From sampling to cellblock: The fully automated journey of cytological specimens.

In recent years, technological innovation have emerged to standardize pathology laboratory processes and reduce the handling of diagnostic samples. Among them is an automatic tissue embedding system that eliminates the need for manual activity in tissue paraffin embedding, thereby improving sample preservation. Unfortunately, this system cannot be used for cytological specimens due to the lack of an effective holder to support the procedure steps. In this study, we evaluated the performance of a commercial polymer matrix to enable and standardize the automatic paraffin embedding of cytological material from different organs and sources. Cytological samples from 40 patients were collected on the matrices and submitted for fully automatic workflow preparation, from formalin fixation until paraffin block, using the Sakura embedding system. Our results demonstrated the feasibility of the automated procedure, from loading cytological sample onto the matrix to obtaining the paraffin cellblock, thereby avoiding manual manipulation of cellular material. All samples resulted adequately processed and paraffin-embedded showing satisfactory tissue permeation by processing reagents, optimal preservation of cytoplasmic and nuclear details, and good quality of staining results on paraffin sections. Automated embedding of cytological samples eliminates the risk of lost specimens, reduces laboratory burden, standardizes procedures, increases diagnostic yield, and ultimately improves patients' management.

Programmed Cell Death Pathways in Cholangiocarcinoma: Opportunities for Targeted Therapy.

Cholangiocarcinoma is a highly aggressive cancer arising from the bile ducts. The limited effectiveness of conventional therapies has prompted the search for new approaches to target this disease. Recent evidence suggests that distinct programmed cell death mechanisms, namely, apoptosis, ferroptosis, pyroptosis and necroptosis, play a critical role in the development and progression of cholangiocarcinoma. This review aims to summarize the current knowledge on the role of programmed cell death in cholangiocarcinoma and its potential implications for the development of novel therapies. Several studies have shown that the dysregulation of apoptotic signaling pathways contributes to cholangiocarcinoma tumorigenesis and resistance to treatment. Similarly, ferroptosis, pyroptosis and necroptosis, which are pro-inflammatory forms of cell death, have been implicated in promoting immune cell recruitment and activation, thus enhancing the antitumor immune response. Moreover, recent studies have suggested that targeting cell death pathways could sensitize cholangiocarcinoma cells to chemotherapy and immunotherapy. In conclusion, programmed cell death represents a relevant molecular mechanism of pathogenesis in cholangiocarcinoma, and further research is needed to fully elucidate the underlying details and possibly identify therapeutic strategies.

The Multiple Mitotic Roles of the ASPM Orthologous Proteins: Insight into the Etiology of ASPM-Dependent Microcephaly.

The Drosophila abnormal spindle (asp) gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that asp is highly conserved and that mutations in its human ortholog ASPM (Abnormal Spindle-like Microcephaly-associated; or MCPH5) are the most common cause of autosomal recessive primary microcephaly. This finding greatly stimulated research on ASPM and its fly and mouse (Aspm) orthologs. The three Asp orthologous proteins bind the microtubules (MTs) minus ends during cell division and also function in interphase nuclei. Investigations on different cell types showed that Asp/Aspm/ASPM depletion disrupts one or more of the following mitotic processes: aster formation, spindle pole focusing, centrosome-spindle coupling, spindle orientation, metaphase-to-anaphase progression, chromosome segregation, and cytokinesis. In addition, ASPM physically interacts with components of the DNA repair and replication machineries and is required for the maintenance of chromosomal DNA stability. We propose the working hypothesis that the asp/Aspm/ASPM genes play the same conserved functions in Drosophila, mouse, and human cells. Human microcephaly is a genetically heterogeneous disorder caused by mutations in 30 different genes that play a variety of functions required for cell division and chromosomal DNA integrity. Our hypothesis postulates that ASPM recapitulates the functions of most human microcephaly genes and provides a justification for why ASPM is the most frequently mutated gene in autosomal recessive primary microcephaly.

Cumulus Cell Transcriptome after Cumulus-Oocyte Complex Exposure to Nanomolar Cadmium in an In Vitro Animal Model of Prepubertal and Adult Age.

