Giuseppina Pichiri

“Physiological” renal regenerating medicine in VLBW preterm infants: could a dream come true?

An emerging hypothesis from the recent literature explain how specific adverse factors related with growth retardation as well as of low birth weight (LBW) might influence renal development during fetal life and then the insurgence of hypertension and renal disease in adulthood. In this article, after introducing a brief overview of human nephrogenesis, the most important factors influencing nephron number at birth will be reviewed, focusing on the “in utero” experiences that lead to an increased risk of developing hypertension and/or kidney disease in adult. Since nephrogenesis in preterm human newborns does not stop at birth, but it continues for 4–6 weeks postnatally, a better knowledge of the mechanisms able to accelerate nephrogenesis in the perinatal period, could represent a powerful tool in the hands of neonatologists. We suggest to define this approach to a possible therapy of a deficient nephrogenesis at birth “physiological renal regenerating medicine”. Our goal in preterm infants, especially VLBW, could be to prolong the nephrogenesis not only for 6 weeks after birth but until 36 weeks of post conceptual age, allowing newborn kidneys to restore their nephron endowment, escaping susceptibility to hypertension and to renal disease later in life.

The Efficiency of In-situ Hybridization on Human Chromosomes with Alphoid DNAs is Enhanced by Previous Digestion with AluI and TaqI

Centromeric alphoid DNAs of human chromosomes 6, 9, 16 and Y were employed to obtain information on the molecular mechanism(s) determining cytological effects produced by digestion in situ with AluI and TaqI restriction enzymes, possibly related to the structure of the above-cited areas. The following cytological and biochemical experiments were carried out using the above-mentioned alphoid sequences as probes: (1) standard in-situ hybridization and in-situ hybridization after chromosome cleavage with AluI/TaqI, and (2) filter hybridization on the DNA fractions obtained from the material solubilized and that retained on the slides after digestion in situ with AluI/TaqI. Biochemical data show that cleavage of alphoid DNAs is not prevented by the peculiar organization of centromeric heterochromatin, but such cleavage is not necessarily followed by complete DNA solubilization. The analysis of alphoid sequence cleavage in naked genomic DNA as well as during digestion of fixed chromosomes shows that (1) AluI cuts more efficiently than TaqI, (2) DNA fragments as large as 3–5 kb can be solubilized, and (3) DNA fragments of the same size are found in both fractions of DNA, i.e. that retained on the chromosomes as well as that solubilized from chromosomes. Cytological data show that previous chromosome digestion, mostly with TaqI, increases the hybridization signal area, suggesting that this fact might be due to (1) chromatin reorganization produced by enzyme attack and/or (2) the presence of alphoid DNAs which might be restricted not only to the kinetochore area but also to para/peri-centromeric heterochromatin. Lastly, centromere DNA solubilization as a consequence of restriction enzyme cleavage seems to vary from chromosome to chromosome, thus suggesting that centromeric regions do not represent a homogeneous class of constitutive heterochromatin.

Rapid multiplex real-time PCR by molecular beacons for different BRAF allele detection in papillary thyroid carcinoma.

BRAF is an oncogene that is commonly mutated in both melanomas and papillary thyroid carcinomas (PTCs). Usually, mutations in the codons 600 or 601 lead to constitutive activity in the Ras-mitogen–activated protein kinase pathway and, recently, the BRAFVK600-1E deletion was described as a relevant risk factor for loco-regional PTC lymph node metastasis. For these reasons, BRAF mutations may be considered a key genetic factor for the metastatic progression of PTC and also for other tumors such as melanoma and colon cancer and a new BRAF-specific therapeutic strategy was already suggested. In this report we describe the development of a rapid qualitative fluorescent real-time polymerase chain reaction assay designed for the detection of BRAFVK600-1E deletion using 2 specific molecular beacons. The assay is able to detect in a single tube the homozygous as well the heterozygous genotypes. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific molecular beacons, and the throughput of a multicolor fluorescence detection procedure. This technique, together with an earlier described real-time test specific for V600E and K601E will be useful for research and molecular diagnostic laboratories involved in the study of BRAF-related neoplasia.

Thymosin beta 4 may translocate from the cytoplasm in to the nucleus in HepG2 cells following serum starvation. An ultrastructural study.

Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases.

Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae).

Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clarified. Few cytogenetic studies have been conducted on this family, and all of them showed the same diploid chromosome number (2n=42) but with conspicuous karyotypic variation among species. The Mediterranean moray eel Gymnothorax unicolor was previously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs) distribution pattern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI) obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromosomes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI fraction was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR). Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new contribution to the karyotype of this species, we found that the major ribosomal genes are localized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly-TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and contribute to the cytogenetic knowledge regarding this fish family, which has not been extensively studied.

