Large Fragments Research Articles

Prone position fibula nail and tibia buttress plate for large Volkmann fracture-dislocations technical note and preliminary results on a short case series

Large posterolateral tibial fragments, known as Volkmann fractures, are common in ankle fracture-dislocations and typically require open reduction and fixation with interfragmentary screws and often buttress plates using a prone posterolateral approach. In this setting, fibula plating often necessitates dissection of the lateral window between the peroneal tendons and the skin, increasing wound-related complications. In recent years, intramedullary nailing of fibula fractures has gained popularity as a minimally invasive technique that allows load-sharing stabilization. However, this procedure has traditionally been performed in the supine position, which can pose challenges if a Volkmann-type fracture is present, requiring repositioning and losing access to the fibula through the posterolateral approach. Our objective is to describe a technique for fixing ankle fracture-dislocations with large posterolateral fragments using tibial buttress plates and fibula nails through a prone posterolateral approach without dissecting the lateral window. Additionally, we will present a case series with preliminary results.

Versatile and efficient mammalian genome editing with Type I-C CRISPR System of Desulfovibrio vulgaris.

CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins. While type I CRISPR systems in Class I may offer greater specificity and versatility, they are not well-developed for genome editing. Here, we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris (Dvu) for efficient and precise genome editing in mammalian cells and animals. We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy. These edited pig cells can serve as donors for generating transgenic cloned piglets. The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair. Additionally, we adapted the Dvu-Cascade effector for cytosine and adenine base editing, developing Dvu-CBE and Dvu-ABE systems. These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks. Off-target analysis confirmed the high specificity of the Dvu type I-C editor. Our findings demonstrate the Dvu type I-C editor's potential for diverse mammalian genome editing applications, including deletions, fragment replacement, and base editing, with high efficiency and specificity for biomedicine and agriculture.

Metabolic responses of the marine mussel Mytilus galloprovincialis after exposure to microplastics of different shapes and sizes

Microplastics (MP) are ubiquitous pollutants with diverse shapes, sizes, and characteristics that pose critical risks to marine organisms and the environment. In this study, we used the Mediterranean mussel Mytilus galloprovincialis as a marine benthic organism model to investigate the metabolic consequences of exposure to different polyethylene terephthalate MP sizes and shapes: round (27–32 μm), small fibers (200–400 μm), large fibers (3000 μm), small fragments (20 μm), medium fragments (45–75 μm), and large fragments (>150 μm). After exposure to high concentrations (100 mg L−1) of MP for 14 days, round and small fiber-type MP were highly accumulated in mussels. Metabolomic analysis revealed that exposure to round and small fiber-type MP induced significant changes in 150 metabolites. Partial least squares-discriminate analysis (PLS-DA) showed that the round and small fiber MP treatment groups displayed similar cluster patterns that differed from those of the control group. In addition, only 22 annotated metabolites related to histidine, valine, leucine, and isoleucine degradation/biosynthesis and vitamin B6 and aminoacyl-tRNA biosynthesis were significantly affected by round or small fiber-type MP. Among the histidine metabolites, round and small fiber-type MP upregulated the levels of L-histidine, L-glutamate, carnosine, imidazole-4-acetaldehyde, 4-imidazolone-5-propanoate, and methylimidazole acetaldehyde and downregulated methylimidazole acetic acid and N-formimino-L-glutamate. These results suggest novel insights into the potential pathways through which MP of specific sizes and shapes affect metabolic processes in mussels.

Enlarging interface reverses the dominance of fungi over bacteria in litter decomposition

