropane) phenyl] propane (1), bisphenol A (2), 5-(2-methylphenyl)-4-pentenoic acid (3), 3-methyl-1H-indole-2-carboxylate (4) and dibutyl phthalate (5). Among them, compounds 2 and 3 showed anti-quorum sensing activity against C. violaceum 12472 and anti-biofilm activity against Staphylococcus aureus 43300. These findings indicated that S. chumphonensis HN2-56 should be a promising candidate for QSIs with potential anti-quorum sensing application. Although BPA was detected in the HN2-56 extract, experimental contamination could not be excluded because of the ubiquitous presence of BPA in plasticware and laboratory reagents.
The inflammatory joint diseases are characterized by high prevalence rates and significant burdens in both Bulgaria and Serbia. Regional differences in pricing policy might influence access to therapy. The goal of our study is to compare and analyze the prices, public spending, and utilization of biological disease modifying drugs for the treatment of antirheumatic diseases (bDMARDs) in Bulgaria and Serbia. It is macro costing, cross-country comparative analysis for the period 2016-2020, which is based on a retrospective review of officially published information about prices, public expenditures, and utilization. The reference prices and public expenditures are retrospectively extracted from the national regulatory bodies. Utilization is calculated as the defined daily dose (DDD)/1000 inh/day. Reference prices per DDD decline in both countries during the observed period, mostly visible for bDMARDs with biosimilar alternatives, mainly infliximab, adalimumab, and etanercept. The price differences are statistically significant for 3 International Nonproprietary Names (INNs): infliximab, golimumab, and secukinumab. Public spending and utilization are almost twice higher in Bulgaria. The medicine with the highest utilization in Serbia is infliximab, while in Bulgaria is adalimumab. Pricing policies and new molecules entrance influence utilization and expenditures for bDMARDs positively but improve patients' access. Utilization, access to new molecules and expenditures are higher in Bulgaria, but prices for most bDMARDs are lower in Serbia. Comparative price analysis supports information about the access to medicines and enlightens pricing policies. Further studies are needed in order to examine all the factors affecting medicine prices and public spending.
Snail mucus is a complex multicomponent mixture of proteoglycans, glycosaminoglycans, enzymes, hyaluronic acid, proteins, antimicrobial peptides, amino acids and metal ions. This study presents the chemical and mineral composition of mucus from the garden snail Cornu aspersum and the effectiveness of its active components on wound healing. Three samples of the mucus of the garden snail C. aspersum were analyzed: native mucus (P1), a fraction with active components with a molecular weight (MW) above 30 kDa (P2) and a MW above 10 kDa (P3). Studies with an amino acid analyser, gas chromatography and mass spectrometric analysis indicate that the snail secretion contains a significant amount of minerals and bioactive organic compounds. The cytotoxicity of the extracts on the viability of skin fibroblasts (BJ) and human keratinocytes (HaCaT) was also evaluated. The presence of important peptides and proteins, such as achacin, collagen, elastin, hemocyanin, superoxide dismutase, catalase, and glutathione peroxidase, etc., with proven healing properties, has been proven. The results showed that P1, P2 and P3 were non-toxic to normal human dermal cells at the concentrations analysed and stimulated the proliferation of BJ and HaCaT cells. Treatment of BJ cells with fraction P3 at a concentration of 120 & micro;g/mL leads to a 40% increase in proliferation and showed the strongest stimulating effect on the synthesis of membrane-associated collagen, which was 33% higher in BJ cells compared to control cells. The results confirmed that the mucus of the garden snail C. aspersum contains active components with proven effectiveness on wound healing.
