D-Bifunctional Protein Deficiency Diagnosis-A Challenge in Low Resource Settings: Case Report and Review of the Literature.

D-bifunctional protein deficiency (D-BPD) is a rare, autosomal recessive peroxisomal disorder that affects the breakdown of long-chain fatty acids. Patients with D-BPD typically present during the neonatal period with hypotonia, seizures, and facial dysmorphism, followed by severe developmental delay and early mortality. While some patients have survived past two years of age, the detectable enzyme activity in these rare cases was likely a contributing factor. We report a D-BPD case and comment on challenges faced in diagnosis based on a narrative literature review. An overview of Romania's first patient diagnosed with D-BPD is provided, including clinical presentation, imaging, biochemical, molecular data, and clinical course. Establishing a diagnosis can be challenging, as the clinical picture is often incomplete or similar to many other conditions. Our patient was diagnosed with type I D-BPD based on whole-exome sequencing (WES) results revealing a pathogenic frameshift variant of the HSD17B4 gene, c788del, p(Pro263GInfs*2), previously identified in another D-BPD patient. WES also identified a variant of the SUOX gene with unclear significance. We advocate for using molecular diagnosis in critically ill newborns and infants to improve care, reduce healthcare costs, and allow for familial counseling.

Colorectal Carcinomas: Searching for New Histological Parameters Associated with Lymph Node Metastases.

Background and Objectives: Colorectal cancer (CRC) continues to be an essential public health problem. Our study aimed to evaluate the prognostic significance of classic prognostic factors and some less-studied histopathological parameters in CRC. Materials and Methods: We performed a retrospective study on 71 colorectal carcinoma patients who underwent surgery at the "Pius Brînzeu" County Clinical Emergency Hospital in Timișoara, Romania. We analyzed the classic parameters but also tumor budding (TB), poorly differentiated clusters (PDCs) of cells, tumor-infiltrating lymphocytes (TILs), and the configuration of the tumor border on hematoxylin-eosin slides. Results: A high degree of malignancy (p = 0.006), deep invasion of the intestinal wall (p = 0.003), an advanced stage of the disease (p < 0.0001), lymphovascular invasion (p < 0.0001), perineural invasion (p < 0.0001), high-grade TB (p < 0.0001), high-grade PDCs (p < 0.0001), infiltrative tumor border configuration (p < 0.0001) showed a positive correlation with lymph node metastases. Conclusions: The analyzed parameters positively correlate with unfavorable prognostic factors in CRC. We highlight the value of classic prognostic factors along with a series of less-known parameters that are more accessible and easier to evaluate using standard staining techniques and that could predict the risk of relapse or aggressive evolution in patients with CRC.

Invasive Cutaneous Melanoma: Evaluating the Prognostic Significance of Some Parameters Associated with Lymph Node Metastases.

Background and Objectives: This study aimed to assess the clinical-pathological profile of patients with invasive cutaneous melanomas and to identify the parameters with a prognostic role in the lymph nodal spread of this malignant tumor. Materials and Methods: We performed a retrospective study on patients with invasive cutaneous melanomas who underwent surgery in the "Pius Brînzeu" County Clinical Emergency Hospital from Timișoara, Romania, and were evaluated for the status of loco-regional lymph nodes. We selected and analyzed some parameters searching for their relationship with lymph node metastases. Results: We identified 79 patients with invasive cutaneous melanomas (29 men and 50 women, mean age 59.36 years). A percentage of 58.3% of melanomas had Breslow tumor thickness >2 mm; 69.6% of melanomas showed a Clark level IV-V. Tumor ulceration was present in 59.5% of melanomas. A mitotic rate of ≥5 mitoses/mm2 was observed in 48.1% of melanomas. Tumor-infiltrating lymphocytes (TILs), non-brisk, were present in 59.5% of cases and 22.8% of patients had satellite/in-transit metastasis (SINTM). Tumor regression was identified in 44.3% of cases. Lymph nodes metastases were found in 43.1% of patients. Statistical analysis showed that lymph node metastases were more frequent in melanomas with Breslow thickness >2 mm (p = 0.0002), high Clark level (p = 0.0026), mitotic rate >5 mitoses/mm2 (p = 0.0044), ulceration (p = 0.0107), lymphovascular invasion (p = 0.0182), SINTM (p = 0.0302), and non-brisk TILs (p = 0.0302). Conclusions: The Breslow thickness >2 mm, high Clark level, high mitotic rate and ulceration are the most important prognostic factors for lymph nodal spread in cutaneous melanomas. However, some melanomas without these clinical-pathological features can have an unexpected, aggressive evolution, which entails the necessity of close and prolonged clinical follow-up of patients, including those with lesions considered without risk.

