Microalgal biofilms are a promising alternative to conventional suspended cultures, offering increased biomass densities with reduced water and energy demands for cultivation and harvesting. However, to fully demonstrate their interest for large-scale production, a better understanding of the impact of operational factors on biofilm structure is required. This study explores the effect of four different photon flux densities (75, 150, 300 and 600 mu mol m- 2 s- 1) on Phaeodactylum tricornutum biofilms cultivated in a millifluidic system. For the first time, biofilm structure and physiology were characterised in situ, non-destructively by complementary imaging tools (Confocal Laser Scanning Microscopy, CLSM, and Optical Coherence Tomography, OCT) and dissolved oxygen measurements. Biofilms cultivated at 150 and 300 mu mol m- 2 s- 1 presented the highest growth rates (0.31 and 0.38 d-1) while higher light intensities (600 mu mol m- 2 s-1) induced photoinhibition. On the other hand, biofilms at 75 mu mol m- 2 s- 1 exhibited the lowest growth rate (0.23 d-1) but they were extremely efficient in converting absorbed light into biomass (2.5 times more efficient than the biofilms grown at 300 mu mol m- 2 s- 1). Interestingly, adjustments in the optical properties of biofilms exposed to different light conditions were observed through changes in the light extinction coefficient with biofilms becoming more transparent when their thickness was higher than 200-300 mu m. These findings reveal important photoacclimation strategies in P. tricornutum biofilms and highlight the potential of exploiting different light intensities and harvesting strategies to optimise the operation of biofilm-based processes.

Background:Rare early-onset lower urinary tract (REOLUT) disorders affect the ureter, urinary bladder, or urethra and manifest before birth or in childhood. Monogenic causes have been reported in a subset of such individuals. Objectives:A possible genetic cause was considered in a child with a megaureter who had syndromic features. Subjects and methods:Whole-exome sequencing was undertaken in individuals with megaureter. Immunohistochemistry was performed in urinary tract tissues of unaffected human fetuses. Results:The index case presented at 6 months with urosepsis and was found to have a unilateral primary non-refluxing megaureter which required stenting of its distal portion. This, together with dysmorphic features and developmental delay, led to a clinical diagnosis of Schinzel-Giedion syndrome (SGS). She was found to carry a de novo missense variant in SET binding protein 1 (SETBP1), c.2613T>G (GenBank: NM_015559.3) (p.Ile871Met), a gene previously implicated in SGS. She was in good general health at 11 years of age, an unusual outcome given that most individuals with SGS die in the first 2 years of life. SETBP1 was detected in the fetal urinary tract, both in the urothelium and in nerve trunks in the kidney hilum and around the ureter. No SETBP1 gene variants were detected in eight further cases of megaureter. Conclusions:This case indicates the value of genetic testing when a REOLUT disorder is accompanied by syndromic signs outside the urinary tract. SETBP1 may drive the functional differentiation of the human fetal ureter.

Biofilm-based microalgal cultivation systems have emerged as a promising alternative to conventional suspended growth methods, offering improved light utilization and biomass productivity. Among these, Rotating Algal Biofilm (RAB) systems are particularly advantageous by subjecting cells to short periodic light/dark (L/D) cycles to mitigate photoinhibition. Through experimental validation and modeling, this study demonstrates that optimized L/D cycles enhance photosynthetic efficiency by temporally diluting high-intensity light. To investigate the impact of light regimes, a model was developed based on Han's photosynthesis framework, incorporating respiration dynamics for broad ranges of cycle times and L/D ratios. Calibrated with experimental data, it accurately predicts biofilm behavior under varying light conditions. A key innovation is the integration of respiration variations during intermittent illumination, providing insights into growth dynamics across frequencies and duty cycles. Key findings show that high light frequencies reduce photoinhibition and enhance growth at given intensities. Increasing the light fraction improves growth rates by reducing peak intensity and shortening dark periods. The model elucidates biofilm responses to fluctuating light and offers strategies for reactor optimization. This study advances algal biofilm photophysiology understanding and provides a predictive tool for optimization and scaling up biofilm-based cultivation systems.

In recent years, microalgal biofilms have garnered significant interest primarily due to their potential to transform wastewater treatment and overcome the limitations associated with conventional microalgal planktonic-based cultivation methods. Advances in imaging technologies have provided insight into the complex dynamics of microalgal biofilms, which could help in understanding both biofilm structure and function, and thus to optimize microalgae biofilm-based technologies. In particular, photonic microscopy allows researchers to study the architectural nuances, physiological responses, as well as spatial organization and interactions within biofilm communities. Despite these significant advances, there is still a lack of consistency in approaches and analytical techniques, which hinders standardized studies. Therefore, there is a need for common protocols and benchmarks in photosynthetic biofilm imaging. This review focuses primarily on key photonic imaging technologies, including confocal laser scanning microscopy (CLSM), light-sheet microscopy, histology coupled with a digital slide scanner, and optical coherence tomography (OCT). Principles, advantages/limitations and current applications of each technique in the context of microalgal biofilm studies, are presented allowing researchers to make reasoned decisions for their specific investigative endeavors.

