Accelerated Vascular Aging in Chronic Kidney Disease: The Potential for Novel Therapies.

The pathophysiology of vascular disease is linked to accelerated biological aging and a combination of genetic, lifestyle, biological, and environmental risk factors. Within the scenario of uncontrolled artery wall aging processes, CKD (chronic kidney disease) stands out as a valid model for detailed structural, functional, and molecular studies of this process. The cardiorenal syndrome relates to the detrimental bidirectional interplay between the kidney and the cardiovascular system. In addition to established risk factors, this group of patients is subjected to a plethora of other emerging vascular risk factors, such as inflammation, oxidative stress, mitochondrial dysfunction, vitamin K deficiency, cellular senescence, somatic mutations, epigenetic modifications, and increased apoptosis. A better understanding of the molecular mechanisms through which the uremic milieu triggers and maintains early vascular aging processes, has provided important new clues on inflammatory pathways and emerging risk factors alike, and to the altered behavior of cells in the arterial wall. Advances in the understanding of the biology of uremic early vascular aging opens avenues to novel pharmacological and nutritional therapeutic interventions. Such strategies hold promise to improve future prevention and treatment of early vascular aging not only in CKD but also in the elderly general population.

Peripheral cord insertion is associated with adverse pregnancy outcome only when accompanied by clinically significant placental pathology.

OBJECTIVE: To examine the relationship between umbilical cord insertion site, placental pathology and adverse pregnancy outcome in a cohort of normal and complicated pregnancies. METHODS: Sonographic measurement of the cord insertion and detailed placental pathology were performed in 309 participants. Associations between cord insertion site, placental pathology and adverse pregnancy outcome (pre-eclampsia, preterm birth, small-for-gestational age) were examined. RESULTS: A total of 93 (30%) participants were identified by pathological examination to have a peripheral cord insertion site. Only 41 of the 93 (44%) peripheral cords were detected by prenatal ultrasound. Peripherally inserted cords were associated significantly (P < 0.0001) with diagnostic placental pathology (most commonly with maternal vascular malperfusion (MVM)); of which 85% had an adverse pregnancy outcome. In cases of isolated peripheral cords, without placental pathology, the incidence of adverse outcome was not statistically different when compared to those with central cord insertion and no placental pathology (31% vs 18%; P = 0.3). A peripheral cord with an abnormal umbilical artery (UA) pulsatility index (PI) corresponded to an adverse outcome in 96% of cases compared to 29% when the UA-PI was normal. CONCLUSIONS: This study demonstrates that peripheral cord insertion is often part of the spectrum of findings of MVM disease and is associated with adverse pregnancy outcome. However, adverse outcome was uncommon when there was an isolated peripheral cord insertion and no placental pathology. Therefore, additional sonographic and biochemical features of MVM should be sought when a peripheral cord is observed. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

Determination of fetal heart rate short-term variation from umbilical artery Doppler waveforms.

OBJECTIVE: To evaluate the feasibility of using umbilical artery (UA) Doppler waveforms to measure fetal heart rate (FHR) short-term variation (STV) across gestation. METHODS: This was a prospective longitudinal study, conducted at two study sites, of 195 pregnancies considered low risk. Pulsed-wave Doppler of the UAs was performed at 4-weekly intervals, between 14 and 40 weeks of gestation, using a standardized imaging protocol. Up to 12 consecutive UA Doppler waveforms were analyzed using offline processing software. FHR STV was calculated using average R-R intervals extracted from the waveforms and baseline corrected for FHR. RESULTS: Baseline-corrected FHR STV increased significantly with gestational age (conditional R2 = 0.37; P < 0.0001) and was correlated inversely with FHR (conditional R2 = 0.54; P < 0.0001). The STV ranged (median (interquartile range)) from 3.5 (2.9-4.1) ms at 14-20 weeks' gestation to 6.3 (4.8-7.7) ms at 34-40 weeks' gestation. The change in heart rate STV did not differ between study sites or individual sonographers. CONCLUSIONS: UA Doppler waveforms offer a robust and feasible method to derive STV of the FHR. It should be emphasized that the UA Doppler-derived STV is not interchangeable with measurements derived with computerized cardiotocography. Accordingly, further investigations are needed to validate associations with outcome, in order to determine the value of concurrent fetal cardiovascular and heart rate evaluations that are possible with the technique described here. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Genetic epidemiology of malignant hyperthermia in the UK.

