DNA Content Research Articles

Exploring floral morphoanatomy and embryology in wild populations of Chrysolaena flexuosa (Vernonia, Asteraceae): a contribution to understanding its ornamental potential.

Chrysolaena flexuosa (Sims.) H. Rob. is a South American species in the tribe Vernonieae, with potential ornamental value: it has attractive inflorescences, is suitable for pot cultivation, and its cypselae are useful for dried flower arrangements. Apart from studies on the growth dynamics of this species under cultivation, chromosome number, DNA content, ploidy level, size, pollen viability, and the characterization of phenotypic and genetic variability, it is noteworthy that other aspects regarding the floral architecture, reproductive mode, and gametophyte formation of C. flexuosa have not yet been studied. For this reason, our study encompasses a floral morphoanatomical survey and a comprehensive assessment of gametophyte development in the species. As a result of this study, we report new floral morphotypes, confirming that the morphological variability of the species might be greater than speculated. The morphoanatomy of the androecium and gynoecium and the male and female gametophyte developmental characteristics are uniform in all the populations studied despite the different ploidy levels. Chrysolaena flexuosa has five tetrasporangiate stamens of the dicotyledonous type of development; all the populations studied displayed a unilocular inferior ovary with a single anatropous, unitegumented, and tenuinucellar ovule. Given that all the embryo sacs observed were of the Polygonum-type development regardless of the ploidy level, we infer that the populations analyzed are fertile and undergo sexual reproduction. Our results not only contribute further research in the field of breeding systems and propagation of this species, but also promote the successful introduction of C. flexuosa to the plant ornamental market.

Harvesting decellularized liver extracellular matrix from rodents for 3D scaffold fabrication

Decellularization is a process to harvest the decellularized extra cellular matrix (dECM) that helps develop 3D scaffolds which mimic the native tissue composition. The decellularized tissues retain the structural and functional properties of the extracellular matrix (ECM) by an efficient decellularization process that retains tissue-specific biochemical and biophysical cues for tissue regeneration. In this study, we report an injection-based decellularization method, without perfusion setup. This study also compares the efficiency of the proposed protocol in the two animal models viz rat and mice. This method harvests rat and mice liver dECM using ethylenediamine tetra acetic acid (EDTA) and sodium dodecyl sulphate (SDS) within 08 h and 02 h respectively and preserved significant amount of ECM proteins. We reported that the harvested mice decellularized extracellular matrix (mdECM) and rat decellularized extracellular matrix (rdECM) had significant reduction in their DNA content (∼97%) and retained structural architecture resembling their native tissue counterparts. The total protein content retained in mdECM was ∼39% while that retained in rdECM was ∼65%. It was also found that the sGAG (sulphated glycosaminoglycan) content showed a no List of Figures.

Cell cycle dynamics of food-entrapping cells of sponges: an experimental approach.

Sponges (phylum Porifera) exhibit surprisingly complex tissue dynamics, maintaining constant cell turnover and migration, rearranging internal structures, and regenerating after severe injuries. Such tissue plasticity relies on the activity of proliferating cells represented primarily by the food-entrapping cells, choanocytes. Although there are plenty of studies regarding the dynamics of regeneration and tissue rearrangement in sponges, cell cycle kinetics of choanocytes in intact tissues remains a controversial issue. This study is devoted to the comparative description of choanocyte cell cycle dynamics in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia corallorrhiza (class Calcarea). We have identified populations of proliferating cells and synchronized them in the S-phase to estimate the growth fraction of cycling cells. Using continuous exposure to labeled thymidine analog ethynyl deoxyuridine (EdU), we calculated choanocyte cell cycle duration and the length of the S phase. We also applied double labeling with EdU and antibodies against phosphorylated histone 3 to estimate the lengths of choanocyte M and G2 phases. Finally, flow-cytometry-based quantitative analysis of DNA content provided us with the lengths of G2 and G1 phases. We found that tissue growth and renewal in the studied sponges are generally maintained by a relatively large population of slowly cycling choanocytes with a total cell cycle duration of 40 h in H. dujardinii and 60 h in L. corallorrhiza. In both species, choanocytes are characterized by an extremely short M-phase and heterogeneity in the duration of the G2 phase.

