Higher education in biosciences is substantiallyinformed by hands-on field trips and practical laboratory skills-training. With the first Covid-19 national lock-down in England in March 2020, on-campus education at higher education institutions was swiftly moved to alternative provisions, including online only options, a mix of synchronous or asynchronous blended, or hybrid adaptions. Students enrolled on an undergraduate bioscience programme have been faced with unprecedented changes and interruptions to their education. This study aimed to evaluate bioscience students' ability to adjust to a fast-evolving learning environment and to capture students' journey building up resilience and graduate attributes. A total of 317 Bioscience undergraduate students in years 1-3 at the biology department at a northwest English university participated in this anonymous, cross-sectional, mixed-method study with open and closed questions evaluating their perception and feedback to remote and blended learning provisions during the Covid-19 pandemic and post pandemic learning capturing academic years 2019/20-2022/23. The Covid-19 pandemic and the consequent restriction of personal social interaction resulted in an significant decrease in the mental wellbeing of undergraduate bioscience students in this study, cumulating in poor or very poor self-rating of wellbeing in spring 2021; while at the same time students showed evidence of advanced adaption to the new learning and social environment by acquisition of additional technical, social and professional graduate-level skills. Post pandemic, bioscience students worry about the increased living costs and are strongly in favour of a mixture of face-to-face and blended learning approaches. Our results show that bioscience students can self-report poor mental health while developing resilience, indicating tailored support can aid in developing students' resilience. Students have adjusted with ease to digital teaching provisions and now expect higher education institutions continue to offer both, face-to-face, and blended teaching, reducing the burden on students' notably risen living costs.

Background Autosomal-recessive congenital ichthyosis (ARCI) is a heterogeneous group of ichthyoses presenting at birth. Self-improving congenital ichthyosis (SICI) is a subtype of ARCI and is diagnosed when skin condition improves remarkably (within years) after birth. So far, there are sparse data on SICI and quality of life (QoL) in this ARCI subtype. This study aims to further delineate the clinical spectrum of SICI as a rather unique subtype of ARCI. Objectives This prospective study included 78 patients (median age: 15 years) with ARCI who were subdivided in SICI (n = 18) and non-SICI patients (nSICI, n = 60) by their ARCI phenotype. Methods Quality of life (QoL) was assessed using the (Children's) Dermatology Life Quality Index. Statistical analysis was performed with chi-squared and t-Tests. Results The genetically confirmed SICI patients presented causative mutations in the following genes: ALOXE3 (8/16; 50.0%), ALOX12B (6/16; 37.5%), PNPLA1 (1/16; 6.3%) and CYP4F22 (1/16; 6.3%). Hypo-/anhidrosis and insufficient vitamin D levels (<30 ng/mL) were often seen in SICI patients. Brachydactyly (a shortening of the 4th and 5th fingers) was statistically more frequent in SICI (P = 0.023) than in nSICI patients. A kink of the ear's helix was seen in half of the SICI patients and tends to occur more frequently in patients with ALOX12B mutations (P = 0.005). QoL was less impaired in patients under the age of 16, regardless of ARCI type. Conclusions SICI is an underestimated, milder clinical variant of ARCI including distinct features such as brachydactyly and kinking of the ears. Clinical experts should be aware of these features when seeing neonates with a collodion membrane. SICI patients should be regularly checked for clinical parameters such as hypo-/anhidrosis or vitamin D levels and monitored for changes in quality of life.

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.

