Inorganic Biomaterials Shape the Transcriptome Profile to Induce Endochondral Differentiation.

Minerals play a vital role, working synergistically with enzymes and other cofactors to regulate physiological functions including tissue healing and regeneration. The bioactive characteristics of mineral-based nanomaterials can be harnessed to facilitate in situ tissue regeneration by attracting endogenous progenitor and stem cells and subsequently directing tissue-specific differentiation. Here, cellular responses of human mesenchymal stem/stromal cells to traditional bioactive mineral-based nanomaterials, such as hydroxyapatite, whitlockite, silicon-dioxide, and the emerging synthetic 2D nanosilicates are investigated. Transcriptome sequencing is utilized to probe the cellular response and determine the significantly affected signaling pathways due to exposure to these inorganic nanomaterials. Transcriptome profiles of stem cells treated with nanosilicates reveals a stabilized skeletal progenitor state suggestive of endochondral differentiation. This observation is bolstered by enhanced deposition of matrix mineralization in nanosilicate treated stem cells compared to control or other treatments. Specifically, use of 2D nanosilicates directs osteogenic differentiation of stem cells via activation of bone morphogenetic proteins and hypoxia-inducible factor 1-alpha signaling pathway. This study provides  insight into impact of nanomaterials on cellular gene expression profile and predicts downstream effects of nanomaterial induction of endochondral differentiation.

Granular Biphasic Colloidal Hydrogels for 3D Bioprinting.

Granular hydrogels composed of hydrogel microparticles are promising candidates for 3D bioprinting due to their ability to protect encapsulated cells. However, to achieve high print fidelity, hydrogel microparticles need to jam to exhibit shear-thinning characteristics, which is crucial for 3D printing. Unfortunately, this overpacking can significantly impact cell viability, thereby negating the primary advantage of using hydrogel microparticles to shield cells from shear forces. To overcome this challenge, a novel solution: a biphasic, granular colloidal bioink designed to optimize cell viability and printing fidelity is introduced. The biphasic ink consists of cell-laden polyethylene glycol (PEG) hydrogel microparticles embedded in a continuous gelatin methacryloyl (GelMA)-nanosilicate colloidal network. Here, it is demonstrated that this biphasic bioink offers outstanding rheological properties, print fidelity, and structural stability. Furthermore, its utility for engineering complex tissues with multiple cell types and heterogeneous microenvironments is demonstrated, by incorporating ß-islet cells into the PEG microparticles and endothelial cells in the GelMA-nanosilicate colloidal network. Using this approach, it is possible to induce cell patterning, enhance vascularization, and direct cellular function. The proposed biphasic bioink holds significant potential for numerous emerging biomedical applications, including tissue engineering and disease modeling.

Stiffness assisted cell-matrix remodeling trigger 3D mechanotransduction regulatory programs.

Engineered matrices provide a valuable platform to understand the impact of biophysical factors on cellular behavior such as migration, proliferation, differentiation, and tissue remodeling, through mechanotransduction. While recent studies have identified some mechanisms of 3D mechanotransduction, there is still a critical knowledge gap in comprehending the interplay between 3D confinement, ECM properties, and cellular behavior. Specifically, the role of matrix stiffness in directing cellular fate in 3D microenvironment, independent of viscoelasticity, microstructure, and ligand density remains poorly understood. To address this gap, we designed a nanoparticle crosslinker to reinforce collagen-based hydrogels without altering their chemical composition, microstructure, viscoelasticity, and density of cell-adhesion ligand and utilized it to understand cellular dynamics. This crosslinking mechanism utilizes nanoparticles as crosslink epicenter, resulting in 10-fold increase in mechanical stiffness, without other changes. Human mesenchymal stem cells (hMSCs) encapsulated in 3D responded to mechanical stiffness by displaying circular morphology on soft hydrogels (5 kPa) and elongated morphology on stiff hydrogels (30 kPa). Stiff hydrogels facilitated the production and remodeling of nascent extracellular matrix (ECM) and activated mechanotransduction cascade. These changes were driven through intracellular PI3AKT signaling, regulation of epigenetic modifiers and activation of YAP/TAZ signaling. Overall, our study introduces a unique biomaterials platform to understand cell-ECM mechanotransduction in 3D for regenerative medicine as well as disease modelling.

