Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations.

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.

Non-destructive monitoring of microalgae biofilms.

Biofilm-based cultivation systems are emerging as a promising technology for microalgae production. However, efficient and non-invasive monitoring routines are still lacking. Here, a protocol to monitor microalgae biofilms based on reflectance indices (RIs) is proposed. This framework was developed using a rotating biofilm system for astaxanthin production by cultivating Haematococcus pluvialis on cotton carriers. Biofilm traits such as biomass, astaxanthin, and chlorophyll were characterized under different light and nutrient regimes. Reflectance spectra were collected to identify the spectral bands and the RIs that correlated the most with those biofilm traits. Robust linear models built on more than 170 spectra were selected and validated on an independent dataset. Astaxanthin content could be precisely predicted over a dynamic range from 0 to 4% of dry weight, regardless of the cultivation conditions. This study demonstrates the strength of reflectance spectroscopy as a non-invasive tool to improve the operational efficiency of microalgae biofilm-based technology.

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

The effect of light intensity on microalgae biofilm structures and physiology under continuous illumination.

The interest by biofilm-based microalgae technologies has increased lately due to productivity improvement, energy consumption reduction and easy harvesting. However, the effect of light, one key factor for system's operation, received less attention than for planktonic cultures. This work assessed the impact of Photon Flux Density (PFD) on Chlorella vulgaris biofilm dynamics (structure, physiology, activity). Microalgae biofilms were cultivated in a flow-cell system with PFD from 100 to 500 [Formula: see text]. In the first stage of biofilm development, uniform cell distribution was observed on the substratum exposed to 100 [Formula: see text] while cell clusters were formed under 500 [Formula: see text]. Though similar specific growth rate in exponential phase (ca. 0.3 [Formula: see text]) was obtained under all light intensities, biofilm cells at 500 [Formula: see text] seem to be ultimately photoinhibited (lower final cell density). Data confirm that Chlorella vulgaris showed a remarkable capability to cope with high light. This was marked for sessile cells at 300 [Formula: see text], which reduce very rapidly (in 2 days) their chlorophyll-a content, most probably to reduce photodamage, while maintaining a high final cell density. Besides cellular physiological adjustments, our data demonstrate that cellular spatial organization is light-dependent.

Bacterial adhesion inhibition by microalgal EPSs from Cylindrotheca closterium and Tetraselmis suecica biofilms.

In the food industry, successful bacterial pathogen colonization and persistence begin with their adhesion to a surface, followed by the spatial development of mature biofilm of public health concerns. Compromising bacterial settlement with natural inhibitors is a promising alternative to conventional anti-fouling treatments typically based on chemical biocides that contribute to the growing burden of antimicrobial resistance. In this study, three extracellular polymeric substance (EPS) fractions extracted from microalgae biofilms of Cylindrotheca closterium (fraction C) and Tetraselmis suecica (fraction Ta rich in insoluble scale structure and fraction Tb rich in soluble EPS) were screened for their anti-adhesive properties, against eight human food-borne pathogens belonging to Escherichia coli, Staphylococcus aureus, Salmonella enterica subsp. enterica, and Listeria monocytogenes species. The results showed that the fraction Ta was the most effective inducing statistically significant reduction for three strains of E. coli, S. aureus, and L. monocytogenes. Overall, EPSs coating on polystyrene surfaces of the different fractions increased the hydrophilic character of the support. Differences in bacterial adhesion on the different coated surfaces could be explained by several dissimilarities in the structural and physicochemical EPS compositions, according to HPLC and ATR-FTIR analysis. Interestingly, while fractions Ta and Tb were extracted from the same microalgal culture, distinct adhesion patterns were observed, highlighting the importance of the extraction process. Overall, the findings showed that EPS extracted from microalgal photosynthetic biofilms can exhibit anti-adhesive effects against food-borne pathogens and could help develop sustainable and non-toxic anti-adhesive surfaces for the food industry. KEY POINTS: •EPSs from a biofilm-based culture of C. closterium/T. suecica were characterized. •Microalgal EPS extracted from T. suecica biofilms showed bacterial anti-adhesive effects. •The anti-adhesive effect is strain-specific and affects both Gram - and Gram + bacteria.

Exploring the dynamics of astaxanthin production in Haematococcus pluvialis biofilms using a rotating biofilm-based system.