Keratosis follicularis spinulosa decalvans (KFSD) is a rare cornification disorder with an X-linked recessive inheritance in most cases. Pathogenic variants causing X-linked KFSD have been described in MBTPS2, the gene for a membrane-bound zinc metalloprotease that is involved in the cleavage of sterol regulatory element binding proteins important for the control of transcription. Few families have been identified with an autosomal dominant inheritance of KFSD. We present two members of an Austrian family with a phenotype of KFSD, a mother and her son. The disease was not observed in her parents, pointing to a dominant inheritance with a de novo mutation in the index patient. Using whole-exome sequencing, we identified a heterozygous missense variant in CST6 in DNA samples from the index patient and her affected son. In line with family history, the variant was not present in samples from her parents. CST6 codes for cystatin M/E, a cysteine protease inhibitor. Patient keratinocytes showed increased expression of cathepsin genes CTSL and CTSV and reduced expression of transglutaminase genes TGM1 and TGM3. A relative gain of active, cleaved transglutaminases was found in patient keratinocytes compared to control cells. The variant found in CST6 is expected to affect protein targeting and results in marked disruption of the balance between cystatin M/E activity and its target proteases and eventually transglutaminases 1 and 3. This disturbance leads to an impairment of terminal epidermal differentiation and proper hair shaft formation seen in KFSD.

Data on vitamin D status of patients with inherited ichthyosis in Europe is scarce and unspecific concerning the genetic subtype. This study determined serum levels of 25-hydroxyvitamin D3 (25(OH)D3) in 87 patients with ichthyosis; 69 patients were additionally analysed for parathyroid hormone. Vitamin D deficiency was pronounced in keratinopathic ichthyosis (n = 17; median 25(OH)D3: 10.5 ng/ml), harlequin ichthyosis (n = 2;7.0 ng/ml) and rare syndromic subtypes (n = 3; 7.0 ng/ml). Vitamin D levels were reduced in TG1-proficient lamellar ichthyosis (n = 15; 8.9 ng/ml), TG1-deficient lamellar ichthyosis (n = 12; 11.7 ng/ml), congenital ichthyosiform erythroderma (n = 13; 12.4 ng/ml), Netherton syndrome (n = 7; 10.7 ng/ml) and X-linked ichthyosis (n = 8; 13.9 ng/ml). In ichthyosis vulgaris 25(OH)D3 levels were higher (n = 10; 19.7 ng/ml). Parathyroid hormone was elevated in 12 patients. Low 25(OH)D3 levels were associated with high severity of scaling (p = 0.03) implicating scaling as a risk factor for vitamin D deficiency. Thus, this study supports our recent guidelines for ichthyoses, which recommend screening for and substituting of vitamin D deficiency.

Autosomal recessive congenital ichthyosis (ARCI) disrupts normal keratinization, resulting in generalized scaling of the skin. There are presently no curative therapies available (Fleckman et al., 2013Fleckman P. Newell B.D. Van Steensel M.A. Yan A.C. Topical treatment of ichthyoses.Dermatol Ther. 2013; 26: 16-25Crossref PubMed Scopus (15) Google Scholar). Local protein replacement is, therefore, an encouraging approach for a more specific treatment. ARCI refers to a heterogeneous group of rare skin keratinization disorders with an estimated prevalence of 1 in 50,000–200,000 (Dreyfus et al., 2014Dreyfus I. Bourrat E. Maruani A. Bessis D. Chiaverini C. Vabres P. et al.Factors associated with impaired quality of life in adult patients suffering from ichthyosis.Acta Derm Venereol. 2014; 94: 344-346Crossref PubMed Scopus (23) Google Scholar). The disease is characterized by notable impairments to the skin's barrier function, resulting in frequent infections and increased transepidermal water loss. ARCI is caused by mutations in 1 of 12 identified genes involved in epidermal differentiation. The most common of these are loss of function mutations in TGM1, affecting approximately 30% of patients (Rodriguez-Pazos et al., 2009Rodriguez-Pazos L. Ginarte M. Vega A. Toribio J. Autosomal recessive congenital ichthyosis.J Invest Dermatol. 2009; 129: 1319-1321Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). TGM1 encodes transglutaminase 1 (TG1), a protein that plays an essential role in the formation of the cornified envelope (Eckert et al., 2005Eckert R.L. Sturniolo M.T. Broome A.M. Ruse M. Rorke E.A. Transglutaminase function in epidermis.J Invest Dermatol. 