Hexaploid triticale (& times;Triticosecale Wittmack), a grain-forage crop, exhibits outstanding stress tolerance and adapts well to the saline soils of the Qaidam Basin in Qinghai Province, China. In this study, we conducted comparative analyses of transcriptomic, phenotypic, and biochemical responses in a salt-tolerant (ST) accession 'QSM2' and a salt-sensitive (SS) accession 'PI429196', following salt stress treatments for 12, 24, 48 h, and 7 d. The ST accession displayed greater tolerance based on root length, seedling height, and antioxidant enzyme activities. The number of DEGs and overlapping DEGs in ST was more than that in SS at three time points (12, 24, and 48 h). Gene co-expression networks were constructed in response to salt stress, and the brown module correlated significantly with ST under salt stress. A total of 25 core genes responsive to salt stress, showed up-regulated expression level in ST but down-regulated expression in SS. Two candidate genes (TsABCF1 and TsLEA14), encoding ABC transporter F and late-embryogenesis abundant (LEA) protein, were obtained based on the conjoint analysis of the transcriptome and genome-wide associated studies. These findings provide valuable insights for the fine-mapping and cloning of salt-tolerant genes and facilitate the development of salt-tolerance triticale cultivars.
Auxin plays an essential role in all stages of plant growth and development. Castor (Ricinus communis L.) is an important oil and bioenergy crop with substantial potential. However, few studies have been conducted on auxin in castor, and the endogenous auxin synthesis process in this plant remains unknown. In this study, the amino acid sequences of the Arabidopsis thaliana YUCCA (AtYUC) proteins were used as query sequences to perform BLAST searches against in the castor genome database. A total of nine YUCCA-like genes (named RcYUCs) were identified in the castor genome. Gene structure, conserved domain, and phylogenetic analyses were then performed on these RcYUCs. The expression pattern of RcYUCs was determined by real-time PCR. The average length and relative molecular weight of the RcYUC proteins were 402 amino acids and 44.99 kDa, respectively, and all were amphoteric proteins. Furthermore, the RcYUC proteins were found to contain conserved flavin monooxygenase (FMO) motifs. High expression of the RcYUC4, 6, and 10a genes was observed in seeds, suggesting an important role in seed development. These findings provide a basis for further investigation into RcYUCs gene function.
The LEGO® coding platform, widely used in education for its modularity and accessibility, has had limited application in laboratory automation. To address limitations of conventional disk diffusion methods, this study developed an automated flame sterilization and inoculation system using LEGO® MINDSTORMS® Education EV3. The system matched the performance of manual methods while improving consistency and cost-effectiveness. Validation through repeated tests confirmed its reliability and reproducibility. Fifteen presumptive multidrug-resistant Enterococci (MRE) isolates were obtained from sewage samples in Korea. PCR analysis targeting 13 resistance genes revealed frequent detection of ermB, poxtA, tetM, and tetL. Species identification via 16S rRNA sequencing and MALDI-TOF MS confirmed all isolates belonged to the genus Enterococcus, grouped into four species. The antibacterial activity of cinnamon bark essential oil (CBEO) was tested using both methods. Inhibition zones ranged from 26.0 to 43.3 mm (mean 31.7 mm), with only a 0.1 mm difference between methods, indicating high reproducibility. Minimum bactericidal concentrations ranged from 1.6% to 12.5%, confirming bactericidal potential. In some cases, CBEO showed stronger inhibition than standard antibiotics such as ampicillin and kanamycin. These findings suggest CBEO's potential as a natural antimicrobial agent against MRE. Further studies are needed to evaluate its efficacy against broader isolate collections and in various applications, including healthcare and environmental settings. Additionally, this study presents the potential of low-cost, LEGO-based automated laboratory equipment for scalable experimental use.
Clostridium butyricum is a strictly anaerobic probiotic widely used in food, medicine, and agriculture. However, its industrial application is limited by low fermentation yields and cultivation difficulties. In this study, a novel strain, C. butyricum CB-a, was isolated from free-range chicken feces and exhibited notable probiotic traits. It maintained 25.0% and 32.5% survival rates after 2-h exposure to simulated gastric and intestinal juices, respectively, and over 42.0% survival in 0.2%–0.5% bile salt solutions. Moreover, it showed clear antimicrobial activity against Escherichia coli and Staphylococcus aureus. To enhance biomass production, one-factor and orthogonal optimization experiments were conducted. Glucose was selected as the optimal carbon source, and soy peptone, yeast extract, and peptone as key nitrogen sources. The optimized medium, combined with suitable pH (6.0), inoculum ratio (3%), and temperature (34 °C), enabled a viable cell count of 4.56 × 10⁹ CFU/mL after 24 h. Although this value is slightly below the conventional high-density threshold, it represents a substantial increase compared to previous C. butyricum reports. This study provides a practical and economical strategy for cultivating C. butyricum with industrial potential and lays the groundwork for its future probiotic application.
