Propranolol reduces IFN-γ driven PD-L1 immunosuppression and improves anti-tumour immunity in ovarian cancer.

The immune system plays an important role in controlling epithelial ovarian cancer (EOC). EOC is considered to be a "cold tumour," a tumour that has not triggered a strong response by the immune system. However, tumour infiltrating lymphocytes (TILs) and the expression of programmed cell death ligand (PD-L1) are used as prognostic indicators in EOC. Immunotherapy such as PD-(L)1 inhibitors have shown limited benefit in EOC. Since the immune system is affected by behavioural stress and the beta-adrenergic signalling pathway, this study aimed to explore the impact of propranolol (PRO), a beta-blocker, on anti-tumour immunity in both in vitro and in vivo EOC models. Noradrenaline (NA), an adrenergic agonist, did not directly regulate PD-L1 expression but PD-L1 was significantly upregulated by IFN-γ in EOC cell lines. IFN-γ also increased PD-L1 on extracellular vesicles (EVs) released by ID8 cells. PRO significantly decreased IFN-γ levels in primary immune cells activated ex vivo and showed increased viability of the CD8+ cell population in an EV-immune cell co-incubation. In addition, PRO reverted PD-L1 upregulation and significantly decreased IL-10 levels in an immune-cancer cell co-culture. Chronic behavioural stress increased metastasis in mice while PRO monotherapy and the combo of PRO and PD-(L)1 inhibitor significantly decreased stress-induced metastasis. The combined therapy also reduced tumour weight compared to the cancer control group and induced anti-tumour T-cell responses with significant CD8 expression in tumour tissues. In conclusion, PRO showed a modulation of the cancer immune response by decreasing IFN-γ production and, in turn, IFN-γ-mediated PD-L1 overexpression. The combined therapy of PRO and PD-(L)1 inhibitor decreased metastasis and improved anti-tumour immunity offering a promising new therapy.

Species-specific primers for the identification of the ectomycorrhizal fungus Tuber macrosporum Vittad.

Limitations in the use of morphological traits to identify ectomycorrhizae have led to the development of species-specific molecular markers. Herein, we report a PCR-based technique for the reliable molecular identification of the ectomycorrhizal fungus Tuber macrosporum Vittad. Species-specific primers were designed from an alignment of internal transcribed spacer rDNA sequences from Tuber spp. and from the most common ectomycorrhizal contaminants found in the root systems of truffle-infected plants. The primers were tested for selective amplification using both different truffles and different ectomycorrhizae and were found to identify T. macrosporum successfully. The application of the primers in certifying the quality of truffle-inoculated seedlings is discussed.

cDNA AFLP-based techniques for studying transcript profiles in horses.

The identification of differentially expressed genes is a fundamental prerequisite for understanding the molecular regulation of most physiological and pathological processes. Among the procedures employed to compare mRNA populations, those that are gel-based appear to hold great promise and are considered excellent tools for studying gene expression in species, such as the equine one, for which little genomic information is available. In the present study, we evaluated two techniques for studying mRNA profiles in horse tissue, one referred to the cDNA-amplified fragment length polymorphism (AFLP) that we called C-AFLP (classical cDNA-AFLP) protocol and the other to ordered differential display (ODD) with some modifications that we named S-AFLP (systematic cDNA-AFLP). Both techniques can be applied in live animals because of the small amount of sample required. We applied the S-AFLP to investigate horse transcript profile modifications during physical exercise. We found two transcripts that are mostly expressed during exercise and immediately after the end of it.

Genomic organization of rDNA loci in natural populations of Medicago truncatula Gaertn.

Medicago truncatula Gaertn. is an annual self-pollinating species characterized by a diploid complement 2n = 16 and low DNA content. It responds very well to transformation methods so it is used as a model species for Leguminosae. In contrast with the advanced studies in molecular biology, cytogenetic research has remained limited even though it is an extremely valuable approach to the analysis of the genome structure. In the present study we examined the chromosomal distribution of rDNA sequences in five natural populations of M. truncatula, explored the genomic diversity of this species and found markers for chromosome identification. FISH experiments revealed three distribution patterns of rDNA sequences, distinguished by one, two and three loci of 5S genes; 18S-5.8S-25S genes were always localized at a single locus. The results add information to the genome structure of M. truncatula, revealing a pattern of distribution of rDNA genes unobserved previously, which consists of 5S genes clustered at a single locus. The physical mapping of rDNA sequences is a first contribution towards the construction of a detailed molecular karyotype of M. truncatula.

