Increased paediatric presentations of severe diabetic ketoacidosis in an Australian tertiary centre during the COVID-19 pandemic.

AIMS: To determine if the frequency of severe diabetic ketoacidosis at presentation of new-onset type 1 diabetes to an Australian tertiary centre increased during the initial period of restrictions resulting from the COVID-19 pandemic (March to May 2020). METHODS: Data were collected on presentations of newly diagnosed type 1 diabetes as well as on all paediatric presentations to the emergency department of a tertiary centre between 2015 and 2020. Data from the period of initial COVID restrictions in Australia (March to May 2020) were compared to the period March to May of the previous 5 years (pre-pandemic periods). RESULTS: The number of new diagnoses of type 1 diabetes was comparable in the pandemic period and pre-pandemic periods (11 in 2020 vs range 6-10 in 2015-2019). The frequency of severe diabetic ketoacidosis was significantly higher in the pandemic period compared to the pre-pandemic periods (45% vs 5%; P <0.003), odds ratio 16.7 (95% CI 2.0, 194.7). The overall frequency of diabetic ketoacidosis was also significantly higher during the pandemic period (73% vs 26%; P <0.007), odds ratio 7.5 (95% CI 1.7, 33.5). None of the individuals tested positive for COVID-19. Presentations of people aged <18 years to the emergency department decreased by 27% in the pandemic period compared to the average of the pre-pandemic periods (4799 vs 6550; range 6268 to 7131). CONCLUSIONS: A significant increase in the frequency of severe diabetic ketoacidosis at presentation of type 1 diabetes was observed during the initial period of COVID-19 restrictions. We hypothesize that concern about presenting to hospital during a pandemic led to a delay in diagnosis. These data have important implications for advocacy of seeking healthcare for non-pandemic-related conditions during a global pandemic.

High-protein meals require 30% additional insulin to prevent delayed postprandial hyperglycaemia.

AIM: To determine the amount of additional insulin required for a high-protein meal to prevent postprandial hyperglycaemia in individuals with type 1 diabetes using insulin pump therapy. METHODS: In this randomized cross-over study, 26 participants aged 8-40 years, HbA1c < 65 mmol/mol (8.1%), received a 50 g protein, 30 g carbohydrate, low-fat (< 1 g) breakfast drink over five consecutive days at home. A standard insulin dose (100%) was compared with additional doses of 115, 130, 145 and 160% for the protein, in randomized order. Doses were commenced 15-min pre-drink and delivered over 3 h using a combination bolus with 65% of the standard dose given up front. Postprandial glycaemia was assessed by 4 h of continuous glucose monitoring. RESULTS: The 100% dosing resulted in postprandial hyperglycaemia. From 120 min, ≥ 130% doses resulted in significantly lower postprandial glycaemic excursions compared with 100% (P < 0.05). A 130% dose produced a mean (sd) glycaemic excursion that was 4.69 (2.42) mmol/l lower than control, returning to baseline by 4 h (P < 0.001). From 120 min, there was a significant increase in the risk of hypoglycaemia compared with control for 145% [odds ratio (OR) 25.4, 95% confidence interval (CI) 5.5-206; P < 0.001) and 160% (OR 103, 95% CI 19.2-993; P < 0.001). Some 81% (n = 21) of participants experienced hypoglycaemia following a 160% dose, whereas 58% (n = 15) experienced hypoglycaemia following a 145% dose. There were no hypoglycaemic events reported with 130%. CONCLUSIONS: The addition of 30% more insulin to a standard dose for a high-protein meal, delivered using a combination bolus, improves postprandial glycaemia without increasing the risk of hypoglycaemia.

Young children, adolescent girls and women with type 1 diabetes are more overweight and obese than reference populations, and this is associated with increased cardiovascular risk factors.

