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One of the most frequently mutated proteins in human B-lineage leukemia is the transcription factor PAX5. These mutations often result in partial rather than complete loss of function of the transcription factor. While the functional dose of PAX5 has a clear connection to human malignancy, there is limited evidence for that heterozygote loss of PAX5 have a dramatic effect on the development and function of B-cell progenitors. One possible explanation comes from the finding that PAX5 mutated B-ALL often display complex karyotypes and additional mutations. Thus, PAX5 might be one component of a larger transcription factor network targeted in B-ALL. To investigate the functional network associated with PAX5 we used BioID technology to isolate proteins associated with this transcription factor in the living cell. This identified 239 proteins out of which several could be found mutated in human B-ALL. Most prominently we identified the commonly mutated IKZF1 and RUNX1, involved in the formation of ETV6-AML1 fusion protein, among the interaction partners. ChIP- as well as PLAC-seq analysis supported the idea that these factors share a multitude of target genes in human B-ALL cells. Gene expression analysis of mouse models and primary human leukemia suggested that reduced function of PAX5 increased the ability of an oncogenic form of IKZF1 or ETV6-AML to modulate gene expression. Our data reveals that PAX5 belong to a regulatory network frequently targeted by multiple mutations in B-ALL shedding light on the molecular interplay in leukemia cells.

Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36 ± 1.28%, 93.40 ± 0.70%, 73.23 ± 1.29% and 46.75 ± 3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65 ± 2.15% and 96.30 ± 1.00% of differentiated cells in comparison to 11.30 ± 0.10% and 19.45 ± 0.55% cells, respectively in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.

The Booroola fecundity (FecB) gene, a mutation in the bone morphogenetic protein receptor-1B (BMPR1B), is known to increase ovulation rate and litter size in sheep. Efficient and accurate, large-scale detection of this single nucleotide polymorphism (SNP) is critical for marker assisted introgression/selection programs for genetic improvement of prolificacy. This study aimed to develop and validate a robust, cost-effective, and high-throughput genotyping assay for detecting the FecB mutation across diverse sheep populations globally. A competitive allele-specific PCR (KASP) assay targeting the A746G nucleotide substitution in BMPR1B gene was designed and tested on a diverse panel of 224 individuals across 22 sheep breeds and validated against direct sequencing. The KASP genotyping results showed 100% concordance with sequence data, with clear fluorescence-based discrimination of homozygous wild-type Fec++ (AA), heterozygous FecB+ (AG), and homozygous mutant FecBB (GG) genotypes. The assay was compatible across three real-time PCR platforms, BioRad CFX96, Roche LightCycler 480 II, and ABI QuantStudio 6 demonstrating reproducible genotyping and cross-platform interoperability. Subsequently, the KASP assay was applied to a global screening of 1471 sheep from 47 breeds in 14 countries for further validation of the assay. The mutation was not observed in the sheep samples investigated from Africa, Europe, Latin America, and West Asia, whereas it was polymorphic or fixed in certain populations from South (India and Bangladesh) and Southeast Asia (Indonesia). Notably, native Bangladeshi sheep exhibited high frequencies of the FecB allele, with some populations (e.g., Bangladesh Central) nearing fixation, reflecting possible selection for high prolificacy. In conclusion, the validated KASP assay offers a powerful, scalable tool for the rapid genotyping of the FecB mutation, enabling efficient screening and incorporation of prolificacy traits in sheep breeding programs worldwide.

Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polymerase chain reaction (PCR) and immunocytochemistry, but did not express haematopoietic markers (CD34, CD45 and CD14). In flow cytometry, the expression of CD29, CD44, CD73 and CD90 was observed in 68.83 ± 1.27, 93.66 ± 1.80, 96.96 ± 0.44 and 93.7 ± 1.89% of AF-MSCs, respectively. Osteogenic, chondrogenic and adipogenic differentiation of MSCs was confirmed by von Kossa and Alizarin red S, Alcian blue and oil red O staining, respectively. Upon supplementation of MSC growth media with 50 ng/ml bone morphogenetic protein (BMP)-12, AF-MSCs differentiated to tenocytes within 14 days. The differentiated cells were more slender, elongated and spindle shaped with thinner and longer cytoplasmic processes and showed expression of tenomodulin and decorin by RT-PCR and immunocytochemistry. In flow cytometry, 96.7 ± 1.90 and 80.9 ± 6.4% of differentiated cells expressed tenomodulin and decorin in comparison to 1.6 and 3.1% in undifferentiated control cells, respectively. Our results suggest that AF is an easily accessible and effective source of MSCs. On BMP-12 supplementation, AF-MSCs can be differentiated to tenocytes, which could be exploited for regeneration of ruptured or damaged tendon in race horses.

SH2 domain containing inositol-5-phosphatase (SHIP1) is an important modulator of innate and adaptive immunity. In mice, loss of SHIP1 provokes severe ileitis resembling Crohn's disease (CD), as a result of deregulated immune responses, altered cytokine production and intestinal fibrosis. Recently, SHIP1 activity was shown to be correlated to the presence of a CD-associated single nucleotide polymorphism in ATG16L1. Here, we studied SHIP1 activity and expression in an adult cohort of CD patients. SHIP1 activity, protein and mRNA expression in peripheral blood mononuclear cells from CD patients in clinical remission were determined by Malachite green assay, Western blotting and qRT-PCR respectively. Genomic DNA was genotyped for ATG16L1 rs2241880. SHIP1 protein levels are profoundly diminished in a subset of patients; however, SHIP1 activity and expression are not correlated to ATG16L1 SNP status in this adult cohort. Aberrant SHIP1 activity can contribute to disease in a subset of adult CD patients, and warrants further investigation.

The role of the innate immunity in the pathogenesis of Crohn's disease (CD), an inflammatory bowel disease, is a subject of increasing interest. Neutrophils (PMN) are key members of the innate immune system which migrate to sites of bacterial infection and initiate the defence against microbes by producing reactive oxygen species (ROS), before undergoing apoptosis. It is believed that impaired innate immune responses contribute to CD, but it is as yet unclear whether intrinsic defects in PMN signal transduction and corresponding function are present in patients with quiescent disease. We isolated peripheral blood PMN from CD patients in remission and healthy controls (HC), and characterised migration, bacterial uptake and killing, ROS production and cell death signalling. Whereas IL8-induced migration and signalling were normal in CD, trans-epithelial migration was significantly impaired. Uptake and killing of E. coli were normal. However, an increased ROS production was observed in CD PMN after stimulation with the bacterial peptide analogue fMLP, which was mirrored by an increased fMLP-triggered ERK and AKT signal activation. Interestingly, cleavage of caspase-3 and caspase-8 during GMCSF-induced rescue from cell-death was decreased in CD neutrophils, but a reduced survival signal emanating from STAT3 and AKT pathways was concomitantly observed, resulting in a similar percentage of end stage apoptotic PMN in CD patients and HC. In toto, these data show a disturbed signal transduction activation and functionality in peripheral blood PMN from patients with quiescent CD, which point toward an intrinsic defect in innate immunity in these patients.