Linkage mapping in apomictic and sexual Kentucky bluegrass ( Poa pratensis L.) genotypes using a two way pseudo-testcross strategy based on AFLP and SAMPL markers.

The high versatility of the mode of reproduction and the retention of a pollen recognition system are the factors responsible for the extreme complexity of the genome in Poa pratensis L. Two genetic maps, one of an apomictic and one of a sexual genotype, were constructed using a two-way pseudo-testcross strategy and multiplex PCR-based molecular markers (AFLP and SAMPL). Due to the high ploidy level and the uncertainty of chromosome pairing-behavior at meiosis, only parent-specific single-dose markers (SDMs) that segregated 1:1 in an F(1) mapping population (161 out of 299 SAMPLs, and 70 out of 275 AFLPs) were used for linkage analysis. A total of 41 paternal (33 SAMPLs and 8 AFLPs) and 47 maternal (33 SAMPLs and 14 AFLPs) SDMs, tested to be linked in coupling phase, were mapped to 7+7 linkage groups covering 367 and 338.4 cM, respectively. The comparison between the two marker systems revealed that SAMPL markers were statistically more efficient than AFLP ones in detecting parent-specific SDMs (75% vs 32.4%). There were no significant differences in the percentages of distorted marker alleles detected by the two marker systems (27.8% of SAMPLs vs 21.3% of AFLPs). The pairwise comparison of co-segregational groups for linkage detection between marker loci suggested that at least some of the P. pratensis chromosomes pair preferentially at meiosis-I.

Genomic relationships between Medicago murex Willd. and Medicago lesinsii E. Small. investigated by in situ hybridization.

Medicago murex Willd. is an annual species (2n = 14) widespread in the wild and of remarkable interest for pastures in regions with a mediterranean climate. It is considered closely related to Medicago lesinsii E. Small (2n = 16) but, up to now, there is no evidence demonstrating their genetic affinity. This research was undertaken to investigate the genomic relationships between M. murex and M. lesinsii by using genomic in situ hybridization (GISH). In this study GISH experiments were performed using both species as sources of chromosomes and genomic probes. To better evaluate the results of the hybridization, the labelled DNA of each species was hybridized to chromosomes of the same species and to chromosomes of the diploid Medicago littoralis (2n = 16). Strong hybridization signals were found on chromosomes of M. murex and M. lesinsii after GISH. Differences in the hybridization strength were not observed when slides from interspecific hybridization were compared with the control preparations. These results suggest that consistent divergences of the DNA sequences did not occur after the separation of the two species. Instead very reduced cross hybridization was found on chromosome spreads of M. littoralis hybridized with the DNA of M. lesinsii or M. murex. The distribution of the ribosomal genes (rDNA) investigated by fluorescent in situ hybridization (FISH) appeared similar in both M. murex and M. lesinsii. The GISH technique may be a valuable approach to obtain information on evolution of the 2n = 14 species and on the origin of the polyploids Medicago rugosa (2n = 30) and Medicago scutellata (2n = 30). The first attempt to investigate the genomic composition of M. scutellata using a genomic probe is reported in this paper.

Apospory and parthenogenesis may be uncoupled in Poa pratensis: a cytological investigation.

Despite the potential that apomixis has for agriculture, there is little information regarding the genetic control of its functional components. We carried out a cytohistological investigation on an F1 segregating population of Poa pratensis obtained from a cross between a sexual and an apomictic parent. About half of the F1 progeny plants were parthenogenic, as adjudicated by an auxin test. The degree of parthenogenesis ranged from 1.44% to 92.9%. Apospory was detected in parthenogenetic plants as well as in two non-parthenogenetic individuals. These results indicate that two distinct genetic factors control apospory and parthenogenesis in P. pratensis and that apospory and parthenogenesis may be developmentally uncoupled.

Age- and trauma-dependent modifications of neuromuscular junction and skeletal muscle structure in the rat. Effects of long-term treatment with Acetyl-L-Carnitine.

The influence of ageing and crushing of the sciatic nerve on the morphology of the neuromuscular junction (NMJ) and on the muscle fiber composition were studied in the rat soleus muscle using histochemical techniques associated with image analysis. The influence of a 6-month treatment with Acetyl-L-Carnitine (ALCAR, 150 mg/kg/day) on the age- and crushing-dependent changes of the NMJ and on age-related modifications of the muscle fiber composition was assessed as well. In control old and injured young rats a loss of complexity of the NMJ was observed. Treatment with ALCAR resulted in an increased endplate complexity both in old rats and in young rats injured by crushing, in comparison with respective controls. The structure of the rat soleus muscle changes with increasing age. Modification mainly consists in a type II fiber atrophy, and in the alteration of the peculiar mosaic organization of the soleus muscle fibers. In ALCAR-treated old rats, the morphology of the soleus muscle fibers was similar to that observed in adult animals. These findings suggest that treatment with ALCAR has a beneficial effect on NMJ and on muscle fiber structure in ageing or after nerve crushing. The possible mechanism of action of this 'trophic' effect of ALCAR-treatment is discussed.