Soil microorganisms are primary decomposers driving carbon and nutrient cycling in terrestrial ecosystems. One prevailing view is that fungi, rather than bacteria, play a predominant role in litter decomposition. However, the roles of bacteria and factors that restrict their activity during decomposition remain unclear. We hypothesized that the limiting activity of bacterial decomposers is associated with litter size beyond chemical quality. To address this gap, we conducted a 180-d decomposition microcosm experiment to investigate the effect of fragment size (large, 1–2 mm; middle, 0.18–0.28 mm; small, <0.07 mm) of oak and pine litters on bacterial or fungal decomposition. Bacterial and fungal decomposition were accelerated with fragment size decrease, suggesting that an interface effect existed between microbial decomposers and litter. Generally, the decomposition ability of bacteria was more sensitive to changes in fragment size compared to fungi. Fungi resulted in faster decomposition of large fragments than bacteria. For small oak litter fragments, bacteria showed faster decomposition than fungi, whereas the opposite was true for small pine litter. Therefore, the decomposition dominance of bacteria and fungi was regulated by fragment size and influenced by the chemical quality of litter, especially the lignin:N ratio. The contrasting decomposition dominances of bacteria versus fungi were likely attributed to filamentous fungi penetrating litter interiors and forming mycelial bridges between scattered litters. Bacteria resided on litter surfaces and even formed biofilms. Consistent with the findings of the microcosm experiment, the proportion of small fragments was greater in the fresh litter layer than in the decomposed layer in the field, and greater in the pine forest than in the oak forest, suggesting the fresh, large, and low-quality litter was preferentially fragmented by fungi. Consequently, the dominance of fungi and bacteria during litter decomposition in the conventional view should be revisited considering the litter size.

High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus.

Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

Posterior Wall Fragments in Acetabular Both-Column Fractures: Morphology, Type, and the Significance of its Projection.

Most both-column acetabular fractures are combined with posterior wall fragments. However, the morphology of this posterior wall is varied, and how to fix this posterior wall remains a controversial topic. To investigate the morphological characteristics of posterior wall fragments of both-column acetabular fractures and select corresponding fixation methods. Data from 352 patients with acetabular fractures admitted to the level one trauma centre in our hospital between January 2006 and December 2022 were collected. The morphology of posterior wall fragments was observed and analyzed in 83 cases of both-column acetabular fractures and classified according to the consistency of posterior wall morphology. A fracture map of the posterior wall was created on a normal template according to the three morphological types of posterior wall fragments. Finally, the high-incidence area of the posterior wall fracture was projected onto the iliac fossa and the medial side of the posterior column to guide the fixation of the posterior wall fragment using the anterior intrapelvic approach. Fractures were divided into four types: I, large posterior wall fragment which was high in the ilium bone (34 cases, 41.0%); II, posterior wall fragment in the acetabular parietal region (18 cases, 21.7%); III, posterior wall marginal fracture (10 cases, 12.0%); and IV, non-combined posterior wall fracture (21 cases, 25.3%). The most common morphologies of the posterior wall fragments of the first two types were mapped and projected onto the anterior iliac inner plate and medial side of the posterior column, where the corresponding area could be used to guide the insertion of the internal fixation. Both-column acetabular fractures combined with posterior wall fractures can be divided into four types according to the morphology of the posterior wall fragment. Understanding the corresponding three-dimensional morphology and projection position of different types of these fragments can help surgeons determine the position and orientation of internal fixation of posterior wall fractures.

MAX germline mutation-associated pheochromocytoma-paraganglioma syndrome: multiple endocrine neoplasia type 5

