Pallister–Hall syndrome diagnosed in a young man after an acute adrenal crisis

Pallister–Hall syndrome (PHS) is a very rare genetic disorder. The diagnosis is usually suspected at the young age when a hypothalamic hamartoma is associated with polydactyly. Endocrine manifestations are mostly related to hypothalamic hamartoma and rarely reveal the disease. We report the case of an 18‐year‐old young man in whom the diagnosis of PHS was delayed until his hospitalization in the endocrinology department for acute adrenal insufficiency.     

High HbA1c levels affect motility parameters and overexpress oxidative stress of human mature spermatozoa

The aim of this study is to investigate, by a validated in vitro model, the effect of diabetic plasma on ejaculated human spermatozoa. Plasma of 51 male diabetic patients (mean age 62.28 ± 9.28 years) was selected according to their HbA_1c level: low HBA_1c ≤ 5% (31 mmol/mol), moderate HBA_1c 6%–8% (42–64 mmol/mol) and high HBA_1c ≥ 10% (86 mmol/mol). The plasma was tested on eighteen normal semen samples by analysing gametes motility using a computer Sperm Class Analyzer® and their corresponding oxidative stress (OS) status using thiobarbituric acid-reactive substances assay. The results indicated that diabetic plasma affected all sperm motility parameters with high HbA_1c showing the most important deleterious effects. Low gametes' straight-line velocity was observed in high HbA_1c level, mainly after 20 min of co-incubation (8.78 ± 0.47 µm/s). Also, the highest lipid peroxidation (nmoles MDA/10⁸ SPZ) was observed in high HbA_1c values (0.92 ± 0.09), higher than those in spermatozoa treated with H₂O₂ (0.85 ± 0.04). Conclusively, a direct impact of diabetic plasma on spermatozoa is revealed with overexpression of OS as the underlying mechanism. These findings suggested that it is strongly recommended to control clinically the glycaemic level and OS in diabetic patients for the maintenance of male fertility.     

Prolonged fever in children. Retrospective study of 67 cases.]

BACKGROUND: The first problem to face in prolonged fever is its etiologic diagnosis. Its incidence varies between 0,5 to 3% of all paediatric hospital-stay. Precise diagnosis need an extensive questionnary, complete physical examination and an algorithm of complementary exams. PURPOSE: To precise the epidemiologic profile and causes of prolonged fever in a children. POPULATION AND METHODS: Retrospective review of 67 children between two and 15 years old admitted in the general paediatrics department of the Fattouma Bourguiba university hospital in Monastir (Tunisia), for prolonged fever between 1(st) January 1988 and 31 December 1998. RESULTS: The incidence of prolonged fever was 1,02%. The mean age was seven years with female predominance. The mean fever duration was 30 days. Fever was isolated in 23,9% of cases. Fever was associated to rheumatic or respiratory signs in respectively 26,9% and 20,9% of cases. Hospital-stay was of 11 days in 50% of cases. Prominent causes were dominated by infectious diseases (56,7%) with predominance of localized infections, followed by inflammatory diseases (20,9%) with predominance of rheumatic fever and neoplasms (3%). Fever remained of unknown origin was seen in 19,4% of cases. CONCLUSION: Prolonged fever is still dominated by infectious and inflammatory diseases and depend on local epidemiological particularities. In fact we noted in this study the relative high-frequency of visceral leishmaniasis, complicated pulmonary hydatic cyst and rheumatic fever. The diagnosis approach should be based on complementary exams of first and second stage because of their high number and cost. Prognosis of fever of unknown origin is often favorable.     

Intracranial stimulation for children with epilepsy

Objectives

To evaluate the efficacy of intracranial stimulation to treat refractory epilepsy in children.

Elucidation of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via MAPK signalling pathway

Melanoma is the most dangerous form of skin cancer with a very poor prognosis. Melanoma develops when unrepaired DNA damage causes to skin cells to multiply and form malignant tumors. The current therapy is limited by the highly ability of this disease to metastasize rapidly. Plumbagin is a naphthoquinone (5-hydroxy-2-methyl-1, 4-naphthoquinone), isolated from the roots of medicinal plant Plumbago zeylanica, and it is widely present in Lawsonia inermis L. It has been shown that plumbagin has an anti-proliferative and anti-invasive activities in various cancer cell lines; however, the anti-cancer and anti-metastatic effects of plumbagin are largely unknown against melanoma cells. In this study, we evaluated the effect of plumbagin on B16F10 murine melanoma cells . Plumbagin decreased B16F10 cell viability as well as the cell migration, adhesion, and invasion. The molecular mechanism was studied, and plumbagin downregulated genes relevant in MAPK pathway, matrix metalloproteinases (MMP's), and cell adhesion. Furthermore, plumbagin elevated the expression of apoptosis and tumors suppressor genes, and genes significant in reactive oxygen species (ROS) response. Taken together, our findings suggest that plumbagin has an anti-invasion and anti-metastasis effect on melanoma cancer cells by acting on MAPK pathway and its related genes.     

