Haploinsufficiency of a spliceosomal GTPase encoded by EFTUD2 causes mandibulofacial dysostosis with microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EFTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the first multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.     

Valeriana wallichii root extract improves sleep quality and modulates brain monoamine level in rats

The present study was performed to investigate the effects of Valeriana wallichi (VW) aqueous root extract on sleep-wake profile and level of brain monoamines on Sprague-Dawley rats. Electrodes and transmitters were implanted to record EEG and EMG in freely moving condition and the changes were recorded telemetrically after oral administration of VW in the doses of 100, 200 and 300 mg/kg body weight. Sleep latency was decreased and duration of non-rapid eye movement (NREM) sleep was increased in a dose dependent manner. A significant decrease of sleep latency and duration of wakefulness were observed with VW at doses of 200 and 300 mg/kg. Duration of NREM sleep as well as duration of total sleep was increased significantly after treatment with VW at the doses of 200 and 300 mg/kg. VW also increased EEG slow wave activity during NREM sleep at the doses of 200 and 300 mg/kg. Level of norepinephrine (NE), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and hydroxy indole acetic acid (HIAA) were measured in frontal cortex and brain stem after VW treatment at the dose of 200mg/kg. NE and 5HT level were decreased significantly in both frontal cortex and brain stem. DA and HIAA level significantly decreased only in cortex. DOPAC level was not changed in any brain region studied. In conclusion it can be said that VW water extract has a sleep quality improving effect which may be dependent upon levels of monoamines in cortex and brainstem.     

Plasmodium falciparum Prp16 homologue and its role in splicing

Large numbers of Plasmodium genes have been predicted to have introns. However, little information exists on the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNA processing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, present in the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is a member of DEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experiments with the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis of ATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associated domain (HA2) reduced these activities considerably, indicating that the helicase-associated domain may regulate the PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N‐terminal domain (1–80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasite cultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16p in splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16 protein, not the full length PfPrp16, can rescue the yeast prp16 temperature‐sensitive mutant. These results suggest that although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtle differences exist with regards to its associated factors or its assembly with spliceosomes.     

Altered regulation of CXCR4 expression during aging contributes to increased CXCL12-dependent chemotactic migration of CD4(+) T cells

Chemokine‐dependent migration of T lymphocytes assures recirculation of naïve T cells to secondary lymphoid organs and tissue‐specific trafficking of memory‐effector T cells. Previous studies carried out in rodents have demonstrated age‐associated modulation of the expression of chemokine receptors such as CXCR4 and CCR5; however, little is known about the molecular mechanisms that regulate receptor expression and turnover in T cells, during advancing age in humans. Our recent results demonstrating increased chemotactic migration in response to CXCL12 in CD4⁺ T cells obtained from the elderly, as compared to those from young donors, led us to hypothesize that increase in surface expression, because of altered endocytic regulation of CXCR4 on T cells during aging, might be directly responsible for increased migration toward CXCL12. Studies presented here demonstrate a significant increase in the surface expression of CXCR4 in CD4⁺ T cells from elderly human donors, relative to those from the young. Additionally, CXCL12‐mediated endocytosis of CXCR4 was differentially regulated during aging, which could be attributed to alterations in the ubiquitination of CXCR4. Thus, altered ubiquitination of CXCR4 may contribute to the increased surface expression and enhanced T‐cell migration to chemotactic stimuli in the elderly.     

A role for Atg8-PE deconjugation in autophagosome biogenesis

Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8-PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8-PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8-PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.     

Cockayne syndrome-xeroderma pigmentosum complex with demyelination: A rare association

Xeroderma pigmentosum-Cockayne syndrome (XP-CS) includes facial freckling and early skin cancers typical of XP and some features typical of CS, such as mental retardation, spasticity, short stature, and hypogonadism. XP-CS does not include skeletal involvement, the facial phenotype of CS, or CNS demyelination and calcifications. We present a rare patient whose genome probably harbored a specific combination of mutations producing a rare double syndrome of XP-CS, with facial phenotype of CS, and CNS demyelination.     

