Variegate porphyria, caused by monoallelic mutations in protoporphyrinogen oxidase (PPOX), causes acute visceral attacks and skin photosensitivity, presenting in blistering and skin fragility. A more severe phenotype is seen with rare biallelic mutations. Fragility is due to accumulation of intermediate porphyrins from the liver, such as 5′-aminolaevulinic acid (ALA), close to the skin surface that are oxidized by sunlight and cause oxidative stress. We hypothesize that PPOX deficiency causes intrinsic pathological changes to keratinocytes that contribute to this skin phenotype. Short hairpin RNA was used to create a model of monoallelic (KD1) and of biallelic hypofunctional variants (KD2). A porphyria-like environment was then induced using ALA and/or an iron chelator. Porphyrin accumulation was measured using intrinsic fluorescent measurement and differentiation was assessed in monolayer and by using three-dimensional (3D) organotypics. Both KD lines displayed reduced PPOX expression (KD1, 50%; KD2, 25%) and significantly reduced proliferation with KD2 displaying reduced migration. Induction of a porphyria-like environment caused accumulation of intermediate porphyrins in both KDs, with significantly more in KD2, and intracellular oxidative stress, evidenced by reduced expression of electron transport complexes and altered glutathione/glutathione disulfide ratios. KD clones displayed reduced differentiation, proportional to KD, and this was replicated by 3D organotypic cultures, while KD2 models also had reduced stratification. Treatment of 3D organoids with ALA further reduced differentiation and stratification in KD models. In conclusion, we show that keratinocytes have an active haem synthetic pathway and contribute to porphyria pathology causing significant changes in keratinocyte proliferation, differentiation, migration and reducing epidermal thickness, which could facilitate the oxidation of porphyrins under the skin. The more severe effects seen with > 75% KD reflects the phenotype seen in individuals with biallelic mutations in PPOX. These 3D organotypics could therefore serve as models to trial therapeutic interventions to improve patient skincare.

Variegate porphyria, due to monoallelic mutations in protoporphyrinogen oxidase (PPOX), causes acute visceral attacks and skin photosensitivity, presenting in blistering and skin fragility, a more severe phenotype is seen with rare biallelic mutations. This is due to accumulation of intermediate porphyrins such as 5'-aminolevulenic acid (ALA), close to the skin surface that are oxidized by sunlight. Oxidized porphyrins cause oxidative stress and degranulation of mast cells resulting in the release of proteases that cause blistering. To elucidate whether reduced PPOX expression causes intrinsic pathological changes to keratinocytes we created two shRNA Knockdown cell lines, KD1 and KD2 (50 & 25% residual expression respectively). Both KD lines displayed significantly reduced proliferation with KD2 also having reduced migratory capacity. KD clones expressed reduced levels of involucrin in monolayer, proportional to the amount of knockdown, suggesting decreased differentiation. This was corroborated by 3D organotypic cultures where KD models expressed less KRT10 and involucrin, proportional to knockdown, while KD2 models also had a thinner epidermis. Keratinocytes were treated with ALA and Deferoxamine (an iron chelator) to mimic porphyrin accumulation. A significant increase was seen in fluorescent porphyrins which caused intracellular oxidative stress evidenced by reduced expression of 4 of the 5 electron transport chain complexes. In conclusion, we show that keratinocytes themselves contribute to porphyria pathology with >50% PPOX loss causing significant changes in keratinocyte proliferation, differentiation, and epidermal stratification, with >75% also affecting migration and reducing epidermal thickness. A thinned epidermis could facilitate the oxidation of porphyrins under the skin. The more severe effects seen with >75% KD reflects the phenotype seen in individuals with biallelic mutations. The 3D organotypics could therefore serve as models to trial therapeutic interventions to improve patient care.

