Protocol for generating three-dimensional induced early ameloblasts using serum-free media and growth factors.

Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta. For complete details on the use and execution of this protocol, please refer to Alghadeer et al.1.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

Highly contractile 3D tissue engineered skeletal muscles from human iPSCs reveal similarities with primary myoblast-derived tissues.

Skeletal muscle research is transitioning toward 3D tissue engineered in vitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease.

Role of Physiotherapy in HPV Proven Cases of Oral Submucous Fibrosis.

Background: To assess the role of physiotherapy in human papillomavirus (HPV) proven cases of oral submucous fibrosis (OSMF). Materials and Methods: Overall, 100 patients got recruited. Only histopathologic confirmed cases of OSMF were enrolled. Purified DNA of tissue blocks was quantified by spectrophotometry. Prevalence of HPV was evaluated. The participants got randomized into 2 cohorts: HPV positive cases and HPV negative cases. Physiotherapy was done and outcome was done and outcome was assessed and compared. Assessment of results was done by SPSS software followed by statistical evaluation. Results: HPV was seen in 80% of the patients. Mean mouth opening pretreatment and postphysiotherapy among patients with HPV positive status was 26.31 mm and 30.12 mm, respectively. Mean mouth opening pretreatment and postphysiotherapy among patients with HPV negative status was 25.11 mm and 29.74 mm, respectively. Nonsignificant results were obtained while comparing the outcome of physiotherapy among HPV positive and negative groups. Conclusion: Outcome of physiotherapy among OSMF patients is independent of HPV status.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Type H blood vessels in coupling angiogenesis-osteogenesis and its application in bone tissue engineering.

One specific capillary subtype, termed type H vessel, has been found with unique functional characteristics in coupling angiogenesis with osteogenesis. Researchers have fabricated a variety of tissue engineering scaffolds to enhance bone healing and regeneration through the accumulation of type H vessels. However, only a limited number of reviews discussed the tissue engineering strategies for type H vessel regulation. The object of this review is to summary the current utilizes of bone tissue engineering to regulate type H vessels through various signal pathways including Notch, PDGF-BB, Slit3, HIF-1α, and VEGF signaling. Moreover, we give an insightful overview of recent research progress about the morphological, spatial and age-dependent characteristics of type H blood vessels. Their unique role in tying angiogenesis and osteogenesis together via blood flow, cellular microenvironment, immune system and nervous system are also summarized. This review article would provide an insight into the combination of tissue engineering scaffolds with type H vessels and identify future perspectives for vasculized tissue engineering research.

Nasal sprays for treating COVID-19: a scientific note.

Clinical management of COVID-19 has been a daunting task. Due to the lack of specific treatment, vaccines have been regarded as the first line of defence. Innate responses and cell-mediated systemic immunity, including serum antibodies, have been the primary focus of practically all studies of the immune response to COVID-19. However, owing to the difficulties encountered by the conventional route, alternative routes for prophylaxis and therapy became the need of the hour. The first site invaded by SARS-CoV-2 is the upper respiratory tract. Nasal vaccines are already in different stages of development. Apart from prophylactic purposes, mucosal immunity can be exploited for therapeutic purposes too. The nasal route for drug delivery offers many advantages over the conventional route. Besides offering a needle-free delivery, they can be self-administered. They present less logistical burden as there is no need for refrigeration. The present article focuses on various aspects of nasal spray for eliminating COVID-19.

Lymphatic vessels in bone support regeneration after injury.

Blood and lymphatic vessels form a versatile transport network and provide inductive signals to regulate tissue-specific functions. Blood vessels in bone regulate osteogenesis and hematopoiesis, but current dogma suggests that bone lacks lymphatic vessels. Here, by combining high-resolution light-sheet imaging and cell-specific mouse genetics, we demonstrate presence of lymphatic vessels in mouse and human bones. We find that lymphatic vessels in bone expand during genotoxic stress. VEGF-C/VEGFR-3 signaling and genotoxic stress-induced IL6 drive lymphangiogenesis in bones. During lymphangiogenesis, secretion of CXCL12 from proliferating lymphatic endothelial cells is critical for hematopoietic and bone regeneration. Moreover, lymphangiocrine CXCL12 triggers expansion of mature Myh11+ CXCR4+ pericytes, which differentiate into bone cells and contribute to bone and hematopoietic regeneration. In aged animals, such expansion of lymphatic vessels and Myh11-positive cells in response to genotoxic stress is impaired. These data suggest lymphangiogenesis as a therapeutic avenue to stimulate hematopoietic and bone regeneration.