Preservation of cGMP-induced relaxation of pulmonary veins of fetal lambs exposed to chronic high altitude hypoxia: role of PKG and Rho kinase

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.     

Identification of PLCL1 gene for hip bone size variation in females in a genome-wide association study

Osteoporosis, the most prevalent metabolic bone disease among older people, increases risk for low trauma hip fractures (HF) that are associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of the key measurable risk factors for HF. Although hip BS is highly genetically determined, genetic factors underlying the trait are still poorly defined. Here, we performed the first genome-wide association study (GWAS) of hip BS interrogating approximately 380,000 SNPs on the Affymetrix platform in 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, PLCL1 (phospholipase c-like 1), that had four SNPs associated with hip BS at, or approaching, a genome-wide significance level in our female subjects; the most significant SNP, rs7595412, achieved a p value of 3.72x10(-7). The gene's importance to hip BS was replicated using the Illumina genotyping platform in an independent UK cohort containing 1,216 Caucasian females. Two SNPs of the PLCL1 gene, rs892515 and rs9789480, surrounded by the four SNPs identified in our GWAS, achieved p values of 8.62x10(-3) and 2.44x10(-3), respectively, for association with hip BS. Imputation analyses on our GWAS and the UK samples further confirmed the replication signals; eight SNPs of the gene achieved combined imputed p values<10(-5) in the two samples. The PLCL1 gene's relevance to HF was also observed in a Chinese sample containing 403 females, including 266 with HF and 177 control subjects. A SNP of the PLCL1 gene, rs3771362 that is only approximately 0.6 kb apart from the most significant SNP detected in our GWAS (rs7595412), achieved a p value of 7.66x10(-3) (odds ratio = 0.26) for association with HF. Additional biological support for the role of PLCL1 in BS comes from previous demonstrations that the PLCL1 protein inhibits IP3 (inositol 1,4,5-trisphosphate)-mediated calcium signaling, an important pathway regulating mechanical sensing of bone cells. Our findings suggest that PLCL1 is a novel gene associated with variation in hip BS, and provide new insights into the pathogenesis of HF.     

Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy

In yeast, approximately 31 autophagy-related (Atg) proteins have been identified. Most of them reside at the phagophore assembly site (PAS), although the function of the PAS mostly remains unclear. One reason for the latter is the lack of stoichiometric information regarding the Atg proteins at this site. We report the application of fluorescence microscopy to study the amount of Atg proteins at the PAS. We find that an increase in the amount of Atg11 at the PAS enhances the recruitment of Atg8 and Atg9 to this site and facilitates the formation of more cytoplasm-to-vacuole targeting vesicles. In response to autophagy induction, the amount of most Atg proteins remains unchanged at the PAS, whereas we see an enhanced recruitment of Atg8 and 9 at this site. During autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes. Application of this method and further analysis will provide more insight into the functions of Atg proteins.     

Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress

Salicylic acid (SA) is an important signal molecule modulating plantresponses to stress. It is recently reported to induce multiple stresstolerancein plants including drought. An experiment was, therefore, conducted toascertain the effect of salicylic acid on the growth and metabolic profile ofwheat seedlings under water stress. Irrespective of the SA concentration(1–3 mM) and water stress, SA treated plants showed, ingeneral, a higher moisture content, dry mass, carboxylase activity of Rubisco,superoxide dismutase (SOD) activity and total chlorophyll compared to those ofuntreated seedlings. SA treatment, under water stress, protected nitratereductase (NR) activity and maintained, especially at 3 mM SAconcentration, the protein and nitrogen content of leaves compared to watersufficient seedlings. Results signify the role of SA in regulating the droughtresponse of plants and suggest that SA could be used as a potential growthregulator, for improving plant growth under water stress.