Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus.

Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.

The Peptidoglycan Recognition Protein 1 confers immune evasive properties on pancreatic cancer stem cells.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies. DESIGN: We used the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent in vitro coculture assays, in vivo mouse and zebrafish tumorigenesis studies, and in silico database approaches were performed. RESULTS: Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC. CONCLUSIONS: This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.

Myopic Regression after FS-LASIK and SMILE.

PURPOSE: To compare the degree of myopic regression after myopia correction with either femtosecond laser-assisted in situ keratomileusis (FS-LASIK) or small-incision lenticule extraction (SMILE) over 18 months. METHODS: Patients undergoing FS-LASIK or SMILE surgery for myopia correction were retrospectively recruited. The propensity scores were used to match patients by age and preoperative manifest spherical equivalent (SEQ) from these 2 groups. Myopic regression was analyzed using the Cox proportional hazard model. RESULTS: A total of 416 eyes of 416 patients undergoing FS-LASIK and 416 eyes of 416 patients undergoing SMILE were matched. Using 1-month SEQ as baseline, the SEQ regression values after FS-LASIK were 0D, -0.17 ± 0.69D, -0.24 ± 0.65D, -0.31 ± 0.65D, -0.32 ± 0.63D, and -0.33 ± 0.62D and the SEQ regression values after SMILE were 0D, -0.07 ± 0.75D, -0.18 ± 0.77D, -0.23 ± 0.82 D, -0.21 ± 0.77D, and -0.24 ± 0.68D at 1, 3, 6, 9, 12, and 18 months, respectively. The Cox proportional hazard model showed that preoperative manifest SEQ (P = 0.021) and designed optical zone (P = 0.048) are significant predictors. The selected surgical procedure had no significant effect on predicting myopic regression (P = 0.470). The cumulative survival rates of myopic regression were 54.74% and 42.10% in the FS-LASIK group and 58.66% and 43.83% in the SMILE group, at 12 and 18 months, respectively (log-rank test, P = 0.11). CONCLUSIONS: After matching based on age and preoperative manifest SEQ, we found that higher myopia and a smaller optical zone contribute significantly to the development of myopic regression after undergoing FS-LASIK or SMILE surgery at 18 months. The selected surgical procedure, however, does not affect the likelihood of myopic regression.

O-GlcNAcylation in ischemic diseases.

Protein glycosylation is an extensively studied field, with the most studied forms being oxygen or nitrogen-linked N-acetylglucosamine (O-GlcNAc or N-GlcNAc) glycosylation. Particular residues on proteins are targeted by O-GlcNAcylation, which is among the most intricate post-translational modifications. Significantly contributing to an organism's proteome, it influences numerous factors affecting protein stability, function, and subcellular localization. It also modifies the cellular function of target proteins that have crucial responsibilities in controlling pathways related to the central nervous system, cardiovascular homeostasis, and other organ functions. Under conditions of acute stress, changes in the levels of O-GlcNAcylation of these proteins may have a defensive function. Nevertheless, deviant O-GlcNAcylation nullifies this safeguard and stimulates the advancement of several ailments, the prognosis of which relies on the cellular milieu. Hence, this review provides a concise overview of the function and comprehension of O-GlcNAcylation in ischemia diseases, aiming to facilitate the discovery of new therapeutic targets for efficient treatment, particularly in patients with diabetes.

Association between concentrations of rare earth elements in chorionic villus and risk for unexplained spontaneous abortion.

Rare earth elements (REEs) exposure during pregnancy may increase the risk of unexplained spontaneous abortion. However, the association between REEs intrauterine exposure and unexplained spontaneous abortion had yet to be studied. In order to conduct this large case-control study, we thus collected chorionic villus from 641 unexplained spontaneous abortion and 299 control pregnant women and detected the concentrations of 15 REEs by inductively coupled plasma mass spectrometer (ICP-MS). Because the detection rates of 10 REEs were less than 80%, the remaining 5 REEs, which were lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd) and yttrium (Y), underwent to further analysis. The association between 5 REEs and unexplained spontaneous abortion was assessed by using the logistic regression, bayesian kernel regression (BKMR) and weighted quantile sum regression (WQS) models. In the adjusted logistic regression model, Pr, Nd and Y enhanced the incidence of unexplained spontaneous abortion in a dose-dependent way and Ce increased the risk only at high concentration group. The result of BKMR model demonstrated that the risk of unexplained spontaneous abortion increased as the percentile of five mixed REEs increased. Y and Nd were both significantly associated with an increased incidence of unexplained spontaneous abortion, but La was correlated with a decrease in the risk of unexplained spontaneous abortion. Pr was substantially associated with an increase in the risk of unexplained spontaneous abortion when other REEs concentrations were fixed at the 25th and 50th percentiles. According to WQS regression analysis, the WQS index was significantly associated with unexplained spontaneous abortion (OR = 3.75, 95% CI:2.40-5.86). Y had the highest weight, followed by Nd and Pr, which was consistent with the analysis results of our other two models. In short, intrauterine exposure to REEs was associated with an increased risk of unexplained spontaneous abortion, with Y, Nd and Pr perhaps playing an essential role.