MVarallo: a new M(Like) alpha 1-antitrypsin-deficient allele

A 73-year-old never-smoker woman with chronic bronchitis, increasing dyspnoea, and airflow limitation with a FEV1 of 49% of predicted value had low serum level of alpha-1-antitrypsin (69 mg/dL, normal range 150–350). Isoelectric focusing showed an Mlike pattern. Direct sequencing showed, in the second exon, a particular DNA alteration localized between codon 41 and codon 51: a region of 30 base pairs (bp) was completely deleted and substituted by a 22-bp sequence. The resulting loss of 8 bp yields, in the second exon, a 70–71 stop codon. This new Mlike variant was denominated MVarallo from the site where it was discovered.

CYTOGENETIC AND MOLECULAR CHARACTERISTICS OF ATLANTIC EELS (ANGUILLA ANGUILLA AND A. ROSTRATA) GENOME

Abstract The Atlantic eels, Anguilla anguilla and A. rostrata, have partially overlapping spawning sites and show incomplete reproductive isolation as testified by the presence of hybrids at low frequency. Nevertheless, significant genetic differences between the two species have been pointed out by data on biochemical polymorphisms and mitochondrial DNA. This study reviews the cyto‐genetic and molecular data and points out differences and similarities between the two species.

Multipotent stem cells of mother's milk

In recent years the presence of stem cells (hBSCs: human breastmilk-derived stem cells) and epithelial progenitors has been demonstrated in mother’s milk (MM). Stem cells present in samples of fresh MM exhibit a high degree of vitality and this makes possible the performance of cell cultures and to evaluate the differentiation capacity of the hBSCs. The most important datum that expresses the enormous potential of the use of MM stem cells is the presence of a cell population capable of differentiating into the three mesoderm, endoderm and ectoderm lines. The small number of studies and MM samples analyzed and the different sampling methods applied suggest standardization in the collection, analysis and culture of MM in future studies, in consideration of the well-known extreme variability of MM composition, also from the standpoint of cells. The analysis of literature data confirms the uniqueness of MM and its enormous potential. Proceedings of the 2 nd International Course on Perinatal Pathology (part of the 11 th International Workshop on Neonatology · October 26 th -31 st , 2015) · Cagliari (Italy) · October 31 st , 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

Breast milk stem cells: four questions looking for an answer

The finding of stem/progenitor cells in the maternal milk and the discovery of their multilineage potential, associated with some evidence regarding the ability of maternal cells to cross the gastrointestinal barrier and integrate into the organs of the breastfed neonate, has opened an intriguing debate, regarding the strict relationship between mother and son in the postnatal period. In particular, thanks to the discovery of the presence in high quantities of mammary stem cells, a new vision of maternal milk is emerging, in which breastfeeding appears as an unique occasion for reinforcing the physiological development of the newborn, putting all the formulas at a different level of relevance for the neonate. In this contribution the authors try to give an answer to the following 4 questions: is there heterogeneity and a hierarchy among breast milk stem cells? can stem cells present in breast milk enter into the newborn organism? can breast milk stem cells integrate in the neonatal organs and differentiate toward different tissues, including neurons and neuroglia? could metabolomics be useful for the study of stem cells in the human milk? Proceedings of the 2 nd International Course on Perinatal Pathology (part of the 11 th International Workshop on Neonatology · October 26 th -31 st , 2015) · Cagliari (Italy) · October 31 st , 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

Human breast milk stem cells: a new challenge for perinatologists

The lactating mammary gland contains a stem cells population with multilineage potentialities. Recently it was also shown that breast milk contains a heterogeneous population of stem cells that have the potential to differentiate in vitro , under the control of specific differentiation conditions, into mammary epithelial as well as into adipogenic, chondrogenic and osteogenic cell lineages. While the different types of cells present in the milk is known, what is less understood is the proportion of different milk cell types, their significance for the mother and the infant and factors influencing them. In this manuscript we summarize some of the latest knowledge from in vivo and in vitro investigations on breast milk stem cells, we discuss their potential functions and applications and we present some of our preliminary data obtained in fresh human breast milk cells. Proceedings of the 2 nd International Course on Perinatal Pathology (part of the 11 th International Workshop on Neonatology · October 26 th -31 st , 2015) · Cagliari (Italy) · October 31 st , 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano