Discovering Genotype Variants in an Infant with VACTERL through Clinical Exome Sequencing: A Support for Personalized Risk Assessment and Disease Prevention

Abstract: Congenital anomalies may have an increased risk of noncommunicable diseases (NCDs) We performed a clinical exome analysis in an infant affected by “Vertebral, Anorectal, Cardiac, Tracheoesophageal, Genitourinary, and Limb” (VACTERL) malformation association to identify potential biomarkers that may be helpful for preventing malignancy risk or other chronic processes. Among the variants, six variants that may be linked with VACTERL were identified in the exome analysis. The variants c.501G>C on OLR1 and c.-8C>G on PSMA6 were previously associated with myocardial infarction. The variants c.1936A>G on AKAP10 and c.575A>G on PON1 are linked to defects in cardiac conduction and artery disease, respectively. Alterations in metabolism were also suggested by the variants c.860G>A on EPHX2 and c.214C>A on GHRL. In addition, three variants associated with colon cancer were discovered. Specifically, the reported variants were c.723G>A on CCND1 and c.91T>A on AURKA proto-oncogenes as well as c.827A>C in the tumor suppressor PTPRJ. A further inspection identified 15 rare variants carried by cancer genes. Specifically, these mutations are located on five tumor suppressors (SDHA, RB1CC1, PTCH1, DMBT1, BCR) and eight proto-oncogenes (MERTK, CSF1R, MYB, ROS1, PCM1, FGFR2, MYH11, BRCC3) and have an allele frequency lower than 0.01 in the Genome Aggregation Database (GnomAD).We observed that the cardiac and metabolic phenotypic traits are linked with the genotype of the patient. In addition, the risk of developing neoplasia cannot be excluded a priori. Long-term surgical issues of patients with VATER syndrome could benefit from the clinical exome sequencing of a personalized risk assessment for the appearance of further disease in pubertal timing and adult age

Biochemical and clinical relevance of alpha lipoic acid: antioxidant and anti-inflammatory activity, molecular pathways and therapeutic potential

The molecular nature of lipoic acid (LA) clarifies its capability of taking part to a variety of biochemical reactions where redox state is meaningful. The pivotal action of LA is the antioxidant activity due to its ability to scavenge and inactivate free radicals. Furthermore, LA has been shown to chelate toxic metals both directly and indirectly by its capability to enhance intracellular glutathione (GSH) levels. This last property is due to its ability to interact with GSH and recycle endogenous GSH. LA exhibits significant antioxidant activity protecting against oxidative damage in several diseases, including neurodegenerative disorders. Interestingly, LA is unique among natural antioxidants for its capability to satisfy a lot of requirements, making it a potentially highly effective therapeutic agent for many conditions related with oxidative damage. In particular, there are evidences showing that LA has therapeutic activity in lowering glucose levels in diabetic conditions. Similarly, LA supplementation has multiple beneficial effects on the regression of the mitochondrial function and on oxidative stress associated with several diseases and aging

Effect of Lipoic Acid on the Biochemical Mechanisms of Resistance to Bortezomib in SH-SY5Y Neuroblastoma Cells

Neuroblastoma (NB) is an extracranial solid cancer and the most common cancer in infancy. Despite the standard treatment for NB is based on the combination of chemotherapeutic drugs such as doxorubicin, vincristine, cyclophosphamide, and cisplatin, chemoresistance occurs over the time. The aim of the present research was to evaluate the effect of bortezomib (BTZ) (50 nM) on NB cell viability and how lipoic acid (ALA) (100 μM) modifies pharmacological response to this chemotherapeutic agent. Cell viability was assessed by ATP luminescence assay whereas expression of oxidative stress marker (i.e., heme oxygenase-1) and endoplasmic reticulum stress proteins was performed by real-time PCR, western blot, and immunofluorescence. Our data showed that BTZ treatment significantly reduced cell viability when compared to untreated cultures (about 40%). Interestingly, ALA significantly reduced the efficacy of BTZ (about 30%). Furthermore, BTZ significantly induced heme oxygenase-1 as a result of increased oxidative stress and such overexpression was prevented by concomitant treatment with ALA. Similarly, ALA significantly reduced BTZ-mediated endoplasmic reticulum stress as measured by reduction in BiP1 and IRE1α, ERO1α, and PDI expression. In conclusion, our data suggest that BTZ efficacy is dependent on cellular redox status and such mechanisms may be responsible of chemoresistance to this chemotherapeutic agent

