Determination of the gram-positive bacterial content of soils and sediments by analysis of teichoic acid components

Many gram-positive bacteria form substituted polymers of glycerol and ribitol phosphate esters known as teichoic acids. Utilizing the relative specificity of cold concentrated hydroflouric acid in the hydrolysis of polyphosphate esters it proved possible to quantitatively assay the teichoic acid-derived glycerol and ribitol from gram-positive bacteria added to various soils and sediments. The lipids are first removed from the soils or sediments with a one phase chloroform-methanol extraction and the lipid extracted residue is hydrolyzed with cold concentrated hydrofluoric acid. To achieve maximum recovery of the teichoic acid ribitol, a second acid hydrolysis of the aqueous extract is required. The glycerol and ribitol are then acetylated after neutralization and analyzed by capillary gas-liquid chromatography. This technique together with measures of the total phospholipid, the phospholipid fatty acid, the muramic acid and the hydroxy fatty acids of the lipopolysaccharide lipid A of the gram-negative bacteria makes it possible to describe the community structure of environmental samples. The proportion of gram-positive bacteria measured as the teichoic acid glycerol and ribitol is higher in soils than in sediments and increases with depth in both.     

PHLPP-mediated dephosphorylation of S6K1 inhibits protein translation and cell growth

PHLPP is a family of Ser/Thr protein phosphatases that contains PHLPP1 and PHLPP2 isoforms. We have shown previously that PHLPP functions as a tumor suppressor by negatively regulating Akt signaling in cancer cells. Here we report the identification of ribosomal protein S6 kinase 1 (S6K1) as a novel substrate of PHLPP. Overexpression of both PHLPP isoforms resulted in a decrease in S6K1 phosphorylation in cells, and this PHLPP-mediated dephosphorylation of S6K1 was independent of its ability to dephosphorylate Akt. Conversely, S6K1 phosphorylation was increased in cells depleted of PHLPP expression. Furthermore, we showed that the insulin receptor substrate 1 (IRS-1) expression and insulin-induced Akt phosphorylation were significantly decreased as the result of activation of the S6K-dependent negative feedback loop in PHLPP knockdown cells. Functionally, the phosphorylation of ribosomal protein S6 (rpS6) and the amount of phosphorylated rpS6 bound to the translation initiation complex were increased in PHLPP-knockdown cells. This correlated with increased cell size, protein content, and rate of cap-dependent translation. Taken together, our results demonstrate that loss of PHLPP expression activates the S6K-dependent negative feedback loop and that PHLPP is a novel player involved in regulating protein translation initiation and cell size via direct dephosphorylation of S6K1.     

In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs’ in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.     

Scribble-mediated membrane targeting of PHLPP1 is required for its negative regulation of Akt

PHLPP1 (PH domain leucine-rich-repeats protein phosphatase) is a Ser/Thr protein phosphatase that acts as a tumour suppressor by negatively regulating Akt. Here, we show that PHLPP1 is recruited to the cell membrane by binding to a scaffolding protein: Scribble. Knockdown of Scribble (Scrib) results in redistribution of PHLPP1 from the membrane to the cytoplasm and an increase in Akt phosphorylation, whereas overexpression of Scrib has the opposite effect. Furthermore, PHLPP1-dependent inhibition of cell proliferation is facilitated by the formation of a Scrib, PHLPP1 and Akt trimeric complex. Thus, our findings identify a functional interaction between PHLPP1 and Scrib in negatively regulating Akt signalling.     

Quinolone Nucleosides: 6,7-Dihalo-N-β- and α-Glycosyl-l 4-dihydro-4-oxo-quinoline-3-carboxylic Acids and Derivatives. Synthesis,Antimicrobial and Antiviral Activity

Abstract

Reaction of the silylated 6,7-dihaloquinoline bases 10–12 with l-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (13) gave ethyl 7-chloro-6-flouro-l,4-dihydro-4-oxo-1 -(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)quinoline-3-carboxylate (14) and the free acids 15 and 16, respectively, which led on deblocking of the sugar moiety to the free nucleosides 17, 18 and 20, respectively. Treatment of 14 with methanolic ammonia afforded the amide derivative 19. Ribosylation of 11 with l,2-di-O-acetyl-3-azido-3-deoxy-5-p-toluoyl-β-D-ribofuranose (21) afforded the azido nucleoside 22, which was again converted into the free nucleoside 23. Analogously, reaction of 11 with the chloro deoxyribose derivative 24 led to a mixture of α /β (2:1) anomers of 25. Deblocking and recrystallization of the product gave mainly the α-anomer 26. Compounds 17–19, 23 and 26 were evaluated against Escherichia coli and found inactive. Compound 16–18 and 22 were inactive aganist HIV-1 (III B) and HIV-2 (ROD) induced cytopathicity in human MT-4 lymphocyte cells.     

Implantation of the Syncardia Total Artificial Heart

With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of such patients are generally well served by left ventricular assist devices (LVADs). However, a subset of patients with late stage biventricular failure or other significant anatomic lesions are not adequately treated by isolated left ventricular mechanical support. Examples of concomitant cardiac pathology that may be better treated by resection and TAH replacement includes: post infarction ventricular septal defect, aortic root aneurysm / dissection, cardiac allograft failure, massive ventricular thrombus, refractory malignant arrhythmias (independent of filling pressures), hypertrophic / restrictive cardiomyopathy, and complex congenital heart disease. Patients often present with cardiogenic shock and multi system organ dysfunction. Excision of both ventricles and orthotopic replacement with a total artificial heart (TAH) is an effective, albeit extreme, therapy for rapid restoration of blood flow and resuscitation. Perioperative management is focused on end organ resuscitation and physical rehabilitation. In addition to the usual concerns of infection, bleeding, and thromboembolism common to all mechanically supported patients, TAH patients face unique risks with regard to renal failure and anemia. Supplementation of the abrupt decrease in brain natriuretic peptide following ventriculectomy appears to have protective renal effects. Anemia following TAH implantation can be profound and persistent. Nonetheless, the anemia is generally well tolerated and transfusion are limited to avoid HLA sensitization. Until recently, TAH patients were confined as inpatients tethered to a 500 lb pneumatic console driver. Recent introduction of a backpack sized portable driver (currently under clinical trial) has enabled patients to be discharged home and even return to work. Despite the profound presentation of these sick patients, there is a 79-87% success in bridge to transplantation.     

Association of a Polymorphism in the BIRC6 Gene with Pseudoexfoliative Glaucoma

Recently an association was observed between alleles in genes of the unfolded protein response pathway and primary open angle glaucoma (POAG). The goal of the current study is to investigate the role of these two genes, protein disulphide isomerase A member 5 (PDIA5) and baculoviral IAP repeat containing 6 (BIRC6), in different forms of glaucoma. 278 patients with POAG, 132 patients with primary angle closure glaucoma (PACG) and 135 patients with pseudoexfoliative glaucoma (PEXG) were genotyped for single nucleotide polymorphisms (SNPs) rs11720822 in PDIA5 and 471 POAG, 184 PACG and 218 PEXG patients were genotyped for rs2754511 in BIRC6. Genotyping was done by allelic discrimination PCR, and genotype and allele frequencies were calculated. Logistic regression analyses were performed using R software to determine the association of these SNPs with glaucoma. The allele and genotype frequencies of rs11720822 in PDIA5 were not associated with POAG, PACG or PEXG. The TT genotype of rs2754511 in BIRC6 was found to be protective for PEXG (p = 0.05, OR 0.42 [0.22–0.81]) in the Pakistani population, but not for POAG or PACG. This study did not confirm a previously reported association of risk alleles in PDIA5 and BIRC6 with POAG, but did demonstrate a protective role of the T allele of rs2754511 in the BIRC6 gene in PEXG. This supports a role for the unfolded protein response pathway and regulation of apoptotic cell death in the pathogenesis of PEXG.     

Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing C_H1 with Cα and C_L with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the C_H1/C_L and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.