Microalgae biofilm emerged as a solid alternative to conventional suspended cultures which present high operative costs and complex harvesting processes. Among several designs, rotating biofilm-based systems stand out for their scalability, although their primary applications have been in wastewater treatment and aquaculture. In this work, a rotating system was utilized to produce a high-value compound (astaxanthin) using Haematococcus pluvialis biofilms. The effect of nitrogen regime, light intensity, and light history on biofilm traits was assessed to better understand how to efficiently operate the system. Our results show that H. pluvialis biofilms follow the classical growth stages described for bacterial biofilms (from adhesion to maturation) and that a two-stage (green and red stages) allowed to reach astaxanthin productivities of 204 mg m-2 d-1 . The higher light intensity applied during the red stage (400 and 800 µmol m-2 s-1 ) combined with nitrogen depletion stimulated similar astaxanthin productivities. However, by training the biofilms during the green stage, using mild-light intensity (200 µmol m-2 s-1 ), a process known as priming, the final astaxanthin productivity was enhanced by 40% with respect to biofilms pre-exposed to 50 µmol m-2 s-1 . Overall, this study shows the possibility of utilizing rotating microalgae biofilms to produce high-value compounds laying the foundation for further biotechnological applications of these emerging systems.

The impact of light/dark regimes on structure and physiology of Chlorella vulgaris biofilms.

Introduction: Biofilm-based microalgae production technologies offer enormous potential for improving sustainability and productivity. However, the light pattern induced by these technologies is a key concern for optimization. Methods: In this work, the effects of light/dark cycles on architecture, growth, and physiology of Chlorella vulgaris biofilms were assessed in a millifluidic flow-cell with different time cycles (15 s to 3 min) keeping the average light constant at 100 µmol·m-2·s-1. Results and discussion: Results showed that photoinhibition can be mitigated by applying a light fraction of 1/3 and a cycle time of 15 s. By contrast, when the cycle time is extended to 90 s and 3 min, photoinhibition is high and photoefficiency dramatically decreases. To cope with light stress, cells acclimate and organize themselves differently in space. A high peak light (500 µmol·m-2·s-1) triggers a stress, reducing cell division and inducing clusters in the biofilm. This work provides guidelines for optimizing rotating microalgae production systems in biofilms and assesses the minimum rotating frequency required to maintain the net growth rate close to that of continuous light of the same average intensity, mitigating photo-inhibition. The overall gain in productivity is then provided by the total surface of the biofilm turning in the illuminated surface area.

Phenotypic Characterization of Oral Mucor Species from Eurasian Vultures: Pathogenic Potential and Antimicrobial Ability.

Due to poisoning and decline in the food resources of Eurasian vultures, there has been a rise in the number of Griffon (Gyps fulvus) and Cinereous vultures (Aegypius monachus) needing veterinary care. In captivity, vultures often develop oral and other infectious diseases which can affect their survival and the probability of reintroduction in the wild. Therefore, it is important to characterize relevant microbial species present in the oral cavity of vultures, such as Mucor spp. In this work, seven Mucor spp. isolates previously obtained from Gyps fulvus and Aegypius monachus oral swabs collected at two rehabilitation centers in Portugal were characterized regarding their pathogenic enzymatic profile and antimicrobial activity. Isolates were identified by macro and microscopic observation, and PCR and ITS sequencing. Their antimicrobial activity was determined using a collection of pathogenic bacteria and two yeast species. Results showed that 86% of the isolates produced α-hemolysis, 71% expressed DNase, 57% produce lecithinase and lipase, 29% expressed gelatinase, and 29% were biofilm producers. Four isolates showed inhibitory activity against relevant human and veterinary clinical isolates, including Escherichia coli, Enterococcus faecium, Neisseria zoodegmatis, and Staphylococcus aureus. In conclusion, accurate management programs should consider the benefits and disadvantages of Mucor spp. presence in the oral mucosa.

Exploring Temporal and Sex-Linked Dysregulation in Alzheimer's Disease Phospho-Proteome.