The Booroola fecundity (Fec.sup.B) gene, a mutation in the bone morphogenetic protein receptor-1B (BMPR1B), is known to increase ovulation rate and litter size in sheep. Efficient and accurate, large-scale detection of this single nucleotide polymorphism (SNP) is critical for marker assisted introgression/selection programs for genetic improvement of prolificacy. This study aimed to develop and validate a robust, cost-effective, and high-throughput genotyping assay for detecting the FecB mutation across diverse sheep populations globally. A competitive allele-specific PCR (KASP) assay targeting the A746G nucleotide substitution in BMPR1B gene was designed and tested on a diverse panel of 224 individuals across 22 sheep breeds and validated against direct sequencing. The KASP genotyping results showed 100% concordance with sequence data, with clear fluorescence-based discrimination of homozygous wild-type Fec.sup.++ (AA), heterozygous Fec.sup.B+ (AG), and homozygous mutant Fec.sup.BB (GG) genotypes. The assay was compatible across three real-time PCR platforms, BioRad CFX96, Roche LightCycler 480 II, and ABI QuantStudio 6 demonstrating reproducible genotyping and cross-platform interoperability. Subsequently, the KASP assay was applied to a global screening of 1471 sheep from 47 breeds in 14 countries for further validation of the assay. The mutation was not observed in the sheep samples investigated from Africa, Europe, Latin America, and West Asia, whereas it was polymorphic or fixed in certain populations from South (India and Bangladesh) and Southeast Asia (Indonesia). Notably, native Bangladeshi sheep exhibited high frequencies of the Fec.sup.B allele, with some populations (e.g., Bangladesh Central) nearing fixation, reflecting possible selection for high prolificacy. In conclusion, the validated KASP assay offers a powerful, scalable tool for the rapid genotyping of the Fec.sup.B mutation, enabling efficient screening and incorporation of prolificacy traits in sheep breeding programs worldwide.

We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.

Colon cancer is a serious health problem, and novel preventive and therapeutical avenues are urgently called for. Delivery of proteins with anticancer activity through genetically modified bacteria provides an interesting, potentially specific, economic and effective approach here. Interestingly, bone morphogenetic protein 2 (BMP-2) is an important and powerful tumour suppressor in the colon and is thus an attractive candidate protein for delivery through genetically modified bacteria. It has not been shown, however, that BMP production in the bacterial context is effective on colon cancer cells. Here we demonstrate that transforming E. coli with a cDNA encoding an ileal-derived mature human BMP-2 induces effective apoptosis in an in vitro model system for colorectal cancer, whereas the maternal organism was not effective in this respect. Furthermore, these effects were sensitive to cotreatment with the BMP inhibitor Noggin. We propose that prevention and treatment of colorectal cancer using transgenic bacteria is feasible.

The Booroola fecundity (Fec B ) gene, a mutation in the bone morphogenetic protein receptor-1B (BMPR1B), is known to increase ovulation rate and litter size in sheep. Efficient and accurate, large-scale detection of this single nucleotide polymorphism (SNP) is critical for marker assisted introgression/selection programs for genetic improvement of prolificacy. This study aimed to develop and validate a robust, cost-effective, and high-throughput genotyping assay for detecting the FecB mutation across diverse sheep populations globally. A competitive allele-specific PCR (KASP) assay targeting the A746G nucleotide substitution in BMPR1B gene was designed and tested on a diverse panel of 224 individuals across 22 sheep breeds and validated against direct sequencing. The KASP genotyping results showed 100% concordance with sequence data, with clear fluorescence-based discrimination of homozygous wild-type Fec ++ (AA), heterozygous Fec B+ (AG), and homozygous mutant Fec BB (GG) genotypes. The assay was compatible across three real-time PCR platforms, BioRad CFX96, Roche LightCycler 480 II, and ABI QuantStudio 6 demonstrating reproducible genotyping and cross-platform interoperability. Subsequently, the KASP assay was applied to a global screening of 1471 sheep from 47 breeds in 14 countries for further validation of the assay. The mutation was not observed in the sheep samples investigated from Africa, Europe, Latin America, and West Asia, whereas it was polymorphic or fixed in certain populations from South (India and Bangladesh) and Southeast Asia (Indonesia). Notably, native Bangladeshi sheep exhibited high frequencies of the Fec B allele, with some populations (e.g., Bangladesh Central) nearing fixation, reflecting possible selection for high prolificacy. In conclusion, the validated KASP assay offers a powerful, scalable tool for the rapid genotyping of the Fec B mutation, enabling efficient screening and incorporation of prolificacy traits in sheep breeding programs worldwide.