Impact of DNA on interactions between core proteins of Hepatitis B virus-like particles comprising different C-terminals

Hepatitis B virus (HBV) virus-like particles (VLPs) are promising therapeutic agents derived from HBV core proteins (Cp). This study investigates the assembly dynamics of HBV VLPs, which is crucial for their potential as drug carriers or gene delivery systems. Coarse-grained molecular dynamics simulations explore the impact of C-terminal domain length (in the Cp ranging from Cp149 to wild-type Cp183) on Cp assembly and stability, particularly in the presence of DNA. Our findings reveal that the C-terminal nucleic acid binding region significantly influences Cp assembly and stability of trimers comprising Cp dimers. Shorter C-terminal domains (Cp164, Cp167) enhance stability and protein-protein interactions, while interactions between naturally occurring Cp183 are destabilized in the absence of DNA. Interestingly, DNA addition further stabilizes Cp assemblies, and this effect is influenced by the length of the nucleic acid binding region. Shorter C-terminal domains show less dependency on DNA content. This stabilization is attributed to electrostatic forces between positively charged C-terminal chains and negatively charged nucleic acids. Our study sheds light on the molecular mechanisms governing protein-protein and protein-DNA interactions in HBV VLP assembly, providing insights into Cp processability and informing the development of efficient gene therapy carriers using VLP technology.

Tissue-specific compensatory mechanisms maintain tissue architecture and body size independent of cell size in polyploid zebrafish

Genome duplications and ploidy transitions have occurred in nearly every major taxon of eukaryotes, but they are far more common in plants than in animals. Due to the conservation of the nuclear:cytoplasmic volume ratio increased DNA content results in larger cells. In plants, polyploid organisms are larger than diploids as cell number remains relatively constant. Conversely, vertebrate body size does not correlate with cell size and ploidy as vertebrates compensate for increased cell size to maintain tissue architecture and body size. This has historically been explained by a simple reduction in cell number that matches the increase in cell size maintaining body size as ploidy increases, but here we show that the compensatory mechanisms that maintain body size in triploid zebrafish are tissue-specific: A) erythrocytes respond in the classical pattern with a reduced number of larger erythrocytes in circulation, B) muscle, a tissue comprised of polynucleated muscle fibers, compensates by reducing the number of larger nuclei such that myofiber and myotome size in unaffected by ploidy, and C) vascular tissue compensates by thickening blood vessel walls, possibly at the expense of luminal diameter. Understanding the physiological implications of ploidy on tissue function requires a detailed description of the specific mechanisms of morphological compensation occurring in each tissue to understand how ploidy changes affect development and physiology.

Mature oocyte dysmorphisms may be associated with progesterone levels, mitochondrial DNA content, and vitality in luteal granulosa cells

PurposeTo identify whether follicular environment parameters are associated with mature oocyte quality, embryological and clinical outcomes.MethodsThis retrospective study examined 303 mature oocytes from 51 infertile women undergoing ICSI cycles between May 2018 and June 2021. Exclusion criteria consisted of advanced maternal age (> 36 years old), premature ovarian failure, obesity in women, or use of frozen gametes. Luteal granulosa cells (LGCs) were analyzed for mitochondrial DNA/genomic (g) DNA ratio and vitality. The relationships between hormone levels in the follicular fluid and oocyte features were assessed. Quantitative morphometric measurements of mature oocytes were assessed, and the association of LGC parameters and oocyte features on live birth rate after single embryo transfer was examined.ResultsResults indicated an inverse correlation between the mtDNA/gDNA ratio of LGCs and the size of polar body I (PBI). A 4.0% decrease in PBI size was observed with each one-unit increase in the ratio (p = 0.04). Furthermore, a 1% increase in LGC vitality was linked to a 1.3% decrease in fragmented PBI (p = 0.03), and a 1 ng/mL increase in progesterone levels was associated with a 0.1% rise in oocytes with small inclusions (p = 0.015). Associations were drawn among LGC characteristics, perivitelline space (PVS) debris, cytoplasmic inclusions, PBI integrity, and progesterone levels. Certain dysmorphisms in mature oocytes were associated with embryo morphokinetics; however, live birth rates were not associated with follicular parameters and oocyte quality characteristics.ConclusionFollicular markers may be associated with mature oocyte quality features.

