ROCK2/rasHa cooperation induce malignant conversion via p53 loss, elevated NF-κβ and tenascin C-associated rigidity but p21 inhibits ROCK2/NF-κβ-mediated progression

To study ROCK2 activation in carcinogenesis, mice expressing 4-hydroxytamoxifen (4HT)- activated ROCK2 [K14.ROCKer] were crossed to mice expressing epidermal activated ras Ha [HK1.ras1205]. At 8 weeks, 4HT-treated K14.ROCKer-HK1.ras1205 cohorts exhibited papillomas similar to HK1.ras1205 controls; however, K14.ROCKer-HK1.ras1205 histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma [wdSCC], exhibiting p53 loss, increased proliferation, and novel NF-κβ expression. By 12 weeks, K14.ROCKer-HK1.ras1205 wdSCCs exhibited increased NF-κβ and novel tenascin C, indicative of elevated rigidity; yet despite continued ROCK2 activities /p-Mypt1 inactivation, progression to SCC required loss of compensatory p21 expression. K14.ROCKer -HK1.ras1205 papillomatogenesis also required a wound-promotion stimulus, confirmed by breeding K14.ROCKer into promotion-insensitive HK1.ras1276 mice, suggesting a permissive K14.ROCKer-HK1.ras1205 papilloma context [wound-promoted/NF-κβ+ve/p53-ve/p21+ve] preceded K14.ROCKer-mediated [p-Mypt1/tenascin C/rigidity] malignant conversion. Malignancy depended on ROCKer/p-Mypt1 expression, as cessation of 4HT-treatment induced disorganised tissue architecture and p21-associated differentiation in wdSCCs; yet tenascin C retention in connective tissue ECM suggests the rigidity laid down for conversion persists. Novel papilloma outgrowths appeared expressing intense, basal-layer p21 which confined endogenous ROCK2/p-Mypt1/NF-κβ to supra-basal layers, and was paralleled by restored basal-layer p53. In later SCCs, 4HT-cessation became irrelevant as endogenous ROCK2 expression increased, driving progression via p21 loss, elevated NF-κβ expression and tenascin C-associated rigidity; with p-Mypt1 inactivation/actinomyosin-mediated contractility to facilitate invasion. However, p21-associated inhibition of early-stage malignant progression and the intense expression in papilloma outgrowths, identifies a novel, significant antagonism between p21 and ras Ha/ROCK2/NF-κβ signalling in skin 3 carcinogenesis. Collectively these data show that ROCK2 activation induces malignancy in rasHa-initiated/promoted papillomas in the context of p53 loss and novel NF-κβ expression;whilst increased tissue rigidity and cell motility/contractility help mediate tumour progression

Fos co-operation with PTEN loss elicits keratoacanthoma not carcinoma due to p53/p21^WAF-induced differentiation triggered by GSK3b inactivation and reduced AKT activity

To investigate gene synergism in multistage skin carcinogenesis, the RU486-inducible cre/lox system was employed to ablate PTEN function [K14.cre/D5PTENflx] in mouse epidermis expressing activated v-fos [HK1.fos]. RU486-treated HK1.fos/D5PTENflx mice exhibited hyperplasia, hyperkeratosis and tumours that progressed to highly differentiated keratoacanthomas rather than carcinomas, due to re-expression of high p53 and p21WAF levels. Despite elevated MAP kinase activity, cyclin D1/E2 over expression and increased AKT activity forming areas of highly proliferative, papillomatous keratinocytes, increasing levels of GSK3b inactivation exceeded a threshold that induced p53/p21WAF expression to halt proliferation and accelerate differentiation, giving the hallmark keratosis of keratoacanthomas. A pivotal facet to this GSK3b-triggered mechanism centred on increasing p53 expression in basal layer keratinocytes. This reduced activated AKT expression and released inhibition of p21WAF, which accelerated keratinocyte differentiation, as indicated by unique basal layer expression of differentiation-specific keratin K1, alongside premature filaggrin and loricrin expression. Thus, fos synergism with PTEN loss elicited a benign tumour context where GSK3b-induced, p53/p21WAF expression continually switched AKT-associated proliferation into one of differentiation, preventing further progression. This putative compensatory mechanism required the critical availability of normal p53 and/or p21WAF otherwise deregulated fos, Akt and GSK3b associate with malignant progression

Pharmacokinetic-pharmacodynamic correlation of imipenem in pediatric burn patients using a bioanalytical liquid chromatographic method

