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Abstract Color polymorphic species provide an excellent opportunity to investigate the ecology and evolution of intraspecific niche differences. The red‐backed salamander, Plethodon cinereus, is a fully terrestrial lungless salamander with two common color forms, striped and unstriped. Previous research suggests the morphs may be differentially adapted to surface and subsurface microhabitats, with the unstriped morph being more fossorial. This hypothesis predicts that the unstriped morph should be more sensitive to the risks of surface activity (e.g., thermal stress, dehydration, predation), and therefore be more selective than striped morphs when choosing soil surface microhabitats. To test this hypothesis, we experimentally manipulated leaf litter mass in small forest patches (~0.45 m2). Leaf litter addition reduced soil temperatures, buffered against changes in air temperature, and likely provided physical protection from predators. Over 3 years, we found that unstriped adults responded positively to leaf litter addition, but striped adults did not. In addition, unstriped morphs spent significantly more time in protective refuges (opaque, moistened tubes) than striped morphs in laboratory assays. Taken together, the field and laboratory results support the hypothesis that the unstriped morph is more sensitive to the risks of surface activity, and therefore is more likely to be fossorial. This difference in microhabitat use, combined with spatiotemporal variation in leaf litter accumulation on the forest floor, may play an important role in the maintenance of the polymorphism.

Recent advances in CRISPR-Cas genomic editors have shifted us ever closer to achieving the ultimate therapeutic goal of accomplishing any edit in any cell. However, delivery of this editing machinery to primary cells with high efficiency while avoiding cellular toxicity remains a formidable challenge. Peptide-Assisted Genome Editing (PAGE) provides a simple, modular, and rapid approach for the protein-based delivery of CRISPR-Cas proteins or ribonucleoprotein complexes into primary cells with high efficiency and minimal cytotoxicity. In this chapter, we detail an expression and purification protocol to obtain highly pure Cas9-T6N and opCas12a-T8N PAGE genomic editors. The robustness of this protocol allows for consistent preparations of the purified editors that can be reliably used for the editing of primary and immortalized cells., (Copyright © 2024. Published by Elsevier Inc.)

Background: The optimal use of infliximab depends on the measurement of trough levels with subsequent appropriate dose adjustment. With the introduction of biosimilars, it is important to demonstrate that the biosimilar behaves similarly in the assay used as the originator-infliximab, for which the assays were developed. In this study, the authors aimed to compare the concentrations of SB2-infliximab (Renflexis) with that of originator-infliximab (Remicade) when added to serum from healthy subjects and those with inflammatory bowel disease when measured by commonly used commercial assays., Methods: Sera from 2 healthy controls, 2 patients with ulcerative colitis (1 with quiescent disease and 1 with active disease), and 2 patients with Crohn disease (1 with quiescent disease and 1 with active disease) were spiked with SB2-infliximab or originator-infliximab at 0-20 mcg/mL. Concentrations were measured using 3 commonly used assay kits (Lisa-Tracker, Shikari Q-Inflix, Promonitor IFX) and one rapid test (Quantum Blue). The results were compared using Bland-Altman techniques., Results: Close agreement was observed between measured concentrations for all assays, irrespective of the origin of the serum. Limits of agreement varied between at worst -0.302 and 0.465 mcg/mL, with the mean difference between the molecules being at worst 0.04 mcg/mL (95% confidence intervals, -0.011 to 0.093)., Conclusions: The originator and SB-2 biosimilar-infliximab behaved similarly in several currently used assays in their concentrations in biological fluids. Clinicians can be confident that therapeutic drug monitoring using platforms designed and developed for the originator-infliximab can be applied to SB-2-infliximab., Competing Interests: M. G. Ward has received educational grants or research support from AbbVie and Merck, Sharp & Dohme (Australia) Pty Ltd, received speaker fees from Janssen, AbbVie, Ferring, Takeda, MSD, and Shire. P. R. Gibson has served as a consultant or advisory board member for Anatara, Atmo Biosciences, Immunic Therapeutics, Janssen, Novozymes, Pfizer, Falk Pharma, and Takeda. His institution has received speaking honoraria from Janssen and Pfizer. He received research grants for investigator‐driven studies from Merck, Sharp & Dohme (Australia) Pty Ltd. The remaining authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Scope: Since epithelial barrier dysfunction has been associated with gluten and fermentable oligosaccharide, disaccharide, monosaccharide, and polyols (FODMAPs), the effect of alterations in FODMAP a gluten intake on epithelial barrier function in patients with irritable bowel syndrome (IBS) who self-reported gluten sensitivity., Methods and Results: Circulating concentrations of markers of epithelial injury (syndecan-1 and intestinal fatty acid-binding protein) and bacterial translocation (lipopolysaccharide-binding protein and soluble CD14) are measured while consuming habitual gluten-free diet and during blinded challenges with gluten or placebo on a background of low FODMAP intake. In 33 patients, only syndecan-1 concentrations during their habitual diet are elevated (median 43 ng mL -1 ) compared with 23 ng mL -1 in 49 healthy subjects (p < 0.001). On a low FODMAP diet, symptoms are reduced and levels of syndecan-1 (but not other markers) fell by a median 3335% (p < 0.001) irrespective of whether gluten is present or not., Conclusion: Gluten ingestion has no specific effect on epithelial integrity or symptoms in this cohort, but reducing FODMAP intake concomitantly reduces symptoms and reverses apparent colonic epithelial injury. These findings highlight the heterogeneity of populations self-reporting gluten sensitivity and implicate FODMAPs in colonic injury in IBS., (© 2020 Wiley-VCH GmbH.)