Differentiated sex chromosomes, karyotype evolution, and spontaneous triploidy in carphodactylid geckos.

Geckos exhibit derived karyotypes without a clear distinction between macrochromosomes and microchromosomes and intriguing diversity in sex determination mechanisms. We conducted cytogenetic analyses in six species from the genera Nephrurus, Phyllurus, and Saltuarius of the gecko family Carphodactylidae. We confirmed the presence of a female heterogametic system with markedly differentiated and heteromorphic sex chromosomes in all examined species, typically with the W chromosome notably larger than the Z chromosome. One species, Nephrurus cinctus, possesses unusual multiple Z1Z1Z2Z2/Z1Z2W sex chromosomes. The morphology of the sex chromosomes, along with repetitive DNA content, suggests that the differentiation or emergence of sex chromosomes occurred independently in the genus Phyllurus. Furthermore, our study unveils a case of spontaneous triploidy in a fully grown individual of Saltuarius cornutus (3n = 57) and explores its implications for reproduction in carphodactylid geckos. We revealed that most carphodactylids retain the putative ancestral gekkotan karyotype of 2n = 38, characterized by predominantly acrocentric chromosomes that gradually decrease in size. If present, biarmed chromosomes emerge through pericentric inversions, maintaining the chromosome (and centromere) numbers. However, Phyllurus platurus is a notable exception, with a karyotype of 2n = 22 chromosomes. Its eight pairs of biarmed chromosomes were probably formed by Robertsonian fusions of acrocentric chromosomes. The family underscores a remarkable instance of evolutionary stability in chromosome numbers, followed by a profound transformation through parallel interchromosomal rearrangements. Our study highlights the need to continue generating cytogenetic data in order to test long-standing ideas about reproductive biology and the evolution of genome and sex determination.

Preparation of Decellularized Tissue as Dual Cell Carrier Systems: A Step Towards Facilitating Re-epithelization and Cell Encapsulation for Tracheal Reconstruction.

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers-decellularized scaffold and hydrogel-both containing the native biochemical niche essential for tracheal tissue engineering applications.

The effect of site-specific recombinases XerCD on the removal of over-replicated chromosomal DNA through outer membrane vesicles in bacteria.

Outer membrane vesicles (OMVs) are universally produced by Gram-negative bacteria and play important roles in symbiotic and pathogenic interactions. The DNA from the lumen of OMVs from the Alphaproteobacterium Dinoroseobacter shibae was previously shown to be enriched for the region around the terminus of replication ter and specifically for the recognition sequence dif of the two site-specific recombinases XerCD. These enzymes are highly conserved in bacteria and play an important role in the last phase of cell division. Here, we show that a similar enrichment of ter and dif is found in the DNA inside OMVs from Prochlorococcus marinus, Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli. The deletion of xerC or xerD in E. coli reduced the enrichment peak directly at the dif sequence, while the enriched DNA region around ter became broader, demonstrating that either enzyme influences the DNA content inside the lumen of OMVs. We propose that the intra-vesicle DNA originated from over-replication repair and the XerCD enzymes might play a role in this process, providing them with a new function in addition to resolving chromosome dimers.IMPORTANCEImprecise termination of replication can lead to over-replicated parts of bacterial chromosomes that have to be excised and removed from the dividing cell. The underlying mechanism is poorly understood. Our data show that outer membrane vesicles (OMVs) from diverse Gram-negative bacteria are enriched for DNA around the terminus of replication ter and the site-specific XerCD recombinases influence this enrichment. Clearing the divisome from over-replicated parts of the bacterial chromosome might be a so far unrecognized and conserved function of OMVs.

Distinctive development of embryo and endosperm caused by male gametes irradiated with carbon-ion beam

