Impact of counselling on careseeking behaviour in families with sick children: cluster randomised trial in rural India

Objective To assess whether training doctors in counselling improves careseeking behaviour in families with sick children.Design Pair matched, community randomised trial conducted in 12 primary health centres (six pairs). Doctors in intervention centres were trained in counselling, communication, and clinical skills, using the integrated management of childhood illness approach.Setting Rural district in Rajasthan, India.Participants Children aged under 5 years presenting for curative care and their mothers were recruited and visited monthly at home for six months. A total of 2460 children were recruited (1248 intervention, 1212 control).Main outcome measures Careseeking behaviour of mothers for sick children; mothers'' knowledge and perceptions of seeking care; counselling performance of doctors.Results For episodes of illness with at least one reported danger sign, 15% of intervention group mothers and 10% of control group mothers reported having sought care from an appropriate provider promptly; this difference was not statistically significant (relative risk reduction 5%, 95% confidence interval -0.4% to 11%; P = 0.07). One month after training, intervention site doctors counselled more effectively than control group doctors, but at six months their performance had declined. A greater proportion of mothers in the intervention group than in the control group recalled having had at least one danger sign explained (45% v 8%; P = 0.02).Conclusions Mothers'' appreciation of the need to seek prompt and appropriate care for severe episodes of childhood illness increased, but their careseeking behaviour did not improve significantly.     

Cyclic peptides incorporating 4-carboxyphenylalanine and phosphotyrosine are potent inhibitors of pp60(c-)(src)

The protein tyrosine kinase, pp60(c-)(src), is involved in cellular signaling and is activated during mitosis and in various tumors. We have been employing cyclic decapeptides to identify the determinants for substrate binding and phosphorylation to develop inhibitors competitive with protein substrates of Src. A structure-activity study [McMurray, J. S., Budde, R. J. A., Ke, S., Obeyesekere, O. U., Wang, W., Ramdas, L., and Lewis, C. A. (1998) Arch. Biochem. Biophys. 355, 124] revealed that, at the position 3 residues C-terminal to the phosphorylated tyrosine (Y + 3), both glutamic acid and phenylalanine gave identical K(i), K(m), and V(max) values. We hypothesized that the area of Src that binds the Y + 3 residue contains either a positively charged lysine or an arginine, capable of ionic interactions with glutamic acid or cation-pi interactions with phenylalanine. To test this hypothesis, a series of phenylalanine analogues were substituted at position 7 (the Y + 3 residue) in cyclo(Asp(1)-Asn(2)-Glu(3)-Tyr(4)-Ala(5)-Phe(6)-Phe(7)-Gln(8)-D-Phe(9 )-Pro(10)). Of these, 4-carboxyphenylalanine (4-Cpa) and phosphotyrosine resulted in high affinity peptides exhibiting K(i) values of 0.85 and 1.1 microM, respectively, 180- and 130-fold increases in potency over the parent cyclic peptide (K(i) = 150 microM). These peptides were noncompetitive with respect to ATP and competitive against the phosphate-accepting substrate, polyGlu(4)Tyr. The truncated cyclic peptide, cyclo(Phe-4-Cpa-Gln-D-Phe-Pro-Asp-Aca) (Aca = epsilon-aminocaproic acid), which did not contain tyrosine, was also a competitive inhibitor with a K(i) value of 24 microM. We conclude that these cyclic peptides bind to a positively charged area that is near the phosphate transfer region of the active site of Src but does not necessarily include the tyrosine-binding pocket. Furthermore, the 4-Cpa-containing cyclic decapeptide shows remarkable selectivity in the inhibition of Src versus the src family members Yes and Lck, as well as other protein tyrosine kinases, Ser/Thr kinases, and other ATP-utilizing enzymes.     

SARS-CoV-2中和纳米抗体在里氏木霉中的重组表达北大核心CSCD

基于骆驼科动物单链抗体VHH结构域的纳米抗体具有分子量小、结构简单、溶解性好、稳定性强等多种优势,可通过吸入给药,在呼吸道病毒的防控中具有重要应用价值。里氏木霉是食品级的蛋白质生产宿主,其纤维素酶分泌量可达到80 g/L以上,有望用于药物蛋白的低成本生产。文中在密码子优化的基础上,使用组成型强启动子Pcdna1,实现了SARS-CoV-2中和纳米抗体Nb20在里氏木霉中的重组表达。将Nb20与里氏木霉纤维二糖水解酶CBHⅠ的N端片段融合表达,并在二者间引入胞内KEX2蛋白酶切位点,于葡萄糖培养基中摇瓶发酵48 h可生产出浓度为47.4 mg/L的Nb20蛋白。重组表达的纳米抗体能够与SARS-CoV-2刺突蛋白的受体结合区相结合,有望用于新型冠状病毒的中和。以上结果显示,里氏木霉在纳米抗体的重组表达中具有一定的应用潜力。     

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G₁ to S phase transition of cells by enhancing CDK inhibitor p21^Cip-1/Waf1 expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB^Ser-780) followed by the up-regulation of E2F1 target genes required to promote G₁ to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB^Ser-780 levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G₁ phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21^Cip-1/Waf1 pathway.     