BACKGROUND: Gaps in our understanding of genetic susceptibility to malignant hyperthermia (MH) limit the application and interpretation of genetic diagnosis of the condition. Our aim was to define the prevalence and role of variants in the three genes implicated in MH susceptibility in the largest comprehensively phenotyped MH cohort worldwide. METHODS: We initially included one individual from each positive family tested in the UK MH Unit since 1971 to detect variants in RYR1, CACNA1S, or STAC3. Screening for genetic variants has been ongoing since 1991 and has involved a range of techniques, most recently next generation sequencing. We assessed the pathogenicity of variants using standard guidelines, including family segregation studies. The prevalence of recurrent variants of unknown significance was compared with the prevalence reported in a large database of sequence variants in low-risk populations. RESULTS: We have confirmed MH susceptibility in 795 independent families, for 722 of which we have a DNA sample. Potentially pathogenic variants were found in 555 families, with 25 RYR1 and one CACNA1S variants previously unclassified recurrent variants significantly over-represented (P<1×10-7) in our cohort compared with the Exome Aggregation Consortium database. There was genotype-phenotype discordance in 86 of 328 families suitable for segregation analysis. We estimate non-RYR1/CACNA1S/STAC3 susceptibility occurs in 14-23% of MH families. CONCLUSIONS: Our data provide current estimates of the role of variants in RYR1, CACNA1S, and STAC3 in susceptibility to MH in a predominantly white European population.

Retrospective review of Surgical Availability and Readiness in 8 African countries.

OBJECTIVES: The purpose of this study was to assess surgical availability and readiness in 8 African countries using the WHO's Service Availability and Readiness Assessment (SARA) tool. SETTING: We analysed data for surgical services, including basic and comprehensive surgery, comprehensive obstetric care, blood transfusion, and infection prevention, obtained from the WHO's SARA surveys in Sierra Leone, Uganda, Mauritania, Benin, Zambia, Burkina Faso, Democratic Republic of Congo and Togo. PRIMARY AND SECONDARY OUTCOME MEASURES: Among the facilities that were expected to offer surgical services (N=3492), there were wide disparities between the countries in the number of facilities per 100 000 population that reported offering basic surgery (1.0-12.1), comprehensive surgery (0.1-0.8), comprehensive obstetric care (0.1-0.8) and blood transfusion (0.1-0.8). Only 0.1-0.3 facilities per 100 000 population had all three bellwether procedures available, namely laparotomy, open fracture management and caesarean section. In all the countries, the facilities that reported offering surgical services generally had a shortage of the necessary items for offering the services and this varied greatly between the countries, with the facilities having on average 27-53% of the items necessary for offering basic surgery, 56-83% for comprehensive surgery, 49-72% for comprehensive obstetric care and 54-80% for blood transfusion. Furthermore, few facilities had all the necessary items present. However, facilities that reported offering surgical services had on average most of the necessary items for the prevention of infection. CONCLUSIONS: There are important gaps in the surgical services in the 8 African countries surveyed. Efforts are therefore urgently needed to address deficiencies in the availability and readiness to deliver surgical services in these nations, and this will require commitment from multiple stakeholders. SARA may be used to monitor availability and readiness at regular intervals, which will enable stakeholders to evaluate progress and identify gaps and areas for improvement.

What is the true in vitro potency of oxytetracycline for the pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida?

The pharmacodynamics of oxytetracycline was determined for pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Indices of potency were determined for the following: (i) two matrices, broth and pig serum; (ii) five overlapping sets of twofold dilutions; and (iii) a high strength starting culture. For A. pleuropneumoniae, minimum inhibitory concentration (MIC) was similar for the two matrices, but for P. multocida, differences were marked and significantly different. MIC and minimum bactericidal concentration (MBC) serum: broth ratios for A. pleuropneumoniae were 0.83:1 and 1.22:1, respectively, and corresponding values for P. multocida were 22.0:1 and 7.34:1. For mutant prevention concentration (MPC) serum: broth ratios were 0.79:1 (A. pleuropneumoniae) and 20.9:1 (P. multocida). These ratios were corrected for serum protein binding to yield fraction unbound (fu) serum: broth MIC ratios of 0.24:1 (A. pleuropneumoniae) and 6.30:1 (P. multocida). Corresponding fu serum: broth ratios for MPC were almost identical, 0.23:1 and 6.08:1. These corrections for protein binding did not account for potency differences between serum and broth for either species; based on fu serum MICs, potency in serum was approximately fourfold greater than predicted for A. pleuropneumoniae and sixfold smaller than predicted for P. multocida. For both broth and serum and both bacterial species, MICs were also dependent on initial inoculum strength. The killing action of oxytetracycline had the characteristics of codependency for both A. pleuropneumoniae and P. multocida in both growth media. The in vitro potency of oxytetracycline in pig serum is likely to be closer to the in vivo plasma/serum concentration required for efficacy than potency estimated in broths.