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.DNA double-strand break repair (DSBR) is essential for cell survival and repair-deficient cells are highly sensitive to chromosome breakage. In Escherichia coli, a single unrepaired DNA DSB per replication cycle is lethal, illustrating the critical nature of the repair reaction (1). DSBR in E. coli is mediated by homologous recombination, which relies on the RecA protein to efficiently recognize DNA sequence identity between two molecules. RecA homologs are widely conserved from bacteriophages to mammals, where they are known as the Rad51 proteins (2). The RecA protein plays its central role by binding single-stranded DNA (ssDNA) to form a presynaptic filament that searches for a homologous double-stranded DNA (dsDNA) donor from which to repair. It then catalyzes a strand-exchange reaction to form a joint molecule (3), which is stabilized by the branch migration activities of the RecG and RuvAB proteins (4). The joint molecule is then resolved by cleavage at its four-way Holliday junction by the nuclease activity of RuvABC (5, 6).RecA binding at the site of a DSB is dependent on the activity of the RecBCD enzyme (Fig. 1A). RecBCD is a helicase-nuclease that binds to dsDNA ends, then separates and unwinds the two DNA strands using the helicase activities of the RecB and RecD subunits (see refs. 7 and 8 for recent reviews). RecD is the faster motor of the two and this consequently results in the formation of a ssDNA loop ahead of RecB (Loop 1 in Fig. 1A) (9). As the enzyme translocates along dsDNA, the 3′-terminated strand is continually passed through the Chi-scanning site thought to be located in the RecC protein (10). When a Chi sequence (the octamer 5′-GCTGGTGG-3′) enters this recognition domain, the RecD motor is disengaged and the 3′ strand continues to be unwound by RecB. Under in vitro conditions, where the concentration of magnesium exceeds that of ATP, the 3′ end (unwound by RecB) is rapidly digested before Chi recognition, whereas the 5′ end (unwound by RecD) is intermittently cleaved (11, 12). After Chi recognition the 3′ end is no longer cleaved but the nuclease domain of RecB continues to degrade the 5′ end as it exits the enzyme (11, 12). Under in vitro conditions where the concentration of ATP exceeds that of magnesium, unwinding takes place but the only site of cleavage detected is ∼5 nucleotides 3′ of the Chi sequence (13, 14). Because the RecB motor continues to operate while the RecD motor is disengaged, Loop 1 is converted to a second loop located between the RecB and RecC subunits or to a tail upon release of the Chi sequence from its recognition site. We therefore describe this single-stranded region as Loop/Tail 2 in Fig. 1A. After the whole of Loop 1 is converted to Loop/Tail 2, this second single-stranded region continues to grow as long as the RecB subunit unwinds the dsDNA. The RecBCD enzyme enables RecA protein to load on to Loop/Tail 2 to generate the presynaptic filament necessary to search for homology and initiate strand-exchange (15). Finally, the RecBCD enzyme stops translocation and disassembles as it dissociates from the DNA, releasing a DNA-free RecC subunit (16).Open in a separate windowFig. 1.DSBR in E. coli. (A and B) Schematic representation of DSB processing by the RecBCD complex. (A) Before Chi recognition, both the RecB and RecD motors progress along the DNA. RecD is the faster motor and as a result a loop of ssDNA (Loop 1) is formed ahead of the slower RecB motor. The 3′ ssDNA strand is scanned for the Chi sequence by the RecC protein. (B) After Chi recognition, RecBCD likely undergoes a conformational change so that only the RecB motor is engaged. The RecA protein is recruited by the RecB nuclease domain and loaded onto the ssDNA loop generated by RecB unwinding to promote RecA nucleoprotein filament formation. In this schematic representation, the Chi site is shown held in its recognition site. However, the Chi site will be released either by disassembly of the RecBCD complex or at some point before this and the second single-stranded region on the 3′ terminating strand will be converted from a loop to a tail. Therefore, this region is denoted Loop/Tail 2. The mathematical model described in SI Appendix does not depend on the ATP/magnesium dependent differential cleavage of DNA strands (7, 8), nor does it depend on the precise time that the 3′ end is released from the complex following Chi recognition. (C) The hairpin endonuclease SbcCD is used to cleave a 246-bp interrupted palindrome inserted in the lacZ gene of the E. coli chromosome. Cleavage of this DNA hairpin results in the generation of a site-specific DSB on only one of a pair of replicating sister chromosomes, thus leaving an intact sister chromosome to serve as a template for repair by homologous recombination.Our understanding of the action of RecBCD and RecA has been the result of more than 40 years of genetic analysis and biochemical investigation of these purified proteins in vitro. However, relatively little is known about their activities on the genomic scale. To investigate these reactions in vivo, we have used RecA chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) in an experimental system that allows us to introduce a single and fully repairable DSB into the chromosome of E. coli (1). Because DSBR by homologous recombination normally involves the repair of a broken chromosome by copying the information on an unbroken sister chromosome, our laboratory has previously developed a procedure for the cleavage of only one copy of two genetically identical sister chromosomes (1). We have made use of the observation that the hairpin nuclease SbcCD specifically cleaves only one of the two sister chromosomes following DNA replication through a 246-bp interrupted palindrome to generate a two-ended DSB (1). As shown in Fig. 1B, this break is fully repairable and we have shown that recombination-proficient cells suffer very little loss of fitness in repairing such breaks (17).Here we investigate in vivo and in a quantitative manner the first steps of DSBR: because the outcome of RecBCD action is understood to be the loading of RecA on DNA in a Chi-dependent manner, we use RecA-ChIP to reveal the consequences of RecBCD action on a genomic scale during DSBR. Analyses of most ChIP-Seq datasets focus on the identification of regions of significant enrichment of a given protein but do not take into account the underlying mechanisms giving rise to the binding (18). We reasoned that given the detailed mechanistic understanding of RecBCD in vitro, we could gain a deeper insight into its in vivo functions by developing a mathematical model of RecBCD action that would enable us to estimate the mechanistic parameters of the complex in live cells. Our ChIP data indicate that RecA is indeed loaded on to DNA in a Chi-dependent manner and we have used our mathematical model to infer the parameters of RecBCD action in vivo on a genomic scale. Furthermore, our analysis reveals that DSBR at lacZ induces DSBR in the terminus region of the chromosome, an unanticipated observation illuminated by the genomic scale of our data.