Impaired Mineral Ion Metabolism in a Mouse Model of Targeted Calcium-Sensing Receptor (CaSR) Deletion from Vascular Smooth Muscle Cells.

BACKGROUND: Impaired mineral ion metabolism is a hallmark of CKD-metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. METHODS: To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells ( SM22α CaSR Δflox/Δflox ). RESULTS: Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. CONCLUSIONS: These results suggest that, in addition to CaSR's established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis.

Monoclonal antibody targeting of fibroblast growth factor receptor 1c causes cardiac valvulopathy in rats.

Fibroblast Growth Factors (FGFs) and their receptors (FGFRs) have been proposed as potential drug targets for the treatment of obesity. The aim of this study was to assess the potential toxicity in rats of three anti-FGFR1c mAbs with differential binding activity prior to clinical development. Groups of male rats received weekly injections of either one of two FGFR1c-specific mAbs or an FGFR1c/FGFR4-specific mAb at 10 mg/kg for up to 4 weeks. All three mAbs caused significant reductions in food intake and weight loss leading to some animals being euthanized early for welfare reasons. In all three groups given these mAbs, microscopic changes were seen in the bones and heart valves. In the bones of the femoro-tibial joint, thickening of the diaphyseal cortex of long bones, due to deposition of well organized new lamellar bone, indicated that an osteogenic effect was observed. In the heart, valvulopathy described as an endocardial myxomatous change affecting the mitral, pulmonary, tricuspid and aortic valves was observed in all mAb-treated animals. The presence of FGFR1 mRNA expression in the heart valves was confirmed using in situ hybridization. Targeting the FGF-FGFR1c pathway with anti-FGFR1c mAbs leads to drug induced valvulopathy in rats. In effect, this precluded the development of these mAbs as potential anti-obesity drugs. The valvulopathy observed was similar to that described for fenfluramine and dexafenfluramine. The pathogenesis of the drug-induced valvulopathy is considered FGFR1c-mediated, based on the specificity of the mAbs and FGFR1 mRNA expression in the heart valves.

Case report: Recurrent glossopharyngeal neuralgia after previous glossopharyngeal rhizotomy: Microvascular decompression with intra-operative neurophysiology.

We present a case of recurrent vago-glossopharyngeal neuralgia after previous surgery, treated successfully with microvascular decompression using intra-operative neurophysiology monitoring.

Tissue expression and correlation of a panel of urinary biomarkers following cisplatin-induced kidney injury.

In recent years, there has been considerable activity to identify urinary biomarkers of nephrotoxicity as noninvasive measurements with greater sensitivity and specificity than traditional biomarkers, such as serum creatinine and blood urea nitrogen. Our study aimed to use cisplatin-treated rats to evaluate the use of immunohistochemistry directed at multiple urinary biomarkers in kidney tissue. Tissue levels were compared to urinary levels of these biomarkers to demonstrate tissue specificity and sensitivity. These techniques could also be used in studies where urine samples are not available, such as retrospective studies in drug safety testing, to demonstrate the potential utility of using these biomarkers in future preclinical or clinical studies. All of the biomarkers investigated showed either an increase (kidney injury molecule [KIM-1], osteopontin [OPN], and, clusterin) or a decrease (alpha-glutathione S-transferase and trefoil factor 3) except beta 2 microglobulin (ß2MG) that showed no significant changes 5 days after 1.0 mg/kg or 2.5 mg/kg cisplatin treatment. All of the biomarkers except ß2MG showed utility as tissue biomarkers, but KIM-1 and OPN expression correlated closely with urinary biomarker measurements and reflect tissue damage. Future studies are needed to determine the wider application of these two markers for detecting renal toxicity following administration of other nephrotoxicants.

Awake minimally invasive parafascicular approach to a language eloquent brain tumour-surgical video.

Deep-seated brain tumours are surgically challenging to access. When planning approaches to these lesions, it is important to take into account eloquent cortical areas, grey matter nuclei, and subcortical white matter tracts. Traditionally, access to deep-seated lesions would require brain retraction; however, this is associated with secondary brain damage, which may impair neurological function. A trans-sulcal minimally invasive parafascicular approach allows gentle splitting of brain fibres and is thought to splay rather than sever white matter tracts. This is particularly important when approaching medially located, language-eloquent tumours, which lack brain surface expression. This video describes a minimally invasive approach to a deep-seated, language-eloquent brain tumour. We utilized preoperative cortical and subcortical planning to define a safe surgical corridor. We then demonstrate using intraoperative neuro-monitoring and mapping of the motor and language functions to define the boundaries of surgical resection. We find trans-sulcal minimally invasive parafascicular approach to be a safe and effective technique when approaching language-eloquent lesions medial to the main language subcortical networks.