Background: The impact of pregnancy on levels of biologic agents in patients with IBD is undefined and time to elimination in vedolizumab-exposed infants is unknown., Aims: To determine the effect of pregnancy on infliximab, adalimumab and vedolizumab levels and to study infant vedolizumab clearance METHODS: In a prospective observational study, maternal drug levels were measured pre-conception, in each trimester, at delivery and postpartum. The association between drug levels and gestation in weeks was assessed using generalised estimating equation modelling. Infant vedolizumab levels were performed at birth (cord blood), 6 weeks and 3 months or until undetectable., Results: We included 50 IBD patients (23 on infliximab, 15 on adalimumab and 12 on vedolizumab) with at least two intrapartum observations, plus 5 patients on vedolizumab with only mother and baby samples at delivery. Modelling showed no change in adalimumab levels, an increase in infliximab levels of 0.16 (95% CI 0.08-0.24) µg/L/week (P < 0.001) and a decrease of 0.18 (95% CI: -0.33 to -0.02) µg/L/week (P = 0.03) for vedolizumab. In 17 mother-baby pairs, median infant vedolizumab levels at birth were lower than maternal levels (P < 0.05) with an infant:maternal ratio of 0.7 (IQR 0.5-0.9). Vedolizumab was undetectable between 15 and 16 weeks of age in all 12 infants completing follow-up testing., Conclusions: During pregnancy, adalimumab levels remain stable, while infliximab levels increase and vedolizumab levels decrease. However, the increments were small suggesting that intrapartum therapeutic drug monitoring and dose adjustment are not indicated. Unlike infliximab and adalimumab, infant vedolizumab levels are lower in cord blood than in mothers and appear to clear rapidly., (© 2020 John Wiley & Sons Ltd.)

The protein, zonulin, has emerged as a popular serological marker to assess the integrity of the intestinal mucosal barrier. However, there is limited information on the utility of serum zonulin to indicate gastrointestinal disease and the validity of zonulin detection in widely-used commercial assays. The current study reports differences in zonulin levels across patient groups with gastrointestinal dysfunction compared with healthy individuals, though methodological inconsistencies indicated that actual zonulin protein was not detected by the commercial assays applied. The nature of the assays' detected antigen was investigated using immunoprecipitation followed by mass spectrometric analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining. Top matches of the assays' detected antigen included haptoglobin and complement C3 for the assay manufactured by CUSABIO (Wuhan, China) and complement C3 for the assay manufactured by Immundiagnostik AG (Bensheim, Germany). These findings confirm that current commercial zonulin assays are not detecting the actual protein as prehaptoglobin-2. Until assay methodology is improved, we advise the greater scientific and medical community to exercise caution in considering the measurement of serum zonulin as a marker of mucosal barrier integrity., Competing Interests: The authors have declared that no competing interests exist.

