Halomonas bluephagenesis, which is a bioplastic poly(3-hydroxybutyrate) producer allowing open unsterile and continuous conditions. Here we report a de novo synthesis pathway for ectoine constructed into the chromosome of H. bluephagenesis utilizing two inducible systems, which serve to fine-tune the transcription levels of three clusters related to ectoine synthesis, including ectABC, lysC and asd based on a GFP-mediated transcriptional tuning approach. Combined with bypasses deletion, the resulting recombinant H. bluephagenesis TD-ADEL-58 is able to produce 28 g L−1 ectoine during a 28 h fed-batch growth process. Co-production of ectoine and PHB is achieved to 8 g L−1 ectoine and 32 g L−1 dry cell mass containing 75% PHB after a 44 h growth. H. bluephagenesis demonstrates to be a suitable co-production chassis for polyhydroxyalkanoates and non-polymer chemicals such as ectoine.Ectoine, a compatible solute synthesized by many halophiles for hypersalinity resistance, has been successfully produced by metabolically engineered  Halomonas bluephagenesis is a halophilic platform bacterium for next generation industrial biotechnology. Here, the authors employ a stimulus response-based flux-tuning method for coproduction of bioplastic PHB and ectoine under open unsterile and continuous growth conditions. Ectoine was reported to show protections for living cells by enhancing the stabilities of proteins, nucleic acids, and cell membranes, enabling survival of halophiles under the circumstances of hypersalinity, high temperatures, extreme pH and exposure to chemical agents7. Therefore, ectoine has been exploited for commercial applications as an ingredient for cosmetics8, medicines9, and organ transplantation maintenance10. However, the high production cost is still unacceptable for wider applications11.Ectoine  is one of the best well-known compatible solutes produced by halophiles for hypersalinity resistance12. The major biosynthesis pathway of ectoine containing its operon ectABC encoding l-2,4-diaminobutyrate acetyltransferase (ectA), l-2,4-diaminobutyrate transaminase (ectB) and ectoine synthase (ectC), respectively, has been clearly characterized to be a highly conservative cluster across halophile species, allowing three-step biocatalysis to form ectoine from l-aspartate-β-semialdehyde (ASA), an important intermediate involved in several amino acids synthesis, such as threonine and lysine15. Interestingly, many halophiles also contain ectoine hydrolase encoded by ectD for converting ectoine into 5-hydroxyectoine as both carbon- and nitrogen-source used under nutrient-limited conditions, which is a dominating bypass limiting the high-level intracellular accumulation of ectoine17.Commercially, fermentative production of ectoine by microorganisms under sterile bioprocesses is the major solution to meet its increasing demandsE. coli and Corynebacterium glutamicum employing various synthetic biology strategies. Specifically, halophiles were first grown in media with a high salt concentration for sufficient ectoine synthesis, subsequently cells were quickly transferred into desalted water for effective secretion of ectoine into extracellular space, termed ‘bacterial milking’19. Interestingly, it was reported that the deletion of TeaABC transporter in Halomonas elongata DSM 2581 led to an efficient secretion of ectoine in extracellular space during a high-cell-density fermentation21. Additionally, the heterologous expression of ectABC cluster from halophiles by engineered model microorganisms enabling sufficient ectoine biosynthesis has been achieved: ectABC from Chromohalobacter salexigens and Halomonas elongata DSM 2581, the most studied cluster, were overexpressed in E. coli for enhanced production of ectoine from l-aspartate and glucose22. Remarkably, metabolically engineered E. coli W3110 and C. glutamicum with bypass deletion, transcriptional (promoter) and translational (RBS) tuning based on high-throughput combinatory constructs and bioprocess optimizations, were successfully used to direct metabolic fluxes towards ectoine synthesis from glucose for efficient ectoine synthesis23.Recent progresses have exemplified the enhanced production of ectoine by recombinant halophiles, halophilic archaea, Halomonas bluephagenesis TD01, a natural poly(3-hydroxybutyrate) (PHB) producer, has been successfully developed for achieving cost-effective production under open unsterile and continuous growth conditions27. PHB, a family member of microbial biopolyesters synthesized by many bacteria and archaea including E. coli, Pseudomonas putida and Cupriavidus necator et al., is a model biodegradable polymer for commercial applications32. H. bluephagenesis has been successfully engineered to produce cost-effective PHB35 from lab-scale to pilot-scale (5000-L) fermentation27. It has become highly desirable to engineer H. bluephagenesis as a versatile chassis for productions of valuable chemicals at a competitive cost, or for co-production of PHA  with a small molecular chemical to increase the process economy37.Compared with current industrial biotechnology (CIB) based on model microorganisms, next generation industrial biotechnology (NGIB) based on H. bluephagenesis could be a suitable host to engineer for ectoine production due to its fast growth and high PHB accumulation under high-salt conditions. So far H. bluephagenesis has been manipulated via various approaches including CRISPR/Cas934, stimulus response-based flux-tuning (SR-FT)38, genomic integrations, inducible promoter mining35, and promoter engineering39. These concreted efforts enable rational strain engineering of complexity for synthesizing diverse metabolic targets, strongly expanding the capability of H. bluephagenesis as a versatile production chassis. Generally, static optimization is a widely used strategy to fine-tune the pathways involved in multi-gene regulation largely dependent on high throughput constructs and machine learning-based analysis42, leading to high cost of clone construction with a long R&D cycle.H. bluephagenesis for enhanced production of ectoine from glucose using “stimulus response-based flux-tuning” method  containing 75% PHB after 44 h fed-batch fermentation.This study aims to chromosomally engineer hod Fig. . Specifi46, of which H. bluephagenesis is the one engineered as the chassis for NGIB47. As a metabolic target and compatible solute, cell growth characterization was performed to study the impact between ectoine and NaCl in the presence of various ectoine and NaCl concentrations, respectively  prototyping ectoine production by the start host, H. bluephagenesis TD1.0; (II) heterologous over-expressing ectABC for ectoine production by recombinant E. coli22.It is important to note that the de novo synthesis pathway of ectoine has been identified, it is highly conservative across species6 Fig. . Based osis Fig.  was annoH. bluephagenesis TD1.0 cultivated in a 60MM medium supplemented with 30 g L−1 glucose in shake flask studies. Results showed that the biosynthesis ectoine shared the same retention time of mass spectra with ectoine standard, which is consistent with the secondary analysis results of LC-MS/MS  was applied to assay the accumulation of ectoine by PHB Fig. , confirmPTG Fig. . The titium Fig. , furtherH. bluephagenesis was generally engineered for diverse PHA productions requiring a nitrogen limited medium, or a 60MM medium supplemented with 30 g L−1 glucose for shake flask cultivations. As ectoine is an aspartate derived compound heavily depending on both carbon- and nitrogen-catabolism, it is necessary to reinforce the nitrogen supply via a medium modification. Even more important, transcriptional tuning of ectABC cluster via adding l-aspartate as precursor is significant for channeling more fluxes to ectoine, while avoiding the possibility of endogenous negative feedback control on the ectABC expression.ectABC on a plasmid-based system in recombinant H. bluephagenesis TD1.0 in the presence of various concentrations of IPTG , to ensure the metabolic fluxes flowing towards ectoine synthesis. As a result, the optimal transcriptional level for ectABC expression was determined after the induction of 20 mg L−1 IPTG, the maximum production titer of ectoine reached 2.38 g L−1 . This resulted in a 2.83 g L−1 ectoine produced by recombinant H. bluephagenesis harboring ectABC expression module induced by 20 mg L−1 IPTG in shake flasks  and ectD (EC 1.14.11.55)  and 5-hydroxyectoine, respectively. Both of these two genes were found in the genome of H. bluephagenesis via genomic analysis, they function to reduce ectoine accumulation. It is thus necessary to eliminate these two branches for blocking the degradation of ectoine by deleting doeA and ectD , and double-knockout H. bluephagenesis TD-AD , respectively  allows robust performance of cell growth under antibiotic-free cultivations. Therefore, a GFP-mediated transcriptional mapping approach developed for fine-tuning target genes in the previous studyter Fig. . Specifip35 Fig. . SimultaectABC depends on the T7-like induced system, such as the optimized 20 mg L−1 IPTG induction for the T7-like system, yet IPTG is an expensive chemical not suitable for large scale applications. Thus, a constitutive porin promoter mutant P140, which exhibits a proximal expression level compared to the T7-like promoter induced by 20 mg L−1 IPTG in terms of the fluorescence intensity values  . Since the expression levels of target modules on plasmid pSEVA321 are reported to be 9-fold higher than that of the chromosomal expression, which can easily lead to poor- or non-growth due to the overload of target fluxes in donor cells (E. coli S17-1)38. Ectoine production by ectABC fine-tuned H. bluephagenesis TD-ADE was carried out in shake flasks containing both 60MME and 60MMU media, forming 4.7 g L−1 and 4.3 g L−1 ectoine, respectively  Fig. , formingely Fig. . In contine Fig. .H. bluephagenesis TD-ADE containing the chromosome-based and fine-tuned ectABC system under the control of porin promoter mutant P140, has enhanced the production of ectoine  was constructed on a plasmid-based expression system controlled by the constitutive promoter PJ2311050, together with sfgfp controlled by Plux, for the does-response functional characterization in H. bluephagenesis TD-ADE using flow cytometer analysis  generated 17% improvement on ectoine accumulation although cell growth decreased significantly . Similarly, a fed-batch fermentation was carried out in the 7 L bioreactor to co-produce ectoine and PHB in different growth phases. After a 44 h cultivation, the DCM reached 32 g L−1 containing 75% PHB, together with 8 g L−1 ectoine  based on halophiles provides low-cost and convenient processing conditions for production of chemicalsess Fig. .H. bluephagenesis was clearly identified by heterologous expression in recombinant E. coli, in which strong enzymatic activities of ectABC cluster were found compared with previous studies22 : 10 trypton, 5 yeast extract and 10 NaCl. 60LB medium is derived from LB medium supplemented with 60 g L−1 NaCl. 60MMG medium consists of (g L−1): glucose 30, NaCl 60, yeast extract 1, (NH4)2SO4 0.25, MgSO4 0.2, Na2HPO4·12H2O 9.65, KH2PO4 1.5, trace element solution I 10 mL L−1 and trace element solution II 1 mL L−1. The composition of trace element solution I consists of (g L−1): Fe(III)-NH4-citrate 5, CaCl2 2, HCl 1 M. The trace element solution II (mg L−1) consists of: ZnSO4·7H2O 100, MnCl2·4H2O 30, H3BO3 300, CoCl2·6H2O 200, CuSO4·5H2O 10, NiCl2·6H2O 20, and NaMoO4·2H2O 30. 