Dog leukocyte antigen genotyping across class I and class II genes in beagle dogs as laboratory animals.

Dog leukocyte antigen (DLA) polymorphisms have been found to be associated with inter-individual variations in the risk, susceptibility, and severity of immune-related phenomena. While DLA class II genes have been extensively studied, less research has been performed on the polymorphisms of DLA class I genes, especially in beagle dogs commonly used as laboratory animals for safety evaluations in drug development. We genotyped four DLA class I genes and four DLA class II genes by locus-specific Sanger sequencing using 93 laboratory beagle dogs derived from two different strains: TOYO and Marshall. The results showed that, for DLA class I genes, 11, 4, 1, and 2 alleles, including a novel allele, were detected in DLA-88, DLA-12/88L, DLA-64, and DLA-79, while, for DLA class II genes, 1, 10, 6, and 7 alleles were detected in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. It was estimated that there were 14 DLA haplotypes, six of which had a frequency of ≥ 5%. Furthermore, when comparing the DLA diversity between TOYO and Marshall strains, the most common alleles and haplotypes differed between them. This is the first study to genotype all DLA loci and determine DLA haplotypes including all DLA class I and class II genes in dogs. Integrating information on the DLA diversity of laboratory beagle dogs should reinforce their benefit as an animal model for understanding various diseases associated with a specific DLA type.

Markers of intestinal barrier damage in patients with chronic insomnia disorder.

Objective: Insomnia disorder stands out as one of the prevalent clinical sleep and psychiatric disorders. Prior research has unequivocally demonstrated variations in the diversity and abundance of gut microbiota among individuals with insomnia disorder. These alterations may play a direct or indirect role in the onset and progression of insomnia disorder by compromising the integrity of the intestinal barrier. This study aims to evaluate the impairment of the intestinal barrier in individuals with insomnia disorder by scrutinizing the serum functionality of this barrier. Materials and methods: 45 patients with chronic insomnia disorder and 30 matched healthy volunteers were meticulously selected based on inclusion criteria. ELISA technology was employed to measure serum levels of diamine oxidase (DAO), D-lactic acid (D-LA), intestinal fatty acid binding protein (I-FABP), and endothelin (ET). Spearman correlation analysis was used to explore the relationship between intestinal mucosal markers and clinical characteristics. Data were analyzed using SPSS 26.0. Results: Compared to the healthy control group, the insomnia disorder group exhibited significantly elevated scores on subjective mood and sleep scales (GAD-7, PHQ-9, HAMA, HAMD, PSQI, and ISI) (P < 0.05). Overnight PSG indicated a notable increase in bed time, total wake time, sleep onset latency, and wake after sleep onset in individuals with insomnia disorder. Additionally, there was a decrease in sleep efficiency and alterations in sleep structure (increased proportion of N1 and N3 stages, prolonged N1 stage) (P < 0.05). The chronic insomnia disorder group displayed significantly reduced concentrations of serum DAO, D-LA, I-FABP, and ET (P < 0.05). Furthermore, significant positive correlations were identified between intestinal epithelial barrier markers and sleep efficiency, while negative correlations were found with wake after sleep onset, total wake time, PSQI, HAMA, and HAMD. Additionally, D-LA levels were significantly positively correlated with ET concentrations. Conclusion: Individuals with chronic insomnia disorder manifest disruptions in sleep structure, heightened susceptibility to anxiety and depressive moods, and impaired intestinal barrier function. These findings suggest that the occurrence and development of insomnia disorder may be linked to the impairment of the intestinal barrier.

Ultra-sensitive CTC-based liquid biopsy for pancreatic cancer enabled by large blood volume analysis.