The 2023 Laser Damage conference thin-film damage competition was devoted to a survey on the state-of-the-art broadband near-IR multilayer dielectric (MLD) mirrors designed for ultra-short pulsed laser applications. The requirements for the coatings were a minimum reflection of 99.5% at 45-deg incidence angle for S-polarization from 830 nm to 1010 nm and group delay dispersion (GDD) <+/- 50 fs(2). The participants were allowed to select the coating materials, coating design, and coating deposition method. Samples were damage tested at a single testing facility to enable direct comparison among the participants using a 25 +/- 5 fs optical parametric chirped-pulse amplification (OPCPA) laser system operating at 5 Hz. The testing results from this set of 37 samples showed that dense coatings by ion-beam sputtering (IBS), magnetron sputtering (MS), and electron-beam ion assisted deposition (e-beam IAD) exhibited highest damage initiation onset (laser-induced damage threshold or LIDT) while e-beam coatings were low performers. In addition, multilayer coatings using tantala and/or hafnia as high index materials were top performers. Furthermore, this competition included for the first time the measurement of the damage growth onset (laser-induced damage growth threshold or LDGT). This latter performance metric plays an important role in establishing the safe operational conditions for larger aperture ultrashort pulsed lasers. Information pertaining to the morphology of the damage sites and their evolution under subsequent exposure to different laser fluences leading to damage growth is presented. Finally, not all coating samples in the survey met the GDD requirements stated above and associated measurements are discussed in the context of the present and past thin-film damage competitions focused on similar broadband near-IR MLD coatings.

This year’s competition proposed to survey the state-of-the-art broadband, near-IR multilayer dielectric (MLD) mirrors designed for ultra-short, pulsed laser applications. The requirements for the coatings were a minimum reflection of 99.5% at 45-degree incidence angle for S-polarization from 830 nm to 1010 nm and group delay dispersion (GDD) < ± 50 fs2. The participants in this effort selected the coating materials, coating design, and deposition method. Samples were damage tested at a single testing facility to enable direct comparison among the participants using a 25 ± 5 fs OPCPA laser system operating at 5 Hz. A double blind test assured sample and submitter anonymity. The damage performance results, sample rankings, details of the deposition processes, coating materials and substrate cleaning methods are shared here. We found that multilayer coatings using tantala and/or hafnia as high index materials were top performers within several coating deposition groups. Specifically, dense coatings by ion-beam sputtering (IBS), magnetron sputtering (MS), and electron-beam ion assisted deposition (e-beam IAD) exhibited highest damage initiation onset (LIDT) while e-beam coatings were low performers. In addition, damage growth onset (LDGT) was also examined and the results are reported here for all samples as this performance metric plays an important role in establishing the safe operational conditions for larger aperture, ultrashort pulsed lasers. Lastly, not all coating samples in the survey met the GDD requirements stated above and associated measurements are discussed in the context of the present and past competitions focused on similar broadband, near-IR MLD coatings.

Monolayers containing subnanometer striations of silica and hafnia to form composite materials at varying ratios are explored as a method to develop high-index dielectric layers with increased laser-induced–damage thresholds (LIDTs). These layers can then be used in multilayer dielectric coatings for short-pulse, high-peak-power laser applications, particularly in regions of the highest electric-field intensity. Fabrication is achieved by means of exposure to two different evaporant vapor plumes, where local exposure to each plume is controlled via shielding to prevent simultaneous exposure. The LIDT of the resulting layers has been evaluated at 1053 nm with 600-fs pulses. The results indicate that such hafnia/silica layers exhibit LIDTs similar to silica for a refractive index of ≤1.65. These results suggest that the use of these layers in locations subjected to high electric-field intensity within multilayer dielectric coatings may significantly improve the LIDT, with this deposition process providing particular benefit for scaling to large-aperture, high-fluence components.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Sphingosine-1-phosphate lyase (SGPL1) insufficiency syndrome (SPLIS) is an autosomal recessive multi-system disorder, which mainly incorporates steroid-resistant nephrotic syndrome and primary adrenal insufficiency. Other variable endocrine manifestations are described. In this study, we aimed to comprehensively annotate the endocrinopathies associated with pathogenic SGPL1 variants and assess for genotype–phenotype correlations by retrospectively reviewing the reports of endocrine disease within our patient cohort and all published cases in the wider literature up to February 2022. Glucocorticoid insufficiency in early childhood is the most common endocrine manifestation affecting 64% of the 50 patients reported with SPLIS, and a third of these individuals have additional mineralocorticoid deficiency. While most individuals also have nephrotic syndrome, SGPL1 variants also account for isolated adrenal insufficiency at presentation. Primary gonadal insufficiency, manifesting with microphallus and cryptorchidism, is reported in less than one-third of affected boys, all with concomitant adrenal disease. Mild primary hypothyroidism affects approximately a third of patients. There is paucity of data on the impact of SGPL1 deficiency on growth, and pubertal development, limited by the early and high mortality rate (approximately 50%). There is no clear genotype–phenotype correlation overall in the syndrome, with variable disease penetrance within individual kindreds. However, with regards to endocrine phenotype, the most prevalent disease variant p.R222Q (affecting 22%) is most consistently associated with isolated glucocorticoid deficiency. To conclude, SPLIS is associated with significant multiple endocrine disorders. While endocrinopathy in the syndrome generally presents in infancy, late-onset disease also occurs. Screening for these is therefore warranted both at diagnosis and through follow-up.

