Microb Genom. 2023 Jan;9(1). doi: 10.1099/mgen.0.000937.

ABSTRACT

The increase of Vancomycin-resistant Enterococcus faecium (VREfm) in recent years has been partially attributed to the rise of specific clonal lineages, which have been identified throughout Germany. To date, there is no gold standard for the interpretation of genomic data for outbreak analyses. New genomic approaches such as split k-mer analysis (SKA) could support cluster attribution for routine outbreak investigation. The aim of this project was to investigate frequent clonal lineages of VREfm identified during suspected outbreaks across different hospitals, and to compare genomic approaches including SKA in routine outbreak investigation. We used routine outbreak laboratory data from seven hospitals and three different hospital networks in Berlin, Germany. Short-read libraries were sequenced on the Illumina MiSeq system. We determined clusters using the published Enterococcus faecium-cgMLST scheme (threshold ≤20 alleles), and assigned sequence and complex types (ST, CT), using the Ridom SeqSphere+ software. For each cluster as determined by cgMLST, we used pairwise core-genome SNP-analysis and SKA at thresholds of ten and seven SNPs, respectively, to further distinguish cgMLST clusters. In order to investigate clinical relevance, we analysed to what extent epidemiological linkage backed the clusters determined with different genomic approaches. Between 2014 and 2021, we sequenced 693 VREfm strains, and 644 (93 %) were associated within cgMLST clusters. More than 74 % (n=475) of the strains belonged to the six largest cgMLST clusters, comprising ST117, ST78 and ST80. All six clusters were detected across several years and hospitals without apparent epidemiological links. Core SNP analysis identified 44 clusters with a median cluster size of three isolates (IQR 2-7, min-max 2-63), as well as 197 singletons (41.4 % of 475 isolates). SKA identified 67 clusters with a median cluster size of two isolates (IQR 2-4, min-max 2-19), and 261 singletons (54.9 % of 475 isolates). Of the isolate pairs attributed to clusters, 7 % (n=3064/45 596) of pairs in clusters determined by standard cgMLST, 15 % (n=1222/8500) of pairs in core SNP-clusters and 51 % (n=942/1880) of pairs in SKA-clusters showed epidemiological linkage. The proportion of epidemiological linkage differed between sequence types. For VREfm, the discriminative ability of the widely used cgMLST based approach at ≤20 alleles difference was insufficient to rule out hospital outbreaks without further analytical methods. Cluster assignment guided by core genome SNP analysis and the reference free SKA was more discriminative and correlated better with obvious epidemiological linkage, at least recently published thresholds (ten and seven SNPs, respectively) and for frequent STs. Besides higher overall discriminative power, the whole-genome approach implemented in SKA is also easier and faster to conduct and requires less computational resources.

PMID:36748706 | DOI:10.1099/mgen.0.000937

Int J Syst Evol Microbiol. 2023 Feb;73(1). doi: 10.1099/ijsem.0.005677.

ABSTRACT

A novel Gram-reaction-negative, aerobic, motile, rod-shaped, grey bacterium, strain P4.10XT, was isolated from plastic debris sampled from shallow waters in the Mediterranean Sea (Valencia, Spain). P4.10XT was catalase- and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA gene sequences revealed that P4.10XT was closely related to Maritalea myrionectae DSM 19524T and Maritalea mobilis E6T (98.25 and 98.03 % sequence similarity, respectively). The DNA G+C content of the genome sequence of P4.10XT was 53.66 %. The genomic indexes average nucleotide identity by blast (ANIb) and digital DNA-DNA hybridization (dDDH) confirmed its classification as representing a novel species of the genus Maritalea. The predominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c) and C18 : 1 ω7c 11-methyl. The results of this polyphasic study confirm that P4.10XT represents a novel species of the genus Maritalea, for which the name Maritalea mediterranea sp. nov. is proposed (type strain P4.10XT=CECT 30306T = DSM 112386T).

PMID:36748519 | DOI:10.1099/ijsem.0.005677

Plant Cell. 2023 Feb 7:koad032. doi: 10.1093/plcell/koad032. Online ahead of print.

