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Background Our previous analyses of cardiomyocyte single-nucleus RNA sequencing (snRNAseq) data from the hearts of fetal pigs and pigs that underwent apical resection surgery on postnatal day (P) 1 (ARP1), myocardial infarction (MI) surgery on P28 (MIP28), both ARP1 and MIP28 (ARP1MIP28), or controls (no surgical procedure or CTL) identified 10 cardiomyocyte subpopulations (clusters), one of which appeared to be primed to proliferate in response to MI. However, the clusters composed of primarily proliferating cardiomyocytes still contained noncycling cells, and we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Here, we improved the precision of our assessments by conducting similar analyses with snRNAseq data for only the 1646 genes included under the Gene Ontology term "cell cycle." Methods Two cardiac snRNAseq datasets, one from mice (GEO dataset number GSE130699) and one from pigs (GEO dataset number GSE185289), were evaluated via our cell-cycle–specific analytical pipeline. Cycling cells were identified via the co-expression of 5 proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BIRC5). Results The cell-cycle–specific autoencoder (CSA) algorithm identified 7 cardiomyocyte clusters in mouse hearts (mCM1 and mCM3-mCM8), including one prominent cluster of cycling cardiomyocytes in animals that underwent MI or Sham surgery on P1. Five cardiomyocyte clusters (pCM1, pCM3-pCM6) were identified in pig hearts, 2 of which (pCM1 and pCM4) displayed evidence of cell cycle activity; pCM4 was found primarily in hearts from fetal pigs, while pCM1 comprised a small proportion of cardiomyocytes in both fetal hearts and hearts from ARP1MIP28 pigs during the 2 weeks after MI induction, but was nearly undetectable in all other experimental groups and at all other time points. Furthermore, pseudotime trajectory analysis of snRNAseq data from fetal pig cardiomyocytes identified a pathway that began at pCM3, passed through pCM2, and ended at pCM1, whereas pCM3 was enriched for the expression of a cell cycle activator that regulates the G1/S phase transition (cyclin D2), pCM2 was enriched for an S-phase regulator (CCNE2), and pCM1 was enriched for the expression of a gene that regulates the G2M phase transition and mitosis (cyclin B2). We also identified 4 transcription factors (E2F8, FOXM1, GLI3, and RAD51) that were more abundantly expressed in cardiomyocytes from regenerative mouse hearts than from nonregenerative mouse hearts, from the hearts of fetal pigs than from CTL pig hearts, and from ARP1MIP28 pig hearts than from MIP28 pig hearts during the 2 weeks after MI induction. Conclusions The CSA algorithm improved the precision of our assessments of cell cycle activity in cardiomyocyte subpopulations and enabled us to identify a trajectory across 3 clusters that appeared to track the onset and progression of cell cycle activity in cardiomyocytes from fetal pigs. [ABSTRACT FROM AUTHOR]

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes.sup.1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest.sup.3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes. Hoxb13 acts as a cofactor of Meis1 in regulating cardiomyocyte maturation and cell cycle, and knockout of both proteins enables regeneration of postnatal cardiac tissue in a mouse model of heart injury., Author(s): Ngoc Uyen Nhi Nguyen [sup.1] , Diana C. Canseco [sup.1] , Feng Xiao [sup.1] , Yuji Nakada [sup.1] , Shujuan Li [sup.1] , Nicholas T. Lam [sup.1] , Shalini [...]

Author(s): Yuji Nakada [1]; Diana C. Canseco [1]; SuWannee Thet [1]; Salim Abdisalaam [2]; Aroumougame Asaithamby [2]; Celio X. Santos [3]; Ajay M. Shah [3]; Hua Zhang [4]; James E. [...]

Endometrial cancer is the most common gynecologic malignancy and the fourth most common malignancy in women. For most patients in whom the disease is confined to the uterus, treatment results in successful remission; however, there are no curative treatments for tumors that have progressed beyond the uterus. The serine/threonine kinase LKB1 has been identified as a potent suppressor of uterine cancer, but the biological modes of action of LKB1 in this context remain incompletely understood. Here, we have shown that LKB1 suppresses tumor progression by altering gene expression in the tumor microenvironment. We determined that LKB1 inactivation results in abnormal, cell-autonomous production of the inflammatory cytokine chemokine (C-C motif) ligand 2 (CCL2) within tumors, which leads to increased recruitment of macrophages with prominent tumor-promoting activities. Inactivation of Ccl2 in an Lkbl-driven mouse model of endometrial cancer slowed tumor progression and increased survival. In human primary endometrial cancers, loss of LKB1 protein was strongly associated with increased CCL2 expression by tumor cells as well as increased macrophage density in the tumor microenvironment. These data demonstrate that CCL2 is a potent effector of LKB1 loss in endometrial cancer, creating potential avenues for therapeutic opportunities., Introduction LKB1 (STK11) was initially identified as the tumor suppressor gene mutated in Peutz-Jeghers syndrome (PJS), a hereditary, autosomal dominant condition characterized by a dramatically elevated (15-20x) incidence of cancer [...]

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of [A.sub.single] spermatogonia in mice. (PAX7.sup.+] cells were present in the testis at birth. Compared with the adult testis, [PAX7.sup.+] cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that [PAX7.sup.+] spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, [PAX7.sup.+] spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, [PAX7.sup.+] spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the [PAX7.sup.+] subset of [A.sub.single] spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germ line injury, such as occurs after cancer therapy., Introduction The functional unit of the mammalian testis, the seminiferous tubule, is a multilayered epithelium that matures from spermatogonial precursors located at the basal layer to more advanced cell types [...]