Factors influencing the potency of marbofloxacin for pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida.

For the pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, Minimum Inhibitory Concentration (MIC) of marbofloxacin was determined in recommended broths and pig serum at three inoculum strengths. MICs in both growth matrices increased progressively from low, through medium to high starting inoculum counts, 104, 106 and 108CFU/mL, respectively. P. multocida MIC ratios for high:low inocula were 14:4:1 for broth and 28.2:1 for serum. Corresponding MIC ratios for A. pleuropneumoniae were lower, 4.1:1 (broth) and 9.2:1 (serum). MIC high:low ratios were therefore both growth matrix and bacterial species dependent. The effect of alterations to the chemical composition of broths and serum on MIC were also investigated. Neither adjusting broth or serum pH in six increments over the range 7.0 to 8.0 nor increasing calcium and magnesium concentrations of broth in seven incremental steps significantly affected MICs for either organism. In time-kill studies, the killing action of marbofloxacin had the characteristics of concentration dependency against both organisms in both growth matrices. It is concluded that MIC and time-kill data for marbofloxacin, generated in serum, might be preferable to broth data, for predicting dosages of marbofloxacin for clinical use.

Potency of marbofloxacin for pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida: Comparison of growth media.

Pharmacodynamic properties of marbofloxacin were established for six isolates each of the pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Three in vitro indices of potency were determined; Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Mutant Prevention Concentration (MPC). For MIC determination Clinical Laboratory Standards Institute guidelines were modified in three respects: (1) comparison was made between two growth media, an artificial broth and pig serum; (2) a high inoculum count was used to simulate heavy clinical bacteriological loads; and (3) five overlapping sets of two-fold dilutions were used to improve accuracy of determinations. Similar methods were used for MBC and MPC estimations. MIC and MPC serum:broth ratios for A. pleuropneumoniae were 0.79:1 and 0.99:1, respectively, and corresponding values for P. multocida were 1.12:1 and 1.32:1. Serum protein binding of marbofloxacin was 49%, so that fraction unbound (fu) serum MIC values were significantly lower than those predicted by correction for protein binding; fu serum:broth MIC ratios were 0.40:1 (A. pleuropneumoniae) and 0.50:1 (P. multocida). For broth, MPC:MIC ratios were 13.7:1 (A. pleuropneumoniae) and 14.2:1 (P. multocida). Corresponding ratios for serum were similar, 17.2:1 and 18.8:1, respectively. It is suggested that, for dose prediction purposes, serum data might be preferable to potency indices measured in broths.

APOBEC3A intratumoral DNA electroporation in mice.

Human APOBEC3A (A3A) cytidine deaminase shows pro-apoptotic properties resulting from hypermutation of genomic DNA, induction of double-stranded DNA breaks (DSBs) and G1 cell cycle arrest. Given this, we evaluated the antitumor efficacy of A3A by intratumoral electroporation of an A3A expression plasmid. DNA was repeatedly electroporated into B16OVA, B16Luc tumors of C57BL/6J mice as well as the aggressive fibrosarcoma Sarc2 tumor of HLA-A*0201/DRB1*0101 transgenic mice using noninvasive plate electrodes. Intratumoral electroporation of A3A plasmid DNA resulted in regression of ~50% of small B16OVA melanoma tumors that did not rebound in the following 2 months without treatment. Larger or more aggressive tumors escaped regression when so treated. As APOBEC3A was much less efficient in provoking hypermutation and DSBs in B16OVA cells compared with human or quail cells, it is likely that APOBEC3A would be more efficient in a human setting than in a mouse model.

Activity of florfenicol for Actinobacillus pleuropneumoniae and Pasteurella multocida using standardised versus non-standardised methodology.

Four indices of antimicrobial potency were determined for florfenicol and the pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), mutant prevention concentration (MPC) and time-kill curves were determined in two matrices, broth and pig serum. Five overlapping sets of two-fold dilutions were used to increase accuracy of the measurements. MIC and MBC serum:broth ratios for A. pleuropneumoniae were 0.96:1 and 1.07:1, respectively, and corresponding values for P. multocida were 0.72:1 and 0.50:1. The percentage binding of florfenicol to serum protein was 65.4%, and fraction unbound (fu) serum MICs were significantly lower, by 2.71-fold and 3.82-fold, respectively, than predicted for free serum concentrations for A. pleuropneumoniae and P. multocida. Similar culture medium differences were obtained for MBC and MPC. MICs in serum and broth were increased significantly and progressively for high, medium and low initial inoculum counts. Serum MPC:MIC ratios for A. pleuropneumoniae and P. multocida were 12.5:1 and 13.6:1, respectively; ratios for broth were similar. The killing action of florfenicol had the characteristics of concentration dependency for both species in both growth media. These data indicate the value of using a biological medium, when determining microbiological potency indices, to predict dosage for clinical use.