Background & Aims: Vitamin D at serum 25(OH)D concentrations above 100 nmol/L is associated with disease remission in patients with IBD, suggesting targeted dosing might be anti-inflammatory. This study aimed to assess the effectiveness, safety and predictors of a 12-week regimen of vitamin D supplementation to achieve such a target in patients with active disease., Methods: In a pilot study, patients with active colitis and a serum 25(OH)D concentration <75 nmol/L were prescribed oral liquid vitamin D supplementation over 12 weeks using a specific protocol with dose adjusted 4-weekly to aim for a target level of 100-125 nmol/L., Results: Five patients each with Crohn's colitis or ulcerative colitis (UC) had mean 25(OH)D concentration 52 (range 27-73 nmol/L). Five reached the targeted level and four 89-95 nmol/L. One withdrew after 4 weeks (88 nmol/L). Target dose was met only in those with BMI <30 kg/m 2 and total dose inversely correlated with initial serum 25(OH)D. One patient had developed a high level at 8 weeks (146 nmol/L) and another new hypercalciuria. There were no serious adverse events attributable to the therapy. Clinical disease activity consistently declined, but faecal calprotectin and circulating markers of inflammation did not., Conclusions: A specified oral vitamin D regimen successfully and safely achieved target or near-target levels, improved symptom-based activity scores, but did not alter objective measures of intestinal or systemic inflammation. A modified version of this dose-escalating regimen would be suitable for a randomised placebo-controlled trial, but does require regular safety monitoring., (Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background: The outcomes of acute severe ulcerative colitis [ASUC] appear to be dependent on early intervention with the first and/or further infliximab [IFX] doses, although parameters to guide decision-making remain uncertain., Aim: To assess whether serum/faecal IFX levels and inflammatory biomarkers early after IFX dose can predict ASUC outcomes., Methods: This prospective pilot study consecutively recruited inpatients with steroid-refractory ASUC, who then received 1-3 IFX rescue doses [5 mg/kg per dose] at the discretion of the treating clinician. Serum IFX, C-reactive protein [CRP], albumin and faecal calprotectin [FC] concentrations were measured daily as an inpatient, and then 7, 14, 28 and 42 days post-first IFX. Faecal IFX was measured 1 day post-IFX. The primary end point was clinical remission (partial Mayo [PM] = 0) and CRP ≤3 mg/l at 6 weeks. Secondary end points were 12-week clinical remission or colectomy during follow-up., Results: Of 24 ASUC patients with a median follow-up of 28 months [range 13-44], 10 [42%] achieved remission at 6 weeks, 12 [50%] achieved 12-week remission, six [25%] had colectomy. In total, 97% received either two or three IFX doses. Post-first dose, receiver-operator curve-derived cutoffs of the area-under-curve [AUC, Days 4-7] concentrations for serum IFX, FC and PM scores each predicted the primary end point with 100% sensitivity, and predicted future colectomy with 89-94% sensitivity. In multivariate analyses, faecal IFX >1 µg/g (odds ratio [OR] 0.04 [0.2, 0.9]), PM AUCd1-3 < 20 (OR 20.2 [1.01, 404], each P < 0.05), FC AUCd1-3 < 10000 µg/ml [OR 13.6 [0.6, 294], trend only, p = 0.09) were each associated with clinical and CRP remission [6 weeks]., Conclusions: In ASUC, post-first dose IFX, early assessment of serum/faecal IFX, calprotectin and PM scores can accurately predict future remission and colectomy, and thus potentially aid in decision-making, i.e. accelerated IFX dosing or surgical planning if/when needed., (Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com)

Purpose: The aim of this study was to evaluate a novel technique of tooth preparation in fixed prosthodontics suitable for dental students and neophyte dentists., Materials and Methods: Twenty-four dental students of the sixth-year class were recruited to verify the predicibility of this technique. Each student prepared two mandibular second premolars on a typodont for a dental crown with a 90° shoulder finishing line. One tooth was prepared using standard procedures taught in the prosthodontic dental course; the other tooth was prepared with the new technique. Three Professors of Prosthodontics of the same University evaluated the result on the basis of 10 criteria., Results: A statistically significant difference between the two techniques was found in 8 out of 10 criteria. The new technique showed higher values (p<0.05) in 7 criteria, while the conventional technique had better results in just 1 criterion. Moreover, the total sum of values was higher for the new technique (total 41.2±3.98, p<0.05) compared to the conventional technique (total 38.12±5.18, p<0.05)., Conclusions: This study showed that the results were less dependent on manual abilities and personal experience with the novel technique. It could helps dental students and neophyte dentists in their learning curve.