Single Cell RNA Sequencing Reveals Human Tooth Type Identity and Guides In Vitro hiPSC Derived Odontoblast Differentiation (iOB).

Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Application of this sci-RNA-seq analysis generates a differentiation protocol to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and its biomineralization capacity using the de novo designed FGFR1/2c isoform specific minibinder scaffolded as a C6 oligomer that acts as a pathway agonist. We find that FGFR1c is upregulated in functional odontoblasts and specifically plays a crucial role in driving odontoblast maturity. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.

Outcome of Submucosal Inferior Turbinoplasty in Perennial Allergic Rhinitis.

Pharmacotherapy forms mainstay of treatment for allergic rhinitis, and has adverse effects associated with it. Topical steroid therapy is the preferred medication and considered best for long term prophylaxis but with limited compliance. Submucosal turbinoplasty reduces the duration of treatment in comparison to topical steroid which has to be taken daily for a long time. The aim was to evaluate the outcome of submucosal inferior turbinoplasty in patients with perennial allergic rhinitis. A prospective interventional study was performed on 35 patients diagnosed with perennial allergic rhinitis, diagnosed as per ARIA criteria from July 2016 to July 2018. The severity of the disease was assessed using mini RQLQ scoring system. The patients were then subjected to bilateral submucosal inferior turbinoplasty under endoscopic guidance under local anesthesia. 50% significant improvement (p value < 0.05) seen in symptoms were need to blow nose, sneezing, nasal obstruction, nasal discharge, watery eyes, need to rub eye, regular house work, recreational activities, sore eyes, tiredness, irritability and thirst. 100% improvement (p value < 0.05) seen in symptoms were sleep, need to blow nose, sneezing, nasal discharge, watery eyes, need to rub eye, recreational activities and irritability. Nasal obstruction was not severe in 17 (48.5%) patients giving a very good symptom relief and improving quality of life. This is due to reduction in the erectile tissue and roominess in the nasal cavity. All patients with allergic rhinitis with associated hypertrophied turbinates should invariably be given option of inferior turbinoplasty along with proper counselling regarding its advantages and disadvantages.

Efficacy of Gas Combination Cryotherapy in the Management of Odontogenic Keratocyst of the Maxilla and Mandible: A Pilot Study.

Aim: The aim of this study was to evaluate outcome of management of odontogenic keratocyst (OKC) using gas combination cryotherapy (GCC). GCC is a treatment modality where cyst enucleation is followed by applying an adjuvant agent, a spray containing propane, butane and isobutene gas onto the bony bed. Materials and Methods: This was a prospective interventional study which included patients with radiographic and histopathologic evidence of OKC. All patients underwent enucleation of the cysts followed by spraying of the bony cavity with "ENDOFROST ™"(commercially available agent for GCC). Patients were periodically reviewed and the presence of wound dehiscence, infection and neurosensory deficit was noted. After 15 months, a CBCT was taken to assess the amount of bone formation, the presence of any pathologic fracture or recurrence. Comparison of the preoperative and the postoperative size of the defect was also assessed. Results: A total of 10 patients were included in the study of which only 2 presented with neurosensory deficit in the post-op period. One patient of the two patients recovered completely within 12 months, while the other recovered within 15 months. There was no evidence of any patient developing infection, wound dehiscence, recurrence or pathologic fracture. Conclusion: The results of the study show that enucleation followed by GCC is a safe, effective and low-cost therapy for the management of OKC.

Labyrinthine Fistula-Our Experience at a Tertiary Hospital.