The limited sensitivity of circulating tumor cell (CTC) detection in pancreatic adenocarcinoma (PDAC) stems from their extremely low concentration in the whole circulating blood, necessitating enhanced detection methodologies. This study sought to amplify assay-sensitivity by employing diagnostic leukapheresis (DLA) to screen large blood volumes. Sixty patients were subjected to DLA, with a median processed blood volume of ~ 2.8 L and approximately 5% of the resulting DLA-product analyzed using CellSearch (CS). Notably, DLA significantly increased CS-CTC detection to 44% in M0-patients and 74% in M1-patients, yielding a 60-fold increase in CS-CTC enumeration. DLA also provided sufficient CS-CTCs for genomic profiling, thereby delivering additional genomic information compared to tissue biopsy samples. DLA CS-CTCs exhibited a pronounced negative prognostic impact on overall survival (OS), evidenced by a reduction in OS from 28.6 to 8.5 months (univariate: p = 0.002; multivariable: p = 0.043). Additionally, a marked enhancement in sensitivity was achieved (by around 3-4-times) compared to peripheral blood (PB) samples, with positive predictive values for OS being preserved at around 90%. Prognostic relevance of CS-CTCs in PDAC was further validated in PB-samples from 228 PDAC patients, consolidating the established association between CTC-presence and reduced OS (8.5 vs. 19.0 months, p < 0.001). In conclusion, DLA-derived CS-CTCs may serve as a viable tool for identifying high-risk PDAC-patients and aiding the optimization of multimodal treatment strategies. Moreover, DLA enables comprehensive diagnostic profiling by providing ample CTC material, reinforcing its utility as a reliable liquid-biopsy approach. This high-volume liquid-biopsy strategy presents a potential pathway for enhancing clinical management in this malignancy.

A Coumarin-Imidazothiadiazole Derivative, SP11 Abrogates Tumor Growth by Targeting HSP90 and Its Client Proteins.

Despite several treatment options for blood cancer, mortality remains high due to relapse and the disease's aggressive nature. Elevated levels of HSP90, a molecular chaperone essential for protein folding, are associated with poor prognosis in leukemia and lymphoma. HSP90 as a target for chemotherapy has been met with limited success due to toxicity and induction of heat shock. This study tested the activity of an HSP90 inhibitor, SP11, against leukemic cells, mouse lymphoma allograft, and xenograft models. SP11 induced cytotoxicity in vitro in leukemic cell lines and induced cell death via apoptosis, with minimal effect on normal cells. SP11 induced cell death by altering the status of HSP90 client proteins both in vitro and in vivo. SP11 reduced the tumor burden in allograft and xenograft mouse models without apparent toxicity. The half-life of SP11 in the plasma was approximately 2 h. SP11 binding was observed at both the N-terminal and C-terminal domains of HSP90. C-terminal binding was more potent than N-terminal binding of HSP90 in silico and in vitro using isothermal calorimetry. SP11 bioavailability and minimal toxicity in vivo make it a potential candidate to be developed as a novel anticancer agent.

Crystal Structure of a Classical MHC Class I Molecule in Dogs; Comparison of DLA-88*0 and DLA-88*5 Category Molecules.

DLA-88 is a classical major histocompatibility complex (MHC) class I gene in dogs, and allelic DLA-88 molecules have been divided into two categories named "DLA-88*0" and "DLA-88*5." The defining difference between the two categories concerns an LQW motif in the α2 domain helical region of the DLA-88*5 molecules that includes the insertion of an extra amino acid compared to MHC class I consensus length. We here show that this motif has been exchanged by recombination between different DLA-88 evolutionary lineages. Previously, with pDLA-88*508:01, the structure of a molecule of the DLA-88*5 category was elucidated. The present study is the first to elucidate a structure, using X-ray crystallography, of the DLA-88*0 category, namely DLA-88*001:04 complexed with ß2m and a nonamer peptide derived from canine distemper virus (CDV). The LQW motif that distinguishes DLA-88*5 from DLA-88*0 causes a shallower peptide binding groove (PBG) and a leucine exposed at the top of the α2 domain helix expected to affect T cell selection. Peptide ligand amino acid substitution and pMHC-I complex formation and stability analyses revealed that P2 and P3 are the major anchor residue positions for binding to DLA-88*001:04. We speculate that the distribution pattern of the LQW motif among canine classical MHC class I alleles represents a strategy to enhance allogeneic rejection by T cells of transmissible cancers such as canine transmissible venereal tumor (CTVT).