Facilitators for increasing dental attendance of people from vulnerable groups: a rapid review of evidence relevant to the UK.

Objective To rapidly review facilitators of access for vulnerable groups and to evaluate their effectiveness.Methods Data sources: MEDLINE via Ovid. Publications in English from 2000. DATA SELECTION: Research involving 'vulnerable groups' relevant to UK health systems, with a primary outcome of increasing attendance. DATA EXTRACTION: One author extracted and tabulated data. These were audited by a second author. DATA SYNTHESIS: A narrative synthesis was produced.Results Data from 31 studies were available for ten vulnerable groups: people with learning, physical or sensory disabilities (n = 8); people experiencing homelessness (n = 6); prisoners (n = 4); asylum-seekers and refugees (n = 3); people living in socioeconomically deprived areas (n = 3); people with severe mental health conditions (n = 2); vulnerable children (n = 2); dependent older people (n = 1); Gypsy, Roma or Traveller groups (n = 1); and people with drug dependency (n = 1). Many facilitators involved organisational reform and more integration of health, social and other services. Other facilitators included: modification of premises; team development and skill-mix use; and awareness of needs and flexible services to meet them. Few studies evaluated effectiveness.Conclusion Although facilitators for access for vulnerable groups have been proposed, there is little evidence to support or refute their effectiveness. Efforts are needed to promote access for vulnerable groups in the UK with evaluation plans embedded.

Evaluation of novel urinary renal biomarkers: biological variation and reference change values.

A number of novel urinary biomarkers have been identified and partially qualified for use as markers for renal injury in rats. We used two multiplex assays for these novel biomarkers to quantify biomarker concentration in serial urine collections from rats of both sexes administered varying concentrations of cisplatin. From these data, we calculate inter-individual variation and reference ranges from predose animals and intra-individual variation and reference change values from undosed control animals. The biomarkers evaluated are albumin, α glutathione s-transferase, glutathione S-transferase-yb1, lipocalin-2, kidney injury molecule-1, osteopontin, and renal papillary antigen 1. For any creatinine-corrected novel biomarkers, we found intra-individual variation to be no greater than 44% and inter-individual variation to be no greater than 46%. Reference change values for most corrected analytes (except osteopontin) were 50-100%, indicating that a >100% increase in analyte concentration between serial samples would be unlikely to be associated with inherent analytical or biological variation.

Delayed brain metastasis after 17-years from papillary thyroid cancer without local recurrence: case report and literature review.

Papillary thyroid carcinoma (PTC) is the most common malignancy originating from the thyroid, with a good overall prognosis. However, distant metastasis of such lesions is very rare, with the brain being an incredibly uncommon site for secondary spread. The authors report a case of PTC brain metastasis 17-years after successful treatment of the primary malignancy, with no local or locoregional recurrence. Initial diagnostic uncertainty necessitated the involvement of a multidisciplinary team, and eventually the patient underwent image-guided gross surgical resection with intraoperative neuromonitoring (IOMN).

A landscape review of controlled release urea products: Patent objective, formulation and technology.

Fertiliser has been a vital part of agriculture due to it boosting crop productivity and preventing starvation throughout the world. Despite this huge contribution, the application of nitrogen (N) fertilisers results in N leaching and the formation of greenhouse gases, which threaten the environment and human health. To minimise the impacts, slow/controlled release fertilisers (S/CRFs) have been being developed since the beginning of the 20th century. Despite the efforts made over a century, the basic terminological and classification information of these fertilisers remains vague. The scientific knowledge published in S/CRF patents has also been overlooked since the beginning. This review focused on the information gaps, clarified the definitions, differentiation and classification methods that have been randomly used in previous literature. The objectives, formulations and technologies of 109 controlled release urea patents involving sulphur coated urea, polymer coated urea and urea matrix fertilisers published in the years since these products emerged were also reviewed to 1) highlight the overlooked scientific knowledge in the patents; 2) understand the evolutionary processes and current research states of the products; 3) clarify research preferences and challenges to date; 4) identify remaining gaps for the future direction. It is expected that the organised basic information and the patent knowledge highlighted in this paper can be new resources and foster the development of S/CRFs in the future.

Favipiravir in early symptomatic COVID-19, a randomised placebo-controlled trial.