A bioanalytical method was developed and applied to quantify the free imipenem concentrations for pharmacokinetics and PK/PD correlation studies of the dose adjustments required to maintain antimicrobial effectiveness in pediatric burn patients. A reverse-phase Supelcosil LC18 column (250 x 4.6 mm 5 micra), binary mobile phase consisting of 0.01 M, pH 7.0 phosphate buffer and acetonitrile (99:1, v/v), flow rate of 0.8 mL/min, was applied. The method showed good absolute recovery (above 90%), good linearity (0.25-100.0 µg/mL, r2=0.999), good sensitivity (LLOQ: 0.25 µg/mL; LLOD: 0.12 µg/mL) and acceptable stability. Inter/intraday precision values were 7.3/5.9%, and mean accuracy was 92.9%. A bioanalytical method was applied to quantify free drug concentrations in children with burns. Six pediatric burn patients (median 7.0 years old, 27.5 kg), normal renal function, and 33% total burn surface area were prospectively investigated; inhalation injuries were present in 4/6 (67%) of the patients. Plasma monitoring and PK assessments were performed using a serial blood sample collection for each set, totaling 10 sets. The PK/PD target attained (40%T>MIC) for each minimum inhibitory concentration (MIC: 0.5, 1.0, 2.0, 4.0 mg/L) occurred at a percentage higher than 80% of the sets investigated and 100% after dose adjustment. In conclusion, the purification of plasma samples using an ultrafiltration technique followed by quantification of imipenem plasma measurements using the LC method is quite simple, useful, and requires small volumes for blood sampling. In addition, a small amount of plasma (0.25 mL) is needed to guarantee drug effectiveness in pediatric burn patients. There is also a low risk of neurotoxicity, which is important because pharmacokinetics are unpredictable in these critical patients with severe hospital infection. Finally, the PK/PD target was attained for imipenem in the control of sepsis in pediatric patients with burns.</p

Large-scale discovery of novel genetic causes of developmental disorders

Despite three decades of successful, predominantly phenotype-driven discovery of the genetic causes of monogenic disorders1, up to half of children with severe developmental disorders of probable genetic origin remain without a genetic diagnosis. Particularly challenging are those disorders rare enough to have eluded recognition as a discrete clinical entity, those with highly variable clinical manifestations, and those that are difficult to distinguish from other, very similar, disorders. Here we demonstrate the power of using an unbiased genotype-driven approach2 to identify subsets of patients with similar disorders. By studying 1,133 children with severe, undiagnosed developmental disorders, and their parents, using a combination of exome sequencing3,4,5,6,7,8,9,10,11 and array-based detection of chromosomal rearrangements, we discovered 12 novel genes associated with developmental disorders. These newly implicated genes increase by 10% (from 28% to 31%) the proportion of children that could be diagnosed. Clustering of missense mutations in six of these newly implicated genes suggests that normal development is being perturbed by an activating or dominant-negative mechanism. Our findings demonstrate the value of adopting a comprehensive strategy, both genome-wide and nationwide, to elucidate the underlying causes of rare genetic disorders

Global age-sex-specific mortality, life expectancy, and population estimates in 204 countries and territories and 811 subnational locations, 1950–2021, and the impact of the COVID-19 pandemic: a comprehensive demographic analysis for the Global Burden of Disease Study 2021