60MME is derived from 60MMG medium supplemented with 100 mM sodium aspartate with additional 100 mM KCl, and 1.7 g L−1 citric acid. Alternatively, 100 mM sodium aspartate in 60MME medium was replaced by 3 and 5 g L−1, respectively, to form 60MMU and 60MMU5 media for de novo synthesis of ectoine for shake flask studies. Ectoine of analytical purity purchased from Sigma-Aldrich  was used as standard for HPLC analysis for fermentative ectoine quantitation.LB medium is composed of  enzymes were deleted, forming H. bluephagenesis TD-AD strain. Then, expression modules including ectABC, lysC and asd cluster were integrated into the chromosome of H. bluephagenesis one-by-one at G4, G7 and G43 locus, respectively, forming H. bluephagenesis strains TD-ADE, TD-ADEL-58, and TD-ADEL-183.All the derivates from 63. All the constructs were assembled by Gibson Assembly kit, and conjugated into H. bluephagenesis derivates. DNA extraction was carried out by using Tiangen Plasmid Extraction kit  and Omega Gel Purification kit (USA). DNA oligonucleotides were synthesized by Zixi Biotechnology . Genetic design and sequence reading were performed by Snap Gene (v2.3.2).Plasmids were constructed to carry functional gene expressions encoded in vector pSEVA32134. Firstly, plasmid pQ08 containing Cas9 expression module was transformed into recombinant H. bluephagenesis by conjugation. Followed by the conjugation of constructs derived from pSEVA241 vector carrying gRNA and homologous arms (500 bp), positive colonies were screened by PCR test and confirmed by DNA sequencing after the discard of plasmids by passaging the cell cultures in 60 LB medium without antibiotics for 3–5 generations.All the plasmids were constructed by Gibson Assembly kit. For gel purification, the Omega gel purification kit was used, and the Tiangen plasmid extraction kit was used to obtain the plasmids. All the plasmids used in this study and genes required fine-tuning to obtain enhanced ectoine production were listed in Supplementary Table E. coli DH5α, subsequently transformed into E. coli S17-1, the donor strain for conjugation. Then, donor cells harboring target constructs were cultivated for 12–14 h in 150-mL shake flasks containing 20 mL LB medium, and recipient cells, recombinant H. bluephagenesis, were cultivated in 60LB medium overnight. Both of the donor and recipient cells were centrifugated and harvested under 4 oC, washed with a fresh medium and recovering to the same volume that sampled. Subsequently, cells were mixed (1:1 volume) and dropped on antibiotic-free 20-LB agar plates, followed by 6–8 h incubation at 37 oC for conjugation. Finally, conjugated cells from the lawn were suspended with 60LB medium and spread on 60LB agar plates containing relevant antibiotics for 36 h incubation to obtain positive colonies.Firstly, plasmids were constructed by H. bluephagenesis TD1.0 harboring plasmid p321-PJ23110-luxR-Plux-sfgfp to characterize the performance in the presence of various AHL  concentrations, respectively. Then, luxR expression module was integrated into the chromosome of H. bluephagenesis TD-ADE at G43 loci, forming TD-LuxR strain, followed by the characterization of LuxR-AHL and T7-like induced systems in H. bluephagenesis TD-LuxR strain harboring plasmids, p321-Plux-sfgfp and p321-PT7-like-sfgfp after conjugation. Cells carrying target expression vessels were plated on 60LB agar plate added with relevant antibiotics for 16–20 h of incubation at 37 °C. Subsequently, single colony was inoculated into 1 mL 60LB medium for 12 h cultivation in 96-deep-well plate covered with sealing films . Then, 5 µL cell culture was inoculated into a new plate supplemented with appropriate antibiotics in presence of a spectrum of inducer. After 12 h incubation, cell cultures were diluted 250-folds using phosphate-buffered saline solution (PBS) for flow cytometry analysis.LuxR-AHL inducible system was first constructed on plasmid-based expression system in −1 for 20 s with at least 30,000 cell captured in total of each sample, and the start host, H. bluephagenesis TD1.0, was used as the negative control groups to modify the fluorescence levels of experimental groups. Intracellular sfGFP protein was excited under 488 nm, and cells were captured on the signal channels of FITC, FSC (forward scatter) and SSC (side scatter). The FlowJo (v7.6) software was used to process the raw data for obtaining mean value of fluorescence intensity and percentage of positive cells.Positive fluorescent cells were recorded by flow cytometer  at the rate of 0.5 µL sH. bluephagenesis TD1.0 was first picked as an inoculum for a 12 h incubation in 1 mL 60LB medium in a 96-deep-well plate  at 37 °C. Subsequently, seed cultures were diluted 200-folds with fresh MM medium supplemented with 20 g L−1 in the presence of various ectoine and NaCl concentrations in a new 96-well plate  for online recording of optical density at 600 nm (OD600) for 48 h.To study the effects of ectoine and NaCl on cell growth, single colony of recombinant H. bluephagenesis in this study, chromosomally engineered strains were pre-cultured in a 60-LB medium in the 96-deep-well plate for 12 h by picking a single colony as an inoculum. Then, seed cultures were diluted 200-folds with a fresh 60LB medium in a new 96-well plate  for online monitoring of optical density at 600 nm (OD600) for 24 h. The cell growth characterization was carried out using a micro-plate reader .To characterize cell growth of recombinant H. bluephagenesis harboring target expression modules on plasmid- or chromosome-based systems were pre-cultured overnight to obtain seed cultures (OD600 reached 2.5 ± 0.2) from a single colony, respectively. Subsequently, 2.5 mL seed culture were inoculated into 50 mL 60MM medium supplemented with 30 g L−1 glucose and nitrogen source (urea or sodium aspartate) whenever necessary. 25 µg mL−1 chloramphenicol was added to the medium grown with H. bluephagenesis harboring the plasmid-carried expression systems. Cell cultures were harvested for dry cell mass (DCM), ectoine and PHB content assays after 48 h cultivation. Additionally, functional verification of gene cluster ectABC from H. bluephagenesis was performed using the same procedure of recombinant H. bluephagenesis grown in LB medium and recombinant E. coli DH5α harboring plasmid p321-J23110-lacI-Ptac-ectABC.For shake flask studies, recombinant 600 reached 2.5 ± 0.2) as inoculums in a fed-batch study conducted in a 7-L bioreactor . A 3 L 60 MM medium supplemented with 3 g L−1 CO(NH2)2, 10 g L−1 yeast extract and 20 g L−1 glucose was used as an initial medium for fermentation. During the fermentation process, dissolved oxygen (DO%) was maintained at ~30% of air saturation by injecting air with a maximum flow rate of 1 VVM (air volume per culture volume per min) by coupling the agitation of less than 800 rpm. pH was adjusted using 5 M NaOH solution. Feed solution of the first phase (Feed-I) is composed of 200 g glucose, 8 g yeast extract and 32 g urea (24 g of urea for co-production purpose). For ectoine production, the second phase feeding solution (Feed-II) consists of 200 g glucose, 4 g yeast extract and 28 g urea, while fed nutrients composed of 200 g glucose, 4 g yeast extract and 8 g urea for co-production of ectoine and PHB. And Feed-III containing only 800 g L−1 glucose was fed when Feed-II ran out during the fermentation for co-production of ectoine and PHB. Feed solutions were fed to maintain the residual glucose at 6–10 g L−1 . All batches of 7-L fermentation were conducted under non-sterilized open condition at 37 °C. Then, cells sampled of every time point were harvested for cell mass, ectoine and PHB assays.For fed-batch studies, cells were grown overnight in 60LB medium in 500 mL shake flasks to obtain 300 mL seed cultures  for 15 min, subsequently suspended and washed twice with distilled water. A dry cell mass was calculated by measuring the mass of harvested cells after 24 h of lyophilization under −60 °C and 100 Pa. For PHB content assays, 30–40 mg lyophilized cells in powder form were sampled for methanolysis under 100 °C for 3.5 h supplemented with 2 mL chloroform and 2 mL methanolysis solution  mixture48. Then, 1 mL water was added to the methanolysis solution after cooling down to room temperature (25 °C) for extraction and phase separation. 1 mL of the heavy (low) phase was sampled for PHB content calculation using GC  analysis. 15 and 20 mg pure PHB (3HB standard from Sigma-Aldrich) were used as standards.First, cells were harvested from 30 mL fermentative broth during a fed-batch study or at the end of the shake flask study via centrifugation at 12,200g for 10 min. Then, the supernatants were filtrated once by 0.22 μm filter membrane for ectoine quantification using high-performance liquid chromatography  equipped with a C18 column based on acetonitrile/water mixture  at a flow rate of 1 mL per min as the mobile phase. UV (ultraviolet) detector (210 nm) was used for ectoine detection. The HPLC-MS analysis was carried out using OrbiTrap Q-Exactive LC-MS (Thermo Scientific) under the same conditions.Five millimeters of fermentative broth from 30 mL cell cultures during fed-batch study or at the end of shake flasks were diluted 10-folds (50-folds for the fed-batch study) in a 50 mL tube. Subsequently, cells were disrupted by a high pressure homogenizer and centrifugated at 12,200Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13


Growing demand for care for Alzheimer’s Disease and related Dementia (ADRD) has resulted in rising use of adult day health centers (ADHCs), which employ teams of professionals including licensed nurses, nursing aides, social workers, and activity directors. This study evaluates the scope of services and staffing models of ADHCs that provide care to persons with ADRD compared to ADHCs that do not, and examines whether there is an association between staffing and client outcomes, measured as rates of hospitalizations, falls, and emergency department visits. We used facility-level data from the 2014 National Study of Long-Term Care Providers (NSLTCP) Adult Day Services Center module. We conducted bivariate comparisons and estimated multivariate regressions to identify ADHC characteristics associated with staffing and client outcomes. ADHCs that offered ADRD services had higher average daily attendance, greater shares of revenue from Medicaid and self-payment, and greater proportions of Blacks and females. They also had greater percentages of enrollees with depression, cardiovascular disease, diabetes, and needing assistance with activities of daily living. There were also greater numbers of registered nurse, licensed practical nurse, and social worker hours per enrollee day, but fewer activity staff hours per enrollee day. Multivariate regressions focused on ADHCs that offered skilled nursing services and revealed that total staff hours per enrollee day were not higher in ADHCs that provided ADRD services, controlling for other characteristics. However, staffing was greater in chain-affiliated ADHCs. Higher staffing levels were associated with lower rates of falls and emergency department visits.