Increased hepatic glucose production, a feature of (non-insulin-dependent diabetes mellitus [NIDDM]), is present at an early age in the New Zealand Obese (NZO) mouse and is associated with impaired suppression of the gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). The aim of this study was to further characterize the abnormality in the regulation of hepatic FBPase in NZO mice versus New Zealand Chocolate (NZC) control mice. At 20 weeks of age, NZO mice have elevated FBPase activity (65.3 +/- 7.9 v 46.7 +/- 5.0 micromol/min/mg protein, P =.07) and protein levels (31.7 +/- 3.1 v 22.5 +/- 2.8 arbitrary units, P < .05), but not mRNA levels (0.18 +/- 0.03 v 0.16 +/- 0.03 arbitrary units). Elevated FBPase activity and protein levels in NZO mice were also shown at 4 to 6 weeks of age, but not in 1-day-old mice, suggesting that the increase occurs between birth and weaning. The Km of the enzyme was the same in NZO and NZC mice (3.7 +/- 0.5 v 5.0 +/- 0.9 micromol/L, NZO v NZC). The regulation of FBPase by the competitive inhibitor, fructose-2,6-bisphosphate ([Fru(2,6)Pz] 5 micromol/L) measured over a range of substrate concentrations (2.5 to 80 micromol/L) was similar between NZO and control mice (Km in the presence of Fru(2,6)Pz, 10.8 +/- v 1.9 v 13.2 +/- 3.3 micromol/L, NZO v NZC). It is concluded that increased FBPase activity in the NZO mouse is due to elevated protein levels, and that this appears to be due to a failure of the normal decrease that occurs following birth in control animals.

The aetiology of non-insulin-dependent diabetes mellitus (NIDDM) is not known. The concordance of NIDDM in identical twins and differences in the prevalence rate of NIDDM between different racial groups suggest a genetic cause. Hyperglycaemia in established diabetes is caused by a combination of hepatic insulin resistance, impaired peripheral (muscle and fat) glucose uptake and a defect in glucose-mediated insulin secretion. However, it is not known if these defects are all inherited or if one can cause the others. This uncertainty is due to the fact that hyperglycaemia per se can cause defects in insulin action and insulin secretion that resemble those found in NIDDM. Furthermore the elevated free fatty acid (FFA) levels found when NIDDM is associated with obesity are known to cause both peripheral and hepatic insulin resistance. Recently we have demonstrated the mechanism by which elevated FFA levels can cause hepatic insulin resistance. However, we also have evidence that the converse holds in that genetically engineered hepatic insulin resistance in a transgenic rat model leads to obesity. Thus an understanding of the pathogenesis of NIDDM is complicated by the fact that hyperglycaemia and obesity can be both causes and consequences of insulin resistance. To overcome these difficulties, studies in young euglycaemic diabetes-prone subjects have been conducted. Results suggest that there may be different causes for NIDDM in different racial groups.

Background/aims: Because the neutral protease urokinase is important in control of cell adhesion and migration, the effects of the physiologically relevant fermentation product butyrate on urokinase secretion by colonic epithelium were examined., Methods: Secreted and cell-associated levels of urokinase and plasminogen activator inhibitor 1 were measured in colonic crypt cells within 24 hours of isolation from macroscopically normal mucosa of normal or cancer-bearing colons., Results: Butyrate caused a concentration-dependent inhibition of both secreted (56% +/- 4% inhibition after 24-hour exposure to 1 mmol/L butyrate; n = 20; mean +/- SEM; P < 0.001) and cell-associated urokinase content (35% +/- 6%; P = 0.003). Acetate and propionate had minimal effects. Butyrate also stimulated plasminogen activator inhibitor 1 secretion by 25% +/- 7% (P = 0.013). Net urokinase activities were suppressed in supernates and cell homogenates by butyrate. Levels of transcripts for urokinase and the inhibitor changed with butyrate exposure in parallel to the levels of secretion of the respective proteins. Cells from the cancer group showed significantly reduced inhibitor secretion and abnormal responses to butyrate (greater inhibition of urokinase secretion and no stimulation of inhibitor secretion), probably reflecting the diffuse disturbance of colonic epithelial biology associated with colorectal cancer., Conclusions: Butyrate has dual effects in markedly reducing colonic epithelial urokinase activity, and these may have important implications to understanding colonic epithelial physiology and the pathogenesis and treatment of colonic diseases.