AIM: Overweight and obesity are frequently reported in young persons with type 1 diabetes, however its relative magnitude in comparison to the general population is not well understood. This study compared the prevalence of overweight and obesity in young persons with type 1 diabetes to a reference population and explored possible associated factors, including gender, age, HbA1c , insulin regimen, age at diagnosis, diabetes duration, socio-economic status and cardiovascular disease risk factors. METHODS: A cross-sectional review was undertaken of data collected from youth (3-17 years) in 2016 and young adults (18-30 years) in 2015 with a diagnosis of type 1 diabetes for > 3 months attending diabetes centres in Newcastle, Australia. Rates of overweight and obesity were compared with matched population survey results. RESULTS: Data from 308 youth and 283 young adults were included. In girls, significantly higher prevalence of overweight and obesity were seen in the 5-8 (43% vs. 18%), 13-16 (41% vs. 27%), 18-24 (46% vs. 34%) and 25-30 (60% vs. 43%) years age groups; whereas in boys increased prevalence was observed in the 5-8 years age group only (41% vs. 18%). Rates of overweight and obesity increased with age across sexes. In youth, BMI standard deviation score was correlated with socio-economic status, insulin regimen, blood pressure and blood lipids (P < 0.05). In adults, BMI was positively associated with blood pressure, and longer diabetes duration (P < 0.02). CONCLUSIONS: Overweight and obesity are over-represented in young persons with type 1 diabetes, particularly girls. As overweight is associated with other cardiovascular disease markers early intervention is paramount.

Increasing the protein quantity in a meal results in dose-dependent effects on postprandial glucose levels in individuals with Type 1 diabetes mellitus.

AIM: To determine the glycaemic impact of increasing protein quantities when consumed with consistent amounts of carbohydrate in individuals with Type 1 diabetes on intensive insulin therapy. METHODS: Participants with Type 1 diabetes [aged 10-40 years, HbA1c ≤ 64 mmol/mol (8%), BMI ≤ 91st percentile] received a 30-g carbohydrate (negligible fat) test drink daily over 5 days in randomized order. Protein (whey isolate 0 g/kg carbohydrate, 0 g/kg lipid) was added in amounts of 0 (control), 12.5, 25, 50 and 75 g. A standardized dose of insulin was given for the carbohydrate. Postprandial glycaemia was assessed by 5 h of continuous glucose monitoring. RESULTS: Data were collected from 27 participants (15 male). A dose-response relationship was found with increasing amount of protein. A significant negative relationship between protein dose and mean excursion was seen at the 30- and 60-min time points (P = 0.007 and P = 0.002, respectively). No significant relationship was seen at the 90- and 120-min time points. Thereafter, the dose-response relationship inverted, such that there was a significant positive relationship for each of the 150-300-min time points (P < 0.004). Mean glycaemic excursions were significantly greater for all protein-added test drinks from 150 to 300 min (P < 0.005) with the 75-g protein load, resulting in a mean excursion that was 5 mmol/l higher when compared with the control test drink (P < 0.001). CONCLUSIONS: Increasing protein quantity in a low-fat meal containing consistent amounts of carbohydrate decreases glucose excursions in the early (0-60-min) postprandial period and then increases in the later postprandial period in a dose-dependent manner.

Differentiation of human epidermal cells transformed by SV40.

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.

Purification of intact microtubules from brain.

Hundred-fold purification of intact microtubules from homogenates of rat brain is reported. The success of purification depends on stabilizing the microtubule structure by the combined effects of hexylene glycol, acidic Ph, and low temperature. A practical, negative stain, electron microscopic assay is used to study purity and stability of microtubule fractions. The purified fractions show a major band which migrates like purified tubulin in the SDS gel electrophoresis system.

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

Association of human papillomavirus types 16 and 18 E6 proteins with p53.

Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product, pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses, the adenoviruses, and SV40 may effect similar cellular pathways in transformation.

Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade.

The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.

Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation.