Tiger nut (Cyperus esculentus L.) is widely recognized as both an oil crop and a high-quality forage. However, the comprehensive utilization of tiger nut meal (TNM) remains underexplored. This study investigates the potential of solid-state fermentation with selected microorganisms to convert TNM into a high-nutritional-value animal feed. After evaluation, fermentation of TNM with Saccharomyces cerevisiae GLLB−3, or the mixture of S. cerevisiae GLLB−3 and Lactiplantibacillus plantarum SCTM−1 (EMIX group) improves the nutrient profiles of TNM. Post-fermentation, the contents of crude protein, crude fiber, total phosphorus, and amino acids increased. Especially, the crude protein content in TNM fermented with S. cerevisiae GLLB−3 increased by 34.71%, whereas TNM fermented with EMIX exhibited a 27.77% increase. Additionally, essential amino acids except histidine showed significant increases, ranging from 6.82% to 119.49%. The fermentation not only enhances TNM’s nutritional values but also contributes to a more balanced amino acid composition suitable for animal feed production. Simultaneously, fermentation of TNM with two Aspergillus strains changes amino acid and other nutrient profiles. The analysis of the fermented products revealed significant increases in the contents of crude protein, crude fiber, total phosphorus, and amino acids. Notably, the levels of eight essential amino acids were substantially elevated, indicating a marked improvement in the nutritional quality of the fermented products as compared to the raw materials. This study demonstrates probiotic fermentation can effectively enhance the nutritional profile of TNM, highlighting it as a promising approach for the comprehensive utilization of tiger nut by-products.
This study evaluates the safety profiles of biosimilar medicinal products (MPs) compared to their reference counterparts within the European Union (EU). Biosimilars, cost-effective alternatives to original biologics, enhance patient access to vital treatments. Despite their similarity to reference products, nuanced differences necessitate rigorous pharmacovigilance. Between April 2006 and November 2022, 73 biosimilars (81%) received EU marketing authorization (MA), 15 (17%) were authorized but later withdrawn, and 2 (2%) had MAs rejected. Notably, none of the 90 biosimilars were withdrawn or rejected for safety reasons, underscoring the EU's stringent drug evaluation and monitoring processes. Our research analyzed six international non-proprietary names (INNs): adalimumab, bevacizumab, trastuzumab, rituximab, infliximab, and etanercept, using European Public Assessment Reports (EPARs), Risk Management Plan (RMP) summaries, and EudraVigilance (EV) data. Of 30 analyzed adverse drug reaction (ADR) groups, 70% showed minor deviations (0 to +/- 10%) between biosimilars and reference products. 17% of the groups were in the +/- 10 to +/- 20% range of difference, and only 13% of the ADR groups were in the range of difference above +/- 20%. The findings demonstrate consistent regulatory approaches, with biosimilars sharing indications and safety risk profiles with their reference products, supporting extrapolation principles in approvals. However, transparency challenges persist in comparing publicly available safety information between biosimilars and reference products. This study highlights the EU's robust regulatory framework ensuring biosimilar safety and efficacy, supporting their therapeutic integration. Long-term observations remain crucial to validate data, build trust, and emphasize interchangeability among biologics and biosimilars.