Background: Well tolerated antivirals administered early in the course of COVID-19 infection when the viremia is highest could prevent progression to severe disease. Favipiravir inhibits SARS-CoV-2 viral replication in vitro with evidence of clinical benefit in open label trials. Placebo controlled studies of people with early symptomatic COVID-19 with regular assessments of SARS-CoV-2 viral load can determine if it has an antiviral effect and improves clinical outcomes. Methods: People with PCR-confirmed COVID-19 and 5 days or less of symptoms were randomised 1:1 to favipiravir 1800 mg on day 1, then 800 mg twice daily or matched placebo for 14 days. SARS-CoV-2 viral load was quantitated from second daily self-collected nose-throat swabs while receiving study drug. The primary endpoint was time to virological cure defined as 2 successive swabs negative for SARS-CoV-2 by PCR and secondary outcomes were progression of disease severity, symptom resolution and safety. Findings: Between 31 July 2020 and 19 September 2021, 200 people were enrolled (199 in the community, 1 in hospital) with 190 receiving one or more doses of drug (modified intention to treat [mITT] population). There was no difference in time to virological cure (Log-rank p=0.6 comparing Kaplan Meier curves), progression to hospitalisation (14 favipiravir, 9 placebo; p=0.38), time to symptom resolution (cough, fever, sore throat) and there were no deaths. 51 people related an adverse event that was possibly drug related, but these were evenly distributed (n=24 favipiravir, n=27 placebo). Sensitivity analyses where the definition of virological cure was changed to: a single negative PCR, exclude datapoints based on the presence or absence of human DNA in the swab, a SARS-CoV-2 viral load < 300 copies/mL being considered negative all demonstrated no difference between arms. Interpretation: Favipiravir does not improve the time to virological cure or clinical outcomes and shows no evidence of an antiviral effect when treating early symptomatic COVID-19 infection. Funding: The study was supported in part by grants from the Commonwealth Bank Australia, the Lord Mayor's Charitable Foundation, Melbourne Australia and the Orloff Family Charitable Trust, Melbourne, Australia. JHM is supported by the Medical Research Future Fund, AYP, JT are supported by the Australian National Health and Medical Research Council.

Glutathione S-transferases as molecular markers of tumour progression and prognosis in renal cell carcinoma.

AIMS: Renal cell carcinoma (RCC) often recurs as distant metastasis; there is thus a need for new indicators to identify high-risk patients. Glutathione S-transferases (GST)-α and -π are involved in the renal bioactivation of toxic metabolites. The aim was to investigate whether their expression is of diagnostic and prognostic value. METHODS AND RESULTS: Western blotting of microdissected normal kidney and immunostaining of histological RCC microarrays shows expression of GST-α in proximal tubular cells, while GST-π was found in the distal nephron. Of the primary 174 RCC cases examined, GST-α immunoreactivity was restricted to conventional RCC (n=76, 68% positive) and was not seen in any other RCC subtypes. The cross-tabulation of the GST-α scores with other prognostic indices demonstrated that GST-α immunostaining was significantly more frequent in low-grade tumours (χ(2): P<0.004), and that conventional GST-α-positive RCC patients had a mean disease-free survival of 6.0 years (95% confidence interval 5.33-6.63), compared with 4.7 years (3.54-5.90) in GST-α-negative tumours (Kaplan-Meier survival analysis, P=0.011, log-rank test). CONCLUSIONS: GST-α is a highly specific diagnostic marker for primary conventional RCC, where it is a prognostic marker if grade is omitted from the multivariate analysis.

Electrospun Membranes as a Porous Barrier for Molecular Transport: Membrane Characterization and Release Assessment.

Electrospun nanofibers have been extensively studied for encapsulated drugs releasing from the inside of the fiber matrix, but have been barely looked at for their potential to control release as a semi-permeable membrane. This study investigated molecular transport behaviors across nanofiber membranes with different micro-structure sizes and compositions. Four types of membranes were made by 5% and 10% poly (ε-caprolactone) (PCL) solutions electro-spun with or without 50 nm calcium carbonate (CaCO3) nanoparticles. The membranes were tested for thickness, fiber diameter, pore size, porosity, tensile strength and elongation, contact angle of water and their impacts on molecular transport behaviors. The presence of the CaCO3 nanoparticles made the 5% membranes stronger and stiffer but the 10% membranes weaker and less stiff due to the different (covering or embedded) locations of the nanoparticles with the corresponding fibers. Solute transport studies using caffeine as the model drug found the 5% membranes further retarded release from the 10% membranes, regardless of only half the amount of material being used for synthesis. The addition of CaCO3 nanoparticles aided the water permeation process and accelerated initial transports. The difference in release profiles between 5% and 10% membranes suggests different release mechanisms, with membrane-permeability dominated release for 5% PCL membranes and solute-concentration-gradient dominated release for 10% PCL membranes.

Metastatic Colon Cancer in the Pituitary: A Case Report with Review of Literature.