APOBEC3A catabolism of electroporated plasmid DNA in mouse muscle.

The mouse is widely used as a model for DNA therapy and vaccination even though the efficiency of DNA delivery in higher mammals and humans is much less. The human APOBEC3 (A3) enzymes impact viral genomes by cytidine deamination, which introduces multiple uridine residues into single-stranded DNA, a process known as genetic editing. This initiates rapid DNA catabolism via a uracil DNA glycosylase dependent pathway. In tissue culture, A3A, A3C and A3B can hyperedit transfected plasmid DNA. We explored plasmid catabolism in vivo initiated by A3A, the most efficient of the human enzymes and one that is functionally conserved across most mammals. As rodents do not encode an A3A enzyme, it was possible to explore DNA degradation in the mouse model. Human A3A genetically edits co-electroporated luciferase plasmid DNA in mouse skeletal muscle that initiates DNA degradation resulting in approximately fourfold decrease in bioluminescence. Part of the degradation occurs in the nucleus as indicated by complex hyperedited DNA molecules. As human A3A is strongly upregulated by interferon α and DNA sensing pathways, it is a strong candidate enzyme for restricting plasmid DNA in higher mammals.

Erroneous identification of APOBEC3-edited chromosomal DNA in cancer genomics.

BACKGROUND: The revolution in cancer genomics shows that the dominant mutations are CG->TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C->T mutations in the signature 5'TpC context. These can be copied as G->A transitions in the 5'GpA context. METHODS: DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA. RESULTS: Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5'GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR. CONCLUSIONS: Differential DNA denaturation PCR cannot be used to identify fixed C->T transitions in cancer genomes. Presently, the overall mutation frequency is ∼10(4)-10(5) base substitutions per cancer genome, or 0.003-0.03 kb(-1). By contrast, the 3DPCR error rate is of the order of 4-20 kb(-1) owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C->T and G->A mutations associated with the 5'GpC represent artefacts.

Polymorphic APOBEC3 modulates chronic hepatitis B in Moroccan population.

The cytidine deaminase apolipoprotein B mRNA editing catalytic subunit-3 (APOBEC3) induces G-to-A hypermutation in hepatitis B virus (HBV) genomes and operates as part of the innate antiviral immune system. We investigated the associations between the presence of APOBEC3 variants and HBV carriage in a case-control study in the Moroccan population. A polymorphic deletion affecting the APOBEC3B gene and the H186R variant of APOBEC3G were genotyped in 179 HBV chronic carriers and 216 healthy control subjects. In addition, to assess the overall impact of APOBEC3 deaminases on circulating HBV, we looked for hyperedited forms of the viral genome using the 3DPCR technique and analysed editing context. Data analysis showed that there was no significant difference in the frequencies of deleted APOBEC3B alleles (P = 0.261) or genotypes (P = 0.333) between patients with chronic hepatitis B and control subjects. By contrast, subjects bearing deleted genotype had a faster progression of liver disease than those with the insertion genotype (adjusted OR, 3.72; 95% CI, 0.38-36.12). The analysis of the APOBEC3G H186R polymorphism revealed that R/R genotype frequencies were not significantly different in HBV infected patients and in healthy subjects. 3DPCR was positive in 26 samples (14%) among 179. Amplified viral segments displayed monomorphic G>A transitions highly reminiscent of APOBEC3G activity. Most intriguingly, hemi/homozygous carriers of the APOBEC3B deletion had significantly lower virus loads than patients with the wild type (median 539 vs. 2213 IU/mL, P = 0.0023). This result suggests that genetic variations in APOBEC3 cytidine deaminases do not predispose to chronicity but may modulate the course of persistent HBV infection.

Galanin acts as a trophic factor to the central and peripheral nervous systems.