This study aims to characterize dysregulation of phosphorylation for the 5XFAD mouse model of Alzheimer's disease (AD). Employing global phosphoproteome measurements, we analyze temporal (3, 6, 9 months) and sex-dependent effects on mouse hippocampus tissue to unveil molecular signatures associated with AD initiation and progression. Our results indicate 1.9 to 4.4 times higher phosphorylation prevalence compared to protein expression across all time points, with approximately 4.5 times greater prevalence in females compared to males at 3 and 9 months. Moreover, our findings reveal consistent phosphorylation of known AD biomarkers APOE and GFAP in 5XFAD mice, alongside novel candidates BIG3, CLCN6 and STX7, suggesting their potential as biomarkers for AD pathology. In addition, we identify PDK1 as a significantly dysregulated kinase at 9 months in females, and the regulation of gap junction activity as a key pathway associated with Alzheimer's disease across all time points. AD-Xplorer, the interactive browser of our dataset, enables exploration of AD-related changes in phosphorylation, protein expression, kinase activities, and pathways. AD-Xplorer aids in biomarker discovery and therapeutic target identification, emphasizing temporal and sex-specific nature of significant phosphoproteomic signatures. Available at: https://yilmazs.shinyapps.io/ADXplorer.

Predicting congenital renal tract malformation genes using machine learning.

Congenital renal tract malformations (RTMs) are the major cause of severe kidney failure in children. Studies to date have identified defined genetic causes for only a minority of human RTMs. While some RTMs may be caused by poorly defined environmental perturbations affecting organogenesis, it is likely that numerous causative genetic variants have yet to be identified. Unfortunately, the speed of discovering further genetic causes for RTMs is limited by challenges in prioritising candidate genes harbouring sequence variants. Here, we exploited the computer-based artificial intelligence methodology of supervised machine learning to identify genes with a high probability of being involved in renal development. These genes, when mutated, are promising candidates for causing RTMs. With this methodology, the machine learning classifier determines which attributes are common to renal development genes and identifies genes possessing these attributes. Here we report the validation of an RTM gene classifier and provide predictions of the RTM association status for all protein-coding genes in the mouse genome. Overall, our predictions, whilst not definitive, can inform the prioritisation of genes when evaluating patient sequence data for genetic diagnosis. This knowledge of renal developmental genes will accelerate the processes of reaching a genetic diagnosis for patients born with RTMs.

Neurogenic Defects Occur in LRIG2-Associated Urinary Bladder Disease.

Introduction: Urofacial, or Ochoa, syndrome (UFS) is an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. It also features an abnormal grimace. Half of individuals with UFS carry biallelic variants in HPSE2, whereas other rare families carry variants in LRIG2.LRIG2 is immunodetected in pelvic ganglia sending autonomic axons into the bladder. Moreover, Lrig2 mutant mice have abnormal urination and abnormally patterned bladder nerves. We hypothesized that peripheral neurogenic defects underlie LRIG2-associated bladder dysfunction. Methods: We describe a new family with LRIG2-associated UFS and studied Lrig2 homozygous mutant mice with ex vivo physiological analyses. Results: The index case presented antenatally with urinary tract (UT) dilatation, and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with UT disease, characterizes UFS. Although HPSE2 sequencing was normal, he carried a homozygous, predicted pathogenic, LRIG2 stop variant (c.1939C>T; p.Arg647∗). Lrig2 mutant mice had enlarged bladders. Ex vivo physiology experiments showed neurogenic smooth muscle relaxation defects in the outflow tract, containing the urethra adjoining the bladder, and in detrusor contractility. Moreover, there were nuanced differences in physiological outflow tract defects between the sexes. Conclusion: Putting this family in the context of all reported UT disease-associated LRIG2 variants, the full UFS phenotype occurs with biallelic stop or frameshift variants, but missense variants lead to bladder-limited disease. Our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function.

Morphological and Molecular Identification of Physaloptera alata (Nematoda: Spirurida) in a Booted Eagle (Aquila pennata) from Portugal.

Physaloptera spp. are parasitic nematodes that infect the gastrointestinal tracts of many carnivores and omnivores. Although they are distributed worldwide, Physaloptera spp. have not been studied in raptors in Portugal. In this study, we report Physaloptera alata in a booted eagle (Aquila pennata) in Portugal. Adult nematodes were discovered in the gizzard of a young booted eagle, and morphological features were consistent with those of the genus Physaloptera. DNA was extracted and a PCR assay performed to amplify a region of the 18S small subunit of the ribosomal RNA gene and the cytochrome c oxidase subunit I gene. The resulting PCR products were Sanger-sequenced, and comparison with the available sequences in the GenBank database confirmed the initial morphological classification as Physaloptera sp. Phylogenetic analysis clustered the sequence within the Physaloptera group. The presence of this parasite in raptors from Portugal is of particular importance to wildlife rehabilitation centers, disease ecologists, and wildlife professionals. Furthermore, we produced a new genetic sequence and have added it to the GenBank database of parasites in birds of prey.