The primary focus of health systems on containing the COVID-19 epidemic impedes access to health care for society’s most vulnerable segments, particularly children. For years, Bulgaria experienced a decline in mandatory vaccine coverage in children, especially for measles, mumps and rubella. The study explored the impact of the COVID-19 pandemic on immunization coverage in Bulgarian children with diphtheria, tetanus, and pertussis-containing vaccine third dose (DTP3) and measles-containing vaccine first dose (MCV1) during the pandemic years, 2020-2022. We used ARIMA model to capture temporal trends in coverage with diphtheria, tetanus, and pertussis-containing vaccine third dose (DTP3) and measles-containing vaccine first dose (MCV1) in children from 2000 to 2019 (pre-pandemic period) to forecast expected coverage in 2020-2022 (pandemic period). The expected vaccination coverage, predicted by ARIMA models, with DTP3 and MCV1 during the whole pandemic period, except for DTP3 in 2022, was higher than the reported vaccination coverage. The observed MCV1 coverage in 2020 and 2021 was slightly below the lower confidence limit, most likely because of the COVID-19 pandemic. The study demonstrates that the COVID-19 pandemic has further exacerbated the problem of vaccine coverage with DTP3 and MCV1 among children in Bulgaria, especially with MCV1.
Piper nigrum or black pepper, is renowned for its medicinal properties and widespread use as a spice. Its essential oil, rich in active compounds, holds promise for combating bacterial contamination, particularly in food and beverages. This study analyzed the antibacterial potential of black pepper essential oil through active compound profiling and computational assessment. Gas chromatography-mass spectrometry (GCMS) analysis was conducted to identify compounds in black pepper essential oil. Toxicity analysis of the identified compounds was performed using ProTox 3.0, while molecular docking and molecular dynamics simulations were employed to assess their interactions with bacterial proteins. GCMS analysis revealed the presence of eleven secondary metabolite compounds in black pepper essential oil, predominantly piperine derivatives. Toxicity analysis indicated low toxicity levels for these compounds. Molecular docking simulations showed strong binding affinity of certain compounds to bacterial proteins, including FtsZ, GyrB, murA, and PTP. Molecular dynamics simulations further confirmed stable interactions between the compounds and their target proteins. Black pepper essential oil, enriched with piperine derivatives, exhibits promising antibacterial activity by targeting FtsZ, GyrB, murA, and PTP proteins. These findings suggest the potential utility of black pepper essential oil as a natural antibacterial agent.
This prospective observational study, conducted at the Affiliated Hospital of Inner Mongolia Medical University, aimed to investigate the effects of metformin on serum Fatty acid-binding protein 4 (FABP4) and Peroxisome Proliferator-activated receptor gamma (PPAR gamma) levels in patients with Type 2 Diabetes Mellitus (T2DM) complicated by Nonalcoholic Fatty Liver Disease (NAFLD). From June 2022 to January 2023, we enrolled patients with T2DM + NAFLD (Group A, n = 31), patients with T2DM treated with metformin monotherapy (Group B, n = 30), and healthy controls (NGT group, n = 37). Serum FABP4 and PPAR gamma levels were quantified using enzyme-linked immunosorbent assay (ELISA). After one month of metformin treatment in the T2DM + NAFLD group, changes in these biomarkers were compared to baseline levels and between groups. The results revealed that both the T2DM and T2DM + NAFLD groups had higher Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and FABP4 levels, and lower PPAR gamma levels compared to the NGT group. Notably, these differences were more pronounced in the T2DM + NAFLD group compared to the T2DM group. Following metformin treatment, HOMA-IR and FABP4 levels decreased, while PPAR gamma levels increased in the T2DM + NAFLD group. These findings suggest that metformin may improve insulin resistance by reducing serum FABP4 and increasing PPAR gamma levels, thereby leading to improved glucose and lipid metabolism in T2DM + NAFLD patients. Thus, FABP4 and PPAR gamma may serve as potential predictors of T2DM + NAFLD.
Terahertz (THz)-based near-field imaging technology exhibits significant potential for applications in biomedical detection. This study investigates biological cells utilizing the rapid imaging capabilities, high resolution, and non-destructive characteristics inherent to terahertz scattering-based scanning near-field optical microscopy (s-SNOM). The findings indicate that the integration of terahertz near-field microscopy with nanoprecision probes and a high-power terahertz radiation source transcends the size and resolution constraints of traditional terahertz far-field imaging systems, which typically achieve resolutions in the range of hundreds to tens of microns for smaller biological tissues. Furthermore, this approach addresses the intrinsic complexities associated with biological cells, such as the presence of numerous intracellular constituents and high water content, thereby facilitating clear and non-destructive imaging of both the surfaces and interiors of individual human sperm at resolutions on the order of tens of nanometers. The simultaneous acquisition of terahertz imaging data is accomplished in approximately 11 min, representing a substantial enhancement in imaging speed. This work not only serves as a compelling example but also presents an effective methodology for imaging medical biological samples using THz near-field microscopy.