Retinal tissues were assessed using high resolution immunohistochemistry to evaluate changes in photoreceptors, glia and microglia in the dorsal, and ventral retina. Photoreceptor dysfunction and death occurred from 1 month of age. There was a striking difference in loss of photoreceptors between the dorsal and ventral retina, with a greater number of photoreceptors surviving in the dorsal retina, despite being adjacent a layer of photoreceptor debris within the subretinal space. Loss of photoreceptors in the ventral retina was associated with fragmentation of the outer limiting membrane, extension of glial processes into the subretinal space that was accompanied by possible adhesion and migration of mononuclear phagocytes in the subretinal space. Overall, these findings highlight that breakdown of the outer limiting membrane could play an important role in exacerbating photoreceptor loss in the ventral retina. Our results also highlight the value of using the RCS rat to model sectorial retinitis pigmentosa, a disease known to predominantly effect the inferior retina.Retinitis pigmentosa is a family of inherited retinal degenerations associated with gradual loss of photoreceptors, that ultimately leads to irreversible vision loss. The Royal College of Surgeon's (RCS) rat carries a recessive mutation affecting mer proto-oncogene tyrosine kinase (merTK), that models autosomal recessive disease. The aim of this study was to understand the glial, microglial, and photoreceptor changes that occur in different retinal locations with advancing disease. Pigmented RCS rats (RCS-p Retinitis pigmentosa (RP) refers to a family of inherited disorders characterized by gradual loss of rod, followed by cone photoreceptors  and with the recommendations of the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research.+/LAV) rats (n = 74) (referred to here as RCS) and age-matched non-dystrophic control (RCS- rdy +p+/LAV) rats (n = 71) (referred to here as rdy) aged postnatal day 18, 1, 2, 4, 6, and 12 months were examined (n = 8–19 per age per strain). Rats were housed at a controlled temperature (22 ± 1°C) on a 12 h light: 12 h dark cycle and at lux levels varying from 3 to 23 lux. Food and water were available ad libitum.Dystrophic pigmented RCS  and xylazine . The corneal reflex was anesthetized via topical administration of Alcaine , while pupil dilation was achieved via Atropine  and phenylephrine hydrochloride . Sterile saline  was applied to the cornea to prevent dehydration. Rats were placed on a heat pad to maintain body temperature while under anesthesia.2 full field flash from a Nikon camera  delivered from a custom made Ganzfeld. Responses were amplified  and digitized at 10 kHz. Two consecutive flashes, with an interval of 0.8 s, elicited a mixed rod and cone pathway response and a cone-only pathway response, respectively . ERGs were recorded by generation of a 2.1-log cds/mSince the responses obtained from dystrophic RCS rats were small, it was not possible to model the ERG responses. Instead, raw ERG data were utilized. The a-wave was defined as the amplitude of the baseline to the trough of the negative slope preceding the leading positive slope. The b-wave was defined as the trough of the a-wave to the peak of the leading positive slope. If no a-wave was present, the b-wave was measured from the pre-stimulus baseline to the peak of the leading slope. The implicit timing of the a- and b-waves were measured from the time of flash to the a-wave trough or b-wave peak, respectively  and fundus images were taken on a Micron III rodent fundus camera  using specialty Micron III (Phoenix Research Labs) and Streampix software . Both RCS and rdy animals were anesthetized as above and then imaged following completion of ERG recording. Throughout imaging, lubricating eye gel  was applied and the pupil further dilated with atropine when required.OCT images that were generated from scans across the retina within two disc diameters of the optic nerve. For quantification of retinal layers, an annular scan centered on the middle of the optic nerve head was used to generate OCT images that were of similar retinal eccentricity. The thickness of individual retinal layers was achieved using a custom Image J script (NIH), that segmented each raw B-san  immediately prior to tissue collection. For orientation, the dorsal region of the eye was marked with a tattoo pen, eyes were removed, the anterior components dissected, and the posterior eye cup placed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 30 min. The eye cups were subsequently cryoprotected in graded sucrose solutions  and snap frozen and stored at −80°C until use.Frozen sections (14 μm) were cut on a cryostat , mounted onto poly-L-lysine coated glass slides  and stored at −80°C until use. All sections were cut from the dorsal to ventral region of the eye, as close to the optic disc as possible, to allow for consistency between eyes.In order to evaluate how photoreceptors, microglia and retinal Müller cells change with increasing degrees of degeneration, vertical sections of the retina were processed for indirect immunofluorescence using protocols previously described . Confocal z-stacks were reconstructed into 3D images in the n = 5 RCS eyes) using the area measurement tool in Image J. In parallel the coverage of GS positive Müller cell sprouting into the debris area was measured the same way and compared.The area and thickness of subretinal debris was measured in confocal images of vertical cryostat sections of dorsal, intermediate and ventral retina of 2 month old RCS rats  detection kit  as previously described . In all figures, statistical significance is expressed as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Statistical Analysis was carried out in Graphpad Prism 9  and data expressed as the Mean ± Standard Error of the Mean (SEM) unless otherwise stated. One or Two way ANOVA was used where comparisons across two factors or more means were evaluated and a Tukey's The central aim of this study was to evaluate how retinal structure and function in the pigmented RCS rat retina changed with both age and location in comparison to age matched congenic control rdy retinae.n = 10–20 rdy rats and 6–18 RCS rats per age) . In particular, the amplitude of a- (B), b- (C), and cone-b (E) waves steadily declined from 1 month of age and was almost undetectable by 6 months of age. The electroretinogram is a serial waveform, and therefore the observed reduction in rod b-wave could be attributed to losses in photoreceptor or alternatively changes in inner retinal function. We compared the percentage loss of the rod a-wave with the rod b wave and found the losses in each waveform to be similar across the different ages , suggesting that the loss in b-wave amplitude could be explained by photoreceptor dysfunction. Finally, we compared the extent of loss in cone mediated and rod mediated function by assessing the relative loss of the cone b- wave with that of the rod b-wave .We first evaluated the time course of retinal functional changes that occur with age using the flash electroretinogram (ERG). Rod and cone mediated function was evaluated by quantifying function in response to a paired-flash stimulus in animals aged postnatal day 18, and 1, 2, 4, and 6 months of age . n = 10–20 rdy and n = 10–15 RCS rats per age; (post-hoc Tukey's test p < 0.001 for comparisons at 4 and 6 months). As shown in post-hoc Tukey's test p < 0.001 for comparisons of 1 and 6 months), there was considerable thinning of the outer nuclear layer in the RCS by 1 month of age and virtually no outer nuclear layer present at 2, 4 or 6 months of age . Instead, a thick layer of hyperreflective material or debris could be measured from 1 month of age in the RCS rat . The decrease in debris thickness corresponded to the reduction in total retinal thickness observed at 4 and 6 months. A comparison of the thickness of the inner and outer retina in the dorsal (superior) and ventral (inferior) retina is shown in Segmentation analysis was used to quantify changes in total retinal thickness and thickness of the inner and outer retina rdy and RCS retinae with advancing age . post-hoc Tukeys test, p < 0.0001 for comparisons at 1 and 2 months of age). A comparison of photoreceptor death in the central, mid-peripheral and peripheral retina was also investigated for all age groups, resulting in no differences between the three retinal locations . Finally, we quantified the number of rows of photoreceptor nuclei in the outer nuclear layer to quantify the change in thickness of the outer nuclear layer more accurately than is possible with OCT. As shown in p < 0.0001). It should be noted, however, that contrary to our observations with OCT . In view of our previous observations that the extent of debris varies across the retina, we used histological sections to correlate the thickness of the outer nuclear layer with the thickness of debris. As shown in r2 = 0.518, p < 0.001).Having established the time course of photoreceptor loss, we next evaluated histologically whether there were any differences in the rate of photoreceptor loss across the retina. Loss of photoreceptors was particularly striking in the ventral retina compared to the dorsal retina . Indeed,Figure 9A) and corresponded to regions where the ONL appeared thicker and more organized. Notably, areas of the ventral and dorsal retina that showed an equivalent number of photoreceptor nuclei . Colocalization of Iba1 with CD3, CD8, and CCR2 was undertaken to potentially identify any subgroups of innate immune cells within the subretinal space  and nestin  of photoreceptor death in the RCS rat retina are not well-understood. Previous studies have suggested that photoreceptor death occurs in the RCS rat as a consequence of the build-up of debris within the subretinal space  in the subretinal space from postnatal 14 (He et al., In conclusion, this study shows that retinal degeneration occurs in a non-uniform fashion across the retina of the RCS rat, with the ventral retina more profoundly affected than the dorsal retina. Importantly, the level of debris was lower in the ventral retina, the number of photoreceptors reduced, whilst mononuclear phagocytes and penetrating gliotic Müller cell processes were more abundant. These findings are significant for several reasons. First, studies evaluating the efficacy of treatments need to be controlled carefully for retinal location, so as to take into account the differences in photoreceptor integrity in the dorsal and ventral retina. Secondly, these results highlight the value of using the RCS rat model as a model to examine sectorial RP. Finally, further work is needed to explore the potential contributing role that breakdown of the outer limiting membrane, protrusion of Müller cells and migration of microglia and other mononuclear phagocytes have in exacerbating photoreceptor death in this and other retinal degenerations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by The University of Melbourne Animal Experimentation Ethics Committee.UG and MH contributed equally to the study design, data collection, data analysis, and writing. GV collected data and undertook data analysis and contributed to writing the manuscript. AJ and KV undertook data analysis and contributed to writing the manuscript. DS, HO'N, and IL contributed to writing the manuscript and analyzing data. EF undertook data analysis, wrote the manuscript, and supervised the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NLRP3 gene is associated with autosomal-dominant sensorineural hearing loss with cochlear autoinflammation in two unrelated families. NLRP3 encodes the NLRP3 protein, a key component of the NLRP3 inflammasome that is expressed in immune cells, including monocytes and macrophages. Gain-of-function mutations of NLRP3 cause abnormal activation of the NLRP3 inflammasome leading to IL-1β secretion in a spectrum of autosomal dominant systemic autoinflammatory phenotypes termed cryopyrin-associated periodic syndromes. The affected subjects of our two families demonstrated atypical phenotypes compared with those reported for subjects with cryopyrin-associated periodic syndromes. These observations led us to test the hypothesis that macrophage/monocyte-like cells in the cochlea can mediate local autoinflammation via activation of the NLRP3 inflammasome. The inflammasome can indeed be activated in macrophage/monocyte-like cells of the mouse cochlea, with secretion of IL-1β. The macrophage/monocyte-like cells in the cochlea were also found to be associated