Prediction of Kinase-Substrate Associations Using The Functional Landscape of Kinases and Phosphorylation Sites.

Protein phosphorylation is a key post-translational modification that plays a central role in many cellular processes. With recent advances in biotechnology, thousands of phosphorylated sites can be identified and quantified in a given sample, enabling proteome-wide screening of cellular signaling. However, for most (> 90%) of the phosphorylation sites that are identified in these experiments, the kinase(s) that target these sites are unknown. To broadly utilize available structural, functional, evolutionary, and contextual information in predicting kinase-substrate associations (KSAs), we develop a network-based machine learning framework. Our framework integrates a multitude of data sources to characterize the landscape of functional relationships and associations among phosphosites and kinases. To construct a phosphosite-phosphosite association network, we use sequence similarity, shared biological pathways, co-evolution, co-occurrence, and co-phosphorylation of phosphosites across different biological states. To construct a kinase-kinase association network, we integrate protein-protein interactions, shared biological pathways, and membership in common kinase families. We use node embeddings computed from these heterogeneous networks to train machine learning models for predicting kinase-substrate associations. Our systematic computational experiments using the PhosphositePLUS database shows that the resulting algorithm, NetKSA, outperforms two state-of-the-art algorithms, including KinomeXplorer and LinkPhinder, in overall KSA prediction. By stratifying the ranking of kinases, NetKSA also enables annotation of phosphosites that are targeted by relatively less-studied kinases.Availability: The code and data are available at compbio.case.edu/NetKSA/.

Prevalence and Characterisation of Patients with Asthma According to Disease Severity in Portugal: Findings from the EPI-ASTHMA Pilot Study.

Objective: To assess the feasibility of the procedures of EPI-ASTHMA. EPI-ASTHMA is a population-based multicentre stepwise study about the prevalence and characterisation of patients with asthma based on disease severity in Portugal. Methods: A pilot study of EPI-ASTHMA was conducted with adults from three primary care centres. We followed a stepwise approach comprising 4 stages: stage 0-invitation phone call (n ~1316); stage 1-telephone interview (n ~658); stage 2-clinical assessment with physical examination, diagnostic tests, and patient-reported outcome measures, to confirm the diagnosis of those with possible asthma at stage 1 (n ~160); stage 3-characterization of a subgroup of asthma patients by collecting data through a telephone interview, patient file review and CARATm app (n ~40), after 3-months. The frequency of asthma was calculated in relation to the entire study population (stage 1) and the frequency of difficult-to-treat/severe asthma in relation to the number of asthma patients (stage 3). Results: From 1305 adults invited, 892 (68%) accepted to participate (stage 0) and 574 (64%; 53[42-67] y; 43% male) were interviewed (stage 1). From those, 148 (26%; 60[46-68] y; 43% male) were assessed at stage 2, and 46 (31%; 51[39-67] y; 44% male) were diagnosed with asthma. Half of these patients (n = 23) accepted to install the app. Stage 3 was completed by 41 (93%) patients, of whom 31 (83%) had asthma confirmed by their file review. A total of 8% of participants had asthma, of those 17% had difficult-to-treat and 5% severe asthma. Conclusion: Attained recruitment rates and the quality of the results confirmed the feasibility of the EPI-ASTHMA stepwise approach. This pilot study provided insight into the improvement of the procedures to be generalized across the country.

Diverse ancestry whole-genome sequencing association study identifies TBX5 and PTK7 as susceptibility genes for posterior urethral valves.

Posterior urethral valves (PUV) are the commonest cause of end-stage renal disease in children, but the genetic architecture of this rare disorder remains unknown. We performed a sequencing-based genome-wide association study (seqGWAS) in 132 unrelated male PUV cases and 23,727 controls of diverse ancestry, identifying statistically significant associations with common variants at 12q24.21 (p=7.8 × 10-12; OR 0.4) and rare variants at 6p21.1 (p=2.0 × 10-8; OR 7.2), that were replicated in an independent European cohort of 395 cases and 4151 controls. Fine mapping and functional genomic data mapped these loci to the transcription factor TBX5 and planar cell polarity gene PTK7, respectively, the encoded proteins of which were detected in the developing urinary tract of human embryos. We also observed enrichment of rare structural variation intersecting with candidate cis-regulatory elements, particularly inversions predicted to affect chromatin looping (p=3.1 × 10-5). These findings represent the first robust genetic associations of PUV, providing novel insights into the underlying biology of this poorly understood disorder and demonstrate how a diverse ancestry seqGWAS can be used for disease locus discovery in a rare disease.