Objectives: The gingiva heals at an accelerated rate with reduced scarring when compared to skin. Potential well -studied factors include immune cell number, angiogenesis disparities and fibroblast gene expression. Differential keratinocyte gene expression, however, remains relatively understudied. This study explored the contrasting healing efficiencies of gingival and skin keratinocytes, alongside their differential gene expression patterns. Methods: 3D organotypic culture models of human gingiva and skin were developed using temporarily immor-talised primary keratinocytes. Models were wounded for visualisation of re-epithelialisation and analysis of keratinocyte migration to close the wound gap. Concurrently, differentially expressed genes between primary gingival and skin keratinocytes were identified, validated, and functionally assessed. Results: Characterisation of the 3D cultures of gingiva and skin showed differentiation markers that recapitulated organisation of the corresponding in vivo tissue. Upon wounding, gingival models displayed a significantly higher efficiency in re-epithelialisation and stratification versus skin, repopulating the wound gap within 24 hours. This difference was likely due to distinct patterns of migration, with gingival cells demonstrating a form of sheet migration, in contrast to skin, where the leading edge was typically 1-2 cells thick. A candidate approach was used to identify several genes that were differentially expressed between gingival and skin keratinocytes. Knockdown of PITX1 resulted in reduced migration capacity of gingival cells. Conclusion: Gingival keratinocytes retain in vivo superior wound healing capabilities in in vitro 2D and 3D en-vironments. Intrinsic gene expression differences could result in gingival cells being 'primed' for healing and play a role in faster wound resolution. Clinical Significance Statement: The successful development of organotypic models, that recapitulate re-epithelialisation, will underpin further studies to analyse the oral response to wound stimuli, and potential therapeutic interventions, in an in vitro environment.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

SummaryThe immunomodulatory activity of the arabinoxylans (AXs) extracts from cereal sources has been reported to impart health benefits in terms of immune enhancement. This study investigated the effect of enzymatic extraction on extraction yield and structure of AXs from wheat flour pentosan fraction. Under the optimised conditions, the extraction yield of AXs reached up to 81.25%. Furthermore, the study determined whether water‐extracted AXs (WEAXs) and enzyme‐extracted AXs (E‐WEAXs) from wheat flour were able to differentially stimulate nitric oxide (NO) secretion through increased levels of inducible nitric oxide synthase (iNOS) in human U937 monocytes. The results indicated that AXs concomitantly induced (P < 0.05) both NO and iNOS productions in U937 monocytes compared to untreated cells. Compared with WEAXs, E‐WEAXs resulted in a higher proportion of low Mw (1–10 KDa) AXs (49.51% vs. 19.11% in WEAXs), a higher A/X ratio (0.83 vs. 0.48 in WEAXs) and a higher yield (12.83 ± 0.35% vs. 7.54 ± 0.47% in WEAXs). Moreover, E‐WEAXs induced significantly (P < 0.05) greater NO and iNOS production per million viable cells (61.8 ± 2.7 μm and 42.41 ± 3.83 ng respectively) than WEAXs (51.6 ± 2.6 μm and 33.46 ± 1.48 ng, respectively). The findings suggest AXs may heighten innate immune activity in the absence of infection or disease through an iNOS‐mediated stimulation of NO production. The immunomodulatory activity of the wheat‐derived AXs was enhanced by enzyme treatment, with low Mw and high A/X ratio associated with elevated NO/iNOS levels in human monocytes compared to water extraction.