The individualized precision management of hereditary pheochromocytoma (PHEO) and paraganglioma (PGL) syndromes (PPGLs) based on molecular diagnosis and molecular subtype is becoming more popular. The newly discovered MAX germline mutation-associated PPGLs are autosomally dominant and rare. To raise awareness and explore the effective management of individual diagnosis and treatment, the relevant literature published between January 2011 and February was systematically reviewed. There were a total of 101 patients in the 77 families, involving all 5 exons, containing 44 types of MAX germline mutations and mostly concentrated in exons 3 and 4 (64.4%), the main mutations were nonsense mutations and missense mutations (72.3%), and some were large fragment deletions or insertions, intron variant, gene fusion mutations were relatively infrequent. Furthermore, about 10% of the patients had a paternal parent-of-origin effect. Among the 101 patients, 96 (95.0%) developed PHEO including 15 metastatic PHEO, 61 bilateral PHEO and 35 unilateral PHEO. The age of diagnosis was (31.7±10.9) years (range: 13 to 80 years). The male to female ratio was 1.2∶1. Eleven were accompanied with chest and abdominal PGL. Eight (7.9%) were accompanied by functional pituitary adenoma. And 12 (11.9%) developed other neuroendocrine tumors (NET), of which 8 were accompanied by PHEO, including 4 hyperparathyroidism, 1 gangliocytoma and neuroblastoma, 1 pancreatic NET, 1 medullary thyroid carcinoma and 1 C cell hyperplasia. Six presented concomitant non-NET, including 1 tongue squamous cell carcinoma, 1 papillary thyroid carcinoma, 1 prostate cancer, 1 renal oncocytoma, 1 breast cancer with renal oncocytoma, and 1 thoracic chondrosarcoma with multifocal adenocarcinoma of lung. The remaining 5 cases (5.0%), including 4 other NET (2 ganglioblastoma, 1 abdominal neuroblastoma and 1 pancreatic NET) and 1 asymptomatic child, did not present PHEO. The MAX germline mutation may cause a novel multiple endocrine neoplasia, which can be described as type 5. A comprehensive baseline assessment of neural crest cell-derived diseases such as PPGL, pituitary adenoma, hyperparathyroidism, and/or gangliocytoma (neuroblastoma) was recommended for all people with MAX germline mutations, and the risk of bilateral and/or metastatic PHEO should also be considered. In contrast, patients with PPGLs combined with other NET, such as functional pituitary adenoma, should undergo genetic testing and pedigree screening that includes at least the MAX gene.

Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking-out the GLB1 gene in SH-SY5Y human neuronal cell line.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of β-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

Effect of the Inter-Ring Delay Time on Rock Fragmentation: Field Tests at the Underground Mine

The effect of inter-ring delay time (IRDT) on rock fragmentation in the tunnel excavation blasting was studied at the Xinjiang Beizhan Iron Mine, China, to improve the rock fragmentation and optimize the blast design. Blasting tests were conducted with an IRDT of 50, 100, 150, 200, and 500 ms; each adjacent ring had an equal IRDT, and two replicate tests were conducted. The blasting plan with IRDT of 100 ms was the original blasting plan used in the mine. Two optimized blasting plans were proposed and implemented based on the experimental results, along with a control experiment using the original blasting plan. The fragment size of each blast test was measured and analyzed by the block-analyzed software. Both collision theoretical and field tests indicated that the IRDT plays an important role in rock fragmentation and that as the IRDT increases, the degree of rock fragmentation increases first and then decreases. For example, the fragment sizes X20, X50, and X80 showed an increase followed by a decrease; the percentage of large fragments (1-P750) showed a decline followed by a rise; the percentage of small fragments P25 showed an increase followed by a decline. The blasting plan with an IRDT of 150 ms had the most optimal rock fragmentation effect, with the lowest percentage of large fragments, the highest percentage of small fragments, and the smallest average fragment size of X50. Furthermore, the two optimized blasting plans demonstrated better control over blasting costs and rock fragmentation compared to the original blasting plan.

Evaluation of artificial intelligence-assisted morphological analysis for platelet count estimation.

This study aims to assess the performance of the platelet count estimation using artificial intelligence technology on the MC-80 digital morphology analyzer. Digital morphology analyzer uses two different computational principles for platelet count estimation: based on PLT/RBC ratio (PLT-M1) and estimate factor (PLT-M2). 977 samples with various platelet counts (low, median, and high) were collected. Out of these, 271 samples were immunoassayed using CD61 and CD41 antibodies. The platelet counts obtained from the hematology analyzer (PLT-I and PLT-O), digital morphology analyzer (PLT-M1 and PLT-M2), and flow cytometry (PLT-IRM) were compared. There was no significant deviation observed before and after verification for both PLT-M1 and PLT-M2 across the analysis range (average bias: -0.845/-0.682, 95% limit of agreement (LOA): -28.675-26.985/-29.420-28.056). When platelet alarms appeared, PLT-M1/PLT-M2 showed the strongest correlation with PLT-IRM than PLT-I with PLT-IRM (r: 0.9814/0.9796 > 0.9601). The correlation between PLT-M1/PLT-M2 and PLT-IRM was strong for samples with interference, such as large platelets or RBC fragments, but relatively weak in small RBCs. The deviation between PLT-M1 and PLT-M2 is related to the number of RBCs. Compared with PLT-I, PLT-M1/PLT-M2 showed higher accuracy for platelet transfusion decisions, especially for samples with low-value PLT. The novel platelet count estimation on the MC-80 digital morphology analyzer provides high accuracy, especially the reviewed result, which can effectively confirm suspicious platelet count.