with hearing loss in a Slc26a4-insufficient mouse model of human deafness. This review addresses our understanding of genetic hearing loss mediated by autoinflammation and macrophage/monocyte-like cells in the cochlea.Sensorineural hearing loss can result from dysfunction of the inner ear, auditory nerve, or auditory pathways in the central nervous system. Sensorineural hearing loss can be associated with age, exposure to ototoxic drugs or noise, or mutations in nuclear or mitochondrial genes. However, it is idiopathic in some patients. Although these disorders are mainly caused by dysfunction of the inner ear, little of the pathophysiology in sensorineural hearing loss is known due to inaccessibility of the living human inner ear for biopsy and pathological analysis. The inner ear has previously been thought of as an immune-privileged organ. We recently showed that a missense mutation of the  Sensorineural hearing loss can result from dysfunction of the inner ear, auditory nerve or higher auditory pathways in the central nervous system. Sensorineural hearing loss includes a wide variety of disorders, such as idiopathic sudden sensorineural hearing loss, age-related hearing loss, hearing loss associated with exposure to ototoxic drugs or noise, and almost all heritable forms of non-syndromic hearing loss. Although these disorders are usually caused by dysfunction of the inner ear, the pathophysiology in sensorineural hearing loss is usually unknown due to inaccessibility of the living human inner ear for biopsy and pathological analysis.NLRP3 mutation. We also review the role of cochlear macrophages in a mouse model of genetic hearing loss phenotypes, non-syndromic recessive deafness DFNB4 and Pendred syndrome, caused by variants of the SLC26A4 gene.In this review, we focus on the hearing loss caused by dysregulation of the NLRP3 inflammasome. The NLRP3 inflammasome is a critical component of a widely studied, canonical inflammatory signaling pathway in the innate immune system. We review syndromic and non-syndromic hearing loss phenotypes caused by an NLRP3 gene  encodes the NLRP3 protein , a key and eponymous component of the NLRP3 inflammasome . The CAPS spectrum includes three classical clinical subtypes: neonatal-onset multisystem inflammatory disease , Muckle-Wells syndrome , and familial cold autoinflammatory syndrome . These phenotypes share signs and symptoms including recurrent fever, rash, headache, conjunctivitis, and arthralgia or arthritis. All of the CAPS subtypes include serologic evidence of systemic inflammation.Gain-of-function mutations of NLRP3 mutations lead to CAPS via constitutive activation of the NLRP3 inflammasome and increased IL-1β production  in a North American Caucasian family (LMG113)  in (LMG113) . There wNLRP3 also causes sensorineural hearing loss with mixed signs and symptoms of systemic autoinflammation in affected members of an unrelated North American family, LMG446, of mixed Caucasian and Hispanic ancestry  mutation of Three affected members of family LMG446 were treated with subcutaneous anakinra. After 5 months of therapy, the pure-tone audiometric thresholds of two members were completely within normal limits . The. TheCx3cmembrane . These GNlrp3 is expressed in cochlea-resident cells expressing Cx3cr1 , we measured levels of IL-1β in supernatants of cultured wild-type mouse cochlear tissues. Higher levels of IL-1β were secreted from cultured cochleae stimulated with LPS and ATP, in comparison to cochleae cultured with LPS but not ATP (mulation . Mature in vivo (The stria vascularis is located in the lateral wall of the cochlea and is composed of three layers of cells: marginal cells, intermediate cells, and basal cells. The stria vascularis contains an extensive vascular network that is physiologically compartmentalized from surrounding strial cells by the blood-labyrinth barrier. The blood-labyrinth barrier includes a layer of endothelial cells connected by tight junctions within the walls of strial blood vessels. The stria vascularis produces endolymph and generates the endocochlear potential that is required for inner ear sensory hair cell function. The normally functioning tight junction-based blood-labyrinth barrier is believed to prevent or restrict the diffusion of immune cells, inner ear antigens, and antibodies between the circulation and the strial tissue , 27. Macin vivo .Slc26a4-null mouse, a model of Pendred syndrome (Slc26a4-insufficient mouse with fluctuation of hearing and endocochlear potential (Cd68 mRNA levels. These data support the hypothesis that macrophage activity is affecting hearing by influencing the function of the stria vascularis. Additionally, we observed that macrophages appeared to phagocytose pigment granules, which are increased in the stria vascularis of Slc26a4-insufficient cochleae (Slc26a4-insufficient cochlea remain undetermined. However, similar observations were reported for macrophages in the iris and ciliary body of the anterior chamber of the eye, presumably to ensure excess pigment granules are retained in the tissue and not released into the anterior chamber of the eye (The origin of the perivascular macrophages in the normal resting stria vascularis is controversial , 31. Howotential . The areotential . Also, tcochleae . The rol the eye .NLRP3 can cause non-syndromic sensorineural hearing loss as well as an atypical presentation of CAPS. In wild-type mouse cochlea, the NLRP3 inflammasome exists and can be activated in the macrophages. These results support the hypothesis that local cochlear activation of the NLRP3 inflammasome can induce cochlear autoinflammation and sensorineural hearing loss in the affected subjects. In an Slc26a4-insufficient mouse model of sensorineural hearing loss, macrophage activity is likely affecting hearing by influencing the function of the stria vascularis. Thus, macrophages in the cochlea can affect cochlear and auditory function in more than one disorder and, possibly, in many disorders of hearing.Macrophage-like cells are scattered throughout all cochlear tissues, including the auditory nerve, spiral ganglion, basilar membrane, stria vascularis, and spiral ligament. The p.Arg918Gln mutation in All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Significant sex differences exist in hearing physiology, while few human studies have investigated sex differences in noise-induced hearing loss (NIHL), and the sex bias in previous studies resulted in inadequate female data. The study aims to investigate sex differences in the characteristics of NIHL to provide insight into sex-specific risk factors, prevention strategies and treatment for NIHL.This cross-sectional study included 2280 industrial noise-exposed shipyard workers  in China. Individual noise exposure levels were measured to calculate the cumulative noise exposure (CNE), and an audiometric test was performed by an experienced technician in a soundproof booth. Sex differences in and influencing factors of low-frequency (LFHL) and high-frequency hearing loss (HFHL) were analyzed using logistic regression models stratified by age and CNE.At comparable noise exposure levels and ages, the prevalence of HFHL was significantly higher in males (34.4%) than in females (13.8%), and males had a higher prevalence of HFHL  after adjusting for age, CNE, and other covariates. Sex differences were constant and highly remarkable among subjects aged 30 to 40 years and those with a CNE of 80 to 95 dB(A). Alcohol consumption might be a risk factor for HFHL in females .This study indicates significant sex differences in NIHL. Males are at higher risk of HFHL than females despite equivalent noise exposure and age. The risk factors for NIHL might be different in males and females. Noise-induced hearing loss (NIHL) is one of the most common occupational diseases worldwide ; it resuThe existence of sex differences in NIHL in humans has been disputed. This is an important issue since exposure to various occupational noises has become increasingly prevalent in females recently . SeveralSeveral large-scale epidemiological surveys that included a number of females noted that occupational NIHL seems to be more prevalent in males than in females –15; neveIn the current study, hearing loss was assessed in 2280 shipyard industrial noise-exposed Chinese workers aged 18–60 years; the participants included 1140 males and 1140 females matched by age, job type, and employment length. Data on body mass index (BMI), noise exposure level, hearing protective device (HPD) usage, community noise, personal earphone usage, smoking, and alcohol consumption were collected to identify true sex differences in NIHL and other influencing factors.This cross-sectional study included 1140 noise-exposed males and 1140 noise-exposed females enrolled at a shipyard in eastern China from August to October 2018. The study was performed in accordance with the principles stated in the Declaration of Helsinki and approved by the Ethics Committee of the Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine. All the participants signed informed consent forms.Recruitment notices were sent to workers in our cooperative shipyard with the help of administrative staffs. After collection of demographic characteristics, information of occupation and hearing related symptoms of subjects by the face-to-face investigation, male and female subjects were matched in 1:1 ratio, according to the following criteria: (1) subjects had an age difference of less than 2 years; (2) subjects had the same job type with comparable task assignments in the same department; and (3) subjects had the same cumulative time of noise-exposure. Subjects then underwent the electro-otoscopy, tympanometry, and air-conduction pure tone audiometry (see the detailed methodology below).LAeq,8h); (5) no exposure to gun shots or bombs.The inclusion criteria were as follows: (1) Chinese Han ethnic population  aged 18–60 years; (2) no history of otological traumas, surgeries or diseases, ototoxic drug use, or a family history of hearing loss; (3) no history of chronic diseases  requiring treatment; (4) a history of work in a single type of job with exposure to industrial noise, with a work-day equivalent A-weighted sound pressure level of at least 80 dB(A) for a continuous 8 h  were initially recruited, and 1140 pairs  from which were included finally.LAeq,8h was assessed using a wearable personal exposure dosimeter  in accordance with the standards of IEC 61672:2002 and IEC 61252:2002. Subjects were asked to wear the dosimeter on their right shoulder from 8:00 am to 4:00 pm on regular work days. Cumulative noise exposure (CNE) was used to estimate the effects of noise exposure for each subject. Since all the subjects included in this study had stable job types, the CNE was calculated using the following formula [Work-day individual  formula :CNE =LAeq,8h+ 10logTT is the occupational noise exposure time-length in years.where Audiometry was performed by an experienced audiological technician using an audiometer with TDH-39P headphones  in a soundproof booth with background noise below 25 dB(A). Pure tone air-conduction audiometric thresholds for both ears at frequencies of 0.5, 1, 2, 3, 4, 6 and 8 kHz were measured in 5-dB steps in accordance with the regulations of ISO 8253-1:2010. Subjects were asked to avoid exposure to occupational noise or loud sounds within 16 h before the hearing examination.In addition to data on demographic characteristics such as biological sex (collected from the identity card), age (years), BMI  and occupational noise exposure time (years), data on individual health-related behavioral covariates were collected during face-to-face interviews; health-related data included (1) community noise exposure ; (2) individual habits that might affect hearing status, such as HPD usage (at least 4 h per weekday was marked as “yes”), personal earphone usage (at least 2 h per day was marked as “yes”), smoking (at least 10 cigarettes per day was marked as “yes”) and alcohol consumption ; and (3) self-reported auditory symptoms, such as hearing difficulty  and tinnitus.In the current study, we calculated the average low-frequency  hearing threshold and high-frequency  hearing threshold of the better ear, and hearing loss was defined as an average hearing threshold > 25 dB HL.t test or the Mann-Whitney U test, and categorical variables were compared by the chi-square test. The association between hearing thresholds and age or CNE was examined with linear regression analysis, and a binary logistic regression model was constructed to analyze the