Abstract Key message In Cyrtanthus mackenii, development of embryo and endosperm were differentially affected by fertilization of male gametes with DNA damage and mutations. Abstract Pollen irradiation with ionizing radiations has been applied in plant breeding and genetic research, and haploid plant induction has mainly been performed by male inactivation with high-dose irradiation. However, the fertilization process of irradiated male gametes and the early development of embryo and endosperm have not received much attention. Heavy-ion beams, a type of radiation, have been widely applied as effective mutagens for plants and show a high mutation rate even at low-dose irradiation. In this study, we analyzed the effects of male gametes of Cyrtanthus mackenii irradiated with a carbon-ion beam at low doses on fertilization. In immature seeds derived from the pollination of irradiated pollen grains, two types of embryo sacs were observed: embryo sac with a normally developed embryo and endosperm and embryo sac with an egg cell or an undivided zygote and an endosperm. Abnormalities in chromosome segregation, such as chromosomal bridges, were observed only in the endosperm nuclei, irrespective of the presence or absence of embryogenesis. Therefore, in Cyrtanthus, embryogenesis is strongly affected by DNA damage or mutations in male gametes. Moreover, various DNA contents were detected in the embryo and endosperm nuclei, and endoreduplication may have occurred in the endosperm nuclei. As carbon-ion irradiation causes chromosomal rearrangements even at low doses, pollen irradiation can be an interesting tool for studying double fertilization and mutation heritability.

Paenibacillus baimaensis sp. nov., a bacterium isolated from mountain soil in the habitat of Rhinopithecus bieti.

A novel Gram-positive strain WQ 127069T that was isolated from the soil of Baima Snow Mountain, a habitat of highly endangered Yunnan snub-nosed monkeys (Rhinopithecus bieti), was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolate belongs to the genus Paenibacillus, showing 98.4 and 96.08 % sequence similarity to the type strains Paenibacillus periandrae PM10T and Paenibacillus foliorum LMG 31456T, respectively. The G+C content of the genomic DNA of strain WQ127069T was 45.6 mol%. The predominant isoprenoid quinone was MK-7, and meso-diaminopimelic acid was present in peptidoglycan. The major cellular fatty acids were antiiso-C15 : 0, iso-C15 : 0 and C16 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylmonomethylethanolamine. The whole genome average nucleotide identity and digital DNA-DNA hybridization values between strain WQ 127069T and strain PM10T were 93.2 and 52.5 %, respectively. Growth occurred at 5-40 °C (optimally at 20-35 °C), pH 6-8 (optimally at pH7.0) and with 0.5-2 % (w/v) NaCl (optimally at 0.5 %). On the basis of the taxonomic evidence, a novel species, Paenibacillus baimaensis sp. nov., is proposed. The type strain is WQ 127069T (=KCTC 43480T=CCTCC AB 2022381T).

Foraminifera as a model of eukaryotic genome dynamism.

In contrast to the canonical view that genomes cycle only between haploid and diploid states, many eukaryotes have dynamic genomes that change content throughout an individual's life cycle. However, the few detailed studies of microeukaryotic life cycles render our understanding of eukaryotic genome dynamism incomplete. Foraminifera (Rhizaria) are an ecologically important, yet understudied, clade of microbial eukaryotes with complex life cycles that include changes in ploidy and genome organization. Here, we apply fluorescence microscopy and image analysis techniques to over 2,800 nuclei in 110 cells to characterize the life cycle of Allogromia laticollaris strain Cold Spring Harbor (CSH), one of few cultivable foraminifera species. We show that haploidy and diploidy are brief moments in the A. laticollaris life cycle and that A. laticollaris nuclei endoreplicate up to 12,000 times the haploid genome size. We find that A. laticollaris reorganizes a highly endoreplicated nucleus into thousands of haploid genomes through a non-canonical mechanism called Zerfall, in which the nuclear envelope degrades and extrudes chromatin into the cytoplasm. Based on these findings, along with changes in nuclear architecture across the life cycle, we believe that A. laticollaris uses spatio-temporal mechanisms to delineate germline and somatic DNA within a single nucleus. The analyses here extend our understanding of the genome dynamics across the eukaryotic tree of life.IMPORTANCEIn traditional depictions of eukaryotes (i.e., cells with nuclei), life cycles alternate only between haploid and diploid phases, overlooking studies of diverse microeukaryotic lineages (e.g., amoebae, ciliates, and flagellates) that show dramatic variation in DNA content throughout their life cycles. Endoreplication of genomes enables cells to grow to large sizes and perhaps to also respond to changes in their environments. Few microeukaryotic life cycles have been studied in detail, which limits our understanding of how eukaryotes regulate and transmit their DNA across generations. Here, we use microscopy to study the life cycle of Allogromia laticollaris strain CSH, an early-diverging lineage within the Foraminifera (an ancient clade of predominantly marine amoebae). We show that DNA content changes significantly throughout their life cycle and further describe an unusual process called Zerfall, by which this species reorganizes a large nucleus with up to 12,000 genome copies into hundreds of small gametic nuclei, each with a single haploid genome. Our results are consistent with the idea that all eukaryotes demarcate germline DNA to pass on to offspring amidst more flexible somatic DNA and extend the known diversity of eukaryotic life cycles.

Glycomyces niveus sp. nov., a novel actinomycete isolated from sandy soil.

A novel mycelium-forming actinomycete, designated strain NEAU-S30T, was isolated from the sandy soil of a sea beach in Shouguang city, Shandong province, PR China. The strain developed long chains of non-motile cylindrical spores with smooth surfaces on aerial mycelia. The results of a polyphasic taxonomic study indicated that NEAU-S30T represented a member of the genus Glycomyces. The results of 16S rRNA gene sequence analysis indicated that NEAU-S30T was closely related to 'Glycomycesluteolus' (98.97 % sequence similarity), Glycomycesalgeriensis (98.90 %), 'Glycomyces tritici' (98.83 %) and Glycomyces lechevalierae (98.76 %). The average nucleotide identity (ANI) values between NEAU-S30T and 'G. luteolus' NEAU-A15, G. algeriensis DSM 44727T, 'G. tritici' NEAU-C2 and G. lechevalierae DSM 44724T were 87.77, 87.53, 87.41 and 87.80 %, respectively. The digital DNA G+C content of the genomic DNA was 70.5 %. The whole-cell sugars contained ribose and xylose. The predominant menaquinones were MK-10(H2), MK-10(H4) and MK-10(H6). The predominant fatty acids were anteiso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0 and iso-C15 : 0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified glycolipid. On the basis of the results of comparative analysis of genotypic, phenotypic and chemotaxonomic data, the novel actinomycete strain NEAU-S30T (=JCM 33975T=CGMCC 4.7890T) represents the type strain of a novel species within the genus Glycomyces, for which the name Glycomyces niveus sp. nov. is proposed.

Protein production dynamics and physiological adaptation of recombinant Komagataella phaffii at near-zero growth rates

BackgroundSpecific productivity (qP) in yeast correlates with growth, typically peaking at intermediate or maximum specific growth rates (μ). Understanding the factors limiting productivity at extremely low μ might reveal decoupling strategies, but knowledge of production dynamics and physiology in such conditions is scarce. Retentostats, a type of continuous cultivation, enable the well-controlled transition to near-zero µ through the combined retention of biomass and limited substrate supply. Recombinant Komagataella phaffii (syn Pichia pastoris) secreting a bivalent single domain antibody (VHH) was cultivated in aerobic, glucose-limited retentostats to investigate recombinant protein production dynamics and broaden our understanding of relevant physiological adaptations at near-zero growth conditions.ResultsBy the end of the retentostat cultivation, doubling times of approx. two months were reached, corresponding to µ = 0.00047 h−1. Despite these extremely slow growth rates, the proportion of viable cells remained high, and de novo synthesis and secretion of the VHH were observed. The average qP at the end of the retentostat was estimated at 0.019 mg g−1 h−1. Transcriptomics indicated that genes involved in protein biosynthesis were only moderately downregulated towards zero growth, while secretory pathway genes were mostly regulated in a manner seemingly detrimental to protein secretion. Adaptation to near-zero growth conditions of recombinant K. phaffii resulted in significant changes in the total protein, RNA, DNA and lipid content, and lipidomics revealed a complex adaptation pattern regarding the lipid class composition. The higher abundance of storage lipids as well as storage carbohydrates indicates that the cells are preparing for long-term survival.ConclusionsIn conclusion, retentostat cultivation proved to be a valuable tool to identify potential engineering targets to decouple growth and protein production and gain important insights into the physiological adaptation of K. phaffii to near-zero growth conditions.