A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster.

The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from Streptomyces ceruleus A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from B. subtilis 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources.

Establishing a straightforward I-SceI-mediated recombination one-plasmid system for efficient genome editing in P. putida KT2440.

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever-improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one-plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I-SceI expressing plasmid pSW-2 as a counterselection marker to accelerated plasmid curing; (2) double-strand break introducing gene I-sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG-RIS. The single plasmid of pEMG-RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I-SceI encoding gene. Here, the usability of the pEMG-RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.

Large Felid Tracks and Bones From Chilly Bowl Cave, Arkansas

Multiple large cat tracks were found within Chilly Bowl Cave, Arkansas, representing at least two individuals of the genus Panthera. Large felid bone fragments were also found in a passageway near the tracks. These poorly preserved skeletal remains primarily consist of metapodials and phalanges and represent the North American Pleistocene jaguar, Panthera onca. Fusion of metapodial epiphyses indicate adult foot size had been reached for this large jaguar. One set of tracks is also consistent with the size and morphology of P. onca. The other tracks are much larger than is known for extant or prehistoric jaguars, and are here attributed to Panthera atrox, the American lion. An alternate hypothesis for the smaller tracks is that they could represent a younger P. atrox instead of P. onca. Regardless of the inability to identify the smaller feline prints to species, these remains and tracks from Chilly Bowl Cave record cave utilization by two separate Pleistocene felids, interpreted here as jaguar and American lion.

Integration of large and diverse angiosperm DNA fragments into Asian Gnetum mitogenomes

BackgroundHorizontal gene transfer (HGT) events have rarely been reported in gymnosperms. Gnetum is a gymnosperm genus comprising 25‒35 species sympatric with angiosperms in West African, South American, and Southeast Asian rainforests. Only a single acquisition of an angiosperm mitochondrial intron has been documented to date in Asian Gnetum mitogenomes. We wanted to develop a more comprehensive understanding of frequency and fragment length distribution of such events as well as their evolutionary history in this genus.ResultsWe sequenced and assembled mitogenomes from five Asian Gnetum species. These genomes vary remarkably in size and foreign DNA content. We identified 15 mitochondrion-derived and five plastid-derived (MTPT) foreign genes. Our phylogenetic analyses strongly indicate that these foreign genes were transferred from diverse eudicots—mostly from the Rubiaceae genus Coptosapelta and ten genera of Malpighiales. This indicates that Asian Gnetum has experienced multiple independent HGT events. Patterns of sequence evolution strongly suggest DNA-mediated transfer between mitochondria as the primary mechanism giving rise to these HGT events. Most Asian Gnetum species are lianas and often entwined with sympatric angiosperms. We therefore propose that close apposition of Gnetum and angiosperm stems presents opportunities for interspecific cell-to-cell contact through friction and wounding, leading to HGT.ConclusionsOur study reveals that multiple HGT events have resulted in massive amounts of angiosperm mitochondrial DNA integrated into Asian Gnetum mitogenomes. Gnetum and its neighboring angiosperms are often entwined with each other, possibly accounting for frequent HGT between these two phylogenetically remote lineages.

Preoperative CT improves the assessment of stability in trochanteric hip fractures.