Background: Estimates of demographic metrics are crucial to assess levels and trends of population health outcomes. The profound impact of the COVID-19 pandemic on populations worldwide has underscored the need for timely estimates to understand this unprecedented event within the context of long-term population health trends. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 provides new demographic estimates for 204 countries and territories and 811 additional subnational locations from 1950 to 2021, with a particular emphasis on changes in mortality and life expectancy that occurred during the 2020–21 COVID-19 pandemic period. Methods: 22 223 data sources from vital registration, sample registration, surveys, censuses, and other sources were used to estimate mortality, with a subset of these sources used exclusively to estimate excess mortality due to the COVID-19 pandemic. 2026 data sources were used for population estimation. Additional sources were used to estimate migration; the effects of the HIV epidemic; and demographic discontinuities due to conflicts, famines, natural disasters, and pandemics, which are used as inputs for estimating mortality and population. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate under-5 mortality rates, which synthesised 30 763 location-years of vital registration and sample registration data, 1365 surveys and censuses, and 80 other sources. ST-GPR was also used to estimate adult mortality (between ages 15 and 59 years) based on information from 31 642 location-years of vital registration and sample registration data, 355 surveys and censuses, and 24 other sources. Estimates of child and adult mortality rates were then used to generate life tables with a relational model life table system. For countries with large HIV epidemics, life tables were adjusted using independent estimates of HIV-specific mortality generated via an epidemiological analysis of HIV prevalence surveys, antenatal clinic serosurveillance, and other data sources. Excess mortality due to the COVID-19 pandemic in 2020 and 2021 was determined by subtracting observed all-cause mortality (adjusted for late registration and mortality anomalies) from the mortality expected in the absence of the pandemic. Expected mortality was calculated based on historical trends using an ensemble of models. In location-years where all-cause mortality data were unavailable, we estimated excess mortality rates using a regression model with covariates pertaining to the pandemic. Population size was computed using a Bayesian hierarchical cohort component model. Life expectancy was calculated using age-specific mortality rates and standard demographic methods. Uncertainty intervals (UIs) were calculated for every metric using the 25th and 975th ordered values from a 1000-draw posterior distribution. Findings: Global all-cause mortality followed two distinct patterns over the study period: age-standardised mortality rates declined between 1950 and 2019 (a 62·8% [95% UI 60·5–65·1] decline), and increased during the COVID-19 pandemic period (2020–21; 5·1% [0·9–9·6] increase). In contrast with the overall reverse in mortality trends during the pandemic period, child mortality continued to decline, with 4·66 million (3·98–5·50) global deaths in children younger than 5 years in 2021 compared with 5·21 million (4·50–6·01) in 2019. An estimated 131 million (126–137) people died globally from all causes in 2020 and 2021 combined, of which 15·9 million (14·7–17·2) were due to the COVID-19 pandemic (measured by excess mortality, which includes deaths directly due to SARS-CoV-2 infection and those indirectly due to other social, economic, or behavioural changes associated with the pandemic). Excess mortality rates exceeded 150 deaths per 100 000 population during at least one year of the pandemic in 80 countries and territories, whereas 20 nations had a negative excess mortality rate in 2020 or 2021, indicating that all-cause mortality in these countries was lower during the pandemic than expected based on historical trends. Between 1950 and 2021, global life expectancy at birth increased by 22·7 years (20·8–24·8), from 49·0 years (46·7–51·3) to 71·7 years (70·9–72·5). Global life expectancy at birth declined by 1·6 years (1·0–2·2) between 2019 and 2021, reversing historical trends. An increase in life expectancy was only observed in 32 (15·7%) of 204 countries and territories between 2019 and 2021. The global population reached 7·89 billion (7·67–8·13) people in 2021, by which time 56 of 204 countries and territories had peaked and subsequently populations have declined. The largest proportion of population growth between 2020 and 2021 was in sub-Saharan Africa (39·5% [28·4–52·7]) and south Asia (26·3% [9·0–44·7]). From 2000 to 2021, the ratio of the population aged 65 years and older to the population aged younger than 15 years increased in 188 (92·2%) of 204 nations. Interpretation: Global adult mortality rates markedly increased during the COVID-19 pandemic in 2020 and 2021, reversing past decreasing trends, while child mortality rates continued to decline, albeit more slowly than in earlier years. Although COVID-19 had a substantial impact on many demographic indicators during the first 2 years of the pandemic, overall global health progress over the 72 years evaluated has been profound, with considerable improvements in mortality and life expectancy. Additionally, we observed a deceleration of global population growth since 2017, despite steady or increasing growth in lower-income countries, combined with a continued global shift of population age structures towards older ages. These demographic changes will likely present future challenges to health systems, economies, and societies. The comprehensive demographic estimates reported here will enable researchers, policy makers, health practitioners, and other key stakeholders to better understand and address the profound changes that have occurred in the global health landscape following the first 2 years of the COVID-19 pandemic, and longer-term trends beyond the pandemic. Funding: Bill & Melinda Gates Foundation

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Inducible cre-mediated N-ras activation and PTEN inactivation in transgenic mouse melanocytes requires keratinocyte hyperplasia to elicit a melanocyte pathology