Characterizing substructure via mixture modeling in large-scale genetic summary statistics.

Genetic summary data are broadly accessible and highly useful including for risk prediction, causal inference, fine mapping, and incorporation of external controls. However, collapsing individual-level data into groups masks intra- and inter-sample heterogeneity, leading to confounding, reduced power, and bias. Ultimately, unaccounted substructure limits summary data usability, especially for understudied or admixed populations. Here, we present Summix2, a comprehensive set of methods and software based on a computationally efficient mixture model to estimate and adjust for substructure in genetic summary data. In extensive simulations and application to public data, Summix2 characterizes finer-scale population structure, identifies ascertainment bias, and identifies potential regions of selection due to local substructure deviation. Summix2 increases the robust use of diverse publicly available summary data resulting in improved and more equitable research.

Fractionation of cassava pectins and their detailed structural analyses using various pectinolytic enzymes.

In this study, we analyzed the pectin structure within the pulp of cassava. Cassava pectin, derived from cassava pulp treatment at 120 °C for 90 min, was separated into four fractions (CP-P, CP-SD1, CP-SD2F, and CP-SD2R) based on variations in water solubility, electrical properties, and molecular weights. Sugar composition analysis demonstrated an abundance of homogalacturonan (HG) in CP-P and CP-SD2F, rhamnogalacturonan I (RG-I) in CP-SD2R, and neutral sugars in CP-SD1. Because RG-I possesses a complex structure, we analyzed CP-SD2R using various pectinolytic enzymes. Galactose was the major sugar in CP-SD2R accounting for 49 %, of which 65 % originated from arabinogalactan I, 9 % from galactose and galactooligosaccharides, 5 % from arabinogalactan II, and 11 % from galactoarabinan. Seventy-four percent of arabinose in CP-SD2R was present as galactoarabinan. The methylation (DM) and acetylation (DAc) degrees of cassava pectin were 11 and 15 %, respectively. The HG and RG-I regions exhibited DAc values of 5 and 44 %, respectively, signifying the high DAc of RG-I compared to HG. Information derived from the structural analysis of cassava pectin will enable efficient degradation of pectin and cellulose, leading to the use of cassava pulp as a raw material for biorefineries.

Genotoxicity and cytotoxicity in male reproductive cells caused by sediment pollutants.

In recent years, mounting evidence has highlighted a global decline in male semen quality, paralleling an increase in male infertility problems. Such developments in the male reproductive system are likely due to a range of environmental factors, which could negatively affect the outcomes of pregnancy, reproductive health, and the well-being of fetuses. Different environmental contaminants ultimately accumulate in riverbed sediments due to gravity, so these sediments are frequently considered hotspots for pollutants. Therefore, understanding the detrimental effects of river sediment pollution on human reproductive health is crucial. This study indicates male germ cells' high vulnerability to environmental contaminants. There is a strong positive correlation between the concentration of complex accumulated pollutants from human activities and the reproductive toxicity observed in human testicular embryonic cell lines NCCIT and NTERA-2. This toxicity is characterized by increased levels of reactive oxygen species, disruption of critical cellular functions, genotoxic impacts, and the induction of cell apoptosis. This research marks a significant step in providing in vitro evidence of the damaging effects of environmental pollutants on the human male germline.

A critical role for the nuclear protein Akirin in larval development in Henosepilachna vigintioctopunctata.