Large-Scale Polymorphism Analysis of Dog Leukocyte Antigen Class I and Class II Genes (DLA-88, DLA-12/88L and DLA-DRB1) and Comparison of the Haplotype Diversity between Breeds in Japan.

Polymorphisms of canine leukocyte antigen (DLA) class I (DLA-88 and DLA-12/88L) and class II (DLA-DRB1) genes are important for disease susceptibility studies, but information on the genetic diversity among dog breeds is still lacking. To better elucidate the polymorphism and genetic diversity between breeds, we genotyped DLA-88, DLA-12/88L, and DLA-DRB1 loci using 829 dogs of 59 breeds in Japan. Genotyping by Sanger sequencing identified 89, 43, and 61 alleles in DLA-88, DLA-12/88L, and DLA-DRB1 loci, respectively, and a total of 131 DLA-88-DLA-12/88L-DLA-DRB1 haplotypes (88-12/88L-DRB1) were detected more than once. Of the 829 dogs, 198 were homozygotes for one of the 52 different 88-12/88L-DRB1 haplotypes (homozygosity rate: 23.8%). Statistical modeling suggests that 90% of the DLA homozygotes or heterozygotes with one or other of the 52 different 88-12/88L-DRB1 haplotypes within somatic stem cell lines would benefit graft outcome after 88-12/88L-DRB1-matched transplantation. As previously reported for DLA class II haplotypes, the diversity of 88-12/88L-DRB1 haplotypes varied remarkably between breeds but was relatively conserved within most breeds. Therefore, the genetic characteristics of high DLA homozygosity rate and poor DLA diversity within a breed are useful for transplantation therapy, but they may affect biological fitness as homozygosity progresses.

Observational evidence linking ocean sulfur compounds to atmospheric dimethyl sulfide during Icelandic Sea phytoplankton blooms.

In two Icelandic Sea spring blooms (May 2018 and 2019) in the North Atlantic Ocean (62.9-68.0°N, 9.0-28.0°W), chlorophyll-a and dimethylsulfoniopropionate (DMSP) concentrations and DMSP lyase activity (the DMSP-to-dimethyl sulfide (DMS) conversion efficiency) were measured at 67 stations, and the hourly atmospheric DMS mixing ratios were concurrently measured only in May 2019 at Storhofdi on Heimaey Island, located south of Iceland (63.4°N, 20.3°W). The ocean parameters for biology (i.e., chlorophyll-a, DMSP, and DMSP lyase activity) were broadly associated in distribution; however, the statistical significance of the association differed among four ocean domains and also between 2018 and 2019. Specifically, the widespread dominance of Phaeocystis, coccolithophores, and dinoflagellates (all rich in DMSP and high in DMSP lyase activity) across the study area is a compelling indication that variations in DMSP-rich phytoplankton were likely a main cause of the variations in statistical significance. For all the ocean domains defined here, we found that the DMS production capacity (calculated using the exposures of air masses to ocean biology prior to their arrivals at Heimaey and the atmospheric DMS mixing ratios of those air masses at Heimaey) was surprisingly consistent with in situ ocean S data (i.e., DMSP and DMSP lyase activity). Our study shows that the proposed computational approach enabled the detection of changes in DMS production and emission in association with changes in ocean primary producers.

Genotyping of canine MHC gene DLA-88 by next-generation sequencing reveals high frequencies of new allele discovery and gene duplication.