To investigate the aetiology of melanoma, transgenic models are under development that exploit a RU486-based gene switch regulator, incorporating an inducible cre recombinase expressed from a 2.2kb tyrosinase promoter/200bp enhancer [E-tyr.CreP]. These are bred to mice with transgenes closely associated with human melanoma: activated N-ras&lt;sup&gt;61&lt;/sup&gt;, expressed via eviction of lox-P flanked termination codons [lsl.N-ras], or floxed exon 5 to inactivate PTEN TSG function. To date, systemic or topical RU486 treatment of E-tyr.creP/lsl.N-ras [C57Bl/6] mice failed to induce an obvious cutaneous phenotype, whilst systemic treatment gave rise to ocular RPE hyperplasia and adenoma of the Hardarian gland at 12 months. These data suggest that [keratinocyte-regulated] melanocyte proliferation in being restricted to anagen and rapid apoptosis in catagen, results in few target melanocytes for RU486-induced mutation and highlights a finite temporal window for treatment. However, despite repeated induction of anagen, the persistent lack of phenotype suggests that N-ras activation was insufficient to achieve melanocyte hyperplasia and mice were crossed into a PTEN floxed exon 5 genotype. To date, topically treated E-tyr.creP/N-ras/PTEN&lt;sup&gt;flx/flx&lt;/sup&gt; compound mice remain non-phenotypic, whereas &lt;i&gt;in-vitro&lt;/i&gt; equivalent transgenic melanocytes display altered growth and transformed morphologies. This result may yet reflect additional complications with the &lt;i&gt;in vivo&lt;/i&gt; model: e.g. low regulator expression; hair cycle and lack of interfollicular melanocytes; or the continued dominant regulation of [mutant] melanocytes by keratinocytes. To examine the latter idea, a keratin K14-based regulator, K14.creP was introduced which elicits inducible PTEN-mediated keratinocyte hyperplasia. Whilst E-creP/Nras/PTEN&lt;sup&gt;flx&lt;/sup&gt; genotypes remain stubbornly non-phenotypic, compound K14.creP/E-creP/N-ras/PTEN mice developed cutaneous lesions following 5 months of topical RU486 treatment, several of which were pigmented. Histologically, these are typical papillomas that possessed focal areas of melanocytes, exclusively confined to the basement membrane, as determined by TRP2/K14 double lable immunefluorescence. Additional studies are clearly required to determine if this preliminary observation is a general phenomenon of papillomas arising from the hair follicle, or specific to an indirect PTEN signalling dysfunction effecting melanocyte regulation by keratinocytes. However, if correct, these data suggest that overt melanocyte hyperplasia in this instance requires prior keratinocyte hyperplasia/regulation dysfunction and subsequently leads to an epidermal/dermal junctional pathology rather than dermal invasion typically associated with murine models. Moreover, the observation that UV exposure in childhood manifests as melanoma in adulthood also supports a prerequisite for melanocyte escape from keratinocyte control. These very preliminary data echo this facet and if true, demonstrate a facilitating role for keratinocyte mutation in a seed/soil field cancerisation model of early melanoma development

Activated p-AKT, but not MDM2, drives malignant progression in <i>Ras/Fos/PTEN^null</i> skin carcinogenesis via p53/p21 loss and elevated cyclin D1/E2 expression

Tumour progression depends on a complex combination of the genetic mutation milieu pitted against the sentinel systems that have evolved to resist carcinogenesis at each specific stage. To investigate tumour progression mechanism in transgenic mouse skin carcinogenesis, inducible PTEN ablation [&lt;i&gt;Δ5PTEN&lt;/i&gt;] was introduced into the epidermis of mice expressing activated ras&lt;sup&gt;Ha&lt;/sup&gt;/fos oncogenes. RU486-treated HK1.ras/fos-Δ5PTEN mice exhibited accelerated papillomatogenesis but malignant conversion was delayed due to compensatory p53/p21 expression. Following p53 loss malignant progression was limited to well-differentiated squamous cell carcinoma via persistent p21 expression and down regulation of cyclin E2. Analysis of AKT activity during papillomatogenesis showed reduced p-AKT expression, associated with fos/PTEN feedback, which returned following p53 loss to circumvent/antagonise p21 expression; co-operate with MAPK signalling [i.e. elevated ERK1/2 expression]; and accelerate tumour progression via increased cyclin D1 and E2 expression. In contrast elevated, suprabasal MDM2 expression in p53-positive papillomas was lost in parallel to p53 loss; hence sustained MDM2-mediated p53 ubiquination does not appear to influence this progression mechanism. These data suggest p53/p21 counter deregulated MAPK signalling during papillomatogenesis and help minimise consequences of PTEN loss via p-AKT inhibition. Stepwise p53/p21 loss subsequently facilitates ras/MAPK/fos co-operation with PTEN/AKT activities to accelerate malignant progression via major failures in cell cycle control. The interplay between these common mutations thus create unique contexts that have important implications for therapies geared to reactivating p53/p21 functions or that target ras/MAPK/fos and PTEN/AKT signalling pathways