Poorly differentiated clusters and tumor budding are important prognostic factors in colorectal carcinomas.

The aim of our study was to assess the prognostic value of the two new grading systems based on the quantification of tumor budding - TB (GBd) and poorly differentiated clusters - PDCs (PDCs-G) in colorectal carcinomas (CRC). We performed a retrospective study on 71 CRC patients who underwent surgery at the Emergency County Hospital, Timișoara. CRC cases were classified based on haematoxylin-eosin slides, using the conventional grading system, GBd and PDCs-G, respectively. We used two-tier and three-tier grading schemes for each system. Subsequently,  we evaluated  associations with other prognostic factors in CRC. Based on the three-tier GBd (GBd-3t)  most cases (34/69, 49.27%) were classified as G3Bd-3t, while based on the conventional grading system, the majority of the cases (55/69, 79.71%) were considered G2. On the other hand, based on the three-tier PDCs-G system (PDCs-G-3t), most cases (31/69, 44.93%) were PDCs-G2-3t. We also noted a more significant association of GBd-3t with other prognostic parameters analyzed, as compared to the conventional grading system. Nodal status, tumor stage, and lymphovascular invasion were strongly correlated with GBd-3t (p=0.0001). Furthermore, we noted that PDCs-G-3t correlated more significantly than the conventional grading system with nodal status (p<0.0001), tumor stage (p=0.0003), lymphovascular invasion (p<0.0001), perineural invasion (p=0.005) and the tumor border configuration (p<0.0001). High GBd and PDCs-G grades correlate directly with other negative prognostic factors in CRC.Thus, these new parameters/classification methods could be used as additional tools for risk stratification in patients with CRC.

Cyclin A is a mediator of p120E4F-dependent cell cycle arrest in G1.

E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120E4F expression induces cell cycle arrest in G1. Although p21WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120E4F can induce a G1 block in p21(-/-) cells, suggesting that other proteins are essential for the p120E4F-dependent block in G1. We show here that cyclin A promoter activity can be repressed by p120E4F and that this repression correlates with p120E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120E4F-arrested cells from the G1 block. These data identify the cyclin A gene as a cellular target for p120E4F and suggest a mechanism for p120E4F-dependent cell cycle regulation.

The influence of fetal position on nuchal translucency thickness.

OBJECTIVE: To determine whether nuchal translucency thickness is influenced by the fetal position at ultrasound examination. SUBJECTS: Transabdominal ultrasound examination for pregnancy dating and measurement of nuchal translucency thickness was performed at 10-14 weeks' gestation in all women attending the antenatal clinic of our hospital. During the examination special attention was paid to a change in fetal position from prone to supine or vice versa. METHODS: For each fetus the nuchal translucency measurement was repeated when a positional change from prone to supine or vice versa was recorded. All measurements were recorded on hard copy. An image-scoring method was used and evaluated by three independent reviewers. RESULTS: Eighty-five fetuses were included in this study. The mean nuchal translucency for supine fetuses was 1.91 mm compared with 1.93 mm for prone fetuses. The mean quality-score was 6.54 for supine fetuses and 6.55 for prone fetuses. This difference was not statistically significant. CONCLUSION: Fetal position has no influence on the measurement of nuchal translucency.

Differential effect of Rac and Cdc42 on p38 kinase activity and cell cycle progression of nonadherent primary mouse fibroblasts.

