Liquid resorbable nanofibrous surgical mesh: a proof of a concept.

BACKGROUND: Surgical mesh is widely used not only to treat but also to prevent incisional hernia formation. Despite much effort by material engineers, the 'ideal' mesh mechanically, biologically and surgically easy to use remains elusive. Advances in tissue engineering and nanomedicine have allowed new concepts to be tested with promising results in both small and large animals. Abandoning the concept of a pre-formed mesh completely for a 'pour in liquid mesh' has never been tested before. MATERIALS AND METHODS: Thirty rabbits underwent midline laparotomy with closure using an absorbable suture and small stitch small bites technique. In addition, their abdominal wall closure was reinforced by a liquid nanofibrous scaffold composed of a fibrin sealant and nanofibres of poly-ε-caprolactone with or without hyaluronic acid or the sealant alone, poured in as an 'onlay' over the closed abdominal wall. The animals were killed at 6 weeks and their abdominal wall was subjected to histological and biomechanical evaluations. RESULTS: All the animals survived the study period with no major complication. Histological evaluation showed an eosinophilic infiltration in all groups and foreign body reaction more pronounced in the groups with nanofibres. Biomechanical testing demonstrated that groups treated with nanofibres developed a scar with higher tensile yield strength. CONCLUSION: The use of nanofibres in a liquid form applied to the closed abdominal wall is easy to use and improves the biomechanical properties of healing fascia at 6 weeks after midline laparotomy in a rabbit model.

Needleless electrospun and centrifugal spun poly-ε-caprolactone scaffolds as a carrier for platelets in tissue engineering applications: A comparative study with hMSCs.

The biofunctionalization of scaffolds for tissue engineering is crucial to improve the results of regenerative therapies. This study compared the effect of platelet-functionalization of 2D electrospun and 3D centrifugal spun scaffolds on the osteogenic potential of hMSCs. Scaffolds prepared from poly-ε-caprolactone, using electrospinning and centrifugal spinning technology, were functionalized using five different concentrations of platelets. Cell proliferation, metabolic activity and osteogenic differentiation were tested using hMSCs cultured in differential and non-differential medium. The porous 3D structure of the centrifugal spun fibers resulted in higher cell proliferation. Furthermore, the functionalization of the scaffolds with platelets resulted in a dose-dependent increase in cell metabolic activity, proliferation and production of an osteogenic marker - alkaline phosphatase. The effect was further promoted by culture in an osteogenic differential medium. The increase in combination of both platelets and osteogenic media shows an improved osteoinduction by platelets in environments rich in inorganic phosphate and ascorbate. Nevertheless, the results of the study showed that the optimal concentration of platelets for induction of hMSC osteogenesis is in the range of 900-3000 × 109 platelets/L. The study determines the potential of electrospun and centrifugal spun fibers with adhered platelets, for use in bone tissue engineering.

Whole exome sequencing unravels disease-causing genes in consanguineous families in Qatar.

Whole exome sequencing (WES) has greatly facilitated the identification of causal mutations for diverse human genetic disorders. We applied WES as a molecular diagnostic tool to identify disease-causing genes in consanguineous families in Qatar. Seventeen consanguineous families with diverse disorders were recruited. Initial mutation screening of known genes related to the clinical diagnoses did not reveal the causative mutations. Using WES approach, we identified the definitive disease-causing mutations in four families: (i) a novel nonsense homozygous (c.1034C>G) in PHKG2 causing glycogen storage disease type 9C (GSD9C) in a male with initial diagnosis of GSD3; (ii) a novel homozygous 1-bp deletion (c.915del) in NSUN2 in a male proband with Noonan-like syndrome; (iii) a homozygous SNV (c.1598C>G) in exon 11 of IDUA causing Hurler syndrome in a female proband with unknown clinical diagnosis; (iv) a de novo known splicing mutation (c.1645+1G>A) in PHEX in a female proband with initial diagnosis of autosomal recessive hypophosphatemic rickets. Applying WES as a diagnostic tool led to the unambiguous identification of disease-causing mutations in phenotypically complex disorders or correction of the initial clinical diagnosis in ˜25% of our cases.

Control of human immunodeficiency virus type 1 RNA metabolism: role of splice sites and intron sequences in unspliced viral RNA subcellular distribution.

In the course of examining the various factors which affect the metabolism of human immunodeficiency virus type 1 (HIV-1) RNA, we examined the role of intron sequences and splice sites in determining the subcellular distribution of the RNA. Using in situ hybridization, we demonstrated that in the absence of Rev, unspliced RNA generated with an HIV-1 env expression construct displayed discrete localization in the nucleus, coincident with the location of the gene and not associated with SC35-containing nuclear speckles. Expression of Rev resulted in a disperse signal for the unspliced RNA throughout both the nucleus and the cytoplasm. Subsequent fractionation of the nucleus revealed that the majority of unspliced viral RNA within the nucleus is associated with the nuclear matrix and that upon expression of Rev, a small proportion of the unspliced RNA is found within the nucleoplasm. Mutations which altered splice site utilization did not alter the sequestration of unspliced RNA into discrete nuclear regions. In contrast, a 2.2-kb deletion of intron sequence resulted in a shift from discrete regions within the nucleus to a disperse signal throughout the cell, indicating that intron sequences, and not just splice sites, are required for the observed nuclear sequestration of unspliced viral RNA.