The Role of Preprocedure Genetic Counseling in Pregnancies Interrupted for Fetal Abnormalities.

OBJECTIVE: Congenital birth defects affect 3 to 5% of pregnancies. Genetic counseling can help patients navigate the testing process and understand results. The study objective was to identify predictors and utility of genetic counseling at the time of pregnancy termination. Additionally, we aimed to see what proportion of patients would benefit from additional testing based on the results of the genetic testing. STUDY DESIGN: This was a retrospective cohort review of all terminations performed for fetal anomalies by an academic center from July 2016 to May 2020. Indications were stratified by abnormal serum screening or types of abnormal ultrasound findings. Data were abstracted regarding uptake of genetic counseling and testing results. Abnormal results that warranted additional testing regarding recurrence risks were noted. Multivariable logistic regression was performed to identify predictors of receipt of genetic counseling and testing. RESULTS: Of 387 patients, 57% (n = 220) received preprocedure genetic counseling and 43% (n = 167) did not. Among patients who received diagnostic testing, 62% (n = 194) had genetic counseling compared with 38% (n = 121) without counseling (adjusted odds ratio 2.46, 95% confidence interval [1.41-4.29], p < 0.001). Among the entire cohort, 38% (n = 148) had suspected aneuploidy based on serum screening. Of these, 89% (n = 132/148) had definitive testing, 92% (n = 122/132) confirming the aneuploidy. Among the other 68% (n = 239) with structural anomalies, 76% (n = 183) had diagnostic testing with 29% (n = 53) yielding an abnormal result. Among those fetuses with structural anomalies, 36% (n = 19/53) of genetic diagnoses warranted additional parental testing because of risk of recurrence compared with only 2% (n = 2/122) of patients with abnormal serum screening results alone. CONCLUSION: Genetic counseling was associated with increased uptake of diagnostic testing, which yielded useful information and prompted additional testing. This is important for determining etiology and recurrence risk and should be offered to patients presenting for termination for fetal indications, as well as providing diagnostic closure for patients. KEY POINTS: · Genetic counseling increases the uptake of diagnostic testing in patients with fetal anomalies.. · Patients with ultrasound anomalies received less diagnostic testing despite actionable results 36% of the time.. · Genetic testing is invaluable for recurrence risk counseling even if patients chose to terminate..

Microorganisms in Organic Food-Issues to Be Addressed.

The review aimed to analyse the latest data on microorganisms present in organic food, both beneficial and unwanted. In conclusion, organic food's microbial quality is generally similar to that of conventionally produced food. However, some studies suggest that organic food may contain fewer pathogens, such as antibiotic-resistant strains, due to the absence of antibiotic use in organic farming practices. However, there is little discussion and data regarding the importance of some methods used in organic farming and the risk of food pathogens presence. Concerning data gaps, it is necessary to plan and perform detailed studies of the microbiological safety of organic food, including foodborne viruses and parasites and factors related to this method of cultivation and specific processing requirements. Such knowledge is essential for more effective management of the safety of this food. The use of beneficial bacteria in organic food production has not yet been widely addressed in the scientific literature. This is particularly desirable due to the properties of the separately researched probiotics and the organic food matrix. The microbiological quality of organic food and its potential impact on human health is worth further research to confirm its safety and to assess the beneficial properties resulting from the addition of probiotics.

Lung Ultrasound: A Complementary Imaging Tool for Chest X-Ray in the Evaluation of Dyspnea.

The present study was designed to evaluate the spectrum of imaging findings seen on chest ultrasonography in patients presenting with dyspnea and verify the concordance between chest X-ray and chest ultrasound. Methods Fifty-three patients presenting with dyspnea were included in this study. Patients with known/suspected cardiac disease were excluded from the study. All patients underwent chest X-ray and chest ultrasound, reported by two different investigators. The concordance was analyzed using Cohen's kappa value with a ' p -value' less than 0.05 considered statistically significant. Results Among the fifty-three patients with dyspnea, five diagnostic pathologies were evaluated. Concordance between lung ultrasound and chest X-ray for diagnosis of pneumonia, pneumothorax, acute exacerbation of COPD/severe asthma, and diffuse alveolar interstitial syndrome was found to be high with Cohen's kappa value > 0.8 ( p < 0.01). Ultrasound was able to correctly diagnose more cases of pneumothorax and pulmonary edema compared with chest X-ray with sensitivity and negative predictive value of 100%. Chest X-ray was found to be superior in correctly diagnosing COPD. The difference was, however, not statistically significant. Similarly, no statistically significant difference could be inferred between the diagnostic value of ultrasound and Chest X-ray in the diagnosis of pneumonia or pleural effusion. Conclusions A high concordance was noted between ultrasound and chest X-ray for diagnosis of all pathologies studied ( p < 0.01), the highest noted in pneumonia/pleural effusion and diffuse interstitial syndrome (κ = 0.9). Hence, ultrasound may be considered a complimentary imaging modality for Chest-X-ray in the evaluation of dyspnea.

Lung ultrasound for evaluation of dyspnea: a pictorial review.

Lung ultrasound is based on the analysis of ultrasound artifacts generated by the pleura and air within the lungs. In recent years, lung ultrasound has emerged as an important alternative for quick evaluation of the patient at the bedside. Several techniques and protocols for performing lung ultrasound have been described in the literature, with the most popular one being the Bedside Lung Ultrasound in Emergency (BLUE) protocol which can be utilized to diagnose the cause of acute dyspnea at the bedside. We attempt to provide a simplified approach to understanding the physics behind the artifacts used in lung ultrasound, the imaging techniques, and the application of the BLUE protocol to diagnose the commonly presenting causes of acute dyspnea.

Dissociation of nanosilicates induces downstream endochondral differentiation gene expression program.

Bioactive materials harness the body's innate regenerative potential by directing endogenous progenitor cells to facilitate tissue repair. Dissolution products of inorganic biomaterials provide unique biomolecular signaling for tissue-specific differentiation. Inorganic ions (minerals) are vital to biological processes and play crucial roles in regulating gene expression patterns and directing cellular fate. However, mechanisms by which ionic dissolution products affect cellular differentiation are not well characterized. We demonstrate the role of the inorganic biomaterial synthetic two-dimensional nanosilicates and its ionic dissolution products on human mesenchymal stem cell differentiation. We use whole-transcriptome sequencing (RNA-sequencing) to characterize the contribution of nanosilicates and its ionic dissolution products on endochondral differentiation. Our study highlights the modulatory role of ions in stem cell transcriptome dynamics by regulating lineage-specific gene expression patterns. This work paves the way for leveraging biochemical characteristics of inorganic biomaterials to direct cellular processes and promote in situ tissue regeneration.

Genetics of Ataxias in Indian Population: A Collative Insight from a Common Genetic Screening Tool.

Cerebellar ataxias (CAs) represent a group of autosomal dominant and recessive neurodegenerative disorders affecting cerebellum with or without spinal cord. Overall, CAs have preponderance for tandem nucleotide repeat expansions as an etiological factor (10 TREs explain nearly 30-40% of ataxia cohort globally). The experience of 10 years of common genetic ataxia subtypes for ≈5600 patients' referrals (Pan-India) received at a single center is shared herein. Frequencies (in %, n) of SCA types and FRDA in the sample cohort are observed as follows: SCA12 (8.6%, 490); SCA2 (8.5%, 482); SCA1 (4.8%, 272); SCA3 (2%, 113); SCA7 (0.5%, 28); SCA6 (0.1%, 05); SCA17 (0.1%, 05), and FRDA (2.2%, 127). A significant amount of variability in TRE lengths at each locus is observed, we noted presence of biallelic expansion, co-occurrence of SCA-subtypes, and the presence of premutable normal alleles. The frequency of mutated GAA-FRDA allele in healthy controls is 1/158 (0.63%), thus an expected FRDA prevalence of 1:100 000 persons. The data of this study are relevant not only for clinical decision making but also for guidance in direction of genetic investigations, transancestral comparison of genotypes, and lastly provide insight for policy decision for the consideration of SCAs under rare disease category.

Emerging 2D Nanomaterials for Biomedical Applications.