Akirin is a nuclear protein that controls development in vertebrates and invertebrates. The function of Akirin has not been assessed in any Coleopteran insects. We found that high levels of akirin transcripts in Henosepilachna vigintioctopunctata, a serious Coleopteran potato defoliator (hereafter Hvakirin), were present at prepupal, pupal and adult stages, especially in larval foregut and fat body. RNA interference (RNAi) targeting Hvakirin impaired larval development. The Hvakirin RNAi larvae arrested development at the final larval instar stage. They remained as stunted larvae, gradually blackened and finally died. Moreover, the remodelling of gut and fat body was inhibited in the Hvakirin depleted larvae. Two layers of cuticles, old and newly formed, were noted in the dsegfp-injected animals. In contrast, only a layer of cuticle was found in the dsakirin-injected beetles, indicating the arrest of larval development. Furthermore, the expression of three transforming growth factor-ß cascade genes (Hvsmox, Hvmyo and Hvbabo), a 20-hydroxyecdysone (20E) receptor gene (HvEcR) and six 20E response genes (HvHR3, HvHR4, HvE75, HvBrC, HvE93 and Hvftz-f1) was significantly repressed, consistent with decreased 20E signalling. Conversely, the transcription of a juvenile hormone (JH) biosynthesis gene (Hvjhamt), a JH receptor gene (HvMet) and two JH response genes (HvKr-h1 and HvHairy) was greatly enhanced. Our findings suggest a critical role of Akirin in larval development in H. vigintioctopunctata.

Changes in Corneal Epithelial Thickness in Different Areas After Femtosecond Laser-Assisted LASIK in Patients With High Astigmatism.

PURPOSE: To explore changes in corneal epithelial thickness (CET) after femtosecond laser-assisted laser in situ keratomileusis in patients with high astigmatism. METHODS: CET was measured at every intersection of the concentric circles and specific axes using AngioVue optical coherence tomography (Angio-OCT) preoperatively and 1 month postoperatively. The average thickness of corneal central, paracentral, and peripheral regions was the mean of the points within the central 2, 2 to 5, and 5 to 7 mm areas, respectively. Correlation analysis was performed to investigate the association between CET along different axes and other preoperative and postoperative parameters. RESULTS: Forty-two eyes of 28 patients were included. CET along the astigmatic (K1) and perpendicular (K2) axes in the central and paracentral areas increased (P < .001), whereas that along the K2 axis decreased in the peripheral area 1 month postoperatively (P = .001). The amount of CET change in the peripheral area between the K1 and K2 axes was significantly different (P < .001). In the central area, the change in CET along the K2 axis was positively correlated with ablation depth (r = 0.315, P = .042) and negatively with refractive power after surgery (r = -0.347, P = .024). In the peripheral area, the changes in CET along both K1 and K2 axes were negatively correlated with ablation depth (r = -0.431, P = .004; r = -0.387, P = .011, respectively). CONCLUSIONS: Epithelial modeling differed between the different astigmatism axes after refractive surgery. The compensatory response of the corneal epithelium is more pronounced along the steeper axis. [J Refract Surg. 2024;40(4):e239-e244.].

Positive Airway Pressure and Metabolic Markers in Children With Obstructive Sleep Apnea.

BACKGROUND: Increasing evidence suggests an association between childhood obstructive sleep apnea (OSA) and metabolic syndrome, with more research available on the potential impacts of positive airway pressure (PAP) on metabolic markers in children. The purpose of this systematic review is to provide a systematic synthesis of the evidence on the effect of PAP use on metabolic markers in children with OSA. METHODS: A search strategy with terms for "OSA" and metabolic markers in pediatrics was run to systematically assess 5 databases until August 26, 2022. Two reviewers independently screened eligible articles, extracted data, and conducted quality appraisal. Meta-analysis was done using random-effects models. Body mass index (BMI), glycemic, lipid, cardiovascular, and other metabolic and inflammatory markers were reported. RESULTS: Sixteen studies (N = 1,213) were included, 15 observational studies and 1 randomized controlled trial (RCT); most reported outcomes in children with obesity. Meta-analysis of 4 studies found no changes in BMI at median average follow-up of 12 months after PAP initiation. A reduction in heart rate and blood pressure parameters was demonstrated in several studies in children with OSA with and without obesity at a median average follow-up of 4.9 months after PAP initiation. Research in echocardiographic outcomes is limited, including one RCT in children with Down syndrome and OSA showing no changes in heart rate variability parameters. Evidence of improvements in glycemic and/or lipid control, liver enzymes, and inflammatory markers with PAP therapy is even more limited and of limited clinical importance. Risk of bias was moderate to critical and outcome evidence very low. CONCLUSIONS: Although evidence on effects of PAP on metabolic markers in children with OSA is encouraging, available literature is limited. Longitudinal studies are still required to further assess the long-term influence of PAP on metabolic and inflammatory markers, particularly in children with obesity.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Aims: Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. Methods: MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Results: Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and ß1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Conclusion: Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

Design, Manufacture, and Characterization of a Critical-Sized Gradient Porosity Dual-Material Tibial Defect Scaffold.