Levels of the tumor suppressor protein p53 are normally quite low due in part to its short half-life. p53 levels increase in cells exposed to DNA-damaging agents, such as radiation, and this increase is thought to be responsible for the radiation-induced G1 cell cycle arrest or delay. The mechanisms by which radiation causes an increase in p53 are currently unknown. The purpose of this study was to compare the effects of gamma and UV radiation on the stability and ubiquitination of p53 in vivo. Ubiquitin-p53 conjugates could be detected in nonirradiated and gamma-irradiated cells but not in cells which were UV treated, despite the fact that both treatments resulted in the stabilization of the p53 protein. These results demonstrate that UV and gamma radiation have different effects on ubiquitinated p53 and suggest that the UV-induced stabilization of p53 results from a loss of p53 ubiquitination. Ubiquitinated forms of p21, an inhibitor of cyclin-dependent kinases, were detected in vivo, demonstrating that p21 is also a target for degradation by the ubiquitin-dependent proteolytic pathway. However, UV and gamma radiation had no effect on the stability or in vivo ubiquitination of p21, indicating that the radiation effects on p53 are specific.

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.

E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein.

Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

Cell-heritable stages of tumor progression in transgenic mice harboring the bovine papillomavirus type 1 genome.

Tumorigenesis of dermal fibroblasts in a line of transgenic mice carrying the BPV-1 genome was found to involve distinct proliferative stages. Cell cultures derived from normal skin, from benign proliferative fibromatoses, and from malignant fibrosarcomas each evidenced distinguishable, cell-heritable characteristics. The latent viral genome was transcriptionally inactive in normal-appearing skin and was activated in the dermal fibromatoses. Fibrosarcoma cells grew continuously in culture, formed domelike foci, and had a more rounded, anaplastic appearance. Independent cultures derived from the fibromatoses varied in their proliferative characteristics, which correlated well with the levels of viral gene expression. In contrast, progression to malignancy was not accompanied by a further increase in transgene activity, which strongly implicated cellular genetic changes in the later stages of tumorigenesis.

A stable bovine papillomavirus hybrid plasmid that expresses a dominant selective trait.

We describe a bovine papillomavirus hybrid plasmid containing the neomycin resistance gene from Tn5 inserted into a mammalian cell transcriptional unit. This plasmid is maintained as a stable extrachromosomal element (20 to 100 copies per diploid genome) in mouse cells selected either for the transformed phenotype or for resistance to the aminoglycoside G418. Cells selected for G418 resistance initially display a flat, nontransformed phenotype before exhibiting the gross morphological characteristics of transformation. The delay in the appearance of the transformed phenotype indicated that some intracellular event or series of events subsequent to the establishment of transcriptionally active bovine papillomavirus 1 hybrid plasmid is required for the manifestation of the transformed phenotype.

Functional characterization of interferon regulatory factor 3a (IRF-3a), an alternative splice isoform of IRF-3.

Virus infection of numerous cell types results in the transcriptional induction of a subset of virus- and interferon (IFN)-stimulated genes. The beta IFN (IFN-beta) gene is one of these rapidly induced genes; it serves as a fundamental component of the cellular defense response in eliciting potent antiviral, immunomodulatory, and antiproliferative effects. One of the transcription factors involved in the stringent regulation of IFN-beta production following virus infection is interferon regulatory factor (IRF) 3 (IRF-3). We have characterized an alternatively spliced isoform of IRF-3 that we have called IRF-3a. IRF-3a can selectively and potently inhibit virus-induced activation of the IFN-beta promoter. IRF-3a lacks half of the DNA binding domain found in IRF-3 and is unable to bind to the classical IRF binding elements, IFN-stimulated response elements. These studies suggest that IRF-3a may act as a modulator of IRF-3.

Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression.