2005; 124: 481-492Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Because animal models of severe keratinization disorders such as ARCI are not viable and animal skin poorly represents human skin (Gerber et al., 2014Gerber P.A. Buhren B.A. Schrumpf H. Homey B. Zlotnik A. Hevezi P. The top skin-associated genes: a comparative analysis of human and mouse skin transcriptomes.Biol Chem. 2014; 395: 577-591Crossref PubMed Scopus (62) Google Scholar), the use of organotypic skin equivalents has emerged as a valid tool to investigate ARCI. In the present study, full-thickness skin equivalents generated from the fibroblasts and keratinocytes of ARCI patients with mutations in TGM1 were treated topically with TG1. Because biomacromolecules do not normally overcome the skin barrier, owing to their high molecular weight, protein delivery was mediated by use of thermoresponsive nanogels (tNG) (Cuggino et al., 2011Cuggino J.C. Alvarez I.C.I. Strumia M.C. Welker P. Licha K. Steinhilber D. et al.Thermosensitive nanogels based on dendritic polyglycerol and N-isopropylacrylamide for biomedical applications.Soft Matter. 2011; 7: 11259-11266Crossref Scopus (67) Google Scholar). Proteins as large as 150 kDa have been encapsulated within tNGs and subsequently released above a thermal trigger point (Giulbudagian et al., 2018bGiulbudagian M. Yealland G. Hönzke S. Geisendörfer B. Kleuser B. Hedtrich S. et al.Breaking the barrier—potent anti-inflammatory activity following efficient topical delivery of etanercept using thermoresponsive nanogels.Theranostics. 2018; 8: 450-463Crossref PubMed Scopus (49) Google Scholar, Witting et al., 2015Witting M. Molina M. Obst K. Plank R. Eckl K.M. Hennies H.C. et al.Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules.Nanomedicine. 2015; 11: 1179-1187Crossref PubMed Scopus (69) Google Scholar). Our groups previously reported the epidermal delivery of functional TG1 using topically applied tNGs and rescue of barrier defects in TGM1 knockdown skin equivalents (Witting et al., 2015Witting M. Molina M. Obst K. Plank R. Eckl K.M. Hennies H.C. et al.Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules.Nanomedicine. 2015; 11: 1179-1187Crossref PubMed Scopus (69) Google Scholar). However, whether TG1-loaded tNGs are an effective topical treatment for ARCI skin with TGM1 mutations, rather than transiently induced TGM1 knockdowns, was still unclear. The study was approved by the Ethics Committee of the Medical University of Innsbruck, Austria, and samples were taken after obtaining written informed consent of the probands. Full-thickness skin equivalents were generated from fibroblasts plus normal keratinocytes, keratinocytes with transient TGM1 knockdowns, or keratinocytes from ARCI patients with TGM1 mutations (Figure 1). In comparison to normal equivalents, TGM1 knockdown and patient equivalents both demonstrated slightly thinned stratum corneum and epidermis, with reduced cell number within the granular layer. The epidermal differentiation markers keratin 14 and 10 were distributed appropriately. TG1 activity was present in normal skin equivalents but not in those generated from patient cells or TGM1 knockdown keratinocytes, in line with the inactivating mutations found in patient 1, and the absence of persistent TG1 expression in patient 2 and knockdown equivalents. Notably, knockdown equivalents demonstrated increasing TGM1 transcript levels over time (>50% after 10 days cultivation), indicating a loss of effective repression (Supplementary Figure S1 online). To assess their biocompatibility, TG1-loaded tNGs were incubated with normal, patient 1, and patient 2 keratinocytes, as well as fibroblasts for up to 48 hours, resulting in no significant cytotoxicity at any of the tested concentrations (Figure 1b, Supplementary Figures S2 and S3 online). Concordantly, no significant cytotoxicity was observed following the application of tNGs onto skin equivalents (Figure 1g). Additionally, the ability of TG1, alone or loaded in tNGs, to enter keratinocytes was assessed by confocal microscopy. In both cases, TG1 entered the cytoplasm in a time-dependent manner (Supplementary Figure S4 online). Notably, tNGs entered more rapidly than the TG1, which, with their lack of clear intracellular co-localization, would suggest that the tNGs and TG1 enter keratinocytes separately, concordant with the relatively quick release of protein at temperatures ≥35°C. It should be noted, however, that previous evidence indicates tNGs are largely unable to overcome the stratum corneum of even barrier-deficient skin, suggesting that, in most cases, little or no contact will occur between them and viable epidermal cells (Giulbudagian et al., 2018aGiulbudagian M. Hönzke S. Bergueiro J. Işik D. Schumacher F. Saeidpour S. et al.Enhanced topical delivery of dexamethasone by β-cyclodextrin decorated thermoresponsive nanogels.Nanoscale. 