Novel exonic elements that modulate splicing of the human fibronectin EDA exon.

Three exons in the fibronectin primary transcript are alternatively spliced in a tissue- and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3'-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric beta-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context.

Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1.

The requirement of human immunodeficiency virus type 1 to generate numerous proteins from a single primary transcript is met largely by the use of suboptimal splicing to generate over 30 mRNAs. To ensure that appropriate quantities of each protein are produced, there must be a signal(s) that controls the efficiency with which any particular splice site in the RNA is used. To identify this control element(s) and to understand how it operates to generate the splicing pattern observed, we have initially focused on the control of splicing of the tat-rev intron, which spans the majority of the env open reading frame. Previous analysis indicated that a suboptimal branchpoint and polypyridimine tract in this intron contribute to its suboptimal splicing (A. Staffa and A. Cochrane, J. Virol. 68:3071-3079, 1994). In this report, we identify two additional elements within the 3'-terminal exon, an exon-splicing enhancer (ESE) and an exon splicing silencer (ESS), that modulate the overall efficiency with which the 3' tat-rev splice site is utilized. Both elements are capable of functioning independently of one another. Furthermore, while both the ESE and ESS can function in a heterologous context, the function of the ESS is extremely sensitive to the sequence context into which it is placed. In conclusion, it would appear that the presence of a suboptimal branchpoint and a polypyrimidine tract as well as the ESE and ESS operate together to yield the balanced splicing of the tat-rev intron observed in vivo.

Activation of promutagens by endogenous and heterologous sulfotransferases expressed in continuous cell cultures.

Various environmental chemicals are metabolised to chemically reactive sulfuric acid esters, which may covalently bind to cellular macromolecules and induce mutations and tumours. This activation pathway is usually not taken into account in external xenobiotic-metabolising systems used in short-term tests. We therefore analysed the abilities of cytosols from mammalian cell lines to activate benzylic alcohols (1-hydroxymethylpyrene and 9-hydroxymethylanthracene) to mutagens detectable in Salmonella typhimurium TA98. No activation was observed in cell lines which are commonly used in mutagenicity and cell transformation assays, and only low activities were found in epithelial cell lines in culture. We have therefore constructed Chinese hamster V79-derived cell lines which stably express a heterologous sulfotransferase, rat hydroxysteroid sulfotransferase a. Cytosol of these cells effectively activated 1-hydroxymethylpyrene and 9-hydroxymethylanthracene to mutagens detected in S. typhimurium. The hepatocarcinogen 6-hydroxymethylbenzo[a]pyrene induced gene mutations in sulfotransferase-expressing V79-derived cells, whereas it elicited only marginal effects in sulfotransferase-deficient control cells. The new cell lines may allow the detection of novel classes of mutagens, since some externally generated reactive sulfuric acid esters may not readily penetrate target cells due to their short life span and their ionization.

The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site.

Proportional expression of retroviral genes requires that splicing of the viral primary transcript be an inefficient process. Much of our current knowledge about retroviral suboptimal splicing comes from studies with Rous sarcoma virus. In this report, we describe the use of chimeric introns composed of human beta-globin and human immunodeficiency virus type 1 (HIV-1) splice sites to establish the basis for inefficient splicing of the intron which comprises most of the HIV-1 env coding sequences (referred to as the tat/rev intron). S1 RNA analysis of transfected COS-7 cells revealed that the 3' splice site (3' ss) of this region was significantly less efficient than the 3' ss of the first intron of beta-globin. Deletion of sequences flanking the tat/rev intron 3' ss demonstrated that the requirements for its inefficiency reside within the region that is expected to comprise the essential signals for splicing (i.e., the branchpoint region, the polypyrimidine tract, and the AG dinucleotide). Introduction of an exact copy of the efficient beta-globin branchpoint sequence within a highly conserved region rendered the tat/rev intron 3' ss highly efficient. Improvement of the polypyrimidine tract also increased the splicing efficiency, but to a degree slightly less than that obtained with the branchpoint mutation. Subsequent examination of the tat/rev intron 5' splice site in a heterologous context revealed that it is efficiently utilized. These results indicate that both a poor branchpoint region and a poor polypyrimidine tract are responsible for the low splicing efficiency of the HIV-1 tat/rev intron. It is of fundamental interest to establish the basis for inefficient splicing of the HIV-1 tat/rev intron since it may provide the key to understanding why nuclear export of mRNAs encoding HIV-1 structural proteins is Rev dependent.