The wild-type (wt) p53 protein has transcriptional activation functions which may be linked to its tumor suppressor activity. Many mutant p53 proteins expressed in cancers have lost the ability to function as transcriptional activators and furthermore may inhibit wt p53 function. To study the mechanisms by which mutant forms of p53 have lost their transactivation function and can act in a dominant negative manner, a structure-function analysis of both mutant and engineered truncated forms of p53 was carried out. We show that different mutant p53 proteins found in cancers vary in the ability to inhibit the transcriptional transactivation and specific DNA binding activities of wt human p53. This transdominant effect was mediated through the carboxy-terminal oligomerization region. The role of the transactivation activity in transformation suppression by wt p53 was also examined by constructing an N-terminal deletion mutant lacking the transactivation domain. This mutant was unable to transactivate but could bind specifically to DNA. Although it was impaired in its ability to suppress transformation of primary rat embryo fibroblasts by adenovirus E1A plus activated ras, the N-terminal deletion mutant still had some suppression activity, suggesting that additional functions of p53 may contribute to transformation suppression.

Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

Inducible expression of the human interferon beta 1 gene linked to a bovine papilloma virus DNA vector and maintained extrachromosomally in mouse cells.

A 1.6-kilobase DNA segment of the genomic human interferon beta 1 (IF-beta 1) gene was inserted into each of two possible orientations at the single HindIII site of a recombinant plasmid pBPV69T, consisting of the 69% transforming region of the bovine papilloma virus type 1 (BPV-1) and a modified SalI-SalI fragment of plasmid pBR322. After cleavage of the pBR322 sequences from this recombinant, BPV69T-IF-beta 1 hybrid DNAs were transfected onto C127 mouse cells by the standard calcium precipitation technique. Mouse cells transformed by this hybrid DNA produced low levels of human IF-beta 1 constitutively and responded to induction with either inactivated Newcastle disease virus or polyriboinosinic acid-polyribocytidylic acid. The BPV69T-IF-beta 1 hybrid DNA was nonintegrated in the transformed mouse cells but had acquired DNA sequences as a result of the transfection. Accurate transcripts of the IF-beta 1 mRNA were detected in cells only after induction. When the IF-beta 1 gene was oriented in the plasmid in the same direction of transcription as the BPV-1 genome, transcription was promoted from within the BPV-1 sequences. These results indicate that the regulatory sequences responsible for the inducible expression of the human IF-beta 1 gene are present in the 1.6-kilobase genomic segment and that these sequences can function in a free extrachromosomal state linked to BPV-1 sequences.

Enhancer-dependent expression of the rat preproinsulin gene in bovine papillomavirus type 1 vectors.

The effect of position in a bovine papillomavirus type 1 (BPV-1) vector on foreign gene expression was assessed with the rat preproinsulin (rI1) gene. The rI1 gene was inserted at each of the BPV-1/pML2d junctions in either transcriptional orientation in derivatives of the pdBPV-1(142-6) vector which consists of the BamHI linear genome of BPV-1 DNA cloned into pML2d. Transformed lines of C127 cells were established and assayed for rI1 gene expression. Cells containing the rI1 gene at the 3' end of the BPV-1 transforming region expressed rat proinsulin, whereas cells with the gene at the 5' end of the nontransforming region did not. Variability in the plasmid copy number or in the extent of DNA rearrangement could not account for this difference. We conclude that the expression of the rat preproinsulin gene (which is normally tissue specific for pancreatic islet cells) in C127 cells depends on the transcriptional activation afforded by viral enhancer sequences located at the 3' end of the transforming region. Intervening BPV-1 or pML2d sequences appear to block this enhancer-mediated gene activation. In agreement with enhancer-dependent activation, a rat preproinsulin gene located in a blocked position (i.e., not adjacent to the BPV-1 enhancer) could be activated by the insertion of a DNA fragment containing the simian virus 40, Moloney murine sarcoma virus, or BPV-1 enhancer element adjacent to the rI1 gene. Thus, a gene which is normally not expressed in a particular cell may be activated when placed adjacent to a viral enhancer in a BPV-1 vector.