2018; 10: 469-479Crossref Google Scholar). Finally, patient 1 skin equivalents were topically treated with TG1, either in solution or loaded in tNGs, four times over 8 days. Untreated patient 1 equivalents demonstrated decreased barrier function, shown by the significant increases in their apparent permeabilities to testosterone compared to normal equivalents (Figure 2a). Following full treatment regimens with TG1-loaded tNGs, significant decreases in apparent permeabilities—indicating improved barrier function—correlating to TG1 dose were seen (Figure 2a, 2d, Supplementary Figure S5 online). Importantly, permeation was almost unaffected by the application of unloaded tNG or TG1 dissolved in phosphate buffered saline only (Figure 2b, 2c). Activity staining confirmed the delivery of functional TG1 into viable epidermal layers (Figure 2e), and the distribution of activity was comparable to normal equivalents. Improvement of barrier activity was further confirmed by permeability tests with Lucifer yellow (Figure 2f) and N-hydroxy-sulfosuccinimide-LC-biotin (Supplementary Figure S6 online). Compared to equivalents with normal keratinocytes, a 59-fold increase was seen in the amount of Lucifer yellow fully passing through patient 1 equivalents. Similarly, 39-fold and 43-fold increases were respectively seen in patient 1 equivalents treated with unloaded tNG and TG1 dissolved in phosphate buffered saline. However, following treatment with TG1-loaded tNGs, full Lucifer yellow penetration was only 1.2-fold that of the control, clearly corroborating the role of TG1-loaded tNGs in the reconstitution of patient equivalent barrier function. It is highly likely that the majority of TG1 penetrating into the viable epidermis did so independently of the tNGs because they do not overcome the stratum corneum (Giulbudagian et al., 2018aGiulbudagian M. Hönzke S. Bergueiro J. Işik D. Schumacher F. Saeidpour S. et al.Enhanced topical delivery of dexamethasone by β-cyclodextrin decorated thermoresponsive nanogels.Nanoscale. 2018; 10: 469-479Crossref Google Scholar). This study aimed to further characterize the therapeutic potential of TG1-loaded tNGs in ARCI skin, as well as to better understand their mechanism of action, based on a previous proof-of-principle study demonstrating epidermal delivery of TG1 following topical application of TG1-loaded tNGs (Witting et al., 2015Witting M. Molina M. Obst K. Plank R. Eckl K.M. Hennies H.C. et al.Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules.Nanomedicine. 2015; 11: 1179-1187Crossref PubMed Scopus (69) Google Scholar). Overall, these data verify that topical protein substitution could mitigate or even reverse the ARCI disease phenotype. Notably, Aufenvenne et al., 2013Aufenvenne K. Larcher F. Hausser I. Duarte B. Oji V. Nikolenko H. et al.Topical enzyme-replacement therapy restores transglutaminase 1 activity and corrects architecture of transglutaminase-1-deficient skin grafts.Am J Hum Genet. 2013; 93: 620-630Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar previously demonstrated that topical applications of TG1 mixed with cationic liposomes successfully delivered the functional protein to skin equivalents, formed from TGM1 mutant ARCI patient cells, grafted onto humanized mice. In contrast to our system, no changes to barrier function were observed upon treatment, likely a result of their model; unlike the typical ARCI phenotype, the grafted animals demonstrated compact hyperkeratosis and transepidermal water loss levels close to non-ARCI controls. In summary, topical TG1 replacement therapy is a highly promising therapeutic avenue for ARCI patients with disease-causing TGM1 mutations. The work here indicates TG1 delivery to the intercellular spaces between keratinocytes, and possibly their intracellular environments, can produce therapeutic improvements to the skin-barrier function of the ARCI