The ingestion of foods and food-derived substances that may mediate the immune system is widely studied. Evidence suggests cereal arabinoxylans (AXs) have immunomodulatory activities that may impart health benefits in terms of immune enhancement. This study extracted AXs from corn bran using alkali and developed a modification process using three endoxylanases to obtain fractions of lower molecular weight ranges. In vitro studies showed extracted and modified AXs significantly (P < 0.05) elevated nitric oxide (NO) synthesis by the human U937 monocytic cell line (ranging from 53.7 ± 1.1 to 62.9 ± 1.2 μM per million viable cells) at all concentrations tested (5-1000 μg/mL), indicative of immune enhancement compared to an untreated control (43.7 ± 1.9 μM per million viable cells). The study suggested the dose range and Mw distribution of AXs are key determinants of immune-modulatory activity. AXs in the low Mw range (0.1-10 KDa) were the most effective at inducing NO secretion by U937 macrophages at low AX concentration ranges (5-50 μg/mL), with NO production peaking at 62.9 ± 1.2 μM per million viable cells with 5 μg/mL of AX (P = 0.0009). In contrast, AXs in the high Mw range (100-794 kDa) were most effective at inducing NO at high AX concentration ranges (500-1000 μg/mL) with NO production reaching a maximum of 62.7 ± 1.3 μM per million viable cells at 1000 μg/mL of AX (P = 0.0011). The findings suggest that dietary AXs from corn bran may heighten innate immune responses in the absence of infection or disease.

Long-chain per- and polyfluoroalkyl substances (PFASs) are being replaced by short-chain PFASs and fluorinated alternatives. For ten legacy PFASs and seven recently discovered perfluoroalkyl ether carboxylic acids (PFECAs), we report (1) their occurrence in the Cape Fear River (CFR) watershed, (2) their fate in water treatment processes, and (3) their adsorbability on powdered activated carbon (PAC). In the headwater region of the CFR basin, PFECAs were not detected in raw water of a drinking water treatment plant (DWTP), but concentrations of legacy PFASs were high. The U.S. Environmental Protection Agency's lifetime health advisory level (70 ng/L) for perfluorooctanesulfonic acid and perfluorooctanoic acid (PFOA) was exceeded on 57 of 127 sampling days. In raw water of a DWTP downstream of a PFAS manufacturer, the mean concentration of perfluoro-2-propoxypropanoic acid (PFPrOPrA), a replacement for PFOA, was 631 ng/L (n = 37). Six other PFECAs were detected, with three exhibiting chromatographic peak areas up to 15 times that of PFPrOPrA. At this DWTP, PFECA removal by coagulation, ozonation, biofiltration, and disinfection was negligible. The adsorbability of PFASs on PAC increased with increasing chain length. Replacing one CF2 group with an ether oxygen decreased the affinity of PFASs for PAC, while replacing additional CF2 groups did not lead to further affinity changes.

Arabinoxylans (AXs) are one of the most important groups of hemicelluloses in the endosperm and outer layers of cereal grains. However, the macromolecular characteristics of extracted AXs and the extraction yields achieved exhibit huge differences. These differences are apparently dependent on the different extraction and modification methods used. This paper aims to review the extraction and modification methods used in the separation of AXs from cereal by-products as reported in previous studies. The effects of different extraction and modification methods on AX extraction yields, molecular characteristics and properties were evaluated. The influence of various extraction methods including chemical solvent extraction, enzymatic extraction and modification, and mechanically-assisted extraction on molecular structure (the ratio of arabinose to xylose and molecular weight distribution) of AXs are compared and discussed in this paper.