This study proposed a composite tibia defect scaffold with radial gradient porosity, utilizing finite element analysis to assess stress in the tibial region with significant critical-sized defects. Simulations for scaffolds with different porosities were conducted, designing an optimal tibia defect scaffold with radial gradient porosity for repairing and replacing critical bone defects. Radial gradient porosity scaffolds resulted in a more uniform stress distribution, reducing titanium alloy stiffness and alleviating stress shielding effects. The scaffold was manufactured using selective laser melting (SLM) technology with stress relief annealing to simplify porous structure fabrication. The study used New Zealand white rabbits' tibia defect sites as simulation parameters, reconstructing the 3D model and implanting the composite scaffold. Finite element analysis in ANSYS-Workbench simulated forces under high-activity conditions, analyzing stress distribution and strain. In the simulation, the titanium alloy scaffold bore a maximum stress of 122.8626 MPa, while the centrally encapsulated HAp material delivered 27.92 MPa. The design demonstrated superior structural strength, thereby reducing stress concentration. The scaffold was manufactured using SLM, and the uniform design method was used to determine a collection of optimum annealing parameters. Nanoindentation and compression tests were used to determine the influence of annealing on the elastic modulus, hardness, and strain energy of the scaffold.

Association between physician-hospital integration and inpatient care delivery in accountable care organizations: An instrumental variable analysis.

OBJECTIVE: To investigate the relationship between physician-hospital integration within accountable care organizations (ACOs) and inpatient care utilization and expenditure. DATA SOURCES: The primary data were Massachusetts All-Payer Claims Database (2009-2013). STUDY SETTING: Fifteen provider organizations that entered a commercial ACO contract with a major private payer in Massachusetts between 2009 and 2013. STUDY DESIGN: Using an instrumental variable approach, the study compared inpatient care delivery between patients of ACOs demonstrating high versus low integration. We measured physician-hospital integration within ACOs by the proportion of primary care physicians in an ACO who billed for outpatient services with a place-of-service code indicating employment or practice ownership by a hospital. The study sample comprised non-elderly adults who had continuous insurance coverage and were attributed to one of the 15 ACOs. Outcomes of interest included total medical expenditure during an episode of inpatient care, length of stay (LOS) of the index hospitalization, and 30-day readmission. An inpatient episode was defined as 30, 45, and 60 days from the admission date. DATA COLLECTION/EXTRACTION METHODS: Not applicable. PRINCIPAL FINDINGS: The study examined 33,535 admissions from patients served by the 15 ACOs. Average medical expenditure within 30 days of admission was $24,601, within 45 days was $26,447, and within 60 days was $28,043. Average LOS was 3.5 days, and 5.4% of patients were readmitted within 30 days. Physician-hospital integration was associated with a 10.6% reduction in 30-day expenditure (95% CI, -15.1% to -5.9%). Corresponding estimates for 45 and 60 days were - 9.7% (95%CI, -14.2% to -4.9%) and - 9.6% (95%CI, -14.3% to -4.7%). Integration was associated with a 15.7% decrease in LOS (95%CI, -22.6% to -8.2%) but unrelated to 30-day readmission rate. CONCLUSIONS: Our instrumental variable analysis shows physician-hospital integration with ACOs was associated with reduced inpatient spending and LOS, with no evidence of elevated readmission rates.

Hierarchical self-recognition and response in CSC and non-CSC micro-niches for cancer therapy.

Cancer stem cells (CSCs) characterized by self-renewal, invasiveness, tumorigenicity and resistance to treatment are regarded as the thorniest issues in refractory tumors. We develop a targeted and hierarchical controlled release nano-therapeutic platform (SEED-NPs) that self-identifies and responds to CSC and non-CSC micro-niches of tumors. In non-CSC micro-niche, reactive oxygen species (ROS) trigger the burst release of the chemotherapeutic drug and photosensitizer to kill tumor cells and reduce tumor volume by combining chemotherapy and photodynamic therapy (PDT). In CSC micro-niche, the preferentially released differentiation drug induces CSC differentiation and transforms CSCs into chemotherapy-sensitive cells. SEED-NPs exhibit an extraordinary capacity for downregulating the stemness of CD44+/CD24- SP (side population) cell population both in vitro and in vivo, and reveal a 4-fold increase of tumor-targeted accumulation. Also, PDT-generated ROS promote the formation of tunneling nanotubes and facilitate the divergent network transport of drugs in deep tumors. Moreover, ROS in turn promotes CSC differentiation and drug release. This positive-feedback-loop strategy enhances the elimination of refractory CSCs. As a result, SEED-NPs achieve excellent therapeutic effects in both 4T1 SP tumor-bearing mice and regular 4T1 tumor-bearing mice without obvious toxicities and eradicate half of mice tumors. SEED-NPs integrate differentiation, chemotherapy and PDT, which proved feasible and valuable, indicating that active targeting and hierarchical release are necessary to enhance antitumor efficacy. These findings provide promising prospects for overcoming barriers in the treatment of CSCs.