Dogs have served as one of the most reliable preclinical models for a variety of diseases and treatments, including stem/progenitor cell transplantation. At the genetic epicenter of dog transplantation models, polymorphic major histocompatibility complex (MHC) genes are most impactful on transplantation success. Among the canine class I and class II genes, DLA-88 has been best studied in transplantation matching and outcomes, with 129 DLA-88 alleles identified. In this study we developed and tested a next generation (NGS) sequencing protocol for rapid identification of DLA-88 genotypes in dogs and compared the workflow and data generated with an established DLA-88 Sanger sequencing protocol that has been in common prior use for clinical studies. By testing the NGS protocol on a random population of 382 dogs, it was possible to demonstrate superior efficacy based on laboratory execution and overall cost. In addition, NGS proved far more effective at discovering new alleles and detecting multiple alleles associated with gene duplication. A total of 51 new DLA-88 alleles are reported here. This rate of new allele discovery indicates that a large pool of yet un-discovered DLA-88 alleles exists in the domestic dog population. In addition, more than 46% of dogs carried three or more copies of DLA-88, further emphasizing the need for more sensitive and cost-effective DLA typing methodology for the dog clinical model.

Artificial Intelligence and Corneal Confocal Microscopy: The Start of a Beautiful Relationship.

Corneal confocal microscopy (CCM) is a rapid non-invasive in vivo ophthalmic imaging technique that images the cornea. Historically, it was utilised in the diagnosis and clinical management of corneal epithelial and stromal disorders. However, over the past 20 years, CCM has been increasingly used to image sub-basal small nerve fibres in a variety of peripheral neuropathies and central neurodegenerative diseases. CCM has been used to identify subclinical nerve damage and to predict the development of diabetic peripheral neuropathy (DPN). The complex structure of the corneal sub-basal nerve plexus can be readily analysed through nerve segmentation with manual or automated quantification of parameters such as corneal nerve fibre length (CNFL), nerve fibre density (CNFD), and nerve branch density (CNBD). Large quantities of 2D corneal nerve images lend themselves to the application of artificial intelligence (AI)-based deep learning algorithms (DLA). Indeed, DLA have demonstrated performance comparable to manual but superior to automated quantification of corneal nerve morphology. Recently, our end-to-end classification with a 3 class AI model demonstrated high sensitivity and specificity in differentiating healthy volunteers from people with and without peripheral neuropathy. We believe there is significant scope and need to apply AI to help differentiate between peripheral neuropathies and also central neurodegenerative disorders. AI has significant potential to enhance the diagnostic and prognostic utility of CCM in the management of both peripheral and central neurodegenerative diseases.

The Microbiological Drivers of Temporally Dynamic Dimethylsulfoniopropionate Cycling Processes in Australian Coastal Shelf Waters.

The organic sulfur compounds dimethylsulfoniopropionate (DMSP) and dimethyl sulfoxide (DMSO) play major roles in the marine microbial food web and have substantial climatic importance as sources and sinks of dimethyl sulfide (DMS). Seasonal shifts in the abundance and diversity of the phytoplankton and bacteria that cycle DMSP are likely to impact marine DMS (O) (P) concentrations, but the dynamic nature of these microbial interactions is still poorly resolved. Here, we examined the relationships between microbial community dynamics with DMS (O) (P) concentrations during a 2-year oceanographic time series conducted on the east Australian coast. Heterogenous temporal patterns were apparent in chlorophyll a (chl a) and DMSP concentrations, but the relationship between these parameters varied over time, suggesting the phytoplankton and bacterial community composition were affecting the net DMSP concentrations through differential DMSP production and degradation. Significant increases in DMSP were regularly measured in spring blooms dominated by predicted high DMSP-producing lineages of phytoplankton (Heterocapsa, Prorocentrum, Alexandrium, and Micromonas), while spring blooms that were dominated by predicted low DMSP-producing phytoplankton (Thalassiosira) demonstrated negligible increases in DMSP concentrations. During elevated DMSP concentrations, a significant increase in the relative abundance of the key copiotrophic bacterial lineage Rhodobacterales was accompanied by a three-fold increase in the gene, encoding the first step of DMSP demethylation (dmdA). Significant temporal shifts in DMS concentrations were measured and were significantly correlated with both fractions (0.2-2 µm and >2 µm) of microbial DMSP lyase activity. Seasonal increases of the bacterial DMSP biosynthesis gene (dsyB) and the bacterial DMS oxidation gene (tmm) occurred during the spring-summer and coincided with peaks in DMSP and DMSO concentration, respectively. These findings, along with significant positive relationships between dsyB gene abundance and DMSP, and tmm gene abundance with DMSO, reinforce the significant role planktonic bacteria play in producing DMSP and DMSO in ocean surface waters. Our results highlight the highly dynamic nature and myriad of microbial interactions that govern sulfur cycling in coastal shelf waters and further underpin the importance of microbial ecology in mediating important marine biogeochemical processes.