In this study, we report a case of a previously undetected triallelic pattern in the arrangement of structures at the D1S1656 locus in the Bulgarian population. The D1S1656 locus is one of the most widely used targets, present in almost all genetic analyses of DNA in forensic medical examinations. During routine forensic analyses for determining loci, we encountered a triallelic pattern in the genetic profile of biological materials from one individual. The samples were taken from a plastic cup and a bottle used by the suspect and were compared with seized material from the buccal mucosa by swabbing from the same individual. The results of the analyzed STR markers, performed using the PCR Amplification Kit, were compared with the nomenclature values of the analyzed STR markers contained in the AmpFlSTR NGM. We performed the identification with a confirmed genotype for the control DNA and compared with the control sample, in which the amplification product is missing. To better characterize the allele outside the allelic ladder of the size, we performed a re-extraction and re-amplification with the PowerPlex® Fusion 6 C System of the reference material from the registered individual. Analysis of the obtained results showed that the anomaly we observed belongs to the tri-allelic type I models. From the extensive review of the forensic literature, we found that such an anomaly has not been reported to date. This specific genetic anomaly confirmed the identification of the suspect and served the forensic scientists in solving the specific case.
Phage-like particles (PLPs) are fabricated self-assembling nanoparticles derived from the structural elements of bacteriophages. These particles have biotechnological utility because of the ability to easily modify surface chemistry and compartmentalize nucleic acids or other materials. A consequential implementation of PLPs in diagnostics is as process controls in nucleic acid amplification tests, where control RNAs are packaged within the protein capsid and protected from degradation by RNases in the sample matrix. Key developments in PLP controls have enhanced the packing efficiency of RNAs into particles, reduced the complexity of their plasmid expression systems, and shifted purification from ultracentrifugation to affinity chromatography, producing progressively greater yields with higher purity. Expanding on prior improvements, this study establishes a revised set of plasmid vectors for Emesvirus zinderi (MS2) derived PLPs that streamline vector manipulations for rapid prototyping of new particles, provide validation of an alternative affinity tag for purification, and contributes a high-throughput low-volume spin column purification strategy. These advancements are combined with a novel internal fusion site in MS2 maturation protein A, a passive element of the MS2 capsid in prior PLP designs, that is capable of displaying polypeptides on the particles’ surface. The functionality of the chimeric maturation protein’s surface display is verified with an affinity tag fusion and subsequent purification. This advancement increases the number of available peptide display sites for the MS2 PLP platform with wide-ranging implications for future applications.
Paederia lanuginosa WALL. (P. lanuginosa) is a medicinal herb used to treat leprosy, expel worms, detoxify the body, and address stomach-related illnesses. On the other hand, P. lanuginosa leaves can also be used as a culinary spice. This study investigates the chemical composition of P. lanuginosa leaf extracts prepared with alcohol and it also evaluates the potential of these extracts for various biological activities, including antibacterial, antifungal and anticancer properties. The results showed that the alcoholic extract of P. lanuginosa leaves includes organic compounds such as alkaloids, iridoid glycosides, flavonoid, tannin, anthraquinone, and terpenoid. The extract did not contain saponin, coumarin, steroid, deodorised sugars, or carboxylic acids. The extract exhibited good broad-spectrum antibacterial activity at a concentration of 50 μg/mL against the tested bacterial strains Escherichia coli, Bacillus subtilis, Lactobacillus plantarum, and Helicobacter pylori. Furthermore, the extract displayed good antifungal activity against Aspergillus brasiliensis, Aspergillus flavus, and Candida albicans at a concentration of 50 μg/mL. The extract of P. lanuginosa leaves also showed moderate inhibitory activity against the three tested cancer cell lines MKN-7, SW480, and HT29, with IC50 values ranging from 25.26 to 28.83 µg/mL.