Acetyl-L-carnitine prevents age-dependent structural alterations in rat peripheral nerves and promotes regeneration following sciatic nerve injury in young and senescent rats.

Morphologic and morphometric alterations of the sciatic nerve from old Sprague-Dawley rats and of lesioned tibial nerve from young and senescent Sprague-Dawley rats were studied. The possible therapeutical effects of treatment with acetyl-L-carnitine (ALCAR) were also investigated, ALCAR being a compound shown to exert a beneficial pharmacological action on diabetic neuropathies. Nerve sections from animals sacrificed under anesthesia were stained with toluidine blue. In old rats, a 6-month treatment with ALCAR markedly reduced the percentage of myelinated fibers (MF) characterized by age-dependent morphologic alterations (4% in treated rats versus 11% in untreated ones), such as myelin balloons, infolded loops of myelin, myelin reduplication, and ovoids. In the lesioned animals, ALCAR-treatment (15-60 days for young rats and 6-9 months for senescent rats) produced a significant increase versus controls in the density of regenerating myelinated fibers (RMF) at 15 days (young rats) and at 30 days (senescent rats) after crush, as well as an increase in the axon diameter in both young and senescent rats at 60 days after nerve crush. The MF diameter (sheath + axons) was significantly larger in treated senescent rats than in controls at 100 days after nerve crush. In ALCAR-treated rats, both young and senescent, the density of degenerative elements was lower and the RMF ratio (RMF density/RMF density + density of degenerative elements) was higher than that in controls at all detection times.

Morphological and electrophysiological changes of peripheral nerve-muscle unit in the aged rat prevented by levocarnitine acetyl.

The effects of levocarnitine acetyl on structure and function of the sciatic nerve and neuromuscular junctions of the soleus and extensor digitorum longus muscles were studied in the aged rat. To that end, neuromuscular conduction velocity (NMCV) was measured in vivo and morphological and morphometric evaluations were performed. Treatment with levocarnitine acetyl, 150 mg/kg day for six months, restored NMCV values to the levels measured in the young rat; significantly reduced the number of degenerating elements; and increased the number of myelinated fibres having normal structural features. In the soleus and extensor digitorum longus muscles, levocarnitine acetyl increased the complexity of neuromuscular junctions. These experimental findings suggest a neurotrophic action of levocarnitine acetyl on the peripheral nervous system that might have therapeutical applications in age-related peripheral nerve changes.

Levocarnitine acetyl stimulates peripheral nerve regeneration and neuromuscular junction remodelling following sciatic nerve injury.

The effects of levocarnitine acetyl were investigated on both peripheral nerve regeneration and neuromuscular remodelling in male Sprague-Dawley rats, three months of age, following crush of their left sciatic nerve. Levocarnitine acetyl, 150 mg/kg/day in drinking water, was given from one week before to 5, 15, 20, and 60 days after nerve crush. The sciatic nerve was examined morphologically at all given times and morphometrically at 15, 20, and 60 days after the lesion. Morphology, at 5, 15, and 60 days, and morphometry, at 60 days after the nerve crush, were also performed on the neuromuscular junction in the soleus and extensor digitorum longus muscles. Five days after nerve crush, complete axonal degeneration was observed in both control and treated rats. At 15 and 20 days, recovery from injury in treated animals was better than in controls, as shown by a significantly higher increase in the number of regenerating axons. At the same times, denervated endplates were present in both groups. At 60 days, axonal regeneration restored the number of axons to normal values in all injured animals, while their size maturation was greater in treated rats than in controls. A markedly lower number of degenerating elements was found in treated animals. In the neuromuscular junctions of the soleus and extensor digitorum longus muscles, nerve terminal branch points were reduced in the lesioned rats in comparison with uninjured ones. However, morphometric analysis revealed a greater endplate complexity in treated animals in which, at 60 days after nerve crush, nerve terminal branching and sprouting index values were significantly higher than in controls. It is concluded that levocarnitine acetyl exerts a beneficial effect on nerve regeneration processes and synaptic remodelling in crush-induced neuropathies.