Chromosome analysis using different staining techniques and fluorescent in situ hybridization in Cerithium vulgatum (Gastropoda: Cerithiidae)

In the present paper one population of the "large" subtidal mollusc Cerithium vulgatum Bruguiere, 1792 (Gastropoda: Cerithiidae) from the Northwestern coast of Sicily was investigated from a karyological point of view. The chromosome complement was Giemsa stained, conventionally karyotyped in 18 homomorphic chromosome pairs (10 bi-armed and 8 mono-armed), and subsequently analysed using silver, CMA3 and DAPI staining, and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal sites (18S-28S rDNA) in the terminal region of the short arms of one small sized mono-armed chromosome pair. Ribosomal DNA was transciptionally active as indicated by its preferential impregnation with silver nitrate (Ag-NOR) and did not contain a high amount of GC base pairs as suggested by the lack of a bright CMA3 fluorescence. The (TTAGGG)n telomeric probe was hybridized to the termini of nearly all chromosomes, thus demonstrating that, in C. vulgatum, this sequence has been conserved during the genomic evolution. The finding of the telomeric hexanucleotide in six species belonging to the three high taxa of Gastropoda supports the notion that this sequence is widespread within this class. The (GATA)n probe did not label any chromosome regions except for a minute terminal area of a single bivalent at pachytene stage

Ecografia toracica: un nuovo strumento nella valutazione del paziente con dispnea