DNA quality and quantity in adipose tissue: a comparison of the effects of bomb explosion

BACKGROUND Adipose tissue is often overlooked in DNA testing due to misconceptions about its DNA content. However, its shock-absorbing qualities may be useful for high-pressure scenarios like bomb blasts. This study aimed to evaluate DNA quality and quantity in adipose tissue affected by blasts compared to that in unaffected tissue.
 METHODS 10 adipose tissue samples were taken from regions near and far from the blast, representing the blast-exposed and non-blast-exposed groups. The adipose tissue was stored at a low temperature for 5 days, after which an organic extraction method was applied. The purity of the DNA extract was assessed using a NanoDrop spectrophotometer, and its integrity was evaluated using 0.8% concentration gel electrophoresis at 60 V for 90 min. DNA typing was conducted using the GlobalFiler™ kit, and DNA quantity was determined with the Quantifiler™ Trio DNA Quantification kit.
 RESULTS Of 20 DNA extracts from adipose tissue, all samples demonstrated purity, integrity, and complete typing results. Adequate integrity was found in 90% of samples in both groups. A 50% incidence of allele shifting was observed at the D7S820 locus within the blast-exposed group.
 CONCLUSIONS DNA from blast-exposed adipose tissue exhibited no significant quality or quantity differences from non-blast-exposed tissue. This suggested adipose tissue’s potential as an alternative DNA source in a bomb explosion.

Solibacillus palustris sp. nov., isolated from wetland soil of ecology park.

A Gram-staining-positive, motile, aerobic and rod-shaped bacterium, designated strain MA9T was isolated from wetland soil of ecology park, in Seoul, Republic of Korea. This bacterium was characterized to determine its taxonomic position by using the polyphasic approach. Strain MA9T grew at 10-37 °C and at pH 6.0-9.5 on TSB. Menaquinone MK-7 was the predominant respiratory quinone and iso-C15 : 0, iso-C16 : 0 and C16 : 1 ω7c alcohol were the major fatty acids. The main polar lipids were phosphatidylethanolamine (PE), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). The peptidoglycan type of the cell wall was A4α l-Lys-d-Glu. Based on 16S rRNA gene sequencing, strain MA9T clustered with species of the genus Solibacillus and appeared closely related to S. silvestris DSM 12223T (97.8 % sequence similarity), S. cecembensis DSM 21993T (97.6 %), S. isronensis DSM 21046T (97.6 %) and S. kalamii DSM 101595T (96.6 %). The G+C content of the genomic DNA was 37.0 mol%. Digital DNA-DNA hybridization between strain MA9T and type strains of S. silvestris, S. isronensis, S. cecembensis and S. kalamii resulted in values below 70 %. Strain MA9T could be differentiated genotypically and phenotypically from the recognized species of the genus Solibacillus. The isolate therefore represents a novel species, for which the name Solibacillus palustris sp. nov. is proposed, with the type strain MA9T (=KACC 22212T = LMG 32188T).

Cellular Enhancement of Frozen Meniscus Allograft Combining Native Meniscus and Mesenchymal Stromal Cell Injections.

This proof-of-concept study investigated an improved cell-based injection therapy combining mesenchymal stem cells (MSCs) and meniscus cells (MCs) to support superior meniscus allograft repopulation and early revival compared to injecting MSCs alone. In this controlled laboratory study, frozen meniscus allograft samples were injected vertically with a cell suspension containing different ratios of MSCs and MCs or control (lactated ringers) and cultured for 28 days. Samples were analyzed weekly for cell viability, migration, and metabolism using histological and biochemical assays. Tissue medium was analyzed for matrix metalloproteinase (MMP) expression using zymography. Cellular repopulation of frozen allografts injected with different cell suspensions was validated by immunohistochemistry. Significant higher DNA content was evidenced in grafts treated with suspensions of MCs or MC:MSC (1:4 ratio). Cell metabolic activity was significantly different between all treated groups and control group after 1 week. Allografts injected with MCs showed significantly more cell proliferation than injections with MSCs. MMP2 activity was detected in medium of all grafts cellularized with MCs with or without MSCs. Scanning electron microscopy (SEM) analysis showed resolution of the needle puncture, but not in the control group. Cell labeling of MCs upon injection of mixed MC:MSC suspensions revealed a gradual increase in the cell ratio. The findings of this study establish that injection of MCs with or without MSCs enhances the cellularity of meniscus allograft to support early graft revival and remodeling.

