Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach.

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.

Posttranscriptional mechanisms controlling diurnal gene expression cycles by body temperature rhythms.

In mammals, body temperature oscillates in a daily fashion around a set point of 36°C-37°C. These fluctuations are controlled by the circadian master clock residing in the brain's suprachiasmatic nucleus and, despite their small amplitudes, contribute to the diurnal expression of genes throughout the organism. By focusing on the mechanisms underlying the temperature-dependent accumulation of the cold-inducible RNA-binding protein CIRBP - a factor involved in the tuning of amplitude and phase in circadian clocks of peripheral tissues - we have recently identified a novel mechanism governing temperature-dependent gene expression. This mechanism involves the differential spicing efficiency of primary RNA transcripts under different temperature conditions and thereby determines the fraction of Cirbp pre-mRNA processed into mature mRNA. A genome-wide transcriptome analysis revealed that this mechanism affects the output of hundreds of genes. Here we discuss our findings and future directions toward the identification of specific factors and parameters governing temperature-sensitive splicing efficacy.

Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp.

In mammals, body temperature fluctuates diurnally around a mean value of 36°C-37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells' circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide "approach to steady-state" kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression.

Clock-Talk: Interactions between Central and Peripheral Circadian Oscillators in Mammals.

In mammals, including humans, nearly all physiological processes are subject to daily oscillations that are governed by a circadian timing system with a complex hierarchical structure. The central pacemaker, residing in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus, is synchronized daily by photic cues transmitted from the retina to SCN neurons via the retinohypothalamic tract. In turn, the SCN must establish phase coherence between self-sustained and cell-autonomous oscillators present in most peripheral cell types. The synchronization signals (Zeitgebers) can be controlled more or less directly by the SCN. In mice and rats, feeding-fasting rhythms, which are driven by the SCN through rest-activity cycles, are the most potent Zeitgebers for the circadian oscillators of peripheral organs. Signaling through the glucocorticoid receptor and the serum response factor also participate in the phase entrainment of peripheral clocks, and these two pathways are controlled by the SCN independently of feeding-fasting rhythms. Body temperature rhythms, governed by the SCN directly and indirectly through rest-activity cycles, are perhaps the most surprising cues for peripheral oscillators. Although the molecular makeup of circadian oscillators is nearly identical in all cells, these oscillators are used for different purposes in the SCN and in peripheral organs.

Stimulus-induced modulation of transcriptional bursting in a single mammalian gene.

Mammalian genes are often transcribed discontinuously as short bursts of RNA synthesis followed by longer silent periods. However, how these "on" and "off" transitions, together with the burst sizes, are modulated in single cells to increase gene expression upon stimulation is poorly characterized. By combining single-cell time-lapse luminescence imaging with stochastic modeling of the time traces, we quantified the transcriptional responses of the endogenous connective tissue growth factor gene to different physiological stimuli: serum and TGF-ß1. Both stimuli caused a rapid and acute increase in burst sizes. Whereas TGF-ß1 showed prolonged transcriptional activation mediated by an increase of transcription rate, serum stimulation resulted in a large and temporally tight first transcriptional burst, followed by a refractory period in the range of hours. Our study thus reveals how different physiological stimuli can trigger kinetically distinct transcriptional responses of the same gene.

The muscle dystrophy-causing ΔK32 lamin A/C mutant does not impair the functions of the nucleoplasmic lamin-A/C-LAP2α complex in mice.

A-type lamins are components of the nuclear lamina, a filamentous network of the nuclear envelope in metazoans that supports nuclear architecture. In addition, lamin A/C can also be found in the interior of the nucleus. This nucleoplasmic lamin pool is soluble in physiological buffer, depends on the presence of the lamin-binding protein, lamina-associated polypeptide 2α (LAP2α) and regulates cell cycle progression in tissue progenitor cells. ΔK32 mutations in A-type lamins cause severe congenital muscle disease in humans and a muscle maturation defect in Lmna(ΔK32/ΔK32) knock-in mice. Mutant ΔK32 lamin A/C protein levels were reduced and all mutant lamin A/C was soluble and mislocalized to the nucleoplasm. To test the role of LAP2α in nucleoplasmic ΔK32 lamin A/C regulation and functions, we deleted LAP2α in Lmna(ΔK32/ΔK32) knock-in mice. In double mutant mice the Lmna(ΔK32/ΔK32)-linked muscle defect was unaffected. LAP2α interacted with mutant lamin A/C, but unlike wild-type lamin A/C, the intranuclear localization of ΔK32 lamin A/C was not affected by loss of LAP2α. In contrast, loss of LAP2α in Lmna(ΔK32/ΔK32) mice impaired the regulation of tissue progenitor cells as in lamin A/C wild-type animals. These data indicate that a LAP2α-independent assembly defect of ΔK32 lamin A/C is the predominant cause of the mouse pathology, whereas the LAP2α-linked functions of nucleoplasmic lamin A/C in the regulation of tissue progenitor cells are not affected in Lmna(ΔK32/ΔK32) mice.

The ticking tail: daily oscillations in mRNA poly(A) tail length drive circadian cycles in protein synthesis.

In this issue of Genes & Development, Kojima and colleagues (pp. 2724-2736) examined the impact of mRNA poly(A) tail length on circadian gene expression. Their study demonstrates how dynamic changes in transcript poly(A) tail length can lead to rhythmic protein expression, irrespective of whether mRNA accumulation is circadian or constitutive.

Multiple novel functions of lamina associated polypeptide 2α in striated muscle.

