Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2's isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.

Motor axons in peripheral nerves have the capacity to regenerate after injury. However, full functional motor recovery rarely occurs clinically, and this depends on the nature and location of the injury. Recent preclinical findings suggest that there may be a time after nerve injury where, while regrowth to the muscle successfully occurs, there is nevertheless a failure to reestablish motor function, suggesting a possible critical period for synapse reformation. We have now examined the temporal and anatomical determinants for the reestablishment of motor function after prolonged neuromuscular junction (NMJ) denervation in rats and mice. Using both sciatic transection-resuture and multiple nerve crush models in rats and mice to produce prolonged delays in reinnervation, we show that regenerating fibers reach motor endplates and anatomically fully reform the NMJ even after extended periods of denervation. However, in spite of this remarkably successful anatomical regeneration, after 1 month of denervation there is a consistent failure to reestablish functional recovery, as assessed by behavioral and electrophygiological assays. We conclude that this represents a failure in reestablishment of synaptic function, and the possible mechanisms responsible are discussed, as are their clinical implications.

Matrix metalloproteinase-3 (MMP-3) over-expression is associated with tissue destruction in the context of chronic inflammation. Previous studies showed that IL-4 inhibits induction of MMP-3 by IL-1β, and suggested that AP-1 might be involved. Here we show that IL-1 induced binding of transcription factor AP-1 to the MMP-3 promoter consists primarily of c-Jun, JunB, and c-Fos and that binding of c-Jun and c-Fos is inhibited by the combination of cytokines while binding of Jun B is not. Mutation of the AP-1 site in the MMP-3 promoter decreased the ability of IL-4 to inhibit its transcription in transfected MG-63 cells. Western blotting showed that both cytokines activate Jun N-terminal kinase (JNK), but with somewhat different kinetics, and that activation of JNK by both cytokines individually is inhibited by the combination. These results indicate that IL-4 inhibition of MMP-3 expression is associated with reduction of IL-1 induced binding of active forms of the AP-1 dimer, while less active JunB-containing dimers remain, and suggest that these changes are associated with decreased activation of JNK.

A 5T/6T polymorphism in the human MMP-3 promoter affects gene expression and impacts the risk and/or severity of various pathological conditions. Chromatin immunoprecipitation (ChIP) in human fibroblasts homozygous for the 6T site demonstrate that it is bound by NF-κB and ZBP-89 transcription factors in its native chromatin. ChIP in COS-1 cells transfected with plasmids containing the 5T and 6T sites in the context of 2 kb of the MMP-3 promoter showed that NF-κB p50 binds preferentially to the 6T site, while more ZBP-89 binding is detected to the 5T site. Over-expressed ZBP-89 increased transcription from the 5T promoter but not from the 6T, while NF-κB decreased transcription from both promoters, even in the presence of excess ZBP-89. A model is suggested in which the physiological impact of the polymorphism is dependent on the relative levels and activities of these competing factors in various cell types and conditions.

The objective of this study was to assess the efficacy and safety of liposomal heparin spray-a new formula of topical heparin delivery. This was a randomized, multicenter, controlled open clinical trial with 2 parallel groups. Forty-six outpatients with clinical signs of superficial venous thrombosis (SVT) were treated with either topical liposomal heparin spraygel (LHSG) (Lipohep Forte Spraygel, 4 puffs of 458 IU tid (n = 22) or with low-molecular-weight heparin (LMWH) (Clexane 40 mg once a day (n = 24), administered subcutaneously (sc). Main outcome measures were efficacy parameters (improvement of local symptoms-pain control and planimetric evaluation of erythema size, duplex Doppler assessment of thrombus regression) and safety parameters (documentation of adverse events, with particular reference to deep vein thrombosis [DVT] by duplex sonography, and patients' and investigators' assessment of drug tolerance). Patients' and investigators' subjective assessment of efficacy of treatment and change in basic biochemical parameters were defined as secondary outcome measures. Statistical analysis was performed with use of Wilcoxon test, Mann-Whitney U-test and Chi-square test. Regression of SVT-related symptoms, including pain, erythema, and thrombus presence, was shown as comparable in LHSG and LMWH groups. These results were corroborated by efficacy assessment by investigators and patients. Three cases of deep venous thrombosis in heparin spraygel and 1 in heparin sc group were reported. No significant adverse reactions were observed in the spraygel group, but 1 serious allergic reaction was observed in the LMWH group. Tolerance of new formula heparin was assessed as good. Heparin spraygel-a new topical mode of heparin application, seems a promising method of heparin delivery. This initial study has demonstrated comparable efficacy and safety of LHSG and LMWH in local treatment of SVT. These findings should be confirmed by further extensive study that will reach appropriate statistical power to support such conclusion, for despite heparin treatment, significant risk of DVT was demonstrated in both groups.

NeoSTX-Bup and NeoSTX-Bup-Epi hold promise for prolonged-duration local anesthesia.

No abstract

Newborns with intrauterine growth-restriction are at increased risk of mortality and life-long co-morbidities. Insulin-like growth factor-II (IGF2) deficiency in humans as well as in mice leads to intrauterine growth restriction and decreased neonatal glycogen stores. The present study aims to further characterize the metabolic and transcriptional consequences of Igf2 deficiency in the newborn. We found that, despite being born significantly smaller than their wild-type (Igf2) littermates, brain size was preserved in Igf2 knockout (Igf2), consistent with nutritional deficiency. Histological and triglyceride analyses of newborn livers revealed that Igf2 mice are born with hepatic steatosis. Gene expression analysis in Igf2 newborn livers, showed an alteration of genes known to be dysregulated in chronic caloric restriction, including the most up-regulated gene - serine dehydratase. Multiple genes connected with lipid metabolism and/or hepatic steatosis were also up-regulated. Ingenuity Pathway Analysis confirmed that the biological functions most altered in livers of Igf2 newborns are related to lipid metabolism, with the top upstream regulator predicted to be the perixosome proliferator-activated receptor alpha, a master regulator of hepatic lipid and carbohydrate homeostasis. Together, our data indicate that Igf2 deficiency leads to a newborn phenotype strongly reminiscent of nutritional deficiency, including growth retardation, increased brain/body weight ratio, hepatic steatosis, and characteristic changes in hepatic gene expression. We propose that in addition to its growth factor proliferating functions, Igf2 may also regulate growth by altering the expression of genes that control nutrient metabolism in the newborn.