ABSTRACT

In most flowering plants, the female germline is initiated in the subepidermal L2 layer of ovule primordia forming a single megaspore mother cell (MMC). How signaling from the L1 (epidermal) layer could contribute to the gene regulatory network restricting MMC formation to a single cell is unclear. We show that EPIDERMAL PATTERNING FACTOR-like (EPFL) peptide ligands are expressed in the L1 layer, together with their ERECTA family (ERf) receptor kinases, to control female germline specification in Arabidopsis thaliana. EPFL-ERf dependent signaling restricts multiple subepidermal cells from acquiring MMC-like cell identity by activating the expression of the major brassinosteroid (BR) receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the BR-responsive transcription factor BRASSINOZOLE RESISTANT 1 (BZR1). Additionally, BZR1 coordinates female germline specification by directly activating the expression of a nucleolar GTP-binding protein, NUCLEOSTEMIN-LIKE 1 (NSN1), which is expressed in early-stage ovules excluding the MMC. Mutants defective in this gene regulatory network form multiple MMCs resulting in a strong reduction of seed set. In conclusion, we uncovered a ligand/receptor-like kinase-mediated signaling pathway acting upstream and coordinating BR signaling via NSN1 to restrict MMC differentiation to a single subepidermal cell.

PMID:36748257 | DOI:10.1093/plcell/koad032

MicroPubl Biol. 2023 Jan 20;2023. doi: 10.17912/micropub.biology.000709. eCollection 2023.

ABSTRACT

Gene regulation has been studied in C. elegans for over 30 years. In this analysis of 102 publications, we find that most transcriptional cis-regulatory elements are located within 5,000 bp of the transcription start site. Over 75% of studies conclude that transcriptional elements and 5'UTRs activate-, while 3'UTRs repress gene expression. While gene regulatory mutations make up less than 0.8% of alleles in forward genetics screens, recent CRISPR-Cas approaches are increasing the number of tested mutations. This work provides a resource of known gene regulatory sequences in C.elegans .

PMID:36748042 | PMC:PMC9898812 | DOI:10.17912/micropub.biology.000709

MicroPubl Biol. 2023 Jan 21;2023. doi: 10.17912/micropub.biology.000748. eCollection 2023.

ABSTRACT

The C. elegans elt-3 gene encodes a GATA transcription factor that is expressed in the hypodermis and has roles in hypodermal specification and regulation of collagen and stress response genes. The gene encodes short and long isoforms, ELT-3A and ELT-3B respectively, that differ upstream of their DNA-binding domains. Previous work showed that ELT-3A can specify hypodermal cell fates when forcibly overexpressed throughout early embryos. We recently showed that the ELT-3B orthologue from the distantly related species C. angaria can specify endodermal fates when forcibly overexpressed in C. elegans. Here, we show that C. elegans ELT-3B can also specify endoderm.

PMID:36748041 | PMC:PMC9898813 | DOI:10.17912/micropub.biology.000748

Heliyon. 2023 Jan 18;9(1):e13044. doi: 10.1016/j.heliyon.2023.e13044. eCollection 2023 Jan.

ABSTRACT

Metastatic progression and tumor evolution complicates the clinical management of cancer patients. Circulating tumor cell (CTC) characterization is a growing discipline that aims to elucidate tumor metastasis and evolution processes. CTCs offer the clinical potential to monitor cancer patients for therapy response, disease relapse, and screen 'at risk' groups for the onset of malignancy. However, such clinical utility is currently limited to breast, prostate, and colorectal cancer patients. Further understanding of the basic CTC biology of other malignancies is required to progress them towards clinical utility. Unfortunately, such basic clinical research is often limited by restrictive characterization methods and high-cost barrier to entry for CTC isolation and imaging infrastructure. As experimental clinical results on applications of CTC are accumulating, it is becoming clear that a two-tier system of CTC isolation and characterization is required. The first tier is to facilitate basic research into CTC characterization. This basic research then informs a second tier specialised in clinical prognostic and diagnostic testing. This study presented in this manuscript describes the development and application of a low-cost, CTC isolation and characterization pipeline; CTC-5. This approach uses an established 'isolation by size' approach (ScreenCell Cyto) and combines histochemical morphology stains and multiparametric immunofluorescence on the same isolated CTCs. This enables capture and characterization of CTCs independent of biomarker-based pre-selection and accommodates both single CTCs and clusters of CTCs. Additionally, the developed open-source software is provided to facilitate the synchronization of microscopy data from multiple sources (https://github.com/CTC5/). This enables high parameter histochemical and immunofluorescent analysis of CTCs with existing microscopy infrastructure without investment in CTC specific imaging hardware. Our approach confirmed by the number of successful tests represents a potential major advance towards highly accessible low-cost technology aiming at the basic research tier of CTC isolation and characterization. The biomarker independent approach facilitates closing the gap between malignancies with poorly, and well-defined CTC phenotypes. As is currently the case for some of the most commonly occurring breast, prostate and colorectal cancers, such advances will ultimately benefit the patient, as early detection of relapse or onset of malignancy strongly correlates with their prognosis.

