Probiotic Potential of a Folate-Producing Strain Latilactobacillus sakei LZ217 and Its Modulation Effects on Human Gut Microbiota

Folate is a B-vitamin required for DNA synthesis, methylation, and cellular division, whose deficiencies are associated with various disorders and diseases. Currently, most folic acid used for fortification is synthesized chemically, causing undesirable side effects. However, using folate-producing probiotics is a viable option, which fortify folate in situ and regulate intestinal microbiota. In this study, the folate production potential of newly isolated strains from raw milk was analyzed by microbiological assay. Latilactobacillus sakei LZ217 showed the highest folate production in Folic Acid Assay Broth, 239.70 ± 0.03 ng/μL. The folate produced by LZ217 was identified as 5-methyltetrahydrofolate. LZ217 was tolerant to environmental stresses (temperature, pH, NaCl, and ethanol), and was resistant to gastrointestinal juices. Additionally, the in vitro effects of LZ217 on human gut microbiota were investigated by fecal slurry cultures. 16S rDNA gene sequencing indicated that fermented samples containing LZ217 significantly increased the abundance of phylum Firmicutes and genus Lactobacillus, Faecalibacterium, Ruminococcus 2, Butyricicoccus compared to not containing. Short-chain fatty acids (SCFAs) analysis revealed that LZ217 also increased the production of butyric acid by fermentation. Together, L. sakei LZ217 could be considered as a probiotic candidate to fortify folate and regulate intestinal microecology.

Long-lasting and responsive DNA/enzyme-based programs in serum-supplemented extracellular media

DNA molecular programs are emerging as promising pharmaceutical approaches due to their versatility for biomolecular sensing and actuation. However, the implementation of DNA programs has been mainly limited to serum-deprived in vitro assays due to the fast deterioration of the DNA reaction networks by the nucleases present in the serum. Here, we show that DNA/enzyme programs are functional in serum for 24h but are latter disrupted by nucleases that give rise to parasitic amplification. To overcome this, we implement 3-letter code networks that suppress autocatalytic parasites while still conserving the functionality of DNA/enzyme programs for at least 3 days in the presence of 10% serum. In addition, we define a new buffer that further increases the biocompatibility and conserves responsiveness to changes in molecular composition across time. Finally, we demonstrate how serum-supplemented extracellular DNA molecular programs remain responsive to molecular inputs in the presence of living cells, having responses 6-fold faster than cellular division rate and are sustainable for at least 3 cellular divisions. This demonstrates the possibility of implementing in situ biomolecular characterization tools for serum-demanding in vitro models. We foresee that the coupling of chemical reactivity to our DNA programs by aptamers or oligonucleotide conjugations will allow the implementation of extracellular synthetic biology tools, which will offer new biomolecular pharmaceutical approaches and the emergence of complex and autonomous in vitro models.

A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity

The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (51Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate 51Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release 51Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells’ killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.

Stress Relief Techniques: p38 MAPK Determines the Balance of Cell Cycle and Apoptosis Pathways

Protein signaling networks are formed from diverse and inter-connected cell signaling pathways converging into webs of function and regulation. These signaling pathways both receive and conduct molecular messages, often by a series of post-translation modifications such as phosphorylation or through protein–protein interactions via intrinsic motifs. The mitogen activated protein kinases (MAPKs) are components of kinase cascades that transmit signals through phosphorylation. There are several MAPK subfamilies, and one subfamily is the stress-activated protein kinases, which in mammals is the p38 family. The p38 enzymes mediate a variety of cellular outcomes including DNA repair, cell survival/cell fate decisions, and cell cycle arrest. The cell cycle is itself a signaling system that precisely controls DNA replication, chromosome segregation, and cellular division. Another indispensable cell function influenced by the p38 stress response is programmed cell death (apoptosis). As the regulators of cell survival, the BCL2 family of proteins and their dynamics are exquisitely sensitive to cell stress. The BCL2 family forms a protein–protein interaction network divided into anti-apoptotic and pro-apoptotic members, and the balance of binding between these two sides determines cell survival. Here, we discuss the intersections among the p38 MAPK, cell cycle, and apoptosis signaling pathways.

Effects of RTTN gene mutations and the need for complementary cDNA analysis for some transcripts: case report

RTTN is a gene coding for Rotatin protein which has been localized at ciliary body and centriole of human fibroblasts. Its exact function in human is not well identified. However, it is thought to play important role in maintenance of normal structure of primary cilia and hence in left to right organ specification, axial rotation, development of notochord as well as early cellular division in mice. Overall 28 cases have been reported with homozygous pathogenic mutations of RTTN gene till 2019 to our knowledge. They presented with primary microcephaly, short stature, polymicrogyria with or without seizures (MPPS). These symptoms can be summarized in microcephaly, generalized growth delay, malformation of the developing cortex (MDC) and hence neurodevelopmental delay with or without seizures. Meanwhile all of these signs usually appear in late prenatal or even postnatal life which emphasizes the need for other diagnostic tools in case of detection of RTTN variables of unknown significance or in case of accidental detection of multiple transcripts by NGS in prenatal genetic testing. A case with prenatal diagnosis of two different RTTN gene transcripts in fetal WES analysis. One of the transcripts showed RTTN homozygous gene mutation while the other transcript was normal. This article presents the multidisciplinary approach followed in prenatal as well as postnatal period. It also highlights the importance of gene expression analysis in prenatal genetics.