phenotype. It is hypothesized that increasing the concentration or enzymatic activity of TG1 within the tNG will result in improved therapeutic efficacy and is the likely starting point for future development. The ability of tNGs to encapsulate a wide variety of proteins and deliver these past the stratum corneum of barrier-deficient skin makes them a promising platform technology to treat a range of inflammatory and monogenic skin diseases. Sarah Hedtrich: http://orcid.org/0000-0001-6770-3657 Hans Christian Hennies: http://orcid.org/0000-0001-7210-2389 The authors state no conflict of interest. The authors would like to thank Katja Fuchs and Maria Molina for their scientific support, Christian Ploner for providing skin samples, and fu:stat for excellent help with the statistical analysis of the data. Funding from the German Research Foundation (HE7440/2-1) and the Berlin-Brandenburg Research Platform BB3R to SH and the German Research Foundation (HE3119/9-1), the Austrian Science Fund (FWF, I2259-B26), the German Federal Ministry for Education and Research (E-Rare-2 01GM1201), and the Cologne Fortune Program of the Faculty of Medicine, University of Cologne, to HCH is greatly acknowledged. Download .pdf (.8 MB) Help with pdf files Supplementary Data

Background Autosomal recessive congenital ichthyosis (ARCI) is a genetically and phenotypically heterogeneous skin disease, associated with defects in the skin permeability barrier. Several but not all genes with underlying mutations have been identified, but a clear correlation between genetic causes and clinical picture has not been described to date. Methods Our study included 19 families from Saudi Arabia, Yemen, and Pakistan. All patients were born to consanguineous parents and diagnosed with ARCI. Mutations were analyzed by homozygosity mapping and direct sequencing. Results We have detected mutations in all families in five different genes: TGM1, ABCA12, CYP4F22, NIPAL4, and ALOXE3. Five likely pathogenic variants were unknown so far, a splice site and a missense variant in TGM1, a splice site variant in NIPAL4, and missense variants in ABCA12 and CYP4F22. We attributed TGM1 and ABCA12 mutations to the most severe forms of lamellar and erythematous ichthyoses, respectively, regardless of treatment. Other mutations highlighted the presence of a phenotypic spectrum in ARCI. Conclusion Our results contribute to expanding the mutational spectrum of ARCI and revealed new insights into genotype/phenotype correlations. The findings are instrumental for a faster and more precise diagnosis, a better understanding of the pathophysiology, and the definition of targets for more specific therapies for ARCI.

Autosomal recessive congenital ichthyosis (ARCI) caused by mutations in CYP4F22 is very rare. CyP4F22, a protein of the cytochrome-P450 family 4, encodes an epidermal ω-hydroxylase decisive in the formation of acylceramides, which is hypothesized to be crucial for skin-barrier function. We report a girl with consanguineous parents presenting as collodion baby with contractures of the great joints and palmoplantar hyperlinearity. In the course of the disease she developed fine scaling of the skin with erythroderma, the latter disappearing until the age of 6 months. Her sister showed a generalized fine-scaling phenotype, and, interestingly, was born without a collodion membrane. The analysis of all known candidate genes for ARCI in parallel with a next-generation sequencing approach using a newly designed dermatogenetics gene panel revealed a previously unknown homozygous splice-site mutation c.549+5G>C in CYP4F22 in both girls, confirming the diagnosis of ARCI. Ultrastructural analysis by transmission electron microscopy in both patients showed epidermal hyperplasia, orthohyperkeratosis with persistence of corneodesmosomes into the outer stratum corneum layers, fragmented and disorganized lamellar lipid bilayers, which could be ascribed to inhomogeneous lamellar body secretion, as well as lamellar body and lipid entombment in the corneocytes. These findings correlated with increased transepidermal water loss on the functional level. For the first time, we report a collodion baby phenotype and epidermal barrier impairment in CyP4F22-deficient epidermis at both the ultrastructural and functional level, and corroborate the importance of CyP4F22 for epidermal maturation and barrier function.