Permitted daily exposure for diisopropyl ether as a residual solvent in pharmaceuticals

Solvents can be used in the manufacture of medicinal products provided their residual levels in the final product comply with the acceptable limits based on safety data. At worldwide level, these limits are set by the "Guideline Q3C (R6) on impurities: guideline for residual solvents" issued by the ICH. Diisopropyl ether (DIPE) is a widely used solvent but the possibility of using it in the pharmaceutical manufacture is uncertain because the ICH Q3C guideline includes it in the group of solvents for which "no adequate toxicological data on which to base a Permitted Daily Exposure (PDE) was found". We performed a risk assessment of DIPE based on available toxicological data, after carefully assessing their reliability using the Klimisch score approach. We found sufficiently reliable studies investigating subchronic, developmental, neurological toxicity and carcinogenicity in rats and genotoxicity in vitro. Recent studies also investigated a wide array of toxic effects of gasoline/DIPE mixtures as compared to gasoline alone, thus allowing identifying the effects of DIPE itself. These data allowed a comprehensive toxicological evaluation of DIPE. The main target organs of DIPE toxicity were liver and kidney. DIPE was not teratogen and had no genotoxic effects, either in vitro or in vivo. However, it appeared to increase the number of malignant tumors in rats. Therefore, DIPE could be considered as a non-genotoxic animal carcinogen and a PDE of 0.98 mg/day was calculated based on the lowest No Observed Effect Level (NOEL) value of 356 mg/m3 (corresponding to 49 mg/kg/day) for maternal toxicity in developmental rat toxicity study. In a worst-case scenario, using an exceedingly high daily dose of 10 g/day, allowed DIPE concentration in pharmaceutical substances would be 98 ppm, which is in the range of concentration limits for ICH Q3C guideline class 2 solvents. This result might be considered for regulatory decisions

Post-transcriptional regulation of HTLV gene expression: Rex to the rescue

Human T-lymphotropic virus type 1 (HTLV-1) and other members of the Deltaretrovirus genus code for a regulatory protein named Rex that binds to the Rex-responsive element present on viral mRNAs. Rex rescues viral mRNAs from complete splicing or degradation and guides them to the cytoplasm for translation. The activity of Rex is essential for expression of viral transcripts coding for the virion components and thus represents a potential target for virus eradication. We present an overview of the functional properties of the HTLV-1 and HTLV-2 Rex proteins (Rex-1 and Rex-2), outline mechanisms controlling Rex function, and discuss similarities and differences in the sequences of Rex coded by HTLV-1, -2, -3, and -4 that may influence their molecular anatomy and functional properties

HTLV-1 infection and pathogenesis: new insights from cellular and animal models

Since the discovery of the human T-cell leukemia virus-1 (HTLV-1), cellular and animal models have provided invaluable contributions in the knowledge of viral infection, transmission and progression of HTLV-associated diseases. HTLV-1 is the causative agent of the aggressive adult T-cell leukemia/lymphoma and inflammatory diseases such as the HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Cell models contribute to defining the role of HTLV proteins, as well as the mechanisms of cell-to-cell transmission of the virus. Otherwise, selected and engineered animal models are currently applied to recapitulate in vivo the HTLV-1 associated pathogenesis and to verify the effectiveness of viral therapy and host immune response. Here we review the current cell models for studying virus-host interaction, cellular restriction factors and cell pathway deregulation mediated by HTLV products. We recapitulate the most effective animal models applied to investigate the pathogenesis of HTLV-1-associated diseases such as transgenic and humanized mice, rabbit and monkey models. Finally, we summarize the studies on STLV and BLV, two closely related HTLV-1 viruses in animals. The most recent anticancer and HAM/TSP therapies are also discussed in view of the most reliable experimental models that may accelerate the translation from the experimental findings to effective therapies in infected patients

Characterization and functional analysis of cis-acting elements of the human farnesyl diphosphate synthetase (FDPS) gene 5′ flanking region

AbstractFarnesyl diphosphate synthetase (FDPS) is a key enzyme in the isoprenoid pathway responsible for cholesterol biosynthesis, post-translational protein modifications and synthesis of steroid hormones, whose expression is regulated by phorbol esters and polyunsaturated fatty acids.Genomic comparison of the 5′ upstream sequence of the FDPS genes identifies conserved binding sites for NF-Y, SP1, SRE3, and YY1 regulatory elements in rat, mouse, dog and chimpanzee. Two additional specific consensus sequences, upstream of the core promoter that had not been analysed previously, are shared only by human and chimpanzee genomes.The work presented here aimed at characterizing these genomic sequence elements in the human FDPS promoter region and their contribution to gene expression. We have characterized functionally the minimal basal promoter of the human FDPS gene by means of deletion mutants and we have identified two cis-acting elements which modulate the FDPS gene expression and are recognized by Pax5 and OCT-1 transcription factors

The emerging role of the RBM20 and PTBP1 ribonucleoproteins in heart development and cardiovascular diseases