Lysine acetylation regulates moonlighting activity of the E2 subunit of the chloroplast pyruvate dehydrogenase complex in Chlamydomonas.

The dihydrolipoamide acetyltransferase subunit DLA2 of the chloroplast pyruvate dehydrogenase complex (cpPDC) in the green alga Chlamydomonas reinhardtii has previously been shown to possess moonlighting activity in chloroplast gene expression. Under mixotrophic growth conditions, DLA2 forms part of a ribonucleoprotein particle (RNP) with the psbA mRNA that encodes the D1 protein of the photosystem II (PSII) reaction center. Here, we report on the characterization of the molecular switch that regulates shuttling of DLA2 between its functions in carbon metabolism and D1 synthesis. Determination of RNA-binding affinities by microscale thermophoresis demonstrated that the E3-binding domain (E3BD) of DLA2 mediates psbA-specific RNA recognition. Analyses of cpPDC formation and activity, as well as RNP complex formation, showed that acetylation of a single lysine residue (K197) in E3BD induces the release of DLA2 from the cpPDC, and its functional shift towards RNA binding. Moreover, Förster resonance energy transfer microscopy revealed that psbA mRNA/DLA2 complexes localize around the chloroplast's pyrenoid. Pulse labeling and D1 re-accumulation after induced PSII degradation strongly suggest that DLA2 is important for D1 synthesis during de novo PSII biogenesis.

StarDist Image Segmentation Improves Circulating Tumor Cell Detection.

After a CellSearch-processed circulating tumor cell (CTC) sample is imaged, a segmentation algorithm selects nucleic acid positive (DAPI+), cytokeratin-phycoerythrin expressing (CK-PE+) events for further review by an operator. Failures in this segmentation can result in missed CTCs. The CellSearch segmentation algorithm was not designed to handle samples with high cell density, such as diagnostic leukapheresis (DLA) samples. Here, we evaluate deep-learning-based segmentation method StarDist as an alternative to the CellSearch segmentation. CellSearch image archives from 533 whole blood samples and 601 DLA samples were segmented using CellSearch and StarDist and inspected visually. In 442 blood samples from cancer patients, StarDist segmented 99.95% of CTC segmented by CellSearch, produced good outlines for 98.3% of these CTC, and segmented 10% more CTC than CellSearch. Visual inspection of the segmentations of DLA images showed that StarDist continues to perform well when the cell density is very high, whereas CellSearch failed and generated extremely large segmentations (up to 52% of the sample surface). Moreover, in a detailed examination of seven DLA samples, StarDist segmented 20% more CTC than CellSearch. Segmentation is a critical first step for CTC enumeration in dense samples and StarDist segmentation convincingly outperformed CellSearch segmentation.

Modeling & implementation of DRLA based partially shaded solar system integration with 3-ϕ conventional grid using constant current controller.