Lack of the Nlrp3 Inflammasome Improves Mice Recovery Following Traumatic Brain Injury

Treatment for traumatic brain injury (TBI) remains elusive despite compelling evidence from animal models for a variety of therapeutic targets. The activation of the NLRP3 (Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome has been proposed as key point in the brain damage associated with TBI. NLRP3 was tested as potential target for reducing neuronal loss and promoting functional recovery in a mouse model of TBI. Male NLRP3-/- (n = 20) and wild type (n = 27) mice were used. A closed TBI model was performed and inflammatory and apoptotic markers were evaluated. A group of WT mice also received BAY 11-7082, a NLRP3 inhibitor, to further evaluate the role of this pathway. At 24 h following TBI NLRP3-/- animals demonstrated a preserved cognitive function as compared to WT mice, additionally brain damage was less severe and the inflammatory mediators were reduced in brain lysates. The administration of BAY 11-7082 in WT animals subjected to TBI produced overlapping results. At day 7 histology revealed a more conserved brain structure with reduced damage in TBI NLRP3-/- animals compared to WT. Our data indicate that the NLRP3 pathway might be exploited as molecular target for the short-term sequelae of TBI

Effect of alpha lipoic acid and choline-containing compounds on the expression of some astroglial biomarkers during proliferation and differentiation of astrocytes and neuroblastoma cultures

La prima sessione del presente studio è stata finalizzata a valutare gli effetti del trattamento per 24h con l'acetilcolina ed altri precursori colinergici come CDP-colina, colina alphoscerato sull espressione di alcuni markers in colture primarie di astrociti di ratto a 14, 21 e 35 giorni in vitro (DIV). Inoltre, l'acido alpha-lipoico svolge un ruolo fondamentale come componente antiossidante e metabolico di alcuni complessi enzimatici coinvolti nel metabolismo del glucosio di tipi cellulari, nella seconda sessione della ricerca abbiamo valutato l'effetto del trattamento di (+)acido lipoico o ( +/-)acido lipoico e/o 10 mM alpha-GPC su colture astrogliali studiando l'espressione di alcuni marcatori di proliferazione e differenziazione cellulare. Nella terza parte della ricerca, abbiamo studiato gli estrogeni e le attività di fattori di crescita (GFs) come mitogeni in grado di stimolare la proliferazione cellulare, analizzando le interazioni tra il fattore di crescita di competenza bFGF e/o estrogeno 17-beta-estradiolo + fattori di crescita di progressione (EGF o IGF-I o INS) sul DNA labeling oltre che sulla proliferazione e la differenziazione cellulare astrogliale in differenti condizioni sperimentali. Infine, nell'ultima sessione della ricerca abbiamo valutato gli effetti antiossidanti dell acido lipoico (LA) in combinazione con il bortezomib (BTZ) su linee cellulari di neuroblastoma (NB). Abbiamo studiato la modulazione dell emeossigenasi-1 (HO-1), del gene HMOX-1, nonché gli chaperoni BIP1, IRE1, Ero1 e PD1, attivati dalle cellule al fine di contrastare la risposta allo stress da reticolo endoplasmatico (ER). Per quanto riguarda i risultati ottenuti nei nostri colture cellulari astrogliali trattati con acido alpha-lipoico ed alpha-GPC da soli o entrambi in combinazione, è particolarmente importante sottolineare che l'aggiunta di farmaci singoli (acido alpha-lipoico o alpha-GPC) ha indotto un up modulazione espressione di biomarcatori utilizzati nel nostro studio. Al contrario, il co-trattamento con l'acido alpha-lipoico + alpha-GPC ha mostrato nessuna modifica significativa dei biomarcatori utilizzati (GFAP, vimentina, ciclina D1, O.D.C. e MAP-chinasi). Sono necessari ulteriori studi al fine di meglio chiarire il meccanismo specifico evocato dal trattamento di tali agenti neuroprotettivi nei nostri modelli in vitro. Una possibile spiegazione di questo risultato inatteso può dipendere dal fatto che, alpha-lipoico, agendo come agente che mima l azione dell'insulina e stimolando i recettori, potrebbe provocare un meccanismo d'azione che possa interferire sul comune percorso di trasduzione del segnale. Per quanto riguarda l'effetto di LA nei meccanismi di resistenza al bortezomib (BTZ) in cellule di neuroblastoma, i nostri dati indicano che LA, in combinazione con BTZ, agisce come chaperone chimico ridurre la risposta allo stress indotto dal proteasoma. Nelle nostre condizioni sperimentali, è stato osservata una upregulation dell emeossigenasi (HO-1) in seguito al trattamento BTZ su tutte le linee cellulari testate di NB, suggerendo un ruolo protettivo contro gli effetti indotti da BTZ Nel nostro modello sperimentale, non abbiamo osservato l'attivazione delle proteine autofagia legati a cellule di NB trattate con BTZ, quindi supponiamo che viene attivata l autofagia per promuovere la sopravvivenza delle cellule. Inoltre, abbiamo osservato un significativo aumento dell'autofagia in seguito al trattamento sia con la combinazione di BTZ e LA. Inoltre, LA è in grado di aumentare l'autofagia nelle sole cellule NB rispetto al solo trattamento con BTZ. I nostri dati confermano che LA protegge le cellule NB dallo stress ossidativo e riducendo il danno indotto dallo stress da ER dovuto al trattamento con BTZ e attiva autofagia come meccanismo di sopravvivenza cellulare