Cadmium (Cd), a highly toxic pollutant, impairs oocyte fertilization, through oxidative damage on cumulus cells (CCs). This study analysed the transcriptomic profile of CCs of cumulus-oocyte complexes (COCs) from adult and prepubertal sheep, exposed to Cd nanomolar concentration during in vitro maturation. In both age-groups, CCs of matured oocytes underwent RNA-seq, data analysis and validation. Differentially expressed genes (DEGs) were identified in adult (n = 99 DEGs) and prepubertal (n = 18 DEGs) CCs upon Cd exposure. Transcriptomes of adult CCs clustered separately between Cd-exposed and control samples, whereas prepubertal ones did not as observed by Principal Component Analysis. The transcriptomic signature of Cd-induced CC toxicity was identified by gene annotation and literature search. Genes associated with previous studies on ovarian functions and/or Cd effects were confirmed and new genes were identified, thus implementing the knowledge on their involvement in such processes. Enrichment and validation analysis showed that, in adult CCs, Cd acted as endocrine disruptor on DEGs involved in hormone biosynthesis, cumulus expansion, regulation of cell signalling, growth and differentiation and oocyte maturation, whereas in prepubertal CCs, Cd affected DEGs involved in CC development and viability and CC-oocyte communications. In conclusion, these DEGs could be used as valuable non-invasive biomarkers for oocyte competence.

The Drosophila simulans Genome Lacks the crystal-Stellate System.

The cry-Ste system is a genetic interaction system between heterochromatin and euchromatin in Drosophila melanogaster, regulated via the piRNA pathway. Deregulation of this system leads to meiotic defects and male sterility. Although the cry-Ste system is peculiar to D. melanogaster, ancestors of Ste and Su(Ste) elements are present in the three closely related species, D. simulans, D. sechellia, and D. mauritiana. The birth, evolution, and maintenance of this genetic system in Drosophila melanogaster are of interest. We investigate the presence of sequences homologous to cry and Ste elements in the simulans complex and describe their chromosomal distribution. The organization and expression of cry- and Ste-like sequences were further characterized in the D. simulans genome. Our results allow us to conclude that the cry-Ste genetic interaction system is absent in the D. simulans genome.

TGS1 impacts snRNA 3'-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development.

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease.

Drosophila Morgana is an Hsp90-interacting protein with a direct role in microtubule polymerisation.

Morgana (Mora, also known as CHORD in flies) and its mammalian homologue, called CHORDC1 or CHP1, is a highly conserved cysteine and histidine-rich domain (CHORD)-containing protein that has been proposed to function as an Hsp90 co-chaperone. Morgana deregulation promotes carcinogenesis in both mice and humans while, in Drosophila, loss of mora causes lethality and a complex mitotic phenotype that is rescued by a human morgana transgene. Here, we show that Drosophila Mora localises to mitotic spindles and co-purifies with the Hsp90-R2TP-TTT supercomplex and with additional well-known Hsp90 co-chaperones. Acute inhibition of Mora function in the early embryo results in a dramatic reduction in centrosomal microtubule stability, leading to small spindles nucleated from mitotic chromatin. Purified Mora binds to microtubules directly and promotes microtubule polymerisation in vitro, suggesting that Mora directly regulates spindle dynamics independently of its Hsp90 co-chaperone role.

Phenotypic characterization of diamond (dind), a Drosophila gene required for multiple aspects of cell division.

Many genes are required for the assembly of the mitotic apparatus and for proper chromosome behavior during mitosis and meiosis. A fruitful approach to elucidate the mechanisms underlying cell division is the accurate phenotypic characterization of mutations in these genes. Here, we report the identification and characterization of diamond (dind), an essential Drosophila gene required both for mitosis of larval brain cells and for male meiosis. Larvae homozygous for any of the five EMS-induced mutations die in the third-instar stage and exhibit multiple mitotic defects. Mutant brain cells exhibit poorly condensed chromosomes and frequent chromosome breaks and rearrangements; they also show centriole fragmentation, disorganized mitotic spindles, defective chromosome segregation, endoreduplicated metaphases, and hyperploid and polyploid cells. Comparable phenotypes occur in mutant spermatogonia and spermatocytes. The dind gene encodes a non-conserved protein with no known functional motifs. Although the Dind protein exhibits a rather diffuse localization in both interphase and mitotic cells, fractionation experiments indicate that some Dind is tightly associated with the chromatin. Collectively, these results suggest that loss of Dind affects chromatin organization leading to defects in chromosome condensation and integrity, which in turn affect centriole stability and spindle assembly. However, our results do not exclude the possibility that Dind directly affects some behaviors of the spindle and centrosomes.

