Medical management of severe obstructive sleep apnoea in two cases during the coronavirus disease 2019 pandemic.

OBJECTIVE: Severe paediatric obstructive sleep apnoea in typically developing children with adenotonsillar hypertrophy is primarily managed surgically. Non-emergency ENT surgery was paused early in the coronavirus disease 2019 pandemic and children were offered medical management for obstructive sleep apnoea. METHODS: A service evaluation was performed to assess the impact of continuous positive airway pressure alongside medical management for severe obstructive sleep apnoea. RESULTS: Over 5 months during 2020, in a tertiary care setting, two children (one boy and one girl), aged 2.7 years and 4.1 years, were offered continuous positive airway pressure and medical treatments for severe obstructive sleep apnoea whilst surgery was paused during the coronavirus disease 2019 pandemic. Both children failed to establish continuous positive airway pressure therapy because of ongoing disturbed sleep on ventilation, and they proceeded to adenotonsillectomy. Sleep-Related Breathing Disorder scale scores improved following surgical intervention. CONCLUSION: Continuous positive airway pressure therapy is poorly tolerated in children with severe obstructive sleep apnoea secondary to adenotonsillar hypertrophy. Surgery remains the most appropriate treatment.

A nurse-delivered advice intervention can reduce chronic non-steroidal anti-inflammatory drug use in general practice: a randomized controlled trial.

OBJECTIVE: To find out whether a nurse-delivered educational package can reduce chronic oral non-steroidal anti-inflammatory drug (NSAID) usage in general practice. METHOD: A prospective randomized controlled trial with assessment of economic cost/benefits was carried out in five general practices in Nottinghamshire with computerized prescribing systems, representing a mix of rural/urban and fundholding/non-fundholding practices. Patients suffering from non-malignant, non-inflammatory musculoskeletal pain received repeat prescriptions for oral NSAIDs. Two hundred and twenty-two patients were randomized to a control group (simple advice regarding NSAID use) or an intervention group (asked to withdraw their NSAIDs and employ appropriate alternative drug and non-drug therapies). All advice was supported by patient literature and delivered by a nurse practitioner trained in musculoskeletal assessment. The primary outcome measure was change in NSAID use 6 months after the intervention. Secondary outcome measures were changes in health and quality of life (SF-36 and EQ-5D questionnaires) and drug, health service and patient costs. RESULTS: An extra 28% of patients in the intervention group either stopped taking oral NSAIDs or reduced dosage by > or =50% at 6 months compared with controls. There was no detrimental effect on health and well-being. Oral NSAID prescription costs were significantly lowered in the intervention group but not in the control group. A non-significant increase in total drug prescription costs occurred in both groups. CONCLUSIONS: Nurse-based intervention can reduce chronic NSAID usage and costs in primary care and would be cost-effective if maintained in the long term. This intervention package would be readily applicable in primary care.

The introduction of a woman-held record into a hospital antenatal clinic: the bring your own records study.

We report the introduction of a woman-held record into an antenatal clinic in a NSW teaching hospital using a randomized controlled trial. In 1997, 150 women were randomized to either retaining their entire antenatal record through pregnancy (women-held group) or to holding a small, abbreviated card, as was standard practice (control group). A questionnaire was distributed to women to measure sense of control, involvement in care and levels of communication. Availability of records at antenatal visits was also measured. Women in both groups were satisfied with their allocated method of record keeping, however, those in the women-held group were significantly more likely to report feeling in 'control' during pregnancy. Women in the control group were more likely to feel anxious and helpless and less likely to have information on their records explained to them by their caregiver. The number of records available at each clinic was similar in both groups.

Angiostatin binds ATP synthase on the surface of human endothelial cells.

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the alpha/beta-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant alpha-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-alpha-subunit ATP synthase antibody. Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.

"Becoming a mother'--developing a new theory of early motherhood.

This paper explains the methods used in a grounded theory analysis of the experience of 55 first-time mothers in Australia, presented in the first of this series of two papers. The categories identified in the research are realising, readiness, drained, aloneness, loss and working it out, encompassed in the core category becoming a mother. Specifically, this paper extends the analysis and explains the application of a 'paradigm model' and the identification of a Basic Social Process (BSP). The paper links the analysis to the literature on early motherhood from nursing, midwifery, feminist, and sociological research. A substantive theory is proposed to explain women's experience in becoming mothers that demonstrates how, when responsive to the needs of those researched, a grounded theory analysis can provide a framework for nursing and midwifery care.

Becoming a mother--an analysis of women's experience of early motherhood.

This paper presents the results of a qualitative study conducted by midwife researchers into women's experience of new motherhood. Data were collected using focus groups involving 55 first-time mothers and analysed using grounded theory method. The analysis produced six categories: 'realizing', 'unready', 'drained', 'aloneness', 'loss' and 'working it out'. The core category, 'becoming a mother', integrates all other categories and encapsulates the process of change experienced by women. Also explained are factors mediating the often distressing experience of becoming a mother. The analysis provides a conceptualization of early motherhood enabling the development of strategies for midwives, nurses and other helping women negotiate this challenge.

Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants.

The retroviral proteases (PRs) have a structural feature called the flap, which consists of a short anti-parallel beta-sheet with a turn. The flap extends over the substrate binding cleft and must be flexible to allow entry and exit of the polypeptide substrates and products. We analyzed the sequence requirements of the amino acids within the flap region (positions 46-56) of the HIV-1 PR. The phenotypes of 131 substitution mutants were determined using a bacterial expression system. Four of the mutant PRs with mutations in different regions of the flap were selected for kinetic analysis. Our phenotypic analysis, considered in the context of published structures of the HIV-1 PR with a bound substrate analogs, shows that: (i) Met-46 and Phe-53 participate in hydrophobic interactions on the solvent-exposed face of the flap; (ii) Ile-47, Ile-54, and Val-56 participate in hydrophobic interactions on the inner face of the flap; (iii) Ile-50 has hydrophobic interactions at the distance of both the delta and gamma carbons; (iv) the three glycine residues in the beta-turn of the flap are virtually intolerant of substitutions. Among these mutant PRs, we have identified changes in both kcat and Km. These results establish the nature of the side chain requirements at each position in the flap and document a role for the flap in both substrate binding and catalysis.

A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant.

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.

Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease.

Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities.

Identification of temperature-sensitive mutants of the human immunodeficiency virus type 1 protease through saturation mutagenesis. Amino acid side chain requirements for temperature sensitivity.

Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus. An Escherichia coli expression and processing assay system was used to screen 285 protease mutants for temperature-sensitive activity. Fourteen protease mutants had a temperature-sensitive phenotype, and approximately half resulted from conservative amino acid substitutions. Of the 14 substitutions that conferred a temperature-sensitive phenotype, 11 substitutions occurred at 6 positions that represent 3 pairs of residues in the protease that contact each other in the three-dimensional structure. These mutants assist in pinpointing regions of the protease that are important for enzyme activity and stability.

Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structural requirements of the P1 amino acid.

Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products. To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared. Sequence preference was evident in positions P4 through P3' when compared to flanking sequences. Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond. The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes. Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag. In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon. The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site. Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.

The role of peptide growth factors in epithelial ovarian cancer.

We studied the effect of epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta on proliferation of four epithelial ovarian cancer cell lines (OVCA 420, OVCA 429, OVCA 432, and OVCA 433). Epidermal growth factor stimulated growth of OVCA 429 cells (P = .0001) and OVCA 433 cells (P = .0002). Platelet-derived growth factor did not stimulate growth of any of the cell lines. Fibroblast growth factor stimulated growth of OVCA 420 cells (P = .003). Transforming growth factor-beta inhibited growth of OVCA 420 cells (P = .0001), OVCA 432 cells (P = .003), and OVCA 433 cells (P = .004). To detect production of known growth factors by the cancer cell lines, we tested the effect of cancer cell-conditioned media on proliferation of cell lines known to respond to growth factors. Only media exposed to OVCA 433 cells were found to contain activity that mimicked one of the known growth factors (transforming growth factor-beta). These results suggest that individual ovarian cancers vary widely in their response to and production of known peptide growth factors. Finally, we found that OVCA 429-conditioned medium significantly inhibited proliferation of mitogen-stimulated lymphocytes (P less than .0001). The characteristics of this immunosuppressive factor were distinct from those of transforming growth factor-beta. Production of this factor by an immortalized cell line provides a unique opportunity to identify an immunosuppressive substance associated with ovarian cancer.

Complete mutagenesis of the HIV-1 protease.

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.

Heterogeneity of antigen expression in benign and malignant breast and ovarian epithelial cells.

Expression of II distinct antigen families has been studied in normal and malignant epithelial cells from breast and ovary by avidin-biotin immunoperoxidase staining of frozen tissue sections. Each of the antigens was expressed in a fraction of the normal and malignant breast tissues from different individuals. Among breast and ovarian cancers, a similar fraction of tumors expressed each of the determinants. An epitope on a 42-kDa glycoprotein was expressed significantly more often by ovarian carcinomas than by benign ovarian epithelium. Several determinants on 66-, 240-kDa or high-molecular-weight proteins or glycoproteins were expressed significantly more often on benign breast epithelium than on normal ovarian epithelium. Substantial heterogeneity in antigen expression was observed among different tumor specimens. Each of 14 epithelial ovarian cancers expressed a distinctive combination of antigens. Among 18 breast cancers, 16 distinct antigenic phenotypes were observed. Interestingly, comparable heterogeneity was observed in the expression of epitopes on benign breast and ovarian epithelium, compatible with the possibility that the monoclonal reagents recognized polymorphic determinants. Substantial variations in antigen expression were also observed among cells within each neoplasm. However, when antibodies against 5 different determinants were used in combination, a majority of cells could be stained intensely in all 13 breast carcinomas tested and more than 95% of tumor cells could be stained in 10 of the 13.