EPI-ASTHMA study protocol: a population-based multicentre stepwise study on the prevalence and characterisation of patients with asthma according to disease severity in Portugal.

INTRODUCTION: In Portugal as in other countries, data on the epidemiology of asthma are mainly grounded in questionnaire studies. Additionally, the detailed characterisation of asthma in terms of disease severity, control and phenotypes remain scarce. Studies assessing the prevalence of asthma and its subgroups using accurate methods are needed. This study aims to determine the prevalence of asthma, difficult-to-treat asthma and severe asthma, and to evaluate sociodemographic and clinical characteristics of those patients, in mainland Portugal. METHODS AND ANALYSIS: A population-based nationwide study with a multicentre stepwise approach will be conducted between 2021 and 2023 in 38 primary care centres of the Portuguese National Health Service. The stepwise approach will comprise four stages: Stage 0-telephone call invitation to adult subjects (≥18 years) randomly selected (n~15 000); stage 1-telephone screening interview assessing the participants' respiratory symptoms (n~7500); stage 2-diagnostic visit, including physical examination, diagnostic tests (eg, spirometry, fraction of exhaled nitric oxide, blood eosinophil count) and patient-reported outcome measures for diagnostic confirmation of those identified with possible asthma at stage 1 (n~1800); stage 3-further evaluation of patients with asthma and of patients with difficult-to-treat asthma and severe asthma, after 3 months (n~460). At stage 3, data will be collected from a review of the patient's electronic health records, a follow-up telephone call and the CARATm (Caracteristicas Auto-reportadas de Asma em Tecnologias Móveis) app database. The prevalence of asthma, difficult-to-treat asthma and severe asthma will be determined as the percentage of patients with asthma confirmed from the overall population (stage 1). For the analysis of factors associated with asthma, difficult-to-treat asthma and severe asthma, logistic regression models will be explored. ETHICS AND DISSEMINATION: Ethical approvals for the study were obtained from the ethics committee of the local health unit of Matosinhos, Porto (38/CES/JAS), Alto Minho (38/2021/CES) and the regional health administration of Lisbon-Vale do Tejo (035/CES/INV/2021). Results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05169619.

Biologic Agents in Crohn's Patients Reduce CD4+ T Cells Activation and Are Inversely Related to Treg Cells.

Crohn's disease (CD) is a chronic inflammatory disease with a complex interface of broad factors. There are two main treatments for Chron's disease: biological therapy and nonbiological therapy. Biological agent therapy (e.g., anti-TNF) is the most frequently prescribed treatment; however, it is not universally accessible. In fact, in Brazil, many patients are only given the option of receiving nonbiological therapy. This approach prolongs the subsequent clinical relapse; however, this procedure could be implicated in the immune response and enhance disease severity. Our purpose was to assess the effects of different treatments on CD4+ T cells in a cohort of patients with Crohn's disease compared with healthy individuals. To examine the immune status in a Brazilian cohort, we analyzed CD4+ T cells, activation status, cytokine production, and Treg cells in blood of Crohn's patients. Patients that underwent biological therapy can recover the percentage of CD4+CD73+ T cells, decrease the CD4+ T cell activation/effector functions, and maintain the peripheral percentage of regulatory T cells. These results show that anti-TNF agents can improve CD4+ T cell subsets, thereby inducing Crohn's patients to relapse and remission rates.

Temporal and Sex-Linked Protein Expression Dynamics in a Familial Model of Alzheimer's Disease.