PMID:36747925 | PMC:PMC9898658 | DOI:10.1016/j.heliyon.2023.e13044

Plant Cell. 2023 Feb 7:koad028. doi: 10.1093/plcell/koad028. Online ahead of print.

ABSTRACT

Plant roots possess remarkable regenerative potential owing to their ability to replenish damaged or lost stem cells. ETHYLENE RESPONSE FACTOR 115 (ERF115), one of the key molecular elements linked to this potential, plays a predominant role in the activation of regenerative cell divisions. However, the downstream operating molecular machinery driving wound-activated cell division is largely unknown. Here, we biochemically and genetically identified the GRAS-domain transcription factor SCARECROW-LIKE 5 (SCL5) as an interaction partner of ERF115 in Arabidopsis thaliana. Although non-essential under control growth conditions, SCL5 acts redundantly with the related PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) and SCL21 transcription factors to activate the expression of the DNA-BINDING ONE FINGER 3.4 (DOF3.4) transcription factor gene. DOF3.4 expression is wound-inducible in an ERF115-dependent manner, and in turn activates D3-type cyclin expression. Accordingly, ectopic DOF3.4 expression drives periclinal cell division, while its downstream D3-type cyclins are essential for the regeneration of a damaged root. Our data highlight the importance and redundant roles of the SCL5, SCL21 and PAT1 transcription factors in wound-activated regeneration processes and pinpoint DOF3.4 as a key downstream element driving regenerative cell division.

PMID:36747478 | DOI:10.1093/plcell/koad028

Plant Physiol. 2023 Feb 7:kiad073. doi: 10.1093/plphys/kiad073. Online ahead of print.

ABSTRACT

Throughout exploration of the soil, roots interact with their environment and adapt to different conditions. Directional root growth is guided by asymmetric molecular patterns but how these become established or are dynamically regulated is poorly understood. Asymmetric gradients of the phytohormone auxin are established during root gravitropism, mainly through directional transport mediated by polarized auxin transporters. Upon gravistimulation, PIN-FORMED2 (PIN2) is differentially distributed and accumulates at the lower root side to facilitate asymmetric auxin transport up to the elongation zone where it inhibits cell elongation. GOLVEN (GLV) peptides function in gravitropism by affecting PIN2 abundance in epidermal cells. In addition, GLV signaling through ROOT GROWTH FACTOR 1 INSENSITIVE (RGI) receptors regulates root apical meristem maintenance. Here, we show that GLV-RGI signaling in these two processes in Arabidopsis (Arabidopsis thaliana) can be mapped to different cells in the root tip and that, in the case of gravitropism, it operates mainly in the lateral root cap (LRC) to maintain PIN2 levels at the plasma membrane. Furthermore, we found that GLV signaling upregulates the phosphorylation level of PIN2 in an RGI-dependent manner. In addition, we demonstrated that the RGI5 receptor is asymmetrically distributed in the LRC and accumulates in the lower side of the LRC after gravistimulation. Asymmetric GLV-RGI signaling in the root cap likely accounts for differential PIN2 abundance at the plasma membrane to temporarily support auxin transport up to the elongation zone thereby representing an additional level of control on the asymmetrical auxin flux to mediate differential growth of the root.