The Hybrid Incompatibility Genes Lhr and Hmr Are Required for Sister Chromatid Detachment During Anaphase but Not for Centromere Function.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin-heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].

Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and ß-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.

Loss of Pol32 in Drosophila melanogaster causes chromosome instability and suppresses variegation.

Pol32 is an accessory subunit of the replicative DNA Polymerase δ and of the translesion Polymerase ζ. Pol32 is involved in DNA replication, recombination and repair. Pol32's participation in high- and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32rd1 and pol32rds in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutants suppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.

Morgana/chp-1, a ROCK inhibitor involved in centrosome duplication and tumorigenesis.

Centrosome abnormalities lead to genomic instability and are a common feature of many cancer cells. Here we show that mutations in morgana/chp-1 result in centrosome amplification and lethality in both Drosophila and mouse, and that the fly centrosome phenotype is fully rescued by the human ortholog of morgana. In mouse cells, morgana forms a complex with Hsp90 and ROCK I and II, and directly binds ROCK II. Morgana downregulation promotes the interaction between ROCK II and nucleophosmin (NPM), leading to an increased ROCK II kinase activity, which results in centrosome amplification. Morgana(+/-) primary cells and mice display an increased susceptibility to neoplastic transformation. In addition, tumor tissue array histochemical analysis revealed that morgana is underexpressed in a large fraction of breast and lung human cancers. Thus, morgana/chp-1 appears to prevent both centrosome amplification and tumorigenesis.

Melusin gene (ITGB1BP2) nucleotide variations study in hypertensive and cardiopathic patients.

BACKGROUND: Melusin is a muscle specific signaling protein, required for compensatory hypertrophy response in pressure-overloaded heart. The role of Melusin in heart function has been established both by loss and gain of function experiments in murine models. With the aim of verifying the hypothesis of a potential role of the Melusin encoding gene, ITGB1BP2, in the modification of the clinical phenotype of human cardiomyopathies, we screened the ITGB1BP2 gene looking for genetic variations possibly associated to the pathological phenotype in three selected groups of patients affected by hypertension and dilated or hypertrophic cardiomyopathy METHODS: We analyzed ITGB1BP2 by direct sequencing of the 11 coding exons and intron flanking sequences in 928 subjects, including 656 hypertensive or cardiopathic patients and 272 healthy individuals. RESULTS: Only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11. CONCLUSIONS: The three variations of the ITGB1BP2 gene have been detected in families of patients affected either by hypertension or primary hypertrophic cardiomyopathy; however, a clear genotype/phenotype correlation was not evident. Preliminary functional results and bioinformatic analysis seem to exclude a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism.Our analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.

Neocentromeres in 15q24-26 map to duplicons which flanked an ancestral centromere in 15q25.

The existence of latent centromeres has been proposed as a possible explanation for the ectopic emergence of neocentromeres in humans. This hypothesis predicts an association between the position of neocentromeres and the position of ancient centromeres inactivated during karyotypic evolution. Human chromosomal region 15q24-26 is one of several hotspots where multiple cases of neocentromere emergence have been reported, and it harbors a high density of chromosome-specific duplicons, rearrangements of which have been implicated as a susceptibility factor for panic and phobic disorders with joint laxity. We investigated the evolutionary history of this region in primates and found that it contains the site of an ancestral centromere which became inactivated about 25 million years ago, after great apes/Old World monkeys diverged. This inactivation has followed a noncentromeric chromosomal fission of an ancestral chromosome which gave rise to phylogenetic chromosomes XIV and XV in human and great apes. Detailed mapping of the ancient centromere and two neocentromeres in 15q24-26 has established that the neocentromere domains map approximately 8 Mb proximal and 1.5 Mb distal of the ancestral centromeric region, but that all three map within 500 kb of duplicons, copies of which flank the centromere in Old World Monkey species. This suggests that the association between neocentromere and ancestral centromere position on this chromosome may be due to the persistence of recombinogenic duplications accrued within the ancient pericentromere, rather than the retention of "centromere-competent" sequences per se. The high frequency of neocentromere emergence in the 15q24-26 region and the high density of clinically important duplicons are, therefore, understandable in the light of the evolutionary history of this region.

A study of host defence peptide beta-defensin 3 in primates.

We have investigated the molecular evolution of the gene coding for beta-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for beta-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251-257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17-->Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279-8289]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human beta-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.