Mouse models of Alzheimer's disease (AD) show progression through stages reflective of human pathology. Proteomics identification of temporal and sex-linked factors driving AD-related pathways can be used to dissect initiating and propagating events of AD stages to develop biomarkers or design interventions. In the present study, we conducted label-free proteome measurements of mouse hippocampus tissue with variables of time (3, 6, and 9 months), genetic background (5XFAD versus WT), and sex (equal males and females). These time points are associated with well-defined phenotypes with respect to the following: Aß42 plaque deposition, memory deficits, and neuronal loss, allowing correlation of proteome-based molecular signatures with the mouse model stages. Our data show 5XFAD mice exhibit increases in known human AD biomarkers as amyloid-beta peptide, APOE, GFAP, and ITM2B are upregulated across all time points/stages. At the same time, 23 proteins are here newly associated with Alzheimer's pathology as they are also dysregulated in 5XFAD mice. At a pathways level, the 5XFAD-specific upregulated proteins are significantly enriched for DNA damage and stress-induced senescence at 3-month only, while at 6-month, the AD-specific proteome signature is altered and significantly enriched for membrane trafficking and vesicle-mediated transport protein annotations. By 9-month, AD-specific dysregulation is also characterized by significant neuroinflammation with innate immune system, platelet activation, and hyper-reactive astrocyte-related enrichments. Aside from these temporal changes, analysis of sex-linked differences in proteome signatures uncovered novel sex and AD-associated proteins. Pathway analysis revealed sex-linked differences in the 5XFAD model to be involved in the regulation of well-known human AD-related processes of amyloid fibril formation, wound healing, lysosome biogenesis, and DNA damage. Verification of the discovery results by Western blot and parallel reaction monitoring confirm the fundamental conclusions of the study and poise the 5XFAD model for further use as a molecular tool for understanding AD.

Expanding the HPSE2 Genotypic Spectrum in Urofacial Syndrome, A Disease Featuring a Peripheral Neuropathy of the Urinary Bladder.

Urofacial (also called Ochoa) syndrome (UFS) is an autosomal recessive congenital disorder of the urinary bladder featuring voiding dysfunction and a grimace upon smiling. Biallelic variants in HPSE2, coding for the secreted protein heparanase-2, are described in around half of families genetically studied. Hpse2 mutant mice have aberrant bladder nerves. We sought to expand the genotypic spectrum of UFS and make insights into its pathobiology. Sanger sequencing, next generation sequencing and microarray analysis were performed in four previously unreported families with urinary tract disease and grimacing. In one, the proband had kidney failure and was homozygous for the previously described pathogenic variant c.429T>A, p.(Tyr143*). Three other families each carried a different novel HPSE2 variant. One had homozygous triplication of exons 8 and 9; another had homozygous deletion of exon 4; and another carried a novel c.419C>G variant encoding the missense p.Pro140Arg in trans with c.1099-1G>A, a previously reported pathogenic splice variant. Expressing the missense heparanase-2 variant in vitro showed that it was secreted as normal, suggesting that 140Arg has aberrant functionality after secretion. Bladder autonomic neurons emanate from pelvic ganglia where resident neural cell bodies derive from migrating neural crest cells. We demonstrated that, in normal human embryos, neuronal precursors near the developing hindgut and lower urinary tract were positive for both heparanase-2 and leucine rich repeats and immunoglobulin like domains 2 (LRIG2). Indeed, biallelic variants of LRIG2 have been implicated in rare UFS families. The study expands the genotypic spectrum in HPSE2 in UFS and supports a developmental neuronal pathobiology.

The architecture and metabolic traits of monospecific photosynthetic biofilms studied in a custom flow-through system.

Microalgae biofilms have great ecological importance and high biotechnological potential. Nevertheless, an in-depth and combined structural (i.e., the architecture of the biofilm) and physiological characterization of microalgae biofilms is still missing. An approach able to provide the same time physiological and structural information during biofilm growth would be of paramount importance to understand these complex biological systems and to optimize their productivity. In this study, monospecific biofilms of a diatom and a green alga were grown under dynamic conditions in custom flow cells represented by UV/Vis spectroscopic cuvettes. Such flow cells were conceived to characterize the biofilms by several techniques mostly in situ and in a nondestructive way. Physiological traits were obtained by measuring variable chlorophyll a fluorescence by pulse amplitude modulated fluorometry and by scanning the biofilms in a spectrometer to obtain in vivo pigments spectral signatures. The architectural features were obtained by imaging the biofilms with a confocal laser scanning microscopy and an optical coherence tomography. Overall, this experimental setup allowed us to follow the growth of two biofilm-forming microalgae showing that cell physiology is more affected in complex biofilms likely as a consequence of alterations in local environmental conditions.