PMID:36747317 | DOI:10.1093/plphys/kiad073

Nat Cell Biol. 2023 Feb 6. doi: 10.1038/s41556-022-01058-9. Online ahead of print.

ABSTRACT

The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. Here we report that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. We demonstrate this by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. We can thereby engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. Our findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.

PMID:36747081 | DOI:10.1038/s41556-022-01058-9

Sci Rep. 2023 Feb 6;13(1):2084. doi: 10.1038/s41598-023-28978-2.

ABSTRACT

The aims of this study were to perform pre-surgery miRNA profiling of patients who develop Vasoplegic syndrome (VS) after coronary artery bypass grafting (CABG) and identify those miRNAs that could be used as VS prognostic tools and biomarkers. The levels of 754 microRNAs (miRNAs) were measured in whole blood samples from a cohort of patients collected right before the coronary artery bypass grafting (CABG) surgery. We compared the miRNA levels of those who developed VS (VASO group) with those who did not (NONVASO group) after surgery. Six miRNAs (hsa-miR-548c-3p, -199b-5p, -383-5p -571 -183-3p, -30d-5p) were increased and two (hsa-1236-3p, and hsa-miR770-5p) were decreased in blood of VASO compared to NONVASO groups. Receiver Operating Characteristic (ROC) curve analysis revealed that a combination of the miRNAs, hsa-miR-30d-5p and hsa-miR-770-5p can be used as VS predictors (AUC = 0.9615, p < 0.0001). The computational and functional analyses were performed to gain insights into the potential role of these dysregulated miRNAs in VS and have identified the "Apelin Liver Signaling Pathway" as the canonical pathway containing the most target genes regulated by these miRNAs. The expression of the combined miRNAs hsa-miR-30d and hsa-miR-770-5p allowed the ability to distinguish between patients who could and could not develop VS, representing a potential predictive biomarker of VS.

PMID:36747073 | DOI:10.1038/s41598-023-28978-2

Nat Commun. 2023 Feb 6;14(1):634. doi: 10.1038/s41467-023-36364-9.

ABSTRACT

Transposable elements (TEs) are major contributors of genetic material in mammalian genomes. These often include binding sites for architectural proteins, including the multifarious master protein, CTCF, which shapes the 3D genome by creating loops, domains, compartment borders, and RNA-DNA interactions. These play a role in the compact packaging of DNA and have the potential to facilitate regulatory function. In this study, we explore the widespread contribution of TEs to mammalian 3D genomes by quantifying the extent to which they give rise to loops and domain border differences across various cell types and species using several 3D genome mapping technologies. We show that specific families and subfamilies of TEs have contributed to lineage-specific 3D chromatin structures across mammalian species. In many cases, these loops may facilitate sustained interaction between distant cis-regulatory elements and target genes, and domains may segregate chromatin state to impact gene expression in a lineage-specific manner. An experimental validation of our analytical findings using CRISPR-Cas9 to delete a candidate TE resulted in disruption of species-specific 3D chromatin structure. Taken together, we comprehensively quantify and selectively validate our finding that TEs contribute to shaping 3D genome organization and may, in some cases, impact gene regulation during the course of mammalian evolution.

PMID:36746940 | DOI:10.1038/s41467-023-36364-9

Blood Cancer J. 2023 Feb 7;13(1):24. doi: 10.1038/s41408-023-00793-y.

NO ABSTRACT

PMID:36746923 | DOI:10.1038/s41408-023-00793-y

BMJ Open Gastroenterol. 2023 Feb;10(1):e001003. doi: 10.1136/bmjgast-2022-001003.

ABSTRACT

OBJECTIVE: To infer potential mechanisms driving disease subtypes among patients with inflammatory bowel disease (IBD), we profiled the transcriptome of purified circulating monocytes and CD4 T-cells.

DESIGN: RNA extracted from purified monocytes and CD4 T-cells derived from the peripheral blood of 125 endoscopically active patients with IBD was sequenced using Illumina HiSeq 4000NGS. We used complementary supervised and unsupervised analytical methods to infer gene expression signatures associated with demographic/clinical features. Expression differences and specificity were validated by comparison with publicly available single cell datasets, tissue-specific expression and meta-analyses. Drug target information, druggability and adverse reaction records were used to prioritise disease subtype-specific therapeutic targets.