Lamina-associated polypeptide 2α (LAP2α) is a nucleoplasmic protein that interacts with A-type lamins and the retinoblastoma protein (pRb) and affects pRb-mediated cell cycle regulation and chromatin organization. Mutations in lamin A/C and LAP2α cause late onset striated muscle diseases, but the molecular mechanisms are poorly understood. We have recently reported on the striated muscle phenotype of LAP2α-deficient mice, revealing new unexpected roles of LAP2α. Loss of LAP2α in skeletal muscle caused an upregulated stem cell-type gene expression in muscle satellite cell progeny and their delayed myogenic differentiation in vitro. In vivo, the myofiber-associated muscle stem cell pool was increased. In addition, absence of LAP2α promoted muscle remodeling towards fast myofiber types in the soleus muscle of old animals. In cardiac tissue, deletion of LAP2α caused systolic dysfunction in young mice with an increased susceptibility for fibrosis in old animals. The functional impairment in the heart was accompanied by a deregulation of major cardiac transcription factors, GATA4 and MEF2c and activation of compensatory pathways, including the downregulation of ß-adrenergic receptor signaling.Here we discuss potential functions of LAP2α in striated muscle at molecular level and how loss of these functions may cause the diverse muscle phenotypes. We propose that LAP2α serves as a transcriptional co-regulator, which controls muscle specific gene expression during muscle regeneration, muscle remodeling and stress response.

Lamina-associated polypeptide 2alpha loss impairs heart function and stress response in mice.

RATIONALE: Lamina-associated polypeptide (LAP)2alpha is a mammalian chromatin-binding protein that interacts with a fraction of A-type lamins in the nuclear interior. Because mutations in lamins and LAP2alpha lead to cardiac disorders in humans, we hypothesized that these factors may play important roles in heart development and adult tissue homeostasis. OBJECTIVE: We asked whether the presence of LAP2alpha was required for normal cardiac function. METHODS AND RESULTS: To study the molecular mechanisms of the disease, we analyzed heart structure and function in complete and conditional Lap2alpha(-/-) mice as well as Lap2alpha(-/-)/Mdx mutants. Unlike conditional deletion of LAP2alpha in late embryonic striated muscle, its complete knockout caused systolic dysfunction in young mice, accompanied by sporadic fibrosis in old animals, as well as deregulation of major cardiac transcription factors GATA4 and myocyte enhancer factor 2c. Activation of compensatory pathways, including downregulation of beta-adrenergic receptor signaling, resulted in reduced responsiveness of the myocardium to chronic beta-adrenergic stimulation and stalled the progression of LAP2alpha-deficient hearts from hypertrophy toward cardiac failure. Dystrophin deficiency in an Mdx background resulted in a transient rescue of the Lap2alpha(-/-) phenotype. CONCLUSIONS: Our data suggest a novel role of LAP2alpha in the maintenance of cardiac function under normal and stress conditions.

Loss of LAP2 alpha delays satellite cell differentiation and affects postnatal fiber-type determination.

Lamina-associated polypeptide 2 alpha (LAP2 alpha) is a nucleoplasmic protein implicated in cell cycle regulation through its interaction with A-type lamins and the retinoblastoma protein. Mutations in lamin A/C and LAP2 alpha cause late onset striated muscle diseases, but the molecular mechanisms are poorly understood. To study the role of LAP2 alpha in skeletal muscle function and postnatal tissue homeostasis, we generated complete and muscle-specific LAP2 alpha knockout mice. Whereas overall muscle morphology, function, and regeneration were not detectably affected, the myofiber-associated muscle stem cell pool was increased in complete LAP2 alpha knockout animals. At molecular level, the absence of LAP2 alpha preserved the stem cell-like phenotype of Lap2 alpha(-/-) primary myoblasts and delayed their in vitro differentiation. In addition, loss of LAP2 alpha shifted the myofiber-type ratios of adult slow muscles toward fast fiber types. Conditional Cre-mediated late muscle-specific ablation of LAP2 alpha affected early stages of in vitro myoblast differentiation, and also fiber-type determination, but did not change myofiber-associated stem cell numbers in vivo. Our data demonstrate multiple and distinct functions of LAP2 alpha in muscle stem cell maintenance, early phases of myogenic differentiation, and muscle remodeling.

Loss of nucleoplasmic LAP2alpha-lamin A complexes causes erythroid and epidermal progenitor hyperproliferation.

Lamina-associated polypeptide (LAP) 2alpha is a chromatin-associated protein that binds A-type lamins. Mutations in both LAP2alpha and A-type lamins are linked to human diseases called laminopathies, but the molecular mechanisms are poorly understood. The A-type lamin-LAP2alpha complex interacts with and regulates retinoblastoma protein (pRb), but the significance of this interaction in vivo is unknown. Here we address the function of the A-type lamin-LAP2alpha complex with the use of LAP2alpha-deficient mice. We show that LAP2alpha loss causes relocalization of nucleoplasmic A-type lamins to the nuclear envelope and impairs pRb function. This causes inefficient cell-cycle arrest in dense fibroblast cultures and hyperproliferation of epidermal and erythroid progenitor cells in vivo, leading to tissue hyperplasia. Our results support a disease-relevant model in which LAP2alpha defines A-type lamin localization in the nucleoplasm, which in turn affects pRb-mediated regulation of progenitor cell proliferation and differentiation in highly regenerative tissues.

Telomerase activity in HeLa cervical carcinoma cell line proliferation.

Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate (3)H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0-1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-beta-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.