The Rho GTPases play an important role in transducing signals linking plasma membrane receptors to the organization of the cytoskeleton and also regulate gene transcription. Here, we show that expression of constitutively active Ras or Cdc42, but not RhoA, RhoG, and Rac1, is sufficient to cause anchorage-independent cell cycle progression of mouse embryonic fibroblasts. However, in anchorage free conditions, whereas activation of either Cdc42 or Ras results in cyclin A transcription and cell cycle progression, Cdc42 is not required for Ras-mediated cyclin A induction, and the two proteins act in a synergistic manner in this process. Surprisingly, the ability of Cdc42 to induce p38 MAPK activity in suspended mouse embryonic fibroblast was impaired. Moreover, inhibition of p38 activity allowed Rac1 to induce anchorage-independent cyclin A transcription, indicating that p38 MAPK has an inhibitory function on cell cycle progression of primary fibroblasts. Finally, a Rac mutant, which is unable to induce lamellipodia and focal complex formation, promoted cyclin A transcription in the presence of SB203580, suggesting that the organization of the cytoskeleton is not required for anchorage-independent proliferation. This demonstrates a novel function for Cdc42, distinct from that of Rac1, in the control of cell proliferation.

Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein.

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.

CHF: a novel factor binding to cyclin A CHR corepressor element.

Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5' element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3' element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.

A novel calcium signaling pathway targets the c-fos intragenic transcriptional pausing site.

In many cell types, increased intracellular calcium gives rise to a robust induction of c-fos gene expression. Here we show that in mouse Ltk(-) fibroblasts, calcium ionophore acts in synergy with either cAMP or PMA to strongly induce the endogenous c-fos gene. Run-on analysis shows that this corresponds to a substantial increase in active polymerases on downstream gene sequences, i.e. relief of an elongation block by calcium. Correspondingly a chimeric gene, in which the human metallothionein promoter is fused to the fos gene, is strongly induced by ionophore alone, unlike a c-fos promoter/beta-globin coding unit chimeric construct. Internal deletions in the hMT-fos reporter localize the intragenic calcium regulatory element to the 5' portion of intron 1, thereby confirming and extending previous in vitro mapping data. Ionophore induced cAMP response element-binding protein phosphorylation on Ser(133) without affecting the extracellular signal-regulated kinase cascade. Surprisingly, induction involved neither CaM-Ks nor calcineurin, while the calmodulin antagonist W7 activated c-fos transcription on its own. These data suggest that a novel calcium signaling pathway mediates intragenic regulation of c-fos expression via suppression of a transcriptional pause site.

Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent phosphorylation.

Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J. (1994) J. Biol. Chem. 269, 26559-26567). Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower Km value for the co-substrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMP-dependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser32, a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects.

Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb.

Many cells, when cultured in suspension, fail to express cyclin A, a regulatory component of cell cycle kinases cdc2 and cdk2 and as a consequence, do not enter S phase. However, many cell type-specific differences are disclosed between not only normal and transformed cells, but also between cell lines whose proliferation is strictly anchorage-dependent. These apparent discrepancies are seen in established cell lines most probably because of adaptative events that have occurred during cell culture. We have therefore used primary cells to understand how cyclin A transcription is controlled by cell anchorage properties. To this aim, we have used embryonic fibroblasts from either wild type, Rb(-/-) or p107(-/-)/p130(-/-) mice and tested the effect of an ectopic expression of Rb mutants. In the experiments reported here, we show that anchorage-dependent expression of cyclin A (i) is reflected by the in vivo occupancy of a negative DNA regulatory element previously shown to be instrumental in the down regulation of cyclin A transcription in quiescent cells (Cell Cycle Responsive Element: CCRE) (ii) requires a functional Rb but neither p107 nor p130 (iii) mutation of the CCRE abolishes both adhesion-dependent regulation and response to Rb.

[Cyclin A: a good markers for the study of cell cycle control and tumor progression?]. / La cycline A: un bon marqueur pour l'étude du contrôle du cycle cellulaire et de la progression tumorale?

Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies conducted in established cell lines and in primary human T lymphocytes, have demonstrated that the promoter of its gene is under negative transcriptional control in quiescent cells. The DNA sequences mediating this repression have been delineated through in vitro mutagenesis as well as in vivo genomic footprinting experiments. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). Using primary fibroblasts from either pRb(-/-), p107(-/-), p130(-/-) or p107(-/-)/p130(-/-) mice, we show in this work that mutation of the pRb gene has the more profound effect on cyclin A transcription. Finally, normal fibroblasts cultured in suspension fail to express cyclin A and can no longer enter S phase and proliferate, revealing thus a dependence of cyclin A expression on cell anchorage. Our work suggests the existence of at least two sets of regulators controlling cell cycle progression. On the one hand, proteins like cyclin D1, whose expression is a direct consequence of the activation of the ras signalling pathway and on the other hand, proteins like cyclin A which are secondary response effectors. As a result, growth factor stimulation leads to a transcriptional activation of the former set, while the transcription of the latter set is under the control of a repressor whose effect is alleviated after triggering the ras cascade. The status of pRb thus dictates whether cells continue their progression through the cell cycle when ras is mutated, probably by allowing the uncontrolled expression of critical genes like cyclin A.

The retinoblastoma protein is essential for cyclin A repression in quiescent cells.

Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies have demonstrated that the promoter of its gene is under transcriptional repression in quiescent cells. Whereas the DNA sequences mediating this effect have been clearly delineated, the nature of the proteins acting in trans is still debated. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). However, the precise role of these proteins has been difficult to assess, since most experiments designed to analyse their function have been carried out in transformed cell lines. Nevertheless, a current model has emerged whereby the role of the p130 protein would be restricted to resting and early G1 cells and p107, absent in quiescent cells, would be involved later in the control of the G1/S transition, whilst pRb would be effective throughout the cell cycle. We show here that cyclin A transcriptional inhibition is relieved in primary fibroblasts from pRb(-/-) embryos and not in fibroblasts from p13O(-/-), p107(-/-) or even p130(-/-)/p107(-/-) double mutant embryos. This suggests a unique role for pRb in controlling the extinction of specific genes in G0, providing thus the first example of non-overlapping functions achieved by the different pocket proteins.

Cyclin A expression is under negative transcriptional control during the cell cycle.

Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.

c-fos mRNA instability determinants present within both the coding and the 3' non coding region link the degradation of this mRNA to its translation.

The instability of oncogenic mRNA such as c-fos mRNA is controlled in cis by sequences present in both the coding and the 3' untranslated regions (3'UTR). The latter contains AU-rich elements (ARE) which, depending on the cellular context, mediate either their rapid degradation or inhibit their translation. These observations, along with the known increase of the life spans of many unstable mRNA promoted by inhibitors of protein synthesis, raise the possibility that both processes are linked. To investigate further the putative involvement of translation in both coding region and ARE-mediated rapid decay of c-fos mRNA, we designed an expression vector based on the use of the ferritin mRNA iron regulatory element (IRE). The latter structure links translation to intracellular iron concentration when inserted at the proper location within the 5'UTR. Rapid degradation of a beta-globin/c-fos 3'UTR construct was prevented by Desferrioxamine, an iron chelator, and facilitated by ferric ammonium citrate or hemin, while stability of other mRNAs not containing the IRE or the ARE were unchanged. The same conclusion was reached when the stability of a c-fos mRNA devoid of ARE was assessed in function of iron availability.

TGF-beta 1 and cAMP attenuate cyclin A gene transcription via a cAMP responsive element through independent pathways.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of the proliferation of many cell lines. The expression of Cyclin A is down-regulated by TGF-beta 1 in Chinese hamster lung fibroblasts and most of this effect is mediated at the transcriptional level through a cAMP-responsive element (CRE), but does not require a functional cAMP-dependent protein kinase. However, activation of the cAMP pathway in these cells gives rise to a strong inhibition of proliferation, paralleled by a down-regulation of Cyclin A promoter activity. This effect requires the integrity of the CRE, suggesting a role for CRE-binding proteins in late G1/S controls.