possible influencing factors of hearing loss. Sex, age, and CNE groups were stratified in the analyses. A P value < 0.05 was considered statistically significant. Data analyses were performed using SPSS software version 25.0 .Continuous variables are expressed as means ± SDs, and categorical variables are presented as percentages (%). Normality tests of continuous variables were conducted using kurtosis and skewness coefficients. Between males and females, statistical significance for differences in continuous variables was examined using Student’s The characteristics of the 1140 male subjects and 1140 female subjects are presented in Table To preliminarily explore detailed sex-specific differences and characteristics of NIHL, the average audiogram results of each ear in males and females were analyzed. Since noise exposure and age are well-known influencing factors of hearing status, subjects were stratified according to CNE and age. As shown in Fig. P < 0.001) and 11.2% dB HL in females , as well as with a per year age increase of 19.2% dB HL in males (95% CI 0.15 to 0.23) and 13.6% dB HL in females (95% CI 0.09 to 0.18). However, the sex differences were nonsignificant in associations of low-frequency hearing thresholds with CNE  or age , according to the linear slopes of the trends.Figure P < 0.001) and age . Mean high-frequency hearing thresholds were associated with a per unit increase in CNE of 54.1% dB HL in males  but only 31.0% dB HL in females , as well as a per year age increase of 76.4% dB HL in males (95% CI 0.69 to 0.84) and 44.4% dB HL in females (95% CI 0.39 to 0.50).In contrast, males and females had remarkably significantly different linear associations of high-frequency hearing thresholds with CNE  and >=95 dB(A) and age < 30, 30–40, and > 40 years old. Adjusted ORs with 95% CIs for sex differences  in hearing loss among each subgroup of subjects are shown in Fig. In general, the present study revealed that under comparable loud occupational  noise exposure conditions, HFHL was significantly more prevalent in males than females after adjustments for age, noise exposure level, and other potential behavioral confounding factors. Notably, we found that alcohol consumption was a covariate associated with HFHL in only females; this association was nonsignificant in males. Overall, our study results suggested that males and females have different responses to noise exposure damage, in accordance with some findings in previous animal studies , 17, 19.n = 1140) with precise individual noise exposure level measurements and audiograms. To date, the epidemiological characteristics and audiological outcome of occupational noise-induced hearing impairment in females are still unclear in comparison with males. Most studies had to marginalize the sex differences mainly due to the lack of enough female subjects for analyses. For instance, a Norwegian study assessed the risk of NIHL by audiometric notches among 4627 train and track maintenance male workers [In the current study, we recruited a relatively large number of females , occupational environmental exposures other than noise , vibration, heat) and shiftwork might influence the risk of NIHL . In thisWith consideration of those confounding factors, our results showed that in only female workers, consuming at least 50 g of alcohol per day might be a risk factor for HFHL. There are disparate findings on whether alcohol consumption increases the risk of hearing loss . A largeWith the increasing understanding of sex differences in the function and regulation of the auditory system , 35, sevThe primary strength of our study is the inclusion of a relatively large number of occupationally noise-exposed females who were well-matched with males. To our knowledge, this is the first study to focus on sex differences in NIHL in humans by assessing individual noise exposure, audiogram results and detailed information regarding probable confounding factors. One limitation of the study might be the possibility of genetic differences existing among subjects, although we have taken the family history of hearing loss into consideration. Another limitation is that the potential impact of subjects’ cumulative lifetime noise exposure on study results. We only matched for the occupational noise exposure, while the leisure-time noise exposure might be quite different between males and females since their different roles in human society. In addition, we used only the current audiogram results in the association analyses because of the lack of baseline data; however, all the subjects had to demonstrate normal hearing to pass the entry audiometry assessment before being offered the job. A longitudinal study of sex differences in NIHL still needs to be conducted.In summary, previous studies have proved the significant sex differences in auditory system, while few studies investigated sex differences in NIHL, and the sex bias in human studies resulted in inadequate data from females. Here we conducted a study included relatively large-scale occupational noise-exposed females with well-matched males to provide the population-based evidence of sex differences in NIHL. Through assessment of the exact individual noise exposure, audiogram and detailed information on many probable confounding factors, we demonstrated that even under comparable noise exposure conditions, females experienced less HFHL than males. In addition, the risk factors for NIHL might be different in males and females. Our findings indicate that sex differences should be considered, and data should be stratified by sex when conducting risk association studies and therapeutic studies of NIHL in the future.


Hearing loss is estimated annually as part of the Global Burden of Disease study. In 2019, input data came from 308 surveys in 77 countries. Hearing loss was measured by taking the pure-tone average of audiometric thresholds ranging from .5– 4 kHz. We ran severity-specific prevalence models, adjusting the severity for hearing aid use down one level, and separately estimating hearing loss due to meningitis, otitis media, congenital abnormalities, and age-related and other factors. In 2019, 1.5 billion (95% UI=1.5–1.6) people experienced hearing loss, primarily due to age and other factors. Of those with hearing loss, 74% (UI=71–77%) had mild hearing loss and 7% (UI=6–9%) used hearing aids. Globally, age-related hearing loss was the third-ranked cause of Years Lived with Disability after low back pain and migraine. Due to ageing, the hearing loss burden has increased over time, indicating a greater need for hearing healthcare services world-wide.


Primary human vessels (HUVECs and fibroblasts) were conditioned by continuous fluid flow and/or small molecule inhibitors to probe cathepsin expression and activity. Luminal flow (in contrast to static culture) decreases the activity of cathepsins in microvessel systems, despite a total protein increase, due to a concurrent increase in the endogenous inhibitor cystatin C. Observations also demonstrate that cathepsins mostly co-localize with fibroblasts, and that fibrin (the hydrogel substrate) may stabilize cathepsin activity in the system. Inhibitor studies suggest that control over cathepsin-mediated ECM remodeling could contribute to improved maintenance of in vitro microvascular networks; however, further investigation is required. Understanding the role of cathepsin activity in in vitro microvessels and other engineered tissues will be important for future regenerative medicine applications.Throughout the process of vascular growth and remodeling, the extracellular matrix (ECM) concurrently undergoes significant changes due to proteolytic activity—regulated by both endothelial and surrounding stromal cells. The role of matrix metalloproteinases has been well-studied in the context of vascular remodeling, but other proteases, such as cysteine cathepsins, could also facilitate ECM remodeling. To investigate cathepsin-mediated proteolysis in vascular ECM remodeling, and to understand the role of shear flow in this process,  In vitro vascular microenvironments are significantly influenced by their cellular constituents, biochemical gradients, and hydrogel substrates within which they are cultured, as well as the mechanical cues imparted on the systems.Vasculogenesis is the formation of in vitro and in vivo models have demonstrated the importance of MMPs in migration and invasion of endothelial cells.In particular, dynamic changes in the ECM heavily influence both vascular formation and remodeling. For instance, capillary morphogenesis requires proteolytic remodeling of the ECM to allow for endothelial migration and invasion into perivascular tissue, as well as for tubulogenesis (lumen formation) to occur.Cathepsins are amongst the most potent mammalian collagenases and elastases.in vitro microvascular networks and their pericellular niche. To do so, we employ a previously designed microfluidic model to examine cysteine cathepsins S, K, and L, in a co-cultured vascular system of human umbilical vein endothelial cells (HUVECs) and lung fibroblasts. Cathepsins are interrogated in response to shear flow and small molecule inhibition both on-chip and in 2D cultures, by immunostaining, western blotting, and zymography. Our work demonstrates that shear flow decreases overall cathepsin activity through an upregulation of an endogenous inhibitor, cystatin C. Moreover, fibroblasts contribute significantly to the expression of cathepsins, more so than endothelial cells. Exogenous addition of a cathepsin inhibitor is observed to reduce activity in microvessels, suggesting that cathepsin interventions could potentially be used to prolong in vitro microvessel stability. Our results demonstrate the need to further regulate cathepsins, as they contribute to ECM remodeling in microvascular networks.Here, we explore whether cathepsin expression and activity could contribute to remodeling in vitro 3D human vessels on-chip, we are able to control the environment to examine cathepsin activity in response to applied fluid shear stresses. Considering the significant influence that fluid flow  has on vasculature,2 . Pan cathepsin inhibition with E-64 significantly reduced the top active band , active catK , catK/L , and catL  compared to microvascular networks with no inhibitor added  for 30 min. Well-plates were fixed at day 2. Following several washes with DPBS, samples were subsequently permeabilized with Triton-x 100 (0.1% v/v in DPBS) for 10 min and incubation in blocking buffer  for several hours. Primary anti-human antibodies were used at 2 μl gel volume). These microvascular networks were cultured until day 7, prior to the addition of 10 μM E-64  or 1 μM cystatin C (Molecular Innovations),Microvascular networks were grown in a scaled-down version  and run on 12.5% SDS-PAGE gels. Proteins were transferred onto a nitrocellulose membrane, and then immunoblotted using the following antibodies: rabbit polyclonal anti-cathepsin K (Proteintech), goat polyclonal anti-cathepsin L (R&D Systems), goat polyclonal anti-cathepsin S (R&D Systems), rabbit polyclonal anti-cystatin C (EMD Millipore), or goat polyclonal anti-GAPDH (R&D Systems). Immunoblots were imaged using Odyssey CLx, LI-COR. Densitometry was performed with ImageJ (NIH).μM of cathepsin K inhibitor II (1-(N-benzyloxycarbonyl-leucyl)-5- carbohydrazide [Z-L-NHNHCONHNH-LF-Boc], Calbiochem/Millipore Sigma) or 1 μM of cathepsin L inhibitor  was added to the pH6 assay buffer prior to overnight incubation. Cathepsin K and cathepsin L inhibitor concentrations were selected based on previously published work.Multiplex cathepsin zymography was used to assess active cathepsins, as previously described.t test when comparing between endothelial cells and fibroblasts for each cathepsin (since primary and secondary antibodies differed between samples); this is indicated for those experiments. All experiments shown have at least three biological replicates and a p value < 0.05 was considered statistically significant. Error bars in densitometry graphs represent standard error of the mean (SEM).Biological replicates were run in separate gels, and in order to compare data across gels the same amount of RAW264.7 macrophage lysate or recombinant enzyme was used as a normalizing signal among different gels, as we have done in earlier analyses.Ethics approval is not required.supplementary material for additional data for inhibitor experiments and 2D HUVECs and fibroblasts experiments referenced in the article.See the S.A.D. and K.H. contributed equally to this work.