L-tyrosine, an aromatic amino acid, has attracted increasing attention owing to its wide application in industrial settings. Nonetheless, to obtain strains with high efficiency in producing L-tyrosine still faces challenges. In this study, a recombinant bacteria with the ability to accumulate L-tyrosine efficiently was identified after analysis of constructed strains obtained by different genetic modifications. For the first strain (TYR02), aroGfbr was up-regulated and tyrR was knocked out to relieve the repression of multiple genes in the L-tyrosine synthesis pathway by TyrR (encoded by tyrR). For the second set of strains (TYR03 and TYR04), the transcription of tyrAfbrB was enhanced by the trc and T7 promoters based on TY02 respectively. In the third set of strains (TYR05 and TYR06), the transcription of tyrAfbrB was enhanced by the trc and T7 promoters respectively, and aroGfbr was up-regulated. TYR02 exhibited relatively efficient on L-tyrosine accumulation in shake flask cultures after 24 h. Notably, up-regulation of the transcript level of tyrAfbrB did not show the predicted effects, while the manifested traits of knocking out tyrR disappeared after up-regulated expression of tyrAfbrB. After 25 h of fed-batch fermentation in a 30 L fermentor, TYR02 accumulated L-tyrosine about 50.2 g/L, representing the highest concentration in such a short fermentation time.
Secondary metabolites (SMs), organic compounds synthesized by plants, play crucial roles in their own physiology and within their ecological niches, and have extensive usages in industries like pharmaceuticals, cosmetics, and food. Due to the complex nature of these metabolites, utilizing microorganisms has been proposed for their efficient and cost-effective production. In addressing challenges in SM synthesis, retrosynthesis has become essential. Applications such as RetroPath2.0, BioNavi-NP, and RetroBioCat have been developed to predict and design biosynthetic pathways, supporting synthetic biology and metabolic engineering efforts. These applications employ different methodologies to enhance the synthesis of target compounds, yet often face limitations in user-friendliness, functionality, and adaptability. This study evaluates the potential of RetroPath2.0, RetroBioCat, and BioNavi-NP in predicting the production pathways of 11 alkaloids, a class of plant secondary metabolites (PSMs). Through comparative analysis, the efficacy of these applications in proposing alternative production strategies was assessed. Findings highlighted RetroPath2.0’s capability in outlining precise production pathways in host organisms, despite some restrictions in its broader practicability. The study also demonstrated the production pathways of dimethyltryptamine, nicotine, and higenamine in non-plant hosts, illustrating the practical uses of these applications.
Polycystic Kidney Disease (PKD), a highly prevalent hereditary condition, presents significant gaps in understanding its early-stage pathophysiology and lacks reliable biomarkers to capture these early mechanistic changes. In this investigation, we leveraged a bidirectional Mendelian randomization(MR) analysis, utilizing data from comprehensive genome-wide association studies (GWAS) to systematically explore the causal relationships between serum and urine biomarkers and PKD onset. Instrumental variable selection adhered to stringent criteria to ensure methodological rigor and robust causal inference. Our primary analytical method, inverse variance weighted (IVW), was complemented by MR-Egger, Weighted Median, and Weighted Mode sensitivity analyses to account for pleiotropy and other biases. Heterogeneity was tested using the Cochrane Q statistic, while horizontal pleiotropy was probed via the MR-Egger intercept test and MR-PRESSO global test. Sensitivity to instrument selection was evaluated with a Leave-one-out analysis. The MR analysis identified two serum biomarkers—Gamma-Glutamyl Transferase (GGT) and Sex Hormone-Binding Globulin (SHBG)—as exhibiting statistically significant causal associations with PKD risk. However, reverse MR analysis revealed no significant feedback effect of PKD genetic liability on these biomarker levels. These findings highlight GGT and SHBG as potential biomarkers for early PKD detection, offering novel insights into its pathobiological underpinnings and informing future therapeutic target development.