RESULTS: Unsupervised/supervised methods identified significant differences in the expression profiles of CD4 T-cells between patients with ileal Crohn's disease (CD) and ulcerative colitis (UC). Following a pathway-based classification (Area Under Receiver Operating Characteristic - AUROC=86%) between ileal-CD and UC patients, we identified MAPK and FOXO pathways to be downregulated in UC. Coexpression module/regulatory network analysis using systems-biology approaches revealed mediatory core transcription factors. We independently confirmed that a subset of the disease location-associated signature is characterised by T-cell-specific and location-specific expression. Integration of drug-target information resulted in the discovery of several new (BCL6, GPR183, TNFAIP3) and repurposable drug targets (TUBB2A, PRKCQ) for ileal CD as well as novel targets (NAPEPLD, SLC35A1) for UC.

CONCLUSIONS: Transcriptomic profiling of circulating CD4 T-cells in patients with IBD demonstrated marked molecular differences between the IBD-spectrum extremities (UC and predominantly ileal CD, sandwiching colonic CD), which could help in prioritising particular drug targets for IBD subtypes.

PMID:36746519 | DOI:10.1136/bmjgast-2022-001003

ACS Chem Neurosci. 2023 Feb 6. doi: 10.1021/acschemneuro.2c00610. Online ahead of print.

ABSTRACT

A deficiency of the functional α7 nicotinic acetylcholine receptor (α7nAChR) impairs neuronal and immune systems. The SARS-CoV-2 spike protein (S12) facilitates virus cell entry during COVID-19 infection and can also independently disrupt cellular functions. Here, we found that S12 expression significantly downregulated surface expression of α7nAChR in mammalian cells. A helical segment of S12 (L1145-L1152) in the spike neck was identified to be responsible for the downregulation of α7nAChR, as the mutant S12AAA (L1145A-F1148A-L1152A) had minimal effects on surface α7nAChR expression. This S12 segment is homologous to the α7nAChR intracellular helical motif known for binding chaperone proteins RIC3 and Bcl-2 to promote α7nAChR surface expression. Competition from S12 for binding these proteins likely underlies suppression of surface α7nAChR. Considering the critical roles of α7nAChR in cellular functions, these findings provide a new perspective for improving mRNA vaccines and developing treatment options for certain symptoms related to long COVID.

PMID:36745901 | DOI:10.1021/acschemneuro.2c00610

ACS Nano. 2023 Feb 6. doi: 10.1021/acsnano.2c10371. Online ahead of print.

ABSTRACT

Rapid, sensitive, simultaneous quantification of multiple biomarkers in point-of-care (POC) settings could improve the diagnosis and management of sepsis, a common, potentially life-threatening condition. Compared to high-end commercial analytical systems, POC systems are often limited by low sensitivity, limited multiplexing capability, or low throughput. Here, we report an ultrasensitive, multiplexed plasmonic sensing technology integrating chemifluorescence signal enhancement with plasmon-enhanced fluorescence detection. Using a portable imaging system, the dual chemical and plasmonic amplification enabled rapid analysis of multiple cytokine biomarkers in 1 h with sub-pg/mL sensitivities. Furthermore, we also developed a plasmonic sensing chip based on nanoparticle-spiked gold nanodimple structures fabricated by wafer-scale batch processes. We used the system to detect six cytokines directly from clinical plasma samples (n = 20) and showed 100% accuracy for sepsis detection. The described technology could be employed in rapid, ultrasensitive, multiplexed plasmonic sensing in POC settings for myriad clinical conditions.

PMID:36745820 | DOI:10.1021/acsnano.2c10371

Proc Natl Acad Sci U S A. 2023 Feb 14;120(7):e2212909120. doi: 10.1073/pnas.2212909120. Epub 2023 Feb 6.

ABSTRACT

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans, whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans-autoinhibition, followed by activation loop autophosphorylation in cis. We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans-autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis. Phosphorylation serves to increase PKD activity and prevent trans-autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

PMID:36745811 | DOI:10.1073/pnas.2212909120

PLoS One. 2023 Feb 6;18(2):e0272672. doi: 10.1371/journal.pone.0272672. eCollection 2023.