Renewable Energy Resources (RERs) are widely used on the concern of global environment protection. Solar energy systems play an important role in the generation of electrical energy, remarkably minimize the utilization of nonrenewable fuel sources. Solar energy can be extracted and transformed into electrical energy via solar photovoltaic process. Several traditional, soft computing, heuristic, and meta-heuristic maximum power point tracking (MPPT) techniques have been developed to extract Maximum Energy Point (MEP) from the solar photovoltaic modules under different atmospheric conditions. In this manuscript, the combination of reinforcement learning algorithm (RLA) and deep learning algorithm (DLA) called deep Reinforcement Learning Algorithm based MPPT (DRLAMPPT) is proposed under partial shading conditions (PSC) of the solar system. DRLAMPPT can deal with continuous state spaces, in contrast to RL it can be operated only with discrete action state spaces. In this proposed DRLAMPPT, deep deterministic policy gradient (DDPG) solves the problem of continuous state spaces are involved to reach the GMEP in photovoltaic systems especially under PSC. In DRLAMPPT, the representative's strategy is parameterized by an artificial neural network (ANN), which uses sensory information as input and directly sends out control signals. This work develops a 2 kW solar photovoltaic power plant comprises of a photovoltaic array, DC/DC step-up converter, 3-Φ Pulse Width Modulated Voltage Source Inverter (PWM-VSI) integrated with conventional power grid using Constant Current Controller (CCC Effectiveness of the proposed DRLAMPPT with CCC can be validated through an experimental setup and with MATLAB. Simulation and tested at different input conditions of solar irradiance. Experimental results prove that, in comparison to existing MPPTs, suggested DRLAMPPT not only attains the best efficiency and also adopts the change in environmental conditions of the photovoltaic system at a much faster rate and able to reach the GMEP within 0.8 s under PSC. Experimental and simulation results also prove that suggested CCC with LC filter makes the inverter output voltage and the grid voltage are in phase at the lower value of THD i.e. 1.1% and 0.98% respectively.

Haplotype structures and polymorphisms of dog leukocyte antigen (DLA) class I loci shaped by intralocus and interlocus recombination events.

The dog leukocyte antigen (DLA) class I genomic region is located on chromosome 12, and the class I genomic region is composed of at least two distinct haplotypic gene structures, DLA-88-DLA-12 and DLA-88-DLA-88L. However, detailed information of the genomic differences among DLA-88, DLA-12, and DLA-88L are still lacking at the full-length gene level, and therefore, DLA allelic sequences classified for each of these loci are limited in number so far. In this study, we determined the DNA sequence of a 95-kb DLA class I genomic region including DLA-88, DLA-12/88L, and DLA-64 with three DLA homozygous dogs and of 37 full-length allelic gene sequences for DLA-88 and DLA-12/88L loci in 26 DLA class I homozygous dogs. Nucleotide diversity profiles of the 95-kb regions and sequence identity scores of the allelic sequences suggested that DLA-88L is a hybrid gene generated by interlocus and/or intralocus gene conversion between DLA-88 and DLA-12. The putative minimum conversion tract was estimated to be at least an 850-bp segment in length located from the 5´flanking untranslated region to the end of intron 2. In addition, at least one DLA-12 allele (DLA-12*004:01) was newly generated by interlocus gene conversion. In conclusion, the analysis for the occurrence of gene conversion within the dog DLA class I region revealed intralocus gene conversion tracts in 17 of 27 DLA-88 alleles and two of 10 DLA-12 alleles, suggesting that intralocus gene conversion has played an important role in expanding DLA allelic variations.

Unraveling agglomeration and deagglomeration in aqueous colloidal dispersions of very small tin dioxide nanoparticles.

HYPOTHESIS: Understanding deagglomeration, agglomerate formation and structure for very small nanoparticles (NPs), due to their more facile agglomeration, is critical for processing or tailoring agglomerates for nanostructured materials. We propose that by controlling and fine-tuning the interplay of agglomeration (colloidal interaction) and deagglomeration (hydrodynamic forces), the design of agglomerate size, microstructure and morphology is possible even for very small NPs. EXPERIMENTS: Here, we investigate very small SnO2 NPs (10 nm) generated in the gas phase as model system. Small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) are used to study dispersions in aqueous media across the entire pH range (2-12) at various NaCl concentrations treated with ultrasound. Parallel to size and size distribution, agglomerate morphology and microstructure are analyzed by means of the mass fractal dimension, dm and modeled with ab initio shape simulations. The critical coagulation concentration (CCC) is determined for the alkaline region where the SnO2 NPs are highly charged. FINDINGS: Quantitative analysis of SAXS and DLS data reveals that size and size distribution of the agglomerates depend similarly on the electrostatic interaction influenced by pH and salinity as observed by the zeta potential. In contrast dm is mainly influenced by the salt concentration. Ab initio shape simulations support these experimental findings.

