The major pathways of DNA double-strand break (DSB) repair are crucial for maintaining genomic stability. However, if deployed in an inappropriate cellular context, these same repair functions can ...mediate chromosome rearrangements that underlie various human diseases, ranging from developmental disorders to cancer. The two major mechanisms of DSB repair in mammalian cells are non-homologous end joining (NHEJ) and homologous recombination. In this Review, we consider DSB repair-pathway choice in somatic mammalian cells as a series of 'decision trees', and explore how defective pathway choice can lead to genomic instability. Stalled, collapsed or broken DNA replication forks present a distinctive challenge to the DSB repair system. Emerging evidence suggests that the 'rules' governing repair-pathway choice at stalled replication forks differ from those at replication-independent DSBs.
Somatic cells (SCs) in milk, which include epithelial cells from the gland and blood cells, are present in milk during the normal course of milking. Increase in SCs is found in mastitis-affected ...dairy cows and can be a useful indicator for estimating mammary health and milk quality worldwide. The aim of this study was to determine whether there was a pattern of somatic cell count (SCC) in mastitis-affected cows on three different farms. The study was conducted on three dairy farms of high milk-producing cattle breeds in Vojvodina during 2021. Samples were taken from 15 cows from each farm, all of the cows being diagnosed with clinical and subclinical mastitis. The SCC in milk samples was determined by the microscopic reference method according to the standard (SRPS EN ISO 13366-1:2010) of the Institute for Standardization of Serbia. The value of SCC was considered as high if >200.000 cells/mL, as this is the threshold indicating secretion disorder. In order to examine the differences between the observed three farms, one-factor analysis of variance (ANOVA) was applied, while a post-hoc LSD test was used for determination of statistically significant differences between the SCC in cows on three different farms. The mean values of the SCC on Farms 1, 2 and 3 were 7,055,266.67, 2,619,893.33 and 552,000 cells/mL, respectively. Based on the results, a statistically significant difference (p<0.05) was established between Farms 1 and 2, as well as between Farms 1 and 3, while there was no statistically significant difference between Farms 2 and 3. Apart from mastitis, differences in the SCC on the farms could also be influenced by the cows’ productivity, parity, lactation stage and breed, as well as poor management practices. Besides mastitis control, better hygiene and proper nutrition can help in reducing SCs in milk. In conclusion, establishing SCs pattern can provide useful information that may contribute to reducing SCs and developing differential SCs standards to help obtain milk with low SCs and consequently better dairy products with a longer shelf life.
Milk somatic cells (SCs) are a mixture of milk-producing cells and immune cells. These cells are secreted in milk during the normal course of milking and are used as an index for estimating mammary ...health and milk quality of dairy animals worldwide. Milk SC is influenced by cow productivity, health, parity, lactation stage, and breed of an animal. Any change in environmental conditions, poor management practices, and also stressful conditions significantly increases the amount of SC coming in milk. Better hygiene and proper nutrition help in reducing milk SC. Milk with low SC means better milk products with a longer shelf life. The present review describes the role of SCs (both secretory and immune) in milk, their role in maintaining the integrity of the mammary gland, and factors affecting their release in milk. This information may help to reduce milk somatic cell counts (SCCs) and to establish differential SCC standards.
Through the successes of checkpoint blockade and adoptive cellular therapy, immunotherapy has become an established treatment modality for cancer. Cellular metabolism has emerged as a critical ...determinant of the viability and function of both cancer cells and immune cells. In order to sustain prodigious anabolic needs, tumours employ a specialized metabolism that differs from untransformed somatic cells. This metabolism leads to a tumour microenvironment that is commonly acidic, hypoxic and/or depleted of critical nutrients required by immune cells. In this context, tumour metabolism itself is a checkpoint that can limit immune-mediated tumour destruction. Because our understanding of immune cell metabolism and cancer metabolism has grown significantly in the past decade, we are on the cusp of being able to unravel the interaction of cancer cell metabolism and immune metabolism in therapeutically meaningful ways. Although there are metabolic processes that are seemingly fundamental to both cancer and responding immune cells, metabolic heterogeneity and plasticity may serve to distinguish the two. As such, understanding the differential metabolic requirements of the diverse cells that comprise an immune response to cancer offers an opportunity to selectively regulate immune cell function. Such a nuanced evaluation of cancer and immune metabolism can uncover metabolic vulnerabilities and therapeutic windows upon which to intervene for enhanced immunotherapy.