Design of a Humidity Sensor for a PPE Kit Using a Flexible Paper Substrate.

The present work reports the rapid sweat detection inside a PPE kit using a flexible humidity sensor based on hydrothermally synthesized ZnO (zinc oxide) nanoflowers (ZNFs). Physical characterization of ZNFs was done using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transmission infrared spectroscopy (FTIR), UV-visible, particle size analysis, Raman analysis, and X-ray photoelectron spectroscopy (XPS) analysis, and the hydrophilicity was investigated by using contact angle measurement. Fabrication of a flexible sensor was done by deposition on the paper substrate using the spin coating technique. It exhibited high sensitivity and low response and recovery times in the humidity range 10-95%RH. The sensor demonstrated the highest sensitivity of 296.70 nF/%RH within the humidity range 55-95%RH, and the rapid response and recovery times were also calculated and found as 5.10/1.70 s, respectively. The selectivity of the proposed sensor was also analyzed, and it is highly sensitive to humidity. The humidity sensing characteristics were theoretically witnessed in terms of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) and electronic properties of sensing materials in ambient and humid conditions. These theoretical results are evidence of the interaction of ZnO with humidity. Overall, the present study provides a scope of architecture-enabled paper-based humidity sensors for the detection of sweat levels inside PPE kits for health workers.

Continuous microalgal culture module and method of culturing microalgae containing macular pigment.

This study established and investigated continuous macular pigment (MP) production with a lutein (L):zeaxanthin (Z) ratio of 4-5:1 by an MP-rich Chlorella sp. CN6 mutant strain in a continuous microalgal culture module. Chlorella sp. CN6 was cultured in a four-stage module for 10 days. The microalgal culture volume increased to 200 L in the first stage (6 days). Biomass productivity increased to 0.931 g/L/day with continuous indoor white light irradiation during the second stage (3 days). MP content effectively increased to 8.29 mg/g upon continuous, indoor white light and blue light-emitting diode irradiation in the third stage (1 day), and the microalgal biomass and MP concentrations were 8.88 g/L and 73.6 mg/L in the fourth stage, respectively. Using a two-step MP extraction process, 80 % of the MP was recovered with a high purity of 93 %, and its L:Z ratio was 4-5:1.

Genetic diversity, population structure, and taxonomic confirmation in annual medic (Medicago spp.) collections from Crimea, Ukraine.

Annual medic (Medicago spp.) germplasm was collected from the Crimean Peninsula of Ukraine in 2008 to fill gaps in geographic coverage in the United States department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS) temperate-adapted forage legume collection. A total of 102 accessions across 10 Medicago species were collected. To assess genetic diversity, population structure, and to confirm taxonomic identities, the collections were phenotypically and genetically characterized. Phenotyping included the use of 24 descriptor traits while genetic characterization was accomplished using a 3K Diversity Array Technologies (DArTag) panel developed for alfalfa (Medicago sativa L.). For both field and molecular characterizations, a reference set of 92 geographically diverse and species-representative accessions were obtained from the NPGS collection. Phenotypic descriptors showed consistency among replicated plants within accessions, some variation across accessions within species, and evident distinctions between species. Because the DArTag panel was developed for cultivated alfalfa, the transferability of markers to the species being evaluated was limited, resulting in an average of ~1,500 marker loci detected per species. From these loci, 448 markers were present in 95% of the samples. Principal component and phylogenetic analysis based on a larger set of 2,396 selected markers clustered accessions by species and predicted evolutionary relationships among species. Additionally, the markers aided in the taxonomic identity of a few accessions that were likely mislabeled. The genotyping results also showed that sampling individual plants for these mostly self-pollinating species is sufficient due to high reproducibility between single (n=3) and pooled (n=7) biological replicate leaf samples. The phenotyping and the 2,396 Single Nucleotide Polymorphism (SNP) marker set were useful in estimating population structure in the Crimean and reference accessions, highlighting novel and unique genetic diversity captured in the Crimean accessions. This research not only demonstrated the utility of the DArTag marker panel in evaluating the Crimean germplasm but also highlighted its broader application in assessing genetic resources within the Medicago genus. Furthermore, we anticipate that our findings will underscore the importance of leveraging genetic resources and advanced genotyping tools for sustainable crop improvement and biodiversity conservation in annual medic species.