ABSTRACT

Sporotrichosis is a subcutaneous mycosis with a global distribution, also known as "rose gardener's disease". Brazil is experiencing a rapid spread of the zoonotic transmission of of Sporothrix brasiliensis, the main etiological agent of this disease in this country, affecting domestic felines. Cost-effective interventions need to be developed to control this emergent public health problem. To allow for the comparison of alternative control strategies, we propose in this paper, a mathematical model representing the transmission of S. brasiliensis among cats, stratified by age and sex. Analytical properties of the model are derived and simulations show possible strategies for reducing the endemic levels of the disease in the cat population, with a positive impact on human health. The scenarios included mass treatment of infected cats and mass implementation of contact reduction practices, such as neutering. The results indicate that mass treatment can reduce substantially the disease prevalence, and this effect is potentialized when combined with neutering or other contact-reduction interventions. On the other hand, contact-reduction methods alone are not sufficient to reduce prevalence.

PMID:36745585 | DOI:10.1371/journal.pone.0272672

J Proteome Res. 2023 Feb 6. doi: 10.1021/acs.jproteome.2c00711. Online ahead of print.

ABSTRACT

In recent years machine learning has made extensive progress in modeling many aspects of mass spectrometry data. We brought together proteomics data generators, repository managers, and machine learning experts in a workshop with the goals to evaluate and explore machine learning applications for realistic modeling of data from multidimensional mass spectrometry-based proteomics analysis of any sample or organism. Following this sample-to-data roadmap helped identify knowledge gaps and define needs. Being able to generate bespoke and realistic synthetic data has legitimate and important uses in system suitability, method development, and algorithm benchmarking, while also posing critical ethical questions. The interdisciplinary nature of the workshop informed discussions of what is currently possible and future opportunities and challenges. In the following perspective we summarize these discussions in the hope of conveying our excitement about the potential of machine learning in proteomics and to inspire future research.

PMID:36744821 | DOI:10.1021/acs.jproteome.2c00711

J Vis Exp. 2023 Jan 20;(191). doi: 10.3791/64001.

ABSTRACT

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population. A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

PMID:36744801 | DOI:10.3791/64001

Front Med (Lausanne). 2023 Jan 19;10:1051831. doi: 10.3389/fmed.2023.1051831. eCollection 2023.

ABSTRACT

Stem cell therapy, which has promising results in acute disorders such as stroke, supports treatment by providing rehabilitation in the chronic stage patients. In acute stroke, thrombolytic medical treatment protocols are clearly defined in neurologic emergencies, but in neurologic patients who miss the "thrombolytic treatment intervention window," or in cases of hypoxic-ischemic encephalopathy, our hands are tied, and we are still unfortunately faced with hopeless clinical implementations. We consider mesenchymal stem cell therapy a viable option in these cases. In recent years, novel research has focused on neuro-stimulants and supportive and combined therapies for stroke. Currently, available treatment options are limited, and only certain patients are eligible for acute treatment. In the scope of our experience, five stroke patients were evaluated in this study, who was treated with a single dose of 1-2 × 106 cells/kg allogenic umbilical cord-mesenchymal stem cells (UC-MSCs) with the official confirmation of the Turkish Ministry of Health Stem Cell Commission. The patients were followed up for 12 months, and clinical outcomes are recorded. NIH Stroke Scale/Scores (NIHSS) decreased significantly (p = 0.0310), and the Rivermead Assessment Scale (RMA) increased significantly (p = 0.0234) for all patients at the end of the follow-up. All the patients were followed up for 1 year within a rehabilitation program. Major clinical outcome improvements were observed in the overall clinical conditions of the UC-MSC treatment patients. We observed improvement in the patients' upper extremity and muscle strength, spasticity, and fine motor functions. Considering recent studies in the literature together with our results, allogenic stem cell therapies are introduced as promising novel therapies in terms of their encouraging effects on physiological motor outcomes.

PMID:36744151 | PMC:PMC9892908 | DOI:10.3389/fmed.2023.1051831