Somatic cell count (SCC) in milk is an essential indicator for defining and managing udder health. However, analyzing differential SCC (dSCC) can be helpful in determining the type or evolution stage ...of mastitis. A high abundance of polymorphonuclear cells (PMN) is associated with acute mastitis; however, the status of a chronic disease is less well characterized. A method capable of analyzing SCC and dSCC can prove to be a helpful tool for monitoring the status of evolution of mastitis disease in a better way. Therefore, a new direct-flow cytometry method was developed to count and differentiate somatic cells in milk without the steps of centrifugation or washing, avoiding variabilities that occur due to enrichment or loss of specific cell types. In this new method, SCC is analyzed using the method of DNA staining with Hoechst stain, whereas dSCC are analyzed using specific antibodies targeting 2 main cell types associated with mastitis: PMN cells and antigen-presenting cells, which are associated with innate and adaptive immunity. Equivalent SCC values were obtained between the new method and the routine ISO 13366-2 method in a comparison of 240 raw milk samples. Furthermore, dSCC results were confirmed by microscopy after May-Gründwald-Giemsa staining in 165 quarter milk samples from healthy and diseased cows. The method was verified with fluorescence microscopy on the 2 targeted cell types and in raw milk samples. The newly developed method is independent of any instrument and can be further designed to differentiate other cell types and animal species by selecting appropriate antibodies.
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Cellular reprogramming can manipulate the identity of cells to generate the desired cell types
. The use of cell intrinsic components, including oocyte cytoplasm and transcription factors, can ...enforce somatic cell reprogramming to pluripotent stem cells
. By contrast, chemical stimulation by exposure to small molecules offers an alternative approach that can manipulate cell fate in a simple and highly controllable manner
. However, human somatic cells are refractory to chemical stimulation owing to their stable epigenome
and reduced plasticity
; it is therefore challenging to induce human pluripotent stem cells by chemical reprogramming. Here we demonstrate, by creating an intermediate plastic state, the chemical reprogramming of human somatic cells to human chemically induced pluripotent stem cells that exhibit key features of embryonic stem cells. The whole chemical reprogramming trajectory analysis delineated the induction of the intermediate plastic state at the early stage, during which chemical-induced dedifferentiation occurred, and this process was similar to the dedifferentiation process that occurs in axolotl limb regeneration. Moreover, we identified the JNK pathway as a major barrier to chemical reprogramming, the inhibition of which was indispensable for inducing cell plasticity and a regeneration-like program by suppressing pro-inflammatory pathways. Our chemical approach provides a platform for the generation and application of human pluripotent stem cells in biomedicine. This study lays foundations for developing regenerative therapeutic strategies that use well-defined chemicals to change cell fates in humans.
Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction ...among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, "planktonic" state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system.
Accurate discovery of somatic mutations in a cell is a challenge that partially lays in immaturity of dedicated analytical approaches. Approaches comparing a cell's genome to a control bulk sample ...miss common mutations, while approaches to find such mutations from bulk suffer from low sensitivity. We developed a tool, All.sup.2, which enables accurate filtering of mutations in a cell without the need for data from bulk(s). It is based on pair-wise comparisons of all cells to each other where every call for base pair substitution and indel is classified as either a germline variant, mosaic mutation, or false positive. As All.sup.2 allows for considering dropped-out regions, it is applicable to whole genome and exome analysis of cloned and amplified cells. By applying the approach to a variety of available data, we showed that its application reduces false positives, enables sensitive discovery of high frequency mutations, and is indispensable for conducting high resolution cell lineage tracing.
The reprogramming of somatic cells with defined factors, which converts cells from one lineage into cells of another, has greatly reshaped our traditional views on cell identity and cell fate ...determination. Direct reprogramming (also known as transdifferentiation) refers to cell fate conversion without transitioning through an intermediary pluripotent state. Given that the number of cell types that can be generated by direct reprogramming is rapidly increasing, it has become a promising strategy to produce functional cells for therapeutic purposes. This Review discusses the evolution of direct reprogramming from a transcription factor-based method to a small-molecule-driven approach, the recent progress in enhancing reprogrammed cell maturation, and the challenges associated with in vivo direct reprogramming for translational applications. It also describes our current understanding of the molecular mechanisms underlying direct reprogramming, including the role of transcription factors, epigenetic modifications, non-coding RNAs, and the function of metabolic reprogramming, and highlights novel insights gained from single-cell omics studies.
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