Pseudomonas aeruginosa PAO1 has two possible catabolic pathways of spermidine and spermine; one includes the spuA and spuB products with unknown functions and the other involves spermidine dehydrogenase (SpdH; EC 1.5.99.6) encoded by an unknown gene. The properties of SpdH in P. aeruginosa PAO1 were characterized and the corresponding spdH gene in this strain identified. The deduced SpdH (620 residues, calculated Mr of 68 861) had a signal sequence of 28 amino acids at the amino terminal and a potential transmembrane segment between residues 76 and 92, in accordance with membrane location of the enzyme. Purified SpdH oxidatively cleaved spermidine into 1,3-diaminopropane and 4-aminobutyraldehyde with a specific activity of 37 units [(mg protein).sup.-1] and a [K.sub.m] value of 36 [micro]M. The enzyme also hydrolysed spermine into spermidine and 3-aminopropanaldehyde with a specific activity of 25 units [(mg protein).sup.-1] and a [K.sub.m] of 18 [micro]M. Knockout of spdH had no apparent effect on the utilization of both polyamines, suggesting that this gene is minimally involved in polyamine catabolism. However, when spdH was fused to the polyamine-inducible promoter of spuA, it fully restored the ability of a spuA mutant to utilize spermidine. It is concluded that SpdH can perform a catabolic role in vivo, but P. aeruginosa PAO1 does not produce sufficient amounts of the enzyme to execute this function.

Pseudomonas aeruginosa PAO1 utilizes 3-guanidinopropionate (3-GP) and 4-guanidinobutyrate (4-GB), which differ in one methylene group only, via distinct enzymes: guanidinopropionase (EC 3.5.3.17; the gpuA product) and guanidinobutyrase (EC 3.5.3.7; the gbuA product). The authors cloned and characterized the contiguous gpuPAR genes (in that order) responsible for 3-GP utilization, and compared the deduced sequences of their putative protein products, and the potential regulatory mechanisms of gpuPA, with those of the corresponding gbu genes encoding the 4-GB catabolic system. GpuA and GpuR have similarity to GbuA (49% identity) and GbuR (a transcription activator of gbuA; 37% identity), respectively. GpuP resembles PA1418 (58 % identity), which is a putative membrane protein encoded by a potential gene downstream of gbuA. These features of the GpuR and GpuP sequences, and the impaired growth of gpuR and gpuP knockout mutants on 3-GP, support the notion that GpuR and GpuP direct the 3-GP-inducible expression of gpuA, and the uptake of 3-GP, respectively. Northern blots of mRNA from 3-GP-induced PAO1 cells revealed three transcripts of gpuA, gpuP, and gpuP and gpuA together, suggesting that gpuP and gpuA each have a 3-GP-responsible promoter, and that some transcription from the gpuP promoter is terminated after gpuP, or proceeds into gpuA. Knockout of gpuR abolished 3-GP-dependent synthesis of the transcripts, confirming that GpuR activates transcription from these promoters, with 3-GP as a specific co-inducer. The sequence conservation between the three functional pairs of the Gpu and Gbu proteins, and the absence of gpuAPR in closely related species, imply that the triad gpu genes have co-ordinately evolved from origins common to the gbu counterparts, to establish an independent catabolic system of 3-GP in P. aeruginosa.

Ngn1 is a basic helix-loop-helix (bHLH) transcription factor expressed in specific regions within the developing brain and spinal cord, sensory ganglia, and olfactory epithelium. We have identified sequences both 5' and 3' of the mouse ngn1 gene that function in regulating ngn1 expression, and each of these sequences contains distinct regulatory cassettes for different subregions of the expression domain. Enhancers for expression in ngn1 domains of the midbrain, hindbrain, trigeminal ganglia, and ventral-neural tube appear redundant and are spread both 5' and 3' of the ngn1 coding sequence. In contrast, a single discrete dorsal-neural tube enhancer was located in the 5' sequence that is conserved among mouse, human, chick, and zebrafish ngnl genes. Functionally, this enhancer is both necessary and sufficient for driving expression of a heterologous reporter in transgenic mice specifically to the dorsal domain of ngn1 expression in the spinal neural tube. Thus, sequences are identified that can be used to direct temporally and spatially restricted expression of heterologous genes to the developing neural tube. Keywords: Transcriptional regulation; Transgenic mice; Dorsal-neural tube; Ventral-neural tube; Basic helix-loop-helix transcription factor; Neurogenin 1; Spinal cord development

Research reveals structural, organizational, and regulatory diversity in the proline catabolic genes between Pseudomonas aeruginosa and P. putida and the genetic processes involved influence the development of proline catabolism in these bacteria. Data show that the proline utilization system from P. aeruginosa differs from that of P. putida in terms of putA gene.

Research has been conducted on Pseudomonas aeruginosa PAO1 multiple-gene locus's polyamine uptake and utilization. The physiological functions of these genes have been investigated via the use of Tn5 transposon-mediated knockout mutants and the results are reported.

The organization and regulatory mechanism of the arginine dehydrogenase pathway are analyzed using the structural gene, gbuA, for guanidinobutyrase in Pseudomonas aeruginosa PAO1. Research reveals that the LysR-type transcriptional regulator, GbuR, induces the expression of gbuA. Sequence analysis assigns guanidinobutyrase to the arginase/agmatinase family of proteins.

Research has been conducted on the utilization of agmatine by Pseudomonas aeruginosa PAO1. The role of the regulatory mechanism of the arginine decarboxylase pathway in arginine and agmatine utilization by P. aeruginosa has been investigated and the results are reported.