THORACIC ULTRASOUND: A NEW TOOL IN THE ASSESSMENT OF PATIENT DYSPNEA Introduction: Rapid differentiation between the causes of cardiogenic and non cardiogenic dyspnea is an objective of primary importance in the management of patients with dyspnea. The instruments available for assessing the amount of extravascular lung water for the physician are essentially made of phisical findings, conventional radiological techniques (chest radiography and computerized axial tomography) and echocardiography.. Each of them has its own characteristics of sensitivity, specificity, accuracy, speed of use and repeatability. They should be added to the lung ultrasound for parenchyma, an ultrasound technique that has been accompanied in recent years with traditional techniques and which, according to data reported in the literature, agrees, at least in the field of Emergency and Critical Care, a good discrimination of cardiogenic causes of dyspnea. Advanteges of this method are also the characteristic speed of use and absolute repeatability (not based on ionizing radiation). Furthermore, it provides semi-quantitative assessments of the amount of extravascular water. Aim of the study: To evaluate the validity and accuracy of lung ultrasound technique in the field of Internal Medicine (pathological sub-acute), comparing with the classical clinical parameters, radiological and laboratory which are used for the clinical interpretation of dyspnea. Materials and methods: 152 patients hospitalized in the Department of Clinical Medicine 1 with a diagnosis of dyspnea.have been esamine. These patients had already performed a clinical evaluation at the emergency room and a chest radiograph and an assessment of NT-proBNP (Brain Natriuretic Peptide). Upon admission to our department, patients underwent lung ultrasound in order to highlight the presence of pulmonary B lines ( "Comet tail artifacts”). The B lines are vertical reverb artifacts that departured from pleura and extended to the base of the screen. The test was considered positive when the number of B lines was higher 8 and there was no difference in the number of B lines increased by 30% in both lung fields. Ultrasonography was performed by three operators with varying experience in the field of ultrasound lung (an experienced operator in the field of ultrasound lung, an expert in the field of internistic ultrasound and an operator with little experience with ultrasound), each of whom has performed two measurements to analyze the intra-operator variability, the duration of the examination was less than three minutes. It 'was considered as the gold standard the diagnosis made by expert phisicians, reassessing the clinical, radiological and bio-humoral (as per the recommendations of the AHA). The ultrasound report was not however in their possession. The examination was also repeated at 48 hours to assess the changes in the ultrasound picture on the basis of treatment performed. Results: We analyzed the values of sensitivity, specificity, positive predictive value (PPV), negative predictive value (VPN), accuracy of lung ultrasound, as well as NT-proBNP and the main clinical and radiological, compared to clinical diagnosis. The results include the following: Sensitivity Specificity VPP VPN Accuracy Ultrasound t0 97% 79% 79% 97% 87% Ultrasound T48 76% 77% 72% 80% 77% NT-proBNP t0 97% 44% 58% 95% 68% NT-proBNP T48 94% 33% 53% 88% 61% Patients diagnosed with heart failure had a higher number of lines B than in those without heart failure (p <0.05), and the same is evident for the values of NT-proBNP (p <0.05). Furthermore, the reduction in the number of line B was significantly higher (p <0.005) in the group of patients treated for heart failure, but did not differ significantly the value of NT-proBNP (p = 0.37). The intra-operator correlation was demonstrated excellent (98%), the inter-operator also excellent between experienced operators (98%), intermediate (90%) among operators of different experience. Discussion: Lung ultrasound has demonstrated a strong sensitivity value and a fair value of specificity in the diagnosis of acute cardiogenic pulmonary edema, even in sub-acute patients, with values of significance similar to those of NT-proBP but with a reduced execution time. The variations in the ultrasound picture also appear to correlate with the appropriateness of the treatment carried out, suggesting a possible use in monitoring therapeutic response. The false positive ultrasound examination are probably referring to forms of interstitial lung imbibition (eg, ARDS ). The ease of execution is witnessed by the excellent reproducibility both intra-and inter-operator. Conclusion: Lung ultrasonography may be considered a valuable tool in the recognition of the origin of cardiogenic dyspnea. This method appears effective, faster and can be used to supplement the standard assessments conducted in departments of internal medicine.Introduzione: Una rapida differenziazione tra le cause cardiogene di dispnea e quelle non cardiogene costituisce un obiettivo di primaria importanza nella gestione del paziente con dispnea. Gli strumenti a disposizione per la valutazione della quantità di acqua libera extravascolare polmonare a disposizione del clinico sono sostanzialmente costituiti da semeiotica fisica, le tecniche radiologiche convenzionali (radiografia del torace e tomografia assiale computerizzata) e l'ecocardiografia. Ognuna di esse presenta caratteristiche proprie di sensibilità, specificità, accuratezza, rapidità d'impiego e ripetibilità. Ad esse va aggiunta l’ecografia polmonare per parenchima, una metodica ecografica che si è affiancata negli ultimi anni alle tecniche tradizionali e che, secondo i dati riportati dalla letteratura, consente, almeno in ambito di Pronto Soccorso e Terapia intensiva, una ottima discriminazione delle cause cardiogene di dispnea. A vantaggio di tale metodica vi sono inoltre la caratteristica rapidità di impiego e la assoluta ripetibilità (non essendo basata su radiazioni ionizzanti). Inoltre essa fornisce delle valutazioni semi-quantitative della quantità di acqua libera extravascolare. Scopo dello studio: valutare la validità e l’accuratezza della metodica ecografica polmonare in ambito di Medicina Interna (quadri clinici sub-acuti), confrontandola con i classici parametri clinici, radiologici e laboratoristici ai quali si fa ricorso per l’interpretazione clinica della dispnea. Materiali e metodi: Sono stati presi in esame 152 pazienti ricoverati nel reparto di Clinica Medica 1 con diagnosi di dispnea. Tali pazienti avevano già eseguito una valutazione clinica presso il Pronto Soccorso, nonché una radiografia del torace e una valutazione del NT-proBNP (Brain Natriuretic Peptide). Al momento del ricovero presso il nostro reparto, i pazienti sono stati sottoposti ad ecografia polmonare al fine di evidenziare la presenza di linee B polmonari (“Comet tail artifacts”. Le linee B sono degli artefatti verticali da riverbero a partenza pleurica estesi fino alla base dello schermo. L'esame è stato considerato positivo quando il numero di linee B era superiore a 8 e non vi era una differenza nel numero delle linee B maggiore del 30% nei due campi polmonari. L’ecografia è stata eseguita da tre operatori con diversa esperienza nel campo dell'ecografia polmonare (un operatore esperto nel campo dell’ecografia polmonare, uno esperto nel campo dell’ecografia internistica e un operatore con scarsa esperienza ecografica), ognuno dei quali ha eseguito due misurazioni, per analizzare la variabilità intra-operatore; la durata dell’esame è stata inferiore ai tre minuti. E’ stata considerata come gold standard la diagnosi clinica finale effettuata da medici esperti, rivalutando i dati clinici, radiologici e bioumorali (come da raccomandazioni della AHA). Il referto ecografico non era invece in loro possesso. L’esame è stato inoltre ripetuto a distanza di 48 ore al fine di valutare le modificazioni del quadro ecografico sulla base del trattamento effettuato. Risultati: Sono stati analizzati i valori di sensibilità, specificità, valore predittivo positivo (VPP), valore predittivo negativo (VPN), accuratezza dell’ecografia polmonare, nonché di NT-proBNP e dei principali parametri clinici e radiologici, confrontati alla diagnosi clinica. I risultati ottenuti riportano: sensibilità specificità VPP VPN accuratezza Ecografia t0 97% 79% 79% 97% 87% Ecografia t48 76% 77% 72% 80% 77% NT-proBNP t0 97% 44% 58% 95% 68% NT-proBNP t48 94% 33% 53% 88% 61% I pazienti con diagnosi di scompenso cardiaco presentavano un numero di linee B superiore rispetto a quelli senza scompenso cardiaco (p<0,05); lo stesso è evidente per i valori di NT-proBNP (p<0,05). Inoltre, la riduzione nel numero di linee B era significativamente maggiore (p<0,005) nel gruppo di pazienti trattati per scompenso cardiaco; non differiva invece significativamente il valore di NT-proBNP (p=0,37). La concordanza intra-operatore si è dimostrata ottima (98%); quella inter-operatore pure ottima tra operatori esperti (98%), discreta (90 %) tra operatori di diversa esperienza. Discussione: L’ecografia del polmone ha dimostrato un ottimo valore di sensibilità ed un discreto valore di specificità nella diagnosi di edema polmonare acuto cariogeno, anche in pazienti sub-acuti, con valori di significatività sovrapponibili a quelli di NT-proBP ma con un ridotto tempo di esecuzione. Le variazioni del quadro ecografico inoltre sembrano correlare con l’appropriatezza del trattamento effettuato, suggerendone un possibile impiego nel monitoraggio della risposta terapeutica. I falsi positivi dell’esame ecografico sono riferibili verosimilmente a forme di imbibizione interstiziale polmonare di origine non cardiogena (es. ARDS). La facilità di esecuzione è testimoniata dall’ottima riproducibilità sia intra- che inter-operatore. Conclusione: L'ecografia del polmone può essere considerata un valido strumento nel riconoscimento dell'origine cardiogena della dispnea. Tale metodica si presenta efficace e rapida, e può essere utilizzata a complemento delle valutazioni standard eseguite nei reparti di medicina interna