Integration and gene co-expression network analysis of scRNA-seq transcriptomes reveal heterogeneity and key functional genes in human spermatogenesis

Spermatogenesis is a complex process of cellular division and differentiation that begins with spermatogonia stem cells and leads to functional spermatozoa production. However, many of the molecular mechanisms underlying this process remain unclear. Single-cell RNA sequencing (scRNA-seq) is used to sequence the entire transcriptome at the single-cell level to assess cell-to-cell variability. In this study, more than 33,000 testicular cells from different scRNA-seq datasets with normal spermatogenesis were integrated to identify single-cell heterogeneity on a more comprehensive scale. Clustering, cell type assignments, differential expressed genes and pseudotime analysis characterized 5 spermatogonia, 4 spermatocyte, and 4 spermatid cell types during the spermatogenesis process. The UTF1 and ID4 genes were introduced as the most specific markers that can differentiate two undifferentiated spermatogonia stem cell sub-cellules. The C7orf61 and TNP can differentiate two round spermatid sub-cellules. The topological analysis of the weighted gene co-expression network along with the integrated scRNA-seq data revealed some bridge genes between spermatogenesis’s main stages such as DNAJC5B, C1orf194, HSP90AB1, BST2, EEF1A1, CRISP2, PTMS, NFKBIA, CDKN3, and HLA-DRA. The importance of these key genes is confirmed by their role in male infertility in previous studies. It can be stated that, this integrated scRNA-seq of spermatogenic cells offers novel insights into cell-to-cell heterogeneity and suggests a list of key players with a pivotal role in male infertility from the fertile spermatogenesis datasets. These key functional genes can be introduced as candidates for filtering and prioritizing genotype-to-phenotype association in male infertility.

Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

This study aimed to assess the relationship and possible interactions between metallothioneins (MTs) and megalin (LRP-2) in different grades of oral squamous cell carcinoma (OSCC) and premalignant lesions of the oral mucosa (oral leukoplakia and oral lichen planus). The study included archived samples of 114 patients and control subjects. Protein expression was examined by immunohistochemistry and immunofluorescence, and staining quantification was performed by ImageJ software. Protein interaction in cancer tissue was tested and visualized by proximity ligation assay. Mann-Whitney and Kruskal-Wallis tests were used to determine the significance of differences between each group, whereas Pearson correlation coefficient was performed to test correlation. Expression of both proteins differed significantly between each group showing the same pattern of gradual increasing from oral lichen planus to poorly differentiated OSCC. Moreover, MTs and megalin were found to co-express and interact in cancer tissue, and their expression positively correlated within the overall study group. Findings of prominent nuclear and chromosomal megalin expression suggest that it undergoes regulated intramembrane proteolysis upon MTs binding, indicating its ability to directly affect gene expression and cellular division in cancer tissue. The data obtained point to the onco-driving potential of MTs-megalin interaction.

The Application of Single-Cell RNA Sequencing in Mammalian Meiosis Studies

Meiosis is a cellular division process that produces gametes for sexual reproduction. Disruption of complex events throughout meiosis, such as synapsis and homologous recombination, can lead to infertility and aneuploidy. To reveal the molecular mechanisms of these events, transcriptome studies of specific substages must be conducted. However, conventional methods, such as bulk RNA-seq and RT-qPCR, are not able to detect the transcriptional variations effectively and precisely, especially for identifying cell types and stages with subtle differences. In recent years, mammalian meiotic transcriptomes have been intensively studied at the single-cell level by using single-cell RNA-seq (scRNA-seq) approaches, especially through two widely used platforms, Smart-seq2 and Drop-seq. The scRNA-seq protocols along with their downstream analysis enable researchers to accurately identify cell heterogeneities and investigate meiotic transcriptomes at a higher resolution. In this review, we compared bulk RNA-seq and scRNA-seq to show the advantages of the scRNA-seq in meiosis studies; meanwhile, we also pointed out the challenges and limitations of the scRNA-seq. We listed recent findings from mammalian meiosis (male and female) studies where scRNA-seq applied. Next, we summarized the scRNA-seq analysis methods and the meiotic marker genes from spermatocytes and oocytes. Specifically, we emphasized the different features of the two scRNA-seq protocols (Smart-seq2 and Drop-seq) in the context of meiosis studies and discussed their strengths and weaknesses in terms of different research purposes. Finally, we discussed the future applications of scRNA-seq in the meiosis field.

A chromatin phase transition protects mitotic chromosomes against microtubule perforation

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells. Assembly of mitotic chromosomes involves DNA looping by condensin and chromatin compaction by global histone deacetylation. While condensin confers mechanical resistance towards spindle pulling forces, it is not known how histone deacetylation affects material properties and segregation mechanics of mitotic chromosomes. Here, we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with specific characteristics necessary for their precise movement during cellular division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. Hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules, and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin-intrinsic phase separation to genome segregation in dividing cells.

Progress and Challenges in Laboratory-Based Diagnostic and Screening Approaches for Aneuploidy Detection during Pregnancy

Aneuploidy is caused by problems during cellular division and segregation errors during meiosis that lead to an abnormal number of chromosomes and initiate significant genetic abnormalities during pregnancy or the loss of a fetus due to miscarriage. Screening and diagnostic technologies have been developed to detect this genetic condition and provide parents with critical information about their unborn child. In this review, we highlight the complexities of aneuploidy as a disease as well as multiple technological advancements in testing that help to identify aneuploidy at various time points throughout pregnancy. We focus on aneuploidy diagnosis during preimplantation genetic testing that is performed during in vitro fertilization as well as prenatal screening and diagnosis during pregnancy. This review focuses on DNA-based analysis and laboratory techniques for aneuploidy detection through reviewing molecular- and engineering-based technical advancements. We also present key challenges in aneuploidy detection during pregnancy, including sample collection, mosaic embryos, economic factors, and the social implications of this testing. The goal of this review is to synthesize broad information about aneuploidy screening and diagnostic sample collection and analysis during pregnancy and discuss major challenges the field is still facing despite decades of advancements.