Genetic Diversity and Relatedness among Captive African Painted Dogs in North America.

African painted dogs (Lycaon pictus, APD) are highly endangered, with fewer than 7000 remaining in nature. Captive breeding programs can preserve a genetically diverse population and provide a source of individuals for reintroductions. However, most programs are initiated from few founders and suffer from low genetic diversity and inbreeding. The aims of this study were to use molecular markers to assess genetic variation, inbreeding, and relatedness among APDs in the North American captive population, to use these data to realign studbook records, and to compare these data to wild populations and to the European captive population to facilitate the development of a global management plan. We sequenced mitochondrial and major histocompatibility (MHC) class II loci and genotyped 14 microsatellite loci from 109 APDs from 34 institutions in North America. We identified three likely studbook errors and resolved ten cases of uncertain paternity. Overall, microsatellite heterozygosity was higher than reported in Europe, but effective population size estimates were lower. Mitochondrial sequence variation was extremely limited, and there were fewer MHC haplotypes than in Europe or the wild. Although the population did not show evidence of significant inbreeding overall, several individuals shared high relatedness values, which should be incorporated into future breeding programs.

Preparation and characterization of withaferin A loaded pegylated nanoliposomal formulation with high loading efficacy: In vitro and in vivo anti-tumour study.

Withaferin A (WA) is a natural steroidal lactone with promising therapeutic applications. However, its clinical application is limited due to the low bioavailability and hydrophobic nature. In this study, we had prepared PEGylated nanoliposomal withaferin A (LWA) using thin-film hydration method. Dynamic light scattering, Transmission electron microscopy, and HPLC were used to investigate the impact of prepared formulations on the size, charge, morphology, and encapsulation efficiency of the LWA. The prepared nanoliposomal system had spherical vesicles, with the mean particle size of 125 nm and had an encapsulation efficiency of 83.65% with good stability. The characterization results indicated that nanoliposomal formulation is able to improve biocompatibility and bioavailability of WA. In vitro drug release study showed that LWA had an enhanced sustained drug release effect than the free drug. In vitro studies using ascites cell lines (DLA and EAC) showed that LWA treatment could induce apoptosis in ascites cells evidenced by acridine orange/ethidium bromide, Hoechst, and Giemsa staining. In vivo tumour study revealed that LWA treatment significantly reduced tumour growth and improved survival in DLA tumour bearing mice. In vivo results further demonstrated that LWA mitigated solid tumour development by regulating Ki-67 and cyclin D1 protein expression. The overall study results reveal that nanoliposome encapsulated WA exhibits therapeutic efficacy over WA in regulating tumour development as evidenced from ascites cell apoptosis as well as experimental tumour reduction studies.

Whole Genome Sequencing Reveals Multiple Linked Genetic Variants on Canine Chromosome 12 Associated with Risk for Symmetrical Lupoid Onychodystrophy (SLO) in the Bearded Collie.

In dogs, symmetrical lupoid onychodystrophy (SLO) results in nail loss and an abnormal regrowth of the claws. In Bearded Collies, an autoimmune nature has been suggested because certain dog leukocyte antigen (DLA) class II haplotypes are associated with the condition. A genome-wide association study of the Bearded Collie revealed two regions of association that conferred risk for disease: one on canine chromosome (CFA) 12 that encompasses the DLA genes, and one on CFA17. Case-control association was employed on whole genome sequencing data to uncover putative causative variants in SLO within the CFA12 and CFA17 associated regions. Genotype imputation was then employed to refine variants of interest. Although no SLO-associated protein-coding variants were identified on CFA17, multiple variants, many with predicted damaging effects, were identified within potential candidate genes on CFA12. Furthermore, many potentially damaging alleles were fully correlated with the presence of DLA class II risk haplotypes for SLO, suggesting that the variants may reflect DLA class II haplotype association with disease or vice versa. Strong linkage disequilibrium in the region precluded the ability to isolate and assess the individual or combined effect of variants on disease development. Nonetheless, all were predictive of risk for SLO and, with judicious assessment, their application in selective breeding may prove useful to reduce the incidence of SLO in the breed.