To investigate if preoperative CT improves detection of unstable trochanteric hip fractures. A single-centre prospective study was conducted. Patients aged 65 years or older with trochanteric hip fractures admitted to Stavanger University Hospital (Stavanger, Norway) were consecutively included from September 2020 to January 2022. Radiographs and CT images of the fractures were obtained, and surgeons made individual assessments of the fractures based on these. The assessment was conducted according to a systematic protocol including three classification systems (AO/Orthopaedic Trauma Association (OTA), Evans Jensen (EVJ), and Nakano) and questions addressing specific fracture patterns. An expert group provided a gold-standard assessment based on the CT images. Sensitivities and specificities of surgeons' assessments were estimated and compared in regression models with correlations for the same patients. Intra- and inter-rater reliability were presented as Cohen's kappa and Gwet's agreement coefficient (AC1). We included 120 fractures in 119 patients. Compared to radiographs, CT increased the sensitivity of detecting unstable trochanteric fractures from 63% to 70% (p = 0.028) and from 70% to 76% (p = 0.004) using AO/OTA and EVJ, respectively. Compared to radiographs alone, CT increased the sensitivity of detecting a large posterolateral trochanter major fragment or a comminuted trochanter major fragment from 63% to 76% (p = 0.002) and from 38% to 55% (p < 0.001), respectively. CT improved intra-rater reliability for stability assessment using EVJ (AC1 0.68 to 0.78; p = 0.049) and for detecting a large posterolateral trochanter major fragment (AC1 0.42 to 0.57; p = 0.031). A preoperative CT of trochanteric fractures increased detection of unstable fractures using the AO/OTA and EVJ classification systems. Compared to radiographs, CT improved intra-rater reliability when assessing fracture stability and detecting large posterolateral trochanter major fragments.

Genomic characterization revealing the high rate of tet(X4)-positive Escherichia coli in animals associated with successful genetic elements.

The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health. Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China. Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4)-positive E. coli. In this study, a total of 775 nonduplicate samples were collected in Guangdong, China from 2019 to 2020. We screened for tet(X4)-positive E. coli by PCR amplification and species identification. Furthermore, we analyzed the phylogenetics and genetic context of tet(X4)-positive E. coli through whole-genome sequencing and long-reads sequencing. Overall, 146 (18.84%) tet(X4)-positive E. coli were isolated, comprising 2 isolates from humans and 144 isolates from pigs. The majority of tet(X4)-positive E. coli exhibited resistance to multiple antibiotics but all of them were susceptible to amikacin and colistin. Phylogenetic analysis showed that ST877, ST871, and ST195 emerged as the predominant sequence types in tet(X4)-positive E. coli. Further analysis revealed various genetic environments associated with the horizontal transfer of tet(X4). Notably, a 100-kbp large fragment insertion was discovered downstream of tet(X4), containing a replicon and a 40-kbp gene cluster for the bacterial type IV secretion system. The high colonization rate of tet(X4)-positive E. coli in animals suggests that colonization as a key factor in its dissemination to humans. Diverse genetic context may contribute to the transfer of tet(X4). Our findings underline the urgent need for controlling the spread of plasmid-mediated tigecycline resistance.

Fine mapping and transcriptome profiling reveal CpAPRR2 to modulate immature fruit rind color formation in zucchini (Cucurbita pepo).

A large fragment deletion of CpAPRR2, encoding a two-component response regulator-like protein, which influences immature white rind color formation in zucchini (Cucurbita pepo). Fruit rind color is an important agronomic trait that affects commodity quality and consumer choice in zucchini (Cucurbita pepo). However, the molecular mechanism controlling rind color is unclear. We characterized two zucchini inbred lines: '19' (dark green rind) and '113' (white rind). Genetic analysis revealed white immature fruit rind color to be controlled by a dominant locus (CpW). Combining bulked segregant analysis sequencing (BSA-seq) and Kompetitive Allele-Specific PCR (KASP) markers, we mapped the CpW locus to a 100.4kb region on chromosome 5 and then narrow down the candidate region to 37.5kb using linkage analysis of 532 BC1 and 1613 F2 individuals, including 6 coding genes. Among them, Cp4.1LG05g02070 (CpAPRR2), encoding a two-component response regulator-like protein, was regarded to be a promising candidate gene. The expression level of CpAPRR2 in dark green rind was significantly higher than that in white rind and was induced by light. A deletion of 2227bp at the 5' end of CpAPRR2 in '113' might explain the white phenotype. Further analysis of allelic diversity in zucchini germplasm resources revealed rind color to be associated with the deletion of CpAPRR2. Subcellular localization analysis indicated that CpAPRR2 was a nuclear protein. Transcriptome analysis using near-isogenic lines with dark green (DG) and white (W) rind indicated that genes involved in photosynthesis and porphyrin metabolism pathways were enriched in DG compared with W. Additionally, chlorophyll synthesis-related genes were upregulated in DG. These results identify mechanisms of zucchini rind color and provide genetic resources for breeding.