Improving motor performance in Parkinson's disease: a preliminary study on the promising use of the computer assisted virtual reality environment (CAREN)

Background: Parkinson's disease (PD) is a neurodegenerative disorder characterized by various motor symptoms including balance and gait impairment. Several studies have shown that physiotherapy, cueing techniques, treadmill training, and cognitive movement strategies are useful in improving balance and gait in patients with PD. Devices employing virtual reality (VR) have been shown to be promising in neurorehabilitation as they can provide the patients with multisensory stimulation creating a realistic environment and improve the motivation and the adhesion of patients to the rehabilitation program. This preliminary study is aimed at testing the efficacy and feasibility of gait training based on the computer-assisted virtual reality environment (CAREN) in a sample of PD. Methods: In this preliminary study, 22 outpatients affected by PD who attended the Behavioral and Robotic Neurorehab Laboratory of the IRCCS Neurolesi between August 2017 and October 2018 were enrolled. All PD patients underwent 20 conventional physiotherapy sessions followed by 3-month of rest. Then, the patients were provided with 20 sessions of CAREN training. Gait and balance performances were rated before, after each training protocol, and 3 months later. Gait analysis was also performed before and after CAREN training. Results: All patients completed both of the rehabilitation trainings without any adverse event. All considered scales improved significantly at the end of both rehabilitation treatments. However, patients presented with a greater clinical improvement after the CAREN training, compared with conventional physiotherapy. In particular, patients walked faster and with more stability, with wider, longer steps. Conclusions: Even though further neurophysiological details are required to identify the patients who may benefit from CAREN training, our findings suggest that this innovative device is an effective and feasible tool to train balance and gait in patients with PD

Dopamine, vesicular transporters, and dopamine receptor expression in rat major salivary glands

The localization of dopamine stores and the expression and localization of dopamine (DAT) and vesicular monoamine transporters (VMAT) type-1 and -2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat submandibular, sublingual, and parotid salivary glands by HPLC with electrochemical detection, as well as immunochemical and immunohistochemical techniques. Male Wistar rats of 2 mo of age were used. The highest dopamine levels were measured in the parotid gland, followed by the submandibular and sublingual glands. Western blot analysis revealed DAT, VMAT-1, VMAT-2, and dopamine receptors immunoreactivity in membrane preparations obtained from the three glands investigated. Immunostaining for dopamine and transporters was developed within striated ducts. Salivary glands processed for dopamine receptors immunohistochemistry developed an immunoreaction primarily in striated and excretory ducts. In the submandibular gland, acinar cells displayed strong immunoreactivity for the D2 receptor, while cells of the convoluted granular tubules were negative for both D1-like and D2-like receptors. Parotid glands acinar cells displayed the highest immunoreactivity for both D1 and D2 receptors compared with other salivary glands. The above localization of dopamine and dopaminergic markers investigated did not correspond closely with neuron-specific enolase (NSE) localization. This indicates that at least in part, catecholamine stores and dopaminergic markers are independent from glandular innervation. These findings suggest that rat major salivary glands express a dopaminergic system probably involved in salivary secretion. The stronger immunoreactivity for dopamine transporters and receptors in striated duct cells suggests that the dopaminergic system could regulate not only quality, but also volume and ionic concentration of saliva