Positive selection on the MHC class II DLA-DQA1 gene in golden jackals (Canis aureus) from their recent expansion range in Europe and its effect on their body mass index.

BACKGROUND: In Europe, golden jackals (Canis aureus) have been expanding their range out of the southern and southeastern Balkans towards central Europe continually since the 1960s. Here, we investigated the level of functional diversity at the MHC class II DLA-DQA1 exon 2 in golden jackal populations from Bulgaria, Serbia, and Hungary. Specifically, we tested for positive selection on and geographic variation at that locus due to adaptation to supposedly regionally varying pathogenic landscapes. To test for potential fitness effects of different protein variants on individual body condition, we used linear modeling of individual body mass indexes (bmi) and accounted for possible age, sex, geographical, and climatic effects. The latter approach was performed, however, only on Serbian individuals with appropriate data. RESULTS: Only three different DLA-DQA1 alleles were detected, all coding for different amino-acid sequences. The neutrality tests revealed no significant but positive values; there was no signal of spatial structuring and no deviation from the Hardy-Weinberg equilibrium across the studied range of expansion. However, we found a signal of trans-species polymorphism and significant test results for positive selection on three codons. Our information-theory based linear modeling results indicated an effect of ambient temperature on the occurrence of individual DLA-DQA1 genotypes in individuals from across the studied expansion range, independent from geographical position. Our linear modeling results of individual bmi values indicated that yearlings homozygous for DLA-DQA1*03001 reached values typical for adults contrary to yearlings carrying other genotypes (protein combinations). This suggested better growth rates and thus a possible fitness advantage of yearlings homozygous for DLA-DQA1*03001. CONCLUSIONS: Our results indicate a demographic (stochastic) signal of reduced DLA-DQA1 exon 2 variation, in line with the documented historical demographic bottleneck. At the same time, however, allelic variation was also affected by positive selection and adaptation to varying ambient temperature, supposedly reflecting geographic variation in the pathogenic landscape. Moreover, an allele effect on body mass index values of yearlings suggested differential fitness associated with growth rates. Overall, a combination of a stochastic effect and positive selection has shaped and is still shaping the variation at the studied MHC locus.

Addition of Astatine-211-Labeled Anti-CD45 Antibody to TBI as Conditioning for DLA-Identical Marrow Transplantation: A Novel Strategy to Overcome Graft Rejection in a Canine Presensitization Model: "Radioimmunotherapy to Overcome Transfusion-Induced Sensitization".

In a canine model of presensitization using donor blood transfusions, 100% of historical control dogs receiving 9.2 Gy total body irradiation (TBI) conditioning before dog leukocyte antigen (DLA)-identical marrow grafts had graft rejection. In this presensitization model, we investigated whether the addition of monoclonal antibody (mAb)-based targeted radioimmunotherapy (RIT) with astatine-211 (211At) to TBI could overcome graft rejection. 211At is an alpha-particle-emitting isotope that has a short path length, very high energy, and a short t½ of 7.2 hours, which allowed targeting radiation to the T cells responsible for graft rejection. Normal canine recipients were given three preceding transfusions of unirradiated whole blood on days -24, -17, and -10 before transplant from their DLA-identical marrow donors. 211At-anti-CD45 mAb was administered on day -3, and TBI followed by marrow grafts on day 0. Six of the 7 dogs (86%) achieved sustained engraftment as assessed by 100% donor chimerism in mononuclear cells, granulocytes, and CD3+ T cells. One dog receiving the lowest CD34+ cell content (0.35 × 106 cells/kg) rejected the graft. There were no late rejections in dogs followed up to 1 year. Graft-versus-host disease was seen in one dog. 211At-anti-CD45 mAb in combination with TBI as conditioning was successful in abrogating graft rejection in 86% of dogs in this presensitization model. 211At-anti-CD45 mAb conditioning with TBI may serve as a novel promising strategy to overcome graft rejection in heavily transfused patients with red cell disorders.