Phenotypic characterization of liver tissue heterogeneity through a next-generation 3D single-cell atlas

Three-dimensional (3D) geometrical models are potent tools for quantifying complex tissue features and exploring structure–function relationships. However, these models are generally incomplete due to experimental limitations in acquiring multiple (> 4) fluorescent channels in thick tissue sections simultaneously. Indeed, predictive geometrical and functional models of the liver have been restricted to few tissue and cellular components, excluding important cellular populations such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we combined deep-tissue immunostaining, multiphoton microscopy, deep-learning techniques, and 3D image processing to computationally expand the number of simultaneously reconstructed tissue structures. We then generated a spatial single-cell atlas of hepatic architecture (Hep3D), including all main tissue and cellular components at different stages of post-natal development in mice. We used Hep3D to quantitatively study 1) hepatic morphodynamics from early post-natal development to adulthood, and 2) the effect on the liver's overall structure when changing the hepatic environment after removing KCs. In addition to a complete description of bile canaliculi and sinusoidal network remodeling, our analysis uncovered unexpected spatiotemporal patterns of non-parenchymal cells and hepatocytes differing in size, number of nuclei, and DNA content. Surprisingly, we found that the specific depletion of KCs results in morphological changes in hepatocytes and HSCs. These findings reveal novel characteristics of liver heterogeneity and have important implications for both the structural organization of liver tissue and its function. Our next-gen 3D single-cell atlas is a powerful tool to understand liver tissue architecture, opening up avenues for in-depth investigations into tissue structure across both normal and pathological conditions.

On the distribution and diversity of tissue-specific somatic mutations in honey bee (Apis mellifera) drones

Somatic mutations originate from both exogenous (e.g. UV radiation, chemical agents) and endogenous (e.g., DNA replication, defective DNA repair) sources and can have significant impacts on an animal’s reproductive success. This may be especially true for haploid organisms that are susceptible to any deleterious alleles inherited from their parent and any that arise over their lifetime. Unfortunately, little is known about the rate of somatic mutation accumulation across individuals and tissues of haplodiploid animal populations, the functional processes through which they arise, and their distribution across tissues and the genome. Here, we generated short-read whole-genome sequencing data for four tissues of haploid honey bee males. We paired this with estimates of telomere length and tissue-specific DNA content to address three major questions: is there variance in somatic mutational load across haploid individuals and specific tissues therein, does increased DNA content in a tissue contribute to somatic mutational load, and does telomere length correlate with mutational load? Our results suggest that variance in somatic mutational load is better captured across individuals than across tissues, that tissue-specific DNA content is not associated with somatic mutation load, and that variance in telomere length does not correlate with somatic mutation loads across tissues. To our knowledge, this is the first observational study on somatic mutational load in Apoidea and likely Hymenoptera. It serves as a useful advent for additional studies understanding the processes through which haploids tolerate or repair somatic mutations.

Cytomolecular trends in Chamaecrista Moench (Caesalpinioideae, Leguminosae) diversification.

Chamaecrista is a Pantropical legume genus of the tribe Cassieae, which includes six other genera. In contrast to most of the other Cassieae genera, Chamaecrista shows significant variability in chromosome number (from 2n = 14 to 2n = 56), with small and morphologically similar chromosomes. Here, we performed a new cytomolecular analysis on chromosome number, genome size, and rDNA site distribution in a molecular phylogenetic perspective to interpret the karyotype trends of Chamaecrista and other two genera of Cassieae, seeking to understand their systematics and evolution. Our phylogenetic analysis revealed that Chamaecrista is monophyletic and can be divided into four major clades corresponding to the four sections of the genus. Chromosome numbers ranged from 2n = 14, 16 (section Chamaecrista) to 2n = 28 (sections Absus, Apoucouita, and Baseophyllum). The number of 5S and 35S rDNA sites varied between one and three pairs per karyotype, distributed on different chromosomes or in synteny, with no obvious phylogenetic significance. Our data allowed us to propose x = 7 as the basic chromosome number of Cassieae, which was changed by polyploidy generating x = 14 (sections Absus, Apoucouita, and Baseophyllum) and by ascending dysploidy to x = 8 (section Chamaecrista). The DNA content values supported this hypothesis, with the genomes of the putative tetraploids being larger than those of the putative diploids. We hypothesized that ascending dysploidy, polyploidy, and rDNA amplification/deamplification are the major events in the karyotypic diversification of Chamaecrista. The chromosomal marks characterized here may have cytotaxonomic potential in future studies.