We report a rare case of metastatic colonic adenocarcinoma to the pituitary gland in a 58-year-old who presented with visual decline and panhypopituitarism. He underwent urgent transsphenoidal endoscopic surgery with significant improvement of his vision, followed by adjuvant fractionated radiotherapy to the resection cavity. He made a satisfactory recovery, but regrettably died from COVID-19 9 weeks after completion of radiotherapy. A multidisciplinary approach is essential for optimal management of this condition due to its rarity and complexity.

Expression and localization of rat aldo-keto reductases and induction of the 1B13 and 1D2 isoforms by phenolic antioxidants.

The aldo-keto reductase (AKR) phase I drug metabolism enzyme superfamily is implicated in detoxification or bioactivation of a wide variety of carbonyl-bearing compounds. In this study, we have used antibodies raised against purified recombinant rat AKR isoforms 1A3, 1B4, 1C9, 1D2, and 7A1 to characterize the expression profile of these superfamily members in the rat and define their localization by immunohistochemistry. Western blotting showed that AKR1A3, AKR1B4, and AKR1C9 are ubiquitously expressed, whereas AKR1D2 and AKR7A1 are present in liver, adrenal gland, and kidney, with the latter also present in testis, spleen, and stomach. Immunohistochemical analysis of the kidney demonstrated the localization of AKR1A3 in proximal convoluted tubules, AKR1B4 in the loop of Henle, and AKR1C9 in the pars recta S3 segment of proximal tubules. We also report localization of AKR1B4 in the adrenal gland (parenchymal cells of the zona reticularis) and testis (Sertoli cells and late spermatids), of AKR1D2 in the liver (hepatocyte nuclei), and of AKR7A1 in the pancreatic duct and bronchiolar epithelium. Previous studies have shown that expression of AKR7A1 is induced in response to dietary administration of the phenolic antioxidants butylated hydroxyanisole and ethoxyquin. Here we identify AKR1B13 and AKR1D2 as further inducible members of the rat AKR superfamily.

Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers.

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.

Characterization of renal papillary antigen 1 (RPA-1), a biomarker of renal papillary necrosis.

Renal papillary necrosis (RPN) is a relatively common toxicity observed in preclinical drug safety testing. It is also observed in a variety of human diseases. RPN is difficult to diagnose without expensive scanning methods or histopathology. A noninvasive biomarker that could be detected at early stages of kidney damage would be of great value both to preclinical drug safety testing and in the clinic. An antibody raised to an unknown epitope of an antigen in rat kidney papilla was found to be specific for collecting duct cells in the kidney; this was termed renal papillary antigen 1 (RPA-1). In this study, the authors show that RPA-1 is an early biomarker of RPN in two different rat models of toxicity: 2-bromoethanamine (BEA) and N-phenylanthranilic acid (NPAA). RPA-1 can be detected in urine at early stages of toxicity and correlates well with the histopathology observed. We also characterized the biochemical properties of RPA-1 and found that the antigen is a high molecular weight membrane bound glycoprotein, with the epitope likely to be carried on an N-linked carbohydrate structure. This study demonstrates that RPA-1 is an excellent marker of RPN that can be used to detect this toxicity in preclinical safety testing.

Enhanced secretion of interferon-gamma by bovine gammadelta T cells induced by coculture with Mycobacterium bovis-infected dendritic cells: evidence for reciprocal activating signals.

Evidence suggests that gammadelta T cells form part of the innate immune response to Mycobacterium bovis infection. Dendritic cells (DCs) are capable of secreting high levels of interleukin-12 (IL-12) following infection with mycobacteria and can induce interferon-gamma (IFN-gamma) secretion by natural killer and gammadelta T cells We investigated the innate interactions occurring between WC1(+)gammadelta T cells and M. bovis-infected DCs. Following coculture with M. bovis-infected DCs, secretion of IFN-gamma and expression of CD25 and major histocompatibility complex class II on WC1(+)gammadelta T cells were significantly enhanced. Reciprocal enhancement of IL-12 secretion by the DCs was also observed and this interaction was found to be contact dependent. We hypothesize that there is an early, transient signal between the WC1(+)gammadelta T cells and the DCs, which promotes the synthesis of biologically active IL-12, and which is dependent upon cell-cell contact. Reciprocal signals including IL-12 are then delivered to WC1(+)gammadelta cells, which leads to the enhanced secretion of IFN-gamma, and the up-regulation of activation markers and antigen presentation molecules by the WC1(+)gammadelta T cells. These interactions are likely to form a critical part of the T helper type 1-conditioning response of DCs to M. bovis.