Evolutionary History of Endogenous Human Herpesvirus 6 Reflects Human Migration out of Africa

Human herpesvirus 6A and 6B (HHV-6) can integrate into the germline, and as a result, ∼70 million people harbor the genome of one of these viruses in every cell of their body. Until now, it has been largely unknown if 1) these integrations are ancient, 2) if they still occur, and 3) whether circulating virus strains differ from integrated ones. Here, we used next-generation sequencing and mining of public human genome data sets to generate the largest and most diverse collection of circulating and integrated HHV-6 genomes studied to date. In genomes of geographically dispersed, only distantly related people, we identified clades of integrated viruses that originated from a single ancestral event, confirming this with fluorescent in situ hybridization to directly observe the integration locus. In contrast to HHV-6B, circulating and integrated HHV-6A sequences form distinct clades, arguing against ongoing integration of circulating HHV-6A or “reactivation” of integrated HHV-6A. Taken together, our study provides the first comprehensive picture of the evolution of HHV-6, and reveals that integration of heritable HHV-6 has occurred since the time of, if not before, human migrations out of Africa.     

Recombinant pp60 c-src from Baculovirus-Infected Insect Cells: Purification and Characterization

A simple and effective method has been developed to purify the recombinant protein tyrosine kinase pp60^c-^src from a baculovirus-insect cell expression system. The procedure includes affinity chromatography and HPLC. Milligram quantities of protein have been isolated with an activity of 3.9 μmol/min/mg protein using the substrate poly E₄Y. This specific activity is many times higher than any published protocol. The enzyme is stable for months when stored in buffered 10% glycerol at ?70°C. This purification technique is compared to the immuno-affinity technique which is widely used for this enzyme. Enzyme kinetics were characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg⁺² versus Mn⁺² ions. Similar to the enzyme expressed in human cells, the recombinant enzyme demonstrated a higher Vmax and substrate specificity for poly E₄Y over 5V-Agt-II. An activation energy of 14.2 kcal/mol was determined. Inhibition by increasing ionic strength is mostly due to an increase in Km for the poly E₄Y substrate and hence was substrate dependent. The Km(ATP) was pH dependent while the Km(poly E₄Y) was pH independent.     

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA.

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.     

Fitness benefits of low infectivity in a spatially structured population of bacteriophages

For a parasite evolving in a spatially structured environment, an evolutionarily advantageous strategy may be to reduce its transmission rate or infectivity. We demonstrate this empirically using bacteriophage (phage) from an evolution experiment where spatial structure was maintained over 550 phage generations on agar plates. We found that a single substitution in the major capsid protein led to slower adsorption of phage to host cells with no change in lysis time or burst size. Plaques formed by phage isolates containing this mutation were not only larger but also contained more phage per unit area. Using a spatially explicit, individual-based model, we showed that when there is a trade-off between adsorption and diffusion (i.e. less ‘sticky’ phage diffuse further), slow adsorption can maximize plaque size, plaque density and overall productivity. These findings suggest that less infective pathogens may have an advantage in spatially structured populations, even when well-mixed models predict that they will not.     

Heterozygous NTF4 Mutations Impairing Neurotrophin-4 Signaling in Patients with Primary Open-Angle Glaucoma

Glaucoma, a main cause of blindness in the developed world, is characterized by progressive degeneration of retinal ganglion cells (RGCs), resulting in irreversible loss of vision. Although members of the neurotrophin gene family in various species are known to support the survival of numerous neuronal populations, including RGCs, it is less clear whether they are also required for survival and maintenance of adult neurons in humans. Here, we report seven different heterozygous mutations in the Neurotrophin-4 (NTF4) gene accounting for about 1.7% of primary open-angle glaucoma patients of European origin. Molecular modeling predicted a decreased affinity of neurotrophin 4 protein (NT-4) mutants with its specific tyrosine kinase receptor B (TrkB). Expression of recombinant NT-4 carrying the most frequent mutation was demonstrated to lead to decreased activation of TrkB. These findings suggest a pathway in the pathophysiology of glaucoma through loss of neurotrophic function and may eventually open the possibility of using ligands activating TrkB to prevent the progression of the disease.