SOX10 and the homozygous c.235delC (p.L79Cfs∗3) mutation in GJB2 as separate pathogenic mutations in distinct affected family members. The SOX10 c.481C>T (p.R161C) mutation has been previously reported in a Caucasian patient with Kallmann syndrome that features congenital hypogonadotropic hypogonadism with anosmia. In contrast, family members carrying the same p.R161C mutation in this study had variable Waardenburg syndrome-associated phenotypes (hearing loss and/or hair hypopigmentation) without olfactory or reproductive anomalies. Our results highlight the importance of applying comprehensive diagnostic approaches such as NGS in molecular diagnosis of hearing loss and show that the p.R161C mutation in SOX10 may be associated with a wide range of variable clinical manifestations.Hearing loss is the most common sensory deficit in humans. Identifying the genetic cause and genotype-phenotype correlation of hearing loss is sometimes challenging due to extensive clinical and genetic heterogeneity. In this study, we applied targeted next-generation sequencing (NGS) to resolve the genetic etiology of hearing loss in a Chinese Han family with multiple affected family members. Targeted sequencing of 415 deafness-related genes identified the heterozygous c.481C>T (p.R161C) mutation in  Hearing loss is the most prevalent neurosensory impairment in humans, affecting over half a billion people worldwide , 2. In aSOX10 have been associated with various forms of syndromic hearing loss. SOX10 is a transcription factor involved in cell fate determination and cell lineage development, especially in the forming and differentiation of the neural crest .–87.SOX10SOX10 mutations can resemble nonsyndromic hearing loss in young children who do not present with pigmentary abnormality. Targeted NGS has the potential to identify such mutations which would improve the management of hearing loss by genetic counseling for the children and risk assessment of the relatives.Our study also demonstrates that targeted NGS is a powerful strategy to discover causative genes in rare, heterogeneous disorders such as hearing loss. WS caused by


Increasing the accessibility and affordability of hearing healthcare is a public health concern. Because low health literacy is a significant barrier to the use of existing effective healthcare services, it is critical to assess and understand health literacy deficits specific to hearing loss before implementing interventions. The purpose of this cross-sectional study was to identify differences in hearing loss health literacy among older and young adults, which is warranted because older adults are at-risk for lower levels of health literacy compared to their younger counterparts. Adults across the lifespan (n = 170) completed the Hearing Loss Health Literacy Assessment Tool, which includes self-rated ability to access/obtain, understand, and appraise hearing health information, as well as apply information to manage life with hearing loss. Results from an independent samples t-test indicated that older adults  self-reported significantly higher overall hearing health literacy than younger adults , t(168) = 4.22, p < 0.0001. Participants rated their ability to access/obtain information significantly lower than the other subscales. Age-differences in self-rated hearing health literacy exist. Findings from this study receive support from evidence indicating that the readability and suitability of the majority of patient education materials on hearing loss are not appropriate for the average U.S. adult. This investigation provides further evidence that the availability and accessibility of patient education materials on hearing loss is an important barrier that contributes to the limited use of hearing health care.


TC toxicity also positively correlated with the ion bonds ofN4-Glu252, but negatively correlated with the ion bonds of N4-Asp379. Hydrogen bonds and ion bonds for above key sites were closely related to the inhibition effect of TCs on CAT.As potential inhibitors target to biological enzymes, antibiotics may have certain impacts on the biochemical treatment process. With micrococcus catalase (CAT) served as the target molecule, the impact and inhibition mechanism for typical tetracyclines (TCs) were evaluated. Toxicity experiments showed that TCs had significant inhibition on CAT in the sequence of tetracycline>chlortetracycline>oxytetracycline>doxycycline. To clarify the inhibition mechanism between TCs and CAT which was explored with the assistance of fluorescence spectroscopy and MOE molecule simulation. According to fluorescence analysis, TCs quenched the fluorescence signal of CAT by the mode of static quenching. Combined with toxicity data, it could be presumed that TCs combined with the catalytic active center and thus inhibited CAT. Above presumption was further verified by the molecular simulation data. When TCs combined with the catalytic center of CAT, the compounds have increased combination areas and prominent energy change . IBM SPSS statistics showed that TC toxicity positively correlated with the hydrogen bonds such as O TCs have been widely used in animal husbandry and aquaculture as the advantages of low-cost and broad antibacterial spectrum –4. BecauIn view of TC biotoxicity, controls on their levels become of great importance. At present, the main treatment methods are the chemical method and biochemical method –14. Comp2O2 into molecular oxygen and water, removes hydrogen peroxide from the body, and protects cells from H2O2 toxicity  = 1 + Kqτ0[Q] , qK, and τ0 were the quenching constant, the concentration of the quencher, the fluorescence quenching rate constant, and the average lifetime of the fluorescent molecules in the absence of the quencher. For biomacromolecules, τ0 = 10−8 s.Fluorescence spectra were measured by a three-dimensional fluorescence spectrometer . Firstly, 2 mL of 0.2 mol/L phosphate buffer (pH 7.8), 1 mL of 400 mg/L CAT solution, and 1 ml different amounts (0 mg/L-1500 mg/L) of TC solution were sequentially added to 50 mL colorimetric tubes. In addition, 2 mL of 0.2 mol/L phosphate buffer (pH 7.8) and 1 mL of 1500 mg/L TC solution were added to another 50 mL colorimetric tube, then make the final volume to 50 ml with ultra-pure water, and mixed and kept for 30 min. The fluorescence spectra of samples were measured at an excitation wavelength of 276 nm and an emission wavelength range of 287-380 nm. Fluorescence quenching type was calculated by the dynamic quenching formula of Stern-Volmer:  Kqτ0[Q] . F0 and http://www.rcsb.org/pdb/home/home.do)  = 1 + Kqτ0[Q]  . The colBased on the toxicity experiment and fluorescence spectroscopy experiment, the interaction for TCs to CAT could be preliminary explained, but the specific molecular mechanism was not clarified. According to molecular docking simulation, the interaction models between TCs and CAT could be obtained, and the discrepant inhibition mechanism could be clarified.http://www.rcsb.org/pdb/home/home.do) [molecular hole method of MOE software, the interaction models between TCs and CAT in the iron porphyrin catalytic active center and no-catalytic active center were obtained  . Based oThe interaction between TCs and CAT usually involved in changed combination area and energy. To further clarify the combination site, relevant parameters were obtained by MOE, and the rate of combination areas to small molecule areas and energy changes for TC-CAT complexes at different combinatiolizan sites were calculated see . CombinaBased on data in the catalytic activity center, the combination area ratio for different TC-CAT complexes was determined as follows: chlortetracycline>tetracycline=oxytetracycline>doxycycline. Besides, the energy change in the catalytic active center for different TCs combined with CAT had different decreased in the sequence of chlortetracycline>oxytetracycline>tetracycline>doxycycline. Combined with toxicity data, combination area ratio and energy change were not well correlated with TC toxicity. The combination area and energy change of chlortetracycline were the largest, which could be related to its special chlorine atom structure. Furthermore, the correlation between TC toxicity and combination area ratio/energy change was evaluated by IBM SPSS statistics see . The resThe specific interaction sites between TCs and CAT can be seen through the ligand interaction diagram provided by the MOE software see . AccordiSimulation information of the hydrogen bonds for TC-CAT complexes was shown in 13→Glu252, O1←Arg195, O6→Asp249, O10→Glu252, O1←Lys256, 6-ring-Arg195, N21→His368, O11←Asn127, C41→Asp379, C51→Asp379, 6-ring-Gln357, O6→Asn369, O6←His368, O11←Asn369, O13←His368, C5→Asp379, N4→Glu125, O13→Ala366, O13→His354, 6-ring-Asn369, and 6-ring-Gly126, but negatively correlated with O10→Pro363, O10→Lys455, O12→Asn127, O13→Asp379, O13→Thr324, O13←Val367, O21←Arg384, C41→Val367, C42→Asp379, N21→Asp379, and N21→Glu125. By statistical analysis, 379 residues in CAT, including six hydrogen bonds of C41→Asp379, C51→Asp379, O13→Asp379, C5→Asp379, C42→Asp379, abnd N21 → Asp379. Besides, the correlation sequence of residues including 2-3 interaction sites was verified as follows >Asn127 >His368 >Glu125 >Asn369 >Arg195 >Glu252 . 6→Asp249, O1←Lys256, 6-ring-Gln357, O10→Pro363, O10→Lys455, O13→Thr324, O21←Arg384) were also important parameters to evaluate inhibition mechanism of TCs on CAT. However, Gly126, Arg334, Val351, Ser316, Tyr200, Glu416, So4505, Asn361, Asn323, and Leu348 residues were less correlated with TC toxicity >N21 >6-ring >O6 >O1 >O11 >O12 . 21←Arg384, C4A→Val367, C42→Asp379, O1C→Glu252, C41→Asp379, C51→Asp379, C5→Asp379, N4→Glu125), which also cannot be ignored. Combining MOE simulation information and related IBM SPSS statistics, these closely related hydrogen bond changes can be used as a key indicator to evaluate the interaction between TCs and CAT and are also very important for the regulation of TC toxicity.According to statistical analysis, hydrogen bonds related to the key sites of TCs also were ranked see . The int Figures : O10  on CAT was evaluated. The inhibition sequence was verified as follows: tetracycline>chlortetracycline>oxytetracycline>doxycycline. With the assistance of fluorescence spectroscopy and MOE molecule simulation, the interactions between TCs and CAT were further evaluated. Fluorescence spectroscopy showed that the reduced fluorescence intensity of CAT should be attributed to static quenching between TCs and CAT in the catalytic active center. Molecular simulation showed that combination sites for TC-CAT complexes were in the catalytic active center by calculating the combination area ratio and energy change in the catalytic active center/noncatalytic active center. Combined with toxicity data, the combination area ratio and energy change in the catalytic active center had basically positive correlation with TC toxicity. In addition, combined with the simulation information and related IBM SPSS statistics, TC toxicity was positively correlated with the hydrogen bonds of O