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome

Genetic Diagnosis in a Cohort of Adult Patients with Inherited Metabolic Diseases: A Single-Center Experience

Inherited metabolic diseases (IMDs) are genetic conditions that result in metabolism alterations. Although research-based Next Generation Sequencing (NGS) testing for IMD has been recently implemented, its application in a clinical diagnostic setting remains challenging. Thus, we aimed at investigating the genetic diagnostic approach in a cohort of adult patients with IMDs referred to our adult metabolic unit. A retrospective analysis was performed collecting demographic, clinical, and genetic data of patients referred to the Adult Metabolic Unit in Padua from November 2017 to March 2022. In total, 108 adult patients (mean age: 33 years +/- 17, 55% women) were enrolled in the study, and 83 (77%) of the patients transitioned from the pediatric metabolic clinics. The most prevalent groups of IMDs were disorders of complex molecule degradation (32 patients) and disorders of amino acid metabolism (31) followed by disorders of carbohydrates (26). Molecular genetic diagnosis was reported by 69 (64%) patients, with the higher rate reported by patients referred from specialty other than pediatric (88% vs. 55%). Almost all the subjects (92%) with disorders of complex molecule degradation had a genetic diagnosis. Patients with disorders of amino acid metabolism and disorders of carbohydrates had almost the same rate of genetic test (39% and 38%, respectively). Among the patients without a genetic diagnosis that we tested, two novel mutations in disease-associated genes were detected. In our single-center cohort, a consistent proportion (36%) of subjects with IMDs reaches the adulthood without a genetic demonstration of the disease. This lack, even if in some cases could be related to disease-specific diagnostic approach or to different disease onset, could be detrimental to patient management and impact to some of the specific needs of adult subjects

Urine-Derived Epithelial Cells as a New Model to Study Renal Metabolic Phenotypes of Patients with Glycogen Storage Disease 1a