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies

Molecular and lifestyle factors modulating obesity disease

Obesity adversely affects bone health by means of multiple mechanisms, e.g., alterations in bone-regulating hormones, inflammation, and oxidative stress. Substantial evidence supports the relationship between adiposity and bone disorders in overweight/obese individuals. It is well known that the balance between mutually exclusive differentiation of progenitor cells into osteoblasts or adipocytes is controlled by different agents, including growth factors, hormones, genetic and epigenetic factors. Furthermore, an association between vitamin D deficiency and obesity has been reported. On the other hand, regular physical activity plays a key role in weight control, in the reduction of obesity-associated risks and promotes osteogenesis. The aim of this review is to highlight relevant cellular and molecular aspects for over-weight containment. In this context, the modulation of progenitor cells during differentiation as well as the role of epigenetics and microbiota in obesity disease will be discussed. Furthermore, lifestyle changes including an optimized diet as well as targeted physical activity will be suggested as strategies for the treatment of obesity disease

Advanced cellular models for rare disease study: exploring neural, muscle and skeletal organoids

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed

A CRISPR/Cas9 based approach to study the implication of HTLV regulatory proteins in the NF-κB modulation.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive form of T-cell malignancy with no cure. The HTLV-1 oncoprotein Tax plays a key role in CD4+ T-cell transformation, mainly through constitutive activation of both the canonical and the alternative NF-κB pathways. The HTLV-1 basic zipper protein (HBZ), encoded by the antisense viral genome strand, plays an essential role in the oncogenic process in concert with Tax, mediating T-cell proliferation. Unlike HTLV-1, the genetically related retrovirus HTLV-2 is not associated with ATL diseases. Functional comparisons between HTLV-1 regulatory proteins, Tax-1 and HBZ, and the HTLV-2 homologs, Tax-2 and APH-2, may highlight different mechanisms of their oncogenic potential. The aim of this study is to investigate how the antisense proteins HBZ and APH-2 impaired the NF-κB pathway activation. We found that both HBZ and APH-2 antagonized the NF-κB promoter activity mediated by Tax, but not in the same extent. Analyzing the intracellular distribution of the antisense proteins, we found that APH-2 is retained in cytoplasm complexes, whereas HBZ is mainly distributed into the nucleus. We observed that in presence of APH-2 and Tax-2, the degradation of the IκB-α inhibitor was reduced. Moreover, we found that unlike HBZ, APH-2 formed complexes with an upstream inhibitor of the alternative NF-κB pathway, the TNF receptor-associated factor 3, TRAF3. We generated a TRAF3 knock-out cell line applying the CRISPR/Cas9-mediated genome editing. By luciferase assays, we showed that TRAF3 is not required for Tax mediated NFκB promoter activation. Analyses are in progress to test the inhibitory effect of the antisense HBZ and APH-2 proteins on NF-κB promoter activity in absence of TRAF3. The results of this study may contribute to clarify the effect of the alternative NF-κB viral deregulation pathway in the expression of proinflammatory genes

Gospodarski ulov, reprodukcija i prehrambene navike raže zvjezdopjege Raja asterias (Chondrichthyes: Rajidae) u priobalju Tirenskog mora (Italija, sjeverni Mediteran)

A total of 52 “rapido” (towed toothed beam gears) trawls were monitored in late winter-summer of the 1999-2000 period to assess the R. asterias size structure at this time of higher yields as well as 36 fishing operations performed by “volantina” (trawl nets with fairly high vertical opening) during distinct seasons on the continental shelf off the fishing harbour of Fiumicino (central western Italy) to gain data also for that gear. Daily yields recorded for the only boat locally authorised to use “rapido” nets gave median values of 32.0 individuals and 24.35 kg vs. 2.5 rays and 2.80 kg for trawlers fishing at the same time. Comparison of the body sizes at which 50% of the skates had been found mature in our samples (265 gonads examined) showed that most specimens caught by the “rapido” nets were in their juvenile stage. Examination of stomach contents from 129 skates confirmed previous reports that they mainly feed on crustaceans and bony fish and the role of the latter in the diet progressively increases as R. asterias specimens grow older and larger.Istraživana su ukupno 52 potega dredžama (“rapido”) u kasnom zimskom-ljetnom periodu 1999.-2000. godine kako bi se ustanovila veličina raže zvjezdopjege, R. asterias u vrijeme većeg ulova. Obavljeno je 36 ribarstvenih uzorkovanja pomoću “volantina” (koće većeg okomitog raspona) tijekom različitih godišnjih doba na kontinentalnom šelfu pokraj luke Fiumicino (srednji zapadni dio Italije) kako bi se dobili podaci i o ovom ribarskom alatu. Dnevni ulov zabilježen na brodu registriranom za uporabu “rapido” mreža iznosio je 32.0 jedniki i 24.35 kg od toga 2.5 kg raža i 2.80 kg koćarskog ulova istovremeno. Usporedbom veličine tijela uzoraka 50% raža je bilo zrelo (265 ispitanih gonada) što ukazuje na činjenicu da je većina ulovljenih primjeraka “rapido” mrežom bilo u juvenilnom stadiju. Ispitivanje želučanog sadržaja kod 129 raža potvrdilo je dosadašnja izvješća da se pretežito hrane rakovima i koštunjičavim ribama, koje su zastupljenije u prehrani starijih i većih primjeraka R. asterias