The neuropeptide galanin is widely, but not ubiquitously, expressed in the adult nervous system. Its expression is markedly up-regulated in many neuronal tissues after nerve injury or disease. Over the last 10 years, we have demonstrated that the peptide plays a developmental survival role to subsets of neurons in the peripheral and central nervous systems with resulting phenotypic changes in neuropathic pain and cognition. Galanin also appears to play a trophic role to adult sensory neurons following injury, via activation of GalR2, by stimulating neurite outgrowth. Furthermore, galanin also plays a neuroprotective role to the hippocampus following excitotoxic injury, again mediated by activation of GalR2. Most recently, we have shown that galanin expression is markedly up-regulated in multiple sclerosis (MS) lesions and in the experimental autoimmune encephalomyelitis (EAE) model of MS. Over-expression of galanin in transgenic mice abolishes disease in the EAE model, whilst loss-of-function mutations in galanin or GalR2 increase disease severity. In summary, these studies demonstrate that a GalR2 agonist might have clinical utility in a variety of human diseases that affect the nervous system.

Total ankle replacement in patients with significant pre-operative deformity of the hindfoot.

We carried out 123 consecutive total ankle replacements in 111 patients with a mean follow-up of four years (2 to 8). Patients with a hindfoot deformity of up to 10 degrees (group A, 91 ankles) were compared with those with a deformity of 11 degrees to 30 degrees (group B, 32 ankles). There were 18 failures (14.6%), with no significant difference in survival between groups A and B. The clinical outcome as measured by the post-operative American Orthopaedic Foot and Ankle Surgeons score was significantly better in group B (p = 0.036). There was no difference between the groups regarding the post-operative range of movement and complications. Correction of the hindfoot deformity was achieved to within 5 degrees of neutral in 27 ankles (84%) of group B patients. However, gross instability was the most common mode of failure in group B. This was not adequately corrected by reconstruction of the lateral ligament. Total ankle replacement can safely be performed in patients with a hindfoot deformity of up to 30 degrees . The importance of adequate correction of alignment and instability is highlighted.

Galanin acts as a trophic factor to the central and peripheral nervous systems.

The neuropeptide galanin is widely, but not ubiquitously, expressed in the adult nervous system. Its expression is markedly upregulated in many neuronal tissues after nerve injury or disease. Over the last 10 years we have demonstrated that the peptide plays a developmental survival role to subsets of neurons in the peripheral and central nervous systems with resulting phenotypic changes in neuropathic pain and cognition. Galanin also appears to play a trophic role to adult sensory neurons following injury, via activation of GalR2, by stimulating neurite outgrowth. Furthermore, galanin also plays a neuroprotective role to the hippocampus following excitotoxic injury, again mediated by activation of GalR2. In summary, these studies demonstrate that a GalR2 agonist might have clinical utility in a variety of human diseases that affect the nervous system.

Prospective comparison of POSSUM and P-POSSUM with clinical assessment of mortality following emergency surgery.

BACKGROUND: Tools to accurately estimate the risk of death following emergency surgery are useful adjuncts to informed consent and clinical decisions. This prospective study compared the Physiological and Operative Severity Score for the Enumeration of Mortality and Morbidity (POSSUM) and Portsmouth POSSUM (P-POSSUM) scoring systems with clinical judgement in predicting mortality from emergency surgery. METHODS: Data were collected prospectively from 163 patients. Details of the physiological and operative severity scores were recorded for POSSUM and P-POSSUM. The estimates of both the surgeon and anaesthetist for 30-day and in-hospital mortality were also recorded pre-operatively. The accuracies of the four predictions were then compared with actual mortalities using linear and exponential analysis and receiver operator characteristics (ROC). RESULTS: P-POSSUM gave the most accurate prediction of 30-day mortality using linear analysis [observed to expected ratio (O : E) = 1.0]. POSSUM gave the most accurate prediction using exponential analysis (O : E = 1.15). Clinical judgement of mortality from both operating surgeons and anaesthetists compared favourably with the scoring systems for 30-day mortality (O : E = 0.83 and O : E = 0.93, respectively). ROC analyses showed both clinical judgement and the POSSUM scores to be good predictors of 30-day mortality with area under the curve values (AUC) of 0.903, 0.907, 0.946 and 0.940 for surgeons, anaesthetists, POSSUM and P-POSSUM respectively. CONCLUSIONS: POSSUM and P-POSSUM appear to be useful indicators for the prediction of mortality. Clinical judgement compares strongly with scoring systems in predicting post-operative mortality, but may underestimate mortality in very high-risk patients with more than 90% mortality.

Migration of endoscopic biliary stent and small bowel perforation within an incisional hernia.

Long-term complications from biliary stents, such as migration and perforation, are unusual but may occur. Distal perforations most commonly involve the colon or distal ileum. This report describes the case of a perforated small bowel, secondary to a stent trapped within an incisional hernia. Patients with comorbid abdominal pathologies, including colonic diverticulae or abdominal herniae, may be at increased risk of perforation from migrated stents.