Glycogen storage diseases (GSDs) represent a model of pathological accumulation of glycogen disease in the kidney that, in animal models, results in nephropathy due to abnormal autophagy and mitochondrial function. Patients with Glycogen Storage Disease 1a (GSD1a) accumulate glycogen in the kidneys and suffer a disease resembling diabetic nephropathy that can progress to renal failure. In this study, we addressed whether urine-derived epithelial cells (URECs) from patients with GSD1a maintain their biological features, and whether they can be used as a model to study the renal and metabolic phenotypes of this genetic condition. Studies were performed on cells extracted from urine samples of GSD1a and healthy subjects. URECs were characterized after the fourth passage by transmission electron microscopy and immunofluorescence. Reactive oxygen species (ROS), at different glucose concentrations, were measured by fluorescent staining. We cultured URECs from three patients with GSD1a and three healthy controls. At the fourth passage, URECs from GSD1a patients maintained their massive glycogen content. GSD1a and control cells showed the ciliary structures of renal tubular epithelium and the expression of epithelial (E-cadherin) and renal tubular cells (aquaporin 1 and 2) markers. Moreover, URECs from both groups responded to changes in glucose concentrations by modulating ROS levels. GSD1a cells were featured by a specific response to the low glucose stimulus, which is the condition that more resembles the metabolic derangement of patients with GSD1a. Through this study, we demonstrated that URECs might represent a promising experimental model to study the molecular mechanisms leading to renal damage in GSD1a, due to pathological glycogen storage

Relevance of hepatic lipase to the metabolism of triacylglycerol-rich lipoproteins

HL (hepatic lipase) is a glycoprotein that is synthesized and secreted by the liver, and which binds to heparan sulphate proteoglycans on the surface of sinusoidal endothelial cells and on the external surface of parenchymal cells in the space of Disse. HL catalyses the hydrolysis of triacylglycerols and phospholipids in different lipoproteins, contributing to the remodelling of VLDL (very-low-density lipoprotein) remnants, as well as IDL, LDL and HDL (intermediate-, low- and high-density lipoprotein respectively). HL deficiency in humans is associated with diminished conversion of VLDL remnants into IDL and a near-complete absence of IDL-to-LDL conversion. Remnant lipoproteins and IDL are major determinants of coronary artery disease risk, and accumulation of these lipoproteins in the presence of low HL activity might lead to increased atherosclerosis. In addition to and independently of its lipolytic activity, HL participates as a ligand in promoting the hepatic uptake of remnants and IDL particles, and the latter may represent an additional mechanism linking low HL levels to plasma accumulation of these atherogenic lipoproteins. On the other hand, high HL activity may also result in an increased atherosclerotic risk by promoting the formation of atherogenic small, dense LDL particles. Finally, HL is also synthesized by human macrophages, suggesting that, at the arterial wall site, HL may also contribute locally to promote atherosclerosis by enhancing the formation and retention in the subendothelial space of the arterial wall of VLDL remnants, IDL and small, dense LDL. In conclusion, by interfering with the metabolism of apolipoprotein B100-containing lipoproteins, HL may have pro- as well as anti-atherogenic effects. The anti- or pro-atherogenic role of HL is likely to be modulated by the concurrent presence of other lipid abnormalities (i.e. LDL-cholesterol levels), as well as by the genetic regulation of other enzymes involved in lipoprotein metabolism

Dapaglifozin on Albuminuria in Chronic Kidney Disease Patients with FabrY Disease: The DEFY Study Design and Protocol

Fabry disease (FD) is a rare genetic disorder caused by a deficiency in the α-galactosidase A enzyme, which results in the globotriaosylceramide accumulation in many organs, including the kidneys. Nephropathy is a major FD complication that can progress to end-stage renal disease if not treated early. Although enzyme replacement therapy and chaperone therapy are effective, other treatments such as ACE inhibitors and angiotensin receptor blockers can also provide nephroprotective effects when renal damage is also established. Recently, SGLT2 inhibitors have been approved as innovative drugs for treating chronic kidney disease. Thus, we plan a multicenter observational prospective cohort study to assess the effect of Dapagliflozin, a SGLT2 inhibitor, in FD patients with chronic kidney disease (CKD) stages 1–3. The objectives are to evaluate the effect of Dapagliflozin primarily on albuminuria and secondarily on kidney disease progression and clinical FD stability. Thirdly, any association between SGT2i and cardiac pathology, exercise capacity, kidney and inflammatory biomarkers, quality of life, and psychosocial factors will also be evaluated. The inclusion criteria are age ≥ 18; CKD stages 1–3; and albuminuria despite stable treatment with ERT/Migalastat and ACEi/ARB. The exclusion criteria are immunosuppressive therapy, type 1 diabetes, eGFR &lt; 30 mL/min/1.73 m2, and recurrent UTIs. Baseline, 12-month, and 24-month visits will be scheduled to collect demographic, clinical, biochemical, and urinary data. Additionally, an exercise capacity and psychosocial assessment will be performed. The study could provide new insights into using SGLT2 inhibitors for treating kidney manifestations in Fabry disease