Two-dimensional (2D) nanomaterials are an emerging class of biomaterials with remarkable potential for biomedical applications. The planar topography of these nanomaterials confers unique physical, chemical, electronic and optical properties, making them attractive candidates for therapeutic delivery, biosensing, bioimaging, regenerative medicine, and additive manufacturing strategies. The high surface-to-volume ratio of 2D nanomaterials promotes enhanced interactions with biomolecules and cells. A range of 2D nanomaterials, including transition metal dichalcogenides (TMDs), layered double hydroxides (LDHs), layered silicates (nanoclays), 2D metal carbides and nitrides (MXenes), metal-organic framework (MOFs), covalent organic frameworks (COFs) and polymer nanosheets have been investigated for their potential in biomedical applications. Here, we will critically evaluate recent advances of 2D nanomaterial strategies in biomedical engineering and discuss emerging approaches and current limitations associated with these nanomaterials. Due to their unique physical, chemical, and biological properties, this new class of nanomaterials has the potential to become a platform technology in regenerative medicine and other biomedical applications.

Complement Factor H Loss in RPE Cells Causes Retinal Degeneration in a Human RPE-Porcine Retinal Explant Co-Culture Model.

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and lifestyle risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on the Complement Factor H (CFH) gene, leading to an amino acid substitution in the Factor H (FH) protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability toward oxidative stress. In this study, we established a novel co-culture model comprising CFH silenced RPE cells and porcine retinal explants derived from the visual streak of porcine eyes, which closely resemble the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observed changes involving the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE-derived FH for retinal homeostasis and provide a valuable model for AMD research.

Characterisation and validation of the 8-fold quadrant dissected human retinal explant culture model for pre-clinical toxicology investigation.

One of the major challenges in studying ocular toxicology is a lack of clinically-relevant retinal experimental models. In this study we describe the use of an in vitro human retinal explant strategy to generate a reproducible experimental model with utility in neuro-toxicity retinal studies. A retinal dissection strategy, referred to as the 8 fold quadrant dissection, was developed by dissecting human donor retinas into 4 fragments through the fovea in order to obtain 8 experimentally reproducible retinal explants from a single donor. This quadrant dissection gave rise to equivalent proportions of CD73+ photoreceptors and CD90+ ganglion cells in 8 fragments from a single donor and this remained stable for up to 3 days in culture. Major retinal cell types continued to be observed after 8 weeks in culture, despite breakdown of the retinal layers, suggesting the potential to use this model in long-term studies where observation of individual cell types is possible. The utility of this system was examined in a proof of principle neuro-toxicology study. We showed reproducible induction of toxicity in photoreceptors and retinal ganglion cells by glutamate, cobalt chloride and hydrogen peroxide insults, and observed the therapeutic positive effects of the administration of memantine, formononetin and trolox. The quadrant dissected human retinal explants have the potential to be used in toxicology studies in human ocular diseases.

Near-Infrared-Triggered Photodynamic, Photothermal, and on Demand Chemotherapy by Multifunctional Upconversion Nanocomposite.

In an attempt to integrate photodynamic therapy (PDT) with photothermal therapy and chemotherapy for enhanced anticancer activity, we have rationally synthesized a multifunctional upconversion nanoplatform using NaYF4:Yb/Tm/Er/Fe nanoparticles (NPs) as the core and NaYbF4:1% Tm as a shell. The as-synthesized core-shell upconversion (CSU) NPs exhibited diverse and enhanced photoluminescence emissions in a wide range (UV to NIR) consequent upon Fe3+ doping in the core and fabrication of an active shell. Subsequently, CSU was first decorated with titania NPs as photosensitizers. Next, the mesoporous silica (MS) shell loaded with doxorubicin (DOX) via a photocleavable Ru complex as the gating molecule was developed around titania-containing CSU. Finally, gold nanorods (GNRs) with localized surface plasmon resonance (LSPR) at 800 nm were incorporated around the MS layer to obtain the multifunctional nanoplatform. We demonstrated that the UV, blue, and NIR emissions from the CSU produced ROS-mediated PDT through titania activation, induced DOX release through photocleavage of the Ru complex, and generated hyperthermia by LSPR activity of GNRs, respectively, upon a single NIR excitation through FRET. The therapeutic efficacy was validated on HeLa cell lines in vitro by various microscopic and biochemical studies under a significantly milder NIR irradiation and lower dosage of the nanoplatforms, which have been further demonstrated as diagnostic nanoprobes for cell imaging.

Adeno-associated virus neutralising antibodies in type 1 diabetes mellitus.

Recombinant Adeno-associated viruses (AAVs) are an attractive vector for gene therapy delivery which may be blocked by AAV neutralising antibodies (NAbs). As Type 1 Diabetes (T1DM) is an endocrine disease of immunological origin, it is likely that NAb profiles are altered in the disease. In this study NAb to AAV2, AAV5, AAV6, and AAV8 in 72 subjects with T1DM and 45 non-diabetic patients were measured over a 4-year follow-up period. AAV2 NAb titres were significantly lower in non-diabetic subjects (P = 0.036). The T1DM group had more AAV8 NAb activity at baseline (P = 0.019), whilst after 4 years follow-up the T1DM group displayed developed increased AAV 5 (P = 0.03), 6 (P = 0.03) and 8 (P = 0.002) activity relative to the control group, however, overall AAV5 and 8 NAb levels were very low in patients <40. AAV NAb titre activity and prevalence generally appears higher in T1DM, however, low levels of AAV 5 and 8, particular in younger adult age groups at which T1DM can be targeted, could make these attractive vectors to target the disease.

Guar gum modified upconversion nanocomposites for colorectal cancer treatment through enzyme-responsive drug release and NIR-triggered photodynamic therapy.

Multimodal therapeutic approach towards colorectal cancer (CRC) holds great promise. There is, however, no convincing strategy reported to date that employs a multimodal strategy in CRC treatment. The present study reports an intense green-emitting core-shell photoluminescent upconversion (CSGU) nanocrystal engineered to synergistically perform photodynamic and enzyme-triggered delivery of the chemotherapeutic agent for an enhanced therapeutic outcome on HT-29 colon carcinoma cells in vitro. The photodynamic activity is achieved by the energy transfer between CSGU and the chemically conjugated Rose Bengal (RB) molecules that are further protected by a mesoporous silica (MS) layer. The chemical assay demonstrates a remarkable FRET mediated generation of 1O2 under NIR (980 nm) excitation. The outermost MS layer of the nanoplatform is utilized for the loading of the 5FU anticancer drug, which is further capped with a guar gum (GG) polysaccharide polymer. The release of the 5FU is specifically triggered by the degradation of the GG cap by specific enzymes secreted from colonic microflora, which otherwise showed 'zero-release behavior' in the absence of any enzymatic trigger in various simulated gastro-intestinal (GI) conditions. Furthermore, the enhanced therapeutic efficacy of the nanoplatform (CSGUR-MSGG/5FU) was evaluated through in vitro studies using HT-29 CRC cell lines by various biochemical and microscopic assays by the simultaneous triggering effect of colonic enzyme and 980 nm laser excitation. In addition, the strong visible emission from the nanoplatform has been utilized for NIR-induced cellular bioimaging.

Gene therapy and the adeno-associated virus in the treatment of genetic and acquired ophthalmic diseases in humans: Trials, future directions and safety considerations.

Voretigene neparvovec-rzyl was recently approved for the treatment of Leber congenital amaurosis, and the use of gene therapy for eye disease is attracting even greater interest. The eye has immune privileged status, is easily accessible, requires a reduced dosage of therapy due to its size and is highly compartmentalized, significantly reducing systemic spread. Adeno-associated virus (AAV), with its low pathogenicity, prolonged expression profile and ability to transduce multiple cell types, has become the leading gene therapy vector. Target diseases have moved beyond currently untreatable inherited dystrophies to common, partially treatable acquired conditions such as exudative age-related macular degeneration and glaucoma, but use of the technology in these conditions imposes added obligations for caution in vector design. This review discusses the current status of AAV gene therapy trials in genetic and acquired ocular diseases, and explores new scientific developments, which could help ensure effective and safe use of the therapy in the future.

Retinal explant culture: A platform to investigate human neuro-retina.

The retina is the tissue responsible for light detection, in which retinal neurons convert light energy into electrical signals to be transported towards the visual cortex. Damage of retinal neurons leads to neuronal cell death and retinal pathologies, compromising visual acuity and eventually leading to irreversible blindness. Models of retinal neurodegeneration include 2D systems like cell lines, disassociated cultures and co-cultures, and 3D models like organoids, organotypic retinal cultures and animal models. Of these, ex vivo human retinal cultures are arguably the most suitable models for translational research as they retain complex inter-cellular interactions of the retina and precisely mimic in-situ responses. In this review, we summarize the distinguishing features of the human retina which are important to preserve in experimental culture, the historical development of human retinal culture systems, the factors affecting ex vivo human retinal culture and the applications and challenges associated with current methods of human retinal explant culture.