Children's toothache is becoming everybody's business: where do parents go when their children have oral pain in London, England? A cross-sectional analysis.

OBJECTIVES: To assess the number of parents who visited community pharmacies in London seeking pain medications for their children's pain and specifically for oral pain, to identify which health services parents contacted before their pharmacy visit and to estimate the cost to the National Health Service (NHS) when children with oral pain who visit pharmacies also see health professionals outside dentistry. DESIGN: A cross-sectional study. SETTING: 1862 pharmacies in London in November 2016-January 2017. PARTICIPANTS: Parents, carers and adolescents purchasing over-the-counter pain medications or collecting pain prescriptions for children (0-19 years). BRIEF INTERVENTION: A survey administered by pharmacy staff to participants and a guidance pack. MAIN OUTCOME MEASURES: The number of parents who visited pharmacies seeking pain medications for their children's pain and oral pain and the number of parents who contacted health professionals outside dentistry before their pharmacy visit. Estimated costs of visits by children with oral pain to health professionals outside dentistry. RESULTS: One in two (951) pharmacies participated collecting information from 6915 parents seeking pain medications for their children. The majority (65%) of parents sought pain medications to relieve their children's oral pain. Only 30% of children with oral pain had seen a dentist before the pharmacy visit, while 28% of children had seen between one and four different health professionals. The cost to the NHS of children contacting health professionals outside dentistry was £36 573, extrapolated to an annual cost of £373 288. Replicating these findings across all pharmacies in England could mean that the NHS spends an estimated £2.3 million annually when children with oral pain inappropriately use multiple health services. CONCLUSION: Most parents who visited pharmacies for children's pain medications in London sought pain medications for children's oral pain. Children's inappropriate contact with multiple health services when they have oral pain adds significant costs to the NHS.

Gene transfer of an engineered transcription factor promoting expression of VEGF-A protects against experimental diabetic neuropathy.

Peripheral neuropathy is a common, irreversible complication of diabetes. We investigated whether gene transfer of an engineered zinc finger protein transcription factor (ZFP-TF) designed to upregulate expression of the endogenous vascular endothelial growth factor (VEGF)-A gene could protect against experimental diabetic neuropathy. ZFP-TF-driven activation of the endogenous gene results in expression of all of the VEGF-A isoforms, a fact that may be of significance for recapitulation of the proper biological responses stimulated by this potent neuroprotective growth factor. We show here that this engineered ZFP-TF activates VEGF-A in appropriate cells in culture and that the secreted VEGF-A protein induced by the ZFP protects neuroblastoma cell lines from a serum starvation insult in vitro. Importantly, single and repeat intramuscular injections of formulated plasmid DNA encoding the VEGF-A-activating ZFP-TF resulted in protection of both sensory and motor nerve conduction velocities in a streptozotocin-induced rat model of diabetes. These data suggest that VEGF-A-activating ZFP-TFs may ultimately be of clinical utility in the treatment of this disease.

Expression of hexokinase isoforms in the dorsal root ganglion of the adult rat and effect of experimental diabetes.

The effect of streptozotocin (STZ)-induced diabetes on expression and activity of hexokinase, the first enzyme and rate-limiting step in glycolysis, was studied in sensory neurons of lumbar dorsal root ganglia (DRG). The DRG and sciatic nerve of adult rats expressed the hexokinase I isoform only. Immunofluorescent staining of lumbar DRG demonstrated that small-medium neurons and satellite cells exhibited high levels of expression of hexokinase I. Large, mainly proprioceptive neurons, had very low or negative staining for hexokinase I. Intracellular localization and biochemical studies on intact DRG from adult rats and cultured adult rat sensory neurons revealed that hexokinase I was almost exclusively found in the mitochondrial compartment. Duration of STZ-diabetes of 6 or 12 weeks diminished hexokinase activity by 28% and 30%, respectively, in lumbar DRG compared with age matched controls (P<0.05). Quantitative Western blotting showed no effect of diabetes on hexokinase I protein expression in homogenates or mitochondrial preparations from DRG. Immunofluorescent staining for hexokinase I showed no diabetes-dependent change in small-medium neuron expression in DRG, however, large neurons became positive for hexokinase I (P<0.05). Such complex effects of diabetes on hexokinase I expression in the DRG may be due to glucose-driven up-regulation of expression or the result of impaired axonal transport and perikaryal accumulation in the large neuron sub-population. Because hexokinase is the rate-limiting enzyme of glycolysis these results imply that metabolic flux through the glycolytic pathway is reduced in diabetes. This finding, therefore, questions the role of high glucose-induced metabolic flux as a key driving force in reactive oxygen species generation by mitochondria.