Amplifications of EVX2 and HOXD9-HOXD13 on 2q31 in mature cystic teratomas of the ovary identified by array comparative genomic hybridization may explain teratoma characteristics in chondrogenesis and osteogenesis

BackgroundTeratomas are a common type of germ cell tumor. However, only a few reports on their genomic constitution have been published. The study of teratomas may provide a better understanding of their stepwise differentiation processes and molecular bases, which could prove useful for the development of tissue-engineering technologies.MethodsIn the present study, we analyzed the copy number aberrations of nine ovarian mature cystic teratomas using array comparative genomic hybridization in an attempt to reveal their genomic aberrations.ResultsThe many chromosomal aberrations observed on array comparative genomic hybridization analysis reveal the complex genetics of this tumor. Amplifications and deletions of large DNA fragments were observed in some samples, while amplifications of EVX2 and HOXD9-HOXD13 on 2q31.1, NDUFV1 on 11q13.2, and RPL10, SNORA70, DNASE1L1, TAZ, ATP6AP1, and GDI1 on Xq28 were found in all nine mature cystic teratomas.ConclusionsOur results indicated that amplifications of these genes may play an important etiological role in teratoma formation. Moreover, amplifications of EVX2 and HOXD9-HOXD13 on 2q31.1, found on array comparative genomic hybridization, may help to explain the characteristics of teratomas in chondrogenesis and osteogenesis.

Effect of Multi-Mode Divergent Ultrasound Pretreatment on Hardness, Microstructure and Digestion of Acid-Induced Whey Protein Gels.

Whey protein was pretreated with multi-frequency ultrasound in mono-, dual-, and tri-frequency modes. The effect of multi-frequency ultrasound pretreatment on the hardness, chemical forces, and microstructure of acid-induced whey protein gel was investigated. Whey protein gels pretreated with dual- and tri-frequency ultrasound showed higher hardness (p < 0.05) and a denser network than mono-frequency ultrasound and control. Moreover, they had higher hydrophobic interaction and lower disulfide bonds than the control (p < 0.05). The effect of gel properties on digestion was evaluated using an in vitro static model. Whey protein gels pretreated with dual- and tri-frequency ultrasound had a higher proportion of large fragments in the oral boluses than in the control. Large fragments (>3.35 mm) in those samples were resistant to gastric digestion. Moreover, the tri-frequency ultrasound pretreatment of whey protein gel released the least free amino group during gastric digestion. In contrast, whey protein gel with the mono-frequency ultrasound pretreatment released the highest amount of free amino acid group during intestinal digestion. Findings from this study suggests that gel hardness and network density could modulate the digestion behaviors of protein gels.

Mechanical properties of soils in internal waste bank of lignite mines and their primary characteristics

The modern development of urban areas is related to, among others, the location of industrial facilities on the outskirts of cities. More and more often, commercial buildings are founded on areas that have not been used for construction so far. Such areas include, among others: reclaimed lignite mine banks in the Konin region. The man-made soil is a chaotic mixture of fragments of glacial tills and Pliocene clays, often exceeding 20 m in thickness, which is naturally consolidated over time. Due to the method of formation of the embankment, despite the fact that banks are made of natural soil, their strength and deformation characteristics clearly differ from those characteristic of lithologically similar soils deposited as a result of geological processes. In this case, the use of a standard test approach may overestimate the strength and stiffness of the soil. Due to the complex structure of the bank in-situ tests were used for geotechnical exploration: CPTU and FVT, as well as laboratory tests in a triaxial apparatus and an oedometer. The results were compared with the results of studies conducted in similar naturally deposited soils. The obtained results provide valuable geotechnical characteristics of the embankment soil, which in its large fragments is built of natural soil clasts. The obtained results indicate a relatively small change in the geotechnical properties of the soils incorporated into the embankment within individual clasts of the natural soil (on a local scale) and a clear deterioration in the scale of the entire embankment, treated as the impact zone of building structures.