Arabidopsis thaliana K+-Uptake Permease (AtKUP5). Structural and molecular docking analysis revealed that the identified PDE domain occupies the C-terminal of this protein forming a solvent-exposed distinctive pocket that can spatially accommodate the cyclic adenosine monophosphate (cAMP) substrate and importantly, cAMP assumes a binding pose that is favorable for interactions with the key amino acids in the consensus motif. PDE activity was confirmed by the sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Notably, this activity was stimulated by the Ca2+/CaM complex, the binding of which to the PDE center was confirmed by surface plasmon resonance (SPR). Since AtKUP5 also has adenylate cyclase (AC) activity that is essential for K+ transport, we propose that this dual moonlighting AC-PDE architecture, offers modulatory roles that afford intricate intramolecular regulation of cAMP levels thereby enabling fine-tuning of cAMP signaling in K+ homeostasis.Cyclic nucleotide monophosphates (cNMPs) are increasingly recognized as essential signaling molecules governing many physiological and developmental processes in prokaryotes and eukaryotes. Degradation of cNMPs is as important as their generation because it offers the capability for transient and dynamic cellular level regulation but unlike their generating enzymes, the degrading enzymes, cyclic nucleotide phosphodiesterases (PDEs) are somewhat elusive in higher plants. Based on sequence analysis and structural properties of canonical PDE catalytic centers, we have developed a consensus sequence search motif and used it to identify candidate PDEs. One of these is an  To-date, only one PDE which is a cGMP-dependent AtCN-PDE1 (At1g17330), was reported in higher plants. This PDE, which belongs to the HD-domain/PDEase-like protein superfamily with orthologs in bacteria and archaea, responds to UV and is implicated in stomatal movement A. thaliana proteome that remain to be discovered and through a combination of complementary computational and experimental approaches, we present an amino acid consensus search motif that can, in tandem with structural and molecular docking assessments, identify seemingly hidden catalytically active PDE centers in complex proteins.Here we propose that there are more PDEs in the 22.1A. thaliana exist in complex multifunctional proteins masked by much larger primary domains and are thus undetected by homology searches. In order to detect them, we aligned PDE catalytic centers from annotated PDEs from yeast and animals and extracted a consensus search term (motif)  A. The movariants . This ty2.2Physcomitrella patens, Marchantia polymorpha and Arabidopsis thaliana, from which the PDE motif was constructed  in the cytosolic region and assumes a “helix-loop” secondary fold, while at the tertiary level, the PDE domain contains a cavity protected by a ‘latch’ much like in AtKUP5. Importantly, cAMP docks at the domain with a mean binding affinity of −6.72 ± 0.01 kcal/mol and assumes a positive binding pose frequency of 94.4%. The key amino acids in the motif, Y668, H669, F671, R672 and E705 also appear in AtKUP7 with the exception that K705 in AtKUP5 is replaced by R704 in AtKUP7 .Further, we examined two Arabidopsis proteins AtLARP1 (At5g21160) and AtKING1 (At3g48530) which were among seven PDE candidates identified by the PDE motif [YFW]Hx[YFW]Rx{20,40}[HRK][DE] . Docking+ transport, RNA-binding and kinase; however, they could accommodate cAMP substrate with good binding affinities  within clear cavities. We propose that the PDE motif identifies catalytic centers that operate as modulators at moonlighting sites in their respective proteins of varying primary functions Computational analysis suggests that the PDE motif appears in structurally diverse proteins with varying primary functions e.g., K2.3573-855 generates AMP from the substrate cAMP with a Vmax of 1.17 pmole AMP min−1 μg protein−1 (0.25 mmol AMP s−1 mol protein-1), and a Km of 5.35 µM. Cyclic GMP is not a substrate for AtKUP5573-855  method A. AtKUP55573-855 . Compari2+ and CaM isoforms derived from A. thaliana and bovine brain on the PDE activity. All CaM isoforms stimulate cAMP hydrolysis, after the formation of the active CaM/Ca2+ complex (2+ ion 2+ complex causes increases in the Vmax (1.78 pmole min−1 μg protein−1) and the affinity of the enzyme for the substrate (Km 4.24 µM)  to assess the binding affinity of CaM to PDE. 573-855 and CaM9 without, and in the presence of, Ca2+ ions. In both cases, an increase in signal was observed, but for the binding of CaM/Ca2+ complex the increasing µRIUs was higher and signal amplitude did not decline to the baseline level after sample washing (11 µRIU difference). This implies that the CaM/Ca2+ complex binds to AtKUP5573-855.We also assessed the effect of Ca complex C where f4.24 µM) D. The fo2.4+ homeostasis. It was previously speculated that phosphorylation of S81, Y92, T75 and/or S76 located within or close to the AC center of AtKUP5, activates its K+ transport activity + uptake will in turn stimulate the AC activity of AtKUP5, converting ATP into the second messenger cAMP + channels thereby further increasing cytosolic K++ into the cell 2+ activated calmodulin (CaM-Ca2+) is predicted to bind at the C-terminal of AtKUP5 which in turn activates the PDE .For the identification of 3.2Physcomitrella patens, Marchantia polymorpha and Arabidopsis thaliana, from which the PDE motif was constructed (The full-length AtKUP5 structure was modeled against the AtKUP7 template as described in structed . Docking3.3in vitro activity was determined by using LC-MS/MS to determine the rate of AMP formation. The reaction mixture contained: 3 mM Tris-HCl (pH 8.0), 0,1 mM cAMP, 0.1% (v/v) 2-mercaptoethanol, 5 µg of GST-AtKUP5573-855, 0.5 mM MgCl2 and MnCl2. To check if calmodulin regulates the PDE activity, three different CaM isoforms  were added to the reaction in the concentration of 2 µM and the GST-AtKUP5573-855 protein concentration was 0,625 µM. Samples were incubated at 37 °C for 25 min. The enzyme reaction was terminated by incubation at 100 °C for 10 min and the samples were centrifuged at 13,200 × g for 10 min.PDE LC-MS/MS experiments were performed using the Nexera UHPLC and LCMS-8045 integrated system (Shimadzu Corporation). The ionization source parameters were optimized in positive ESI mode using pure AMP dissolved in HPLC-grade water (Sigma). Samples were separated using a ReproSil Star 100 ZIK HiliC column . A gradient of solvent A (0.1% (v/v) formic acid with 25 mM ammonium formate) and solvent B (100% (v/v) acetonitryle) was applied over 6 min: B: 95% − 35% with a flow rate of 0.6 mL/min. The interface voltage was set at 4.0 kV for positive (ES + ) electrospray. Data acquisition and analysis were done with LabSolutions workstation for LCMS-8045.M.K. was supported from the project POWR.03.05.00–00-Z302/17 Universitas Copernicana Thoruniensis in Futuro-IDS “Academia Copernicana”. A.W. was supported by the National Natural Science Foundation of China (31850410470) and the Zhejiang Provincial Natural Science Foundation of China (LQ19C130001).Mateusz Kwiatkowski: Conceptualization, Methodology, Investigation, Writing - original draft, Funding acquisition. Aloysius Wong: Conceptualization, Formal analysis, Visualization, Funding acquisition, Writing - original draft, Writing - review & editing. Anna Kozakiewicz: Methodology, Investigation. Christoph Gehring: Conceptualization, Writing - original draft, Writing - review & editing, Supervision. Krzysztof Jaworski: Conceptualization, Writing - original draft, Writing - review & editing, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Individually, vision and hearing impairments have been linked to higher levels of anxiety and depression. We investigated the effect of dual sensory impairment (difficulty seeing and hearing) in a large representative sample of Spanish adults.Data from a total of 23,089 adults  from the Spanish National Health Survey 2017 were analyzed. Self-reported difficulty of seeing and hearing (exposures), and depression and chronic anxiety (outcomes) were analyzed. Multivariable logistic regression was assessed for difficulty with vision alone, hearing alone and with difficulty with both, adjusting for gender, age, marital status, living as a couple, education, smoking, alcohol consumption, BMI, physical activity, use of glasses/contact lenses, and hearing aid.Visual difficulty, hearing difficulty, and dual difficulties were all associated with significantly higher odds for depression  and for chronic anxiety . Dual sensory difficulty was associated with higher odds ratios for depression and anxiety when compared to either impairment alone.Dual sensory difficulty is associated with significantly higher odds of anxiety and depression when compared to either vision or hearing difficulty alone. Appropriate interventions are needed to address any reversible causes of vision and hearing as well as anxiety and depression in people in these specific groups. Depression and anxiety are among the most prevalent mental health disorders globally. Depression affects around 264 million people globally, and is characterized by persistent sadness, lack of interest or pleasure in previously rewarding or enjoyable activities, problems with sleep and appetite, tiredness and poor concentration ,b. AnxieP = 0.036) and also in those who were blind   . Other s= 0.016)  have fou= 0.016) , whilst = 0.016) . Hearing= 0.016) . Systema= 0.016) .It is likely that the association between sensory impairment and mental health is bidirectional, and this could be explained by several factors associated to both sensory impairment and mental health, including difficulties with socializing ,b, activPrevious studies have analyzed the association between visual impairment alone and hearing impairment alone in depression or anxiety. However, importantly, no study to date has compared the risk of dual vision and hearing visual difficulties (the two sensory impairments at the same time in the same person) with both depression and anxiety, although several systematic reviews provide risk ratios for vision and hearing losses individually. Moreover, as studies cited in the literature are from different countries, direct comparison of vision or hearing losses becomes difficult as environmental and healthcare availability may vary in different countries and this may impact on prevalence rates of depression and anxiety. It is therefore important to carry out studies in different countries. There may be local environmental and healthcare differences in various regions of Spain but there are many country-wide characteristics in Spain that makes important to analyze the Spanish population as a whole in order to allow comparisons with other countries. In addition, it is likely that a stronger association will exist with people with dual difficulty since these individuals will struggle more with relation to day-to-day activities. We aim to investigate, using cross-sectional associations, the risk of anxiety and depression in people with seeing difficulty only, hearing difficulty only and difficulties with both seeing and hearing in a large representative sample of Spanish adults. This is the first study about this topic in Spain and is particularly important as previous data suggests a high prevalence of difficulty seeing (12.1%) and difficulty hearing (12.7%) among Spanish adults , which aSpanish National Health Survey (year 2017) was conducted between October 2016 and October 2017. A detailed description of the Spanish National Health survey is provided in previous literature , b and wAn affirmative answer to the question “Do you have difficulty seeing?” were noted. This group comprised participants who reported difficulty seeing but did not habitually use any visual correction (spectacles/contact lenses) or had difficulty seeing even with their habitual glasses/contact lenses. Self-reported vision impairment is a method widely used and accepted in epidemiological papers to measure vision impairment .People who answered affirmatively to the question “Do you have difficulty hearing what is being said in a conversation with another person in a quiet place?” were considered to have difficulty hearing. This group was composed of people who had difficulty hearing with or without the using a hearing