Biofilm systems present a promising approach for microalgae production by reducing water and energy costs while improving productivity and operational efficiency. However, this technology is still in its infancy, particularly for high-value compounds production. To confirm its potential at large scale, mathematical models are required to better understand biofilm behavior under varying environmental conditions and to predict productivity. In this study, a dynamic model was developed to estimate astaxanthin production by Haematococcus lacustris biofilms on a rotating system. It incorporates well-established dynamics, accounting for nitrogen limitation and photoacclimation, while introducing a novel hypothesis correlating astaxanthin dynamics with those of chlorophyll. The model predicts key biofilm traits, including biomass density, intracellular nitrogen, and pigment quotas, demonstrating its ability to simulate changes in light and nitrogen conditions and assess their impact on biofilm physiology. Furthermore, the possibility of dynamically altering the life cycle of H. lacustris within a biofilm was demonstrated both experimentally and mathematically, enabling reversible transitions between green and red stages. This reversion facilitates continuous astaxanthin production through repeated harvest and regrowth cycles. This was assessed through the development of an optimization strategy that maximized astaxanthin productivity by adjusting light intensity over time and determining the optimal harvest frequency. The model provides a valuable framework for optimizing astaxanthin production in microalgal biofilms, enabling the development of continuous production systems and supporting the scale-up of biofilm technology.

Abstract 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 $${\upmu {\textrm{mol}} \, \textrm{m}^{-2} \, \textrm{s}^{-1}}$$ μ mol m - 2 s - 1 . In the first stage of biofilm development, uniform cell distribution was observed on the substratum exposed to 100 $${\upmu \textrm{mol} \, \textrm{m}^{-2} \, \textrm{s}^{-1}}$$ μ mol m - 2 s - 1 while cell clusters were formed under 500 $${\upmu \textrm{mol} \, \textrm{m}^{-2} \, \textrm{s}^{-1}}$$ μ mol m - 2 s - 1 . Though similar specific growth rate in exponential phase (ca. 0.3 $${\textrm{d}^{-1}}$$ d - 1 ) was obtained under all light intensities, biofilm cells at 500 $${\upmu \textrm{mol} \, \textrm{m}^{-2} \, \textrm{s}^{-1}}$$ μ mol m - 2 s - 1 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 $${\upmu \textrm{mol} \, \textrm{m}^{-2} \, \textrm{s}^{-1}}$$ μ mol m - 2 s - 1 , 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.

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.

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.

Biofilm-based technologies present many advantages (e.g., higher productivity, lower water demand and harvesting costs) compared with the conventional planktonic approaches for microalgae cultivation. A better understanding of photosynthetic biofilm formation is though still needed in order to develop and run biofilm-based systems at large-scale. In this study, for the first time the physiological transition of C. vulgaris cells from planktonic to immobilized state was tracked (cell number, morphology, photosynthetic performance, and cellular composition) during the first 24 h of immobilization. The results clearly confirmed that microalgae rapidly respond to immobilization via physiological adjustments. Over very short time-scales (3 h), cells used photosynthesis to grow (increase in size, 140 %), at expense of cell division, and adjusted their carbon allocation patterns (increase in the relative carbohydrates pool, 135 %). The experiments confirmed that this behavior is specific to cells in the immobilized state. Triggering factors such as water and/or nutrient availability may be responsible for this fast acclimation process. Additionally, lipid content doubled by the end of cultivation, possibly due to imbalanced carbon and nitrogen metabolisms. These results offer new insights for understanding the mechanisms involved in microalgae biofilm formation and development, further helping the operators to optimize the biofilm-based systems.

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. •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.

Rare early-onset lower urinary tract disorders include defects of functional maturation of the bladder. Current treatments do not target the primary pathobiology of these diseases. Some have a monogenic basis, such as urofacial, or Ochoa, syndrome (UFS). Here, the bladder does not empty fully because of incomplete relaxation of its outflow tract, and subsequent urosepsis can cause kidney failure. UFS is associated with biallelic variants of HPSE2, encoding heparanase-2. This protein is detected in pelvic ganglia, autonomic relay stations that innervate the bladder and control voiding. Bladder outflow tracts of Hpse2 mutant mice display impaired neurogenic relaxation. We hypothesized that HPSE2 gene transfer soon after birth would ameliorate this defect and explored an adeno-associated viral (AAV) vector-based approach. AAV9/HPSE2, carrying human HPSE2 driven by CAG, was administered intravenously into neonatal mice. In the third postnatal week, transgene transduction and expression were sought, and ex vivo myography was undertaken to measure bladder function. In mice administered AAV9/HPSE2, the viral genome was detected in pelvic ganglia. Human HPSE2 was expressed and heparanase-2 became detectable in pelvic ganglia of treated mutant mice. On autopsy, wild-type mice had empty bladders, whereas bladders were uniformly distended in mutant mice, a defect ameliorated by AAV9/HPSE2 treatment. Therapeutically, AAV9/HPSE2 significantly ameliorated impaired neurogenic relaxation of Hpse2 mutant bladder outflow tracts. Impaired neurogenic contractility of mutant detrusor smooth muscle was also significantly improved. These results constitute first steps towards curing UFS, a clinically devastating genetic disease featuring a bladder autonomic neuropathy.

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.

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.

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.

Supplementary Table 1 from A Novel, Selective, and Efficacious Nanomolar Pyridopyrazinone Inhibitor of ^V600EBRAF

Supplementary Table 2 from A Novel, Selective, and Efficacious Nanomolar Pyridopyrazinone Inhibitor of V600EBRAF

Contains Supplementary Methods and Table S1 and S2 which summarize the origin, characteristics, provenance and culture conditions of the cell lines used in this study