aid. Self-reported hearing loss is a valid method to measure hearing loss .Those who answered affirmatively to both hearing and vision difficulties were categorized into dual difficulty group.Participants who responded affirmatively to “Have you ever been diagnosed of depression by a physician?”; “Have you ever been diagnosed of chronic anxiety by a physician?” were considered to have depression and chronic anxiety, respectively. The question for depression has been previously validated . The que1 as: (1) Underweight, (2) Normal, (3) Overweight, and (4) Obesity. The International Physical Activity Questionnaire (IPAQ) Short Form was used to measure physical activity. The unit of physical activity used was MET-minutes/week, where MET is the Metabolic Equivalent of Task. Total physical activity MET-minutes/week were calculated through the following formula: sum of Walking + Moderate + Vigorous MET-minutes/week scores  fewk scores . IPAQ hak scores . Particin = 39; 0.17%), living as a couple , smoking , alcohol consumption , BMI , physical activity , hearing aid . Complete-case analysis was carried out. The level of statistical significance was set at p < 0.05.The statistical analysis was performed with SPSS 23.0 . The outcomes: prevalence (frequencies and percentages) of depression and chronic anxiety in Spanish adults by exposure: difficulties seeing and hearing and all other covariates is shown in The data comprised 23,089 Spanish participants aged 15–103 years (mean age 53.4 ± 18.9 years) with roughly equal gender representation (45.9% men). The prevalence of depression in the total sample was 10.7%. Analyzing separate groups, the prevalence of depression was 7.8% in the sample without any difficulties, 20.4% in those with seeing difficulty, 17.4% in those with hearing difficulty and 29% in those with both seeing and hearing difficulties. The overall prevalence of chronic anxiety in the whole sample was 9%, with 7.3% in the those without either seeing/hearing difficulties, 15.6% in those with seeing difficulty, 11.9% in the population with hearing difficulty and 19.7% in those with both seeing and hearing difficulties .In analyzing the prevalence of depression by covariates, it was observed that the following groups had the highest prevalence of depression in each variable: females (14.5%), ≥65 years (16.6%), widowed/divorced/separated (14.5%), not living as a couple (13.2%), no formal education (19.9%), those who never smoked (11.3%), those who did not drink alcohol in the last 12 months (18.3%), obesity (15.3%), <600 MET-minutes/week of physical activity (12.7%), cataracts (23.7%), glasses/contact lenses (12.7%) and hearing aid (15.2%).When analyzing the prevalence of chronic anxiety by covariates, the following groups had the highest prevalence of chronic anxiety in each variable: females (12.1%), ≥65 years (10.3%), widowed/divorced/separated (10.1%), not living as a couple (10.4%), no formal education (13.0%), current smokers (10.5%), those who did not drink alcohol in the last 12 months (14.2%), obesity (12.0%), <600 MET-minutes/week of physical activity (12.2%), cataracts (14.5%), glasses/contact lenses (10.4%) and hearing aid (9.4%) .Multivariable logistic regression showed that seeing difficulty, hearing difficulty and the combination of both were associated with significantly higher odds for depression  and also for chronic anxiety  . With re2(29) = 1,311.771, p < 0.001] and the deviance chi-square indicated good fit . For the model to predict chronic anxiety, the final model was a significant improvement in fit over the null model  and the deviance chi-square indicated good fit .The suitability of the regression models was assessed. The final model to predict depression was a significant improvement in fit over the null model [χIn this large representative sample of Spanish adults, seeing difficulty and hearing difficulty were both associated with significantly higher odds for depression and anxiety, with seeing difficulty showing a slightly higher risk for both mental health indices. However, in people with dual sensory difficulties (both vision and hearing difficulties together) the odds were significantly higher. This is the first study to show that people with dual vision and hearing visual difficulties (the two sensory impairments at the same time in the same person) have a higher risk of both depression and anxiety. It is also the first study showing this effect in a Spanish population.Our data compare well with previous studies that have analyzed the association of visual impairment alone and hearing impairment alone with depression and anxiety. A systematic review, based on 35 papers and 147,148 people reported similar odds ratios  for people with hearing difficulties . In peopThe present study compares the risk of depression and anxiety in people with vision, hearing and dual sensory difficulties in a population of Spanish adults. The data agree with previous literature by confirming the increased risk of depression and anxiety in people with difficulties in seeing and hearing. It adds to the literature through demonstrating the increased risk in those who have both seeing and hearing difficulties, being the first paper to examine and compare single sensory difficulty with dual sensory difficulties for both anxiety and depression.It is likely that the association between sensory impairment and mental health is bidirectional, and this bidirectionality could be explained by several factors associated to both sensory impairment and mental health. It is likely that that depression in people with difficulties in seeing and hearing may have an underlying etiology involving somatization. Somatization is common in depression, and this could influence the perception of vision and hearing difficulties . In peopTargeted assessment and treatment of somatic and positive affect symptoms in older adults with visual and hearing difficulties might improve the wellbeing of people with hearing and vision difficulties . AnticipIt may be appropriate to determine first whether assistive devices such as spectacles and hearing aids can address the visual and hearing difficulties. In 2017, a Lancet Commission report highlighted that hearing losses carried a greater risk of dementia than other potential risk factors such as hypertension and obesity . ResearcThe analysis of covariates in the present study also showed that the population groups with a significantly higher risk of depression and chronic anxiety were females, widowed/divorced/separated, those not living as a couple, those with no tertiary education, those who never smoked, those who did not drink alcohol in the last 12 months, those with obesity, people doing less physical activity, those with cataracts and those with glasses or contact lenses. These results are comparable to those found in previous studies  and indiThe large representative sample of Spanish adults and analysis of seeing and hearing difficulties in the same population are clear strengths of the present study. However, findings of the present study must be interpreted in light of the studies limitations. First, the data are self-reported potentially introducing self-reporting and recall bias into the findings. Second, the study is cross-sectional and thus it is difficult to know whether the reported sensory impairment increases one’s risk of anxiety and depression or whether the state of mind increases one’s perception of sensory difficulty. Finally, as the stem question for anxiety and depression was “have you ever,” it is possible that some participants suffered from anxiety and depression before the existence of their sensory impairment, which suggests bi-directionality.In conclusion, in this large representative sample of Spanish adults, vision difficulty was associated with higher odds of anxiety and depression when compared to hearing difficulty, and importantly a dual sensory difficultly showed the greatest risk. Our results highlight the need for appropriate interventions which may include multidisciplinary programs to improve the mental health status in people with sensory losses. The detection of both sensory difficulty and associated depression represents a priority for clinicians and patients alike. More efforts are needed to identify relevant factors inducing depression and depressive symptoms among people with vision and hearing difficulties and to provide appropriate prevention and treatment for mental disorders.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A piece of pottery showing a Cinta pig was found in the Graffignano castle  dated 15th–16th centuries, spurring us to investigate the diet of the inhabitants. Ancient DNA analysis was carried out on 21 pig specimens on three nuclear SNPs: (1) g.43597545C>T, on the KIT gene, informative for the identification of the Cinta senese breed; (2) rs81460129, on an intergenic region in chr. 16, which discriminates between domestic pigs and wild boars, and; (3) a SNP on the ZFY/ZFX homologous genes, to determine the sex of the individuals. Our results indicate that the Cinta senese was present in Northern Latium in Late Medieval time, although it was not the only breed, and that pigs, including Cinta, interbred with wild boars, suggesting free-range breeding for all types of pigs. Moreover, the unexpected high proportion of young females may be considered as evidence for the wealth of the family inhabiting the castle.The Cinta senese is a pig breed, highly esteemed for its meat and derived products, characterized by a black coat with a typical white “belt” and documented by scant iconography, since the 13 Plant and animal domestication represented a dramatic change in human evolution, determining the shift to food-producing subsistence strategies. Over the past few years, several multidisciplinary studies have tackled the modes and times of dispersal of animal domesticates along with humans, as well as the selective processes that grounded the profound physical and behavioral changes that differentiate wild and domestic animals . AncientSus scrofa) were domesticated for the first time around 11,000–10,000 years ago (ya) in Southwest Asia, together with sheep, goats and cattle . M. M59]. MKIT and WBO) typical of Cinta and WBO. Since the belted allele is dominant [Interestingly, one individual has alleles in both loci  are lacking in the literature, assessment of sex ratio in relation to age at butchering is very rarely reported and often only in general terms, although a selection of younger male pigs is suggested in Italy since Roman times .In this study, by using aDNA techniques, we have obtained a clear picture of the sex ratio. Surprisingly, it is quite the opposite of what was suggested by Medieval sources , strongThe use of aDNA techniques and established archaeological tools allowed us to shed light on the life scenario of a wealthy family of Medieval Ages.We discovered that female individuals, often slaughtered at a young age were part of their diet. Usually females were exploited for reproduction, but the Baglioni family ignored the costs of killing a reproductive sow at young age in favor of splurging in luxury. We might infer that the taste of the family was attracted by tender, juicy, yet tasty meat, as it is also reflected by the young age of sheep/goat and even chicken. On the other hand, genetic data seem to suggest that, in contrast to the present-day situation in which local animal genetic resources are protected, the Cinta breed was only one of those exploited during this period, and its “pureness” was not considered a priority within the managing strategies, as evidenced by the presence of cross-breeding with other domestic breeds as well as with wild boar.