This study has aimed to determine the anatomical site of labyrinthine fistula in patients of chronic suppurative otitis media at our centre. Labyrinthine fistulae (LF) are caused by abnormal communications between the inner ear and surrounding structures resulting in perilymph leakage and hearing loss. Labyrinthine fistula represents as erosive loss of the enchondral bone overlying the semicircular canals without loss of perilymph. The manifestations of fistula like vertigo, hearing loss vary in severity and complexity, commonly ranging from very mild to incapacitating. Cholesteatoma induced fistula most commonly involves lateral semicircular canal probably because of its close proximity to the middle ear, but can involve other semicircular canals and rarely cochlea. This is a retrospective analysis of 36 patients of chronic suppurative otitis media with history of vertigo undergoing tympanomastoid surgery in whom there was an evidence of labyrinthine fistula on HRCT scan of temporal bone. The incidence of patients with labyrinthine fistula presenting with vertigo, nystagmus, sensorineural hearing loss, history of vertigo were analysed. The anatomical location of the fistula was supported by Radiological evidence. Patients underwent either canal wall down mastoidectomy or cortical mastoidectomy. The anatomical site and length of the labyrinthine fistula were analysed. Amongst the 36 patients of chronic suppurative otitis media with labyrinthine fistula 22 (61.1%) patients had atticoantral disease, 4 (11.1%) patients had chronic otitis media with extensive granulation, 2 (5.5%) patients had Tubotympanic disease with polyps, 4 (11.1%) patients had Tuberculous otitis media, 1 (2.77%) patient had Tubotympanic disease with extensive tympanosclerosis eroding the dome of lateral semicircular canal, 1 (2.77%) patient had extensive cholesteatoma with cerebellar abscess, 1 (2.77%) patient had fistula in the promontory following trauma, 1 (2.77%) patient had extensive tympanosclerosis with erosion of promontory. It was noticed that, in 14 (38.88%) patients the fistula was at the centre, in 17 (47.22%) patients the fistula is towards the ampullary end of horizontal semicircular canal and in 5 (13.88%) patients the fistula was towards the non ampullary end of lateral semicircular canal. The maximum length of fistula noticed was 6 mm and the minimum length of the fistula noticed was 2 mm. Labyrinthine fistula are most commonly noticed in the ampullary end of the lateral semicircular canal. The average length of the fistula was found to be 4 mm. Careful elevation of the cholesteatoma matrix over the endosteal membrane and immediate placement of temporal fascia over the exposed fistula is important to avoid injury to the inner ear. Maximum number of fistula were seen in the atticoantral type of Chronic suppurative otitis media. Prior knowledge of anatomical location of the fistulous tract in HRCT temporal bone is important to address the fistula.

Comparision of Allogenic Cartilage and Autologous Cortical Bone in Ossicular Reconstruction: A Comparative Study.

Ossicular discontinuity is the most common cause of conductive hearing loss. The use of ossicular graft material in ossicular chain reconstruction significantly improves the result in hearing. This study was conducted to compare and analyze the outcome of ossicular reconstruction using allogenic septal spur cartilage and autologous cortical bone in terms of hearing results and graft uptake rates. Study design: randomized clinical trial. Study included 112 patients visiting our ENT department. Patients between 16 and 50 years of age with history of chronic ear discharge and air-bone-gap (ABG) of > 35 dB and ossicular involvement were included in the study. The patients underwent detailed ENT examination, audiological and radiological assessment of temporal bone and those patients with evidence of ossicular erosion were subjected to ossiculoplasty with allogenic septal spur cartilage (group I) and autologous cortical bone (group II) randomly. The patients were followed up to 6 months to analyze functional and anatomical results. 50 patients out of 56 patients (90%) from group I who underwent allogenic septal cartilage ossicular reconstruction showed significant improvement in hearing as assessed by pure tone audiogram after 3 months and 6 months. Remaining 10% of patients who did not show hearing improvement on PTA were reopened after 6 months. It was observed that the stapes head got necrosed in them. 40 patients (72%) out of 56 patients (50%) from group II who underwent autologous cortical bone reconstruction showed hearing improvement. Remaining 16 patients (28%) showed no hearing improvement. They were reopened and ankylosis, dislocation of ossicle and extrusion were noted. In our study, graft uptake rates, formation of retraction pockets, and hearing improvements were analyzed. Complications like ankylosis formation, dislocation of ossicle and extrusion rates were more in the group II compared to group I. Hearing results of group I are better compared to group II and the allogenic septal cartilage being readily available is a good option for ossicular reconstruction.

Extensive Cholesteatomas: Presentation, Complications and Management Strategy.

To document the clinical presentation, complications, management strategy and post-operative outcomes of extensive cholesteatomas. Cholesteatoma is a well demarcated cystic lesion derived from an abnormal growth of keratinizing squamous epithelium in the temporal bone. Cholesteatomas commonly involve the middle ear, epitympanum, mastoid antrum and air cells and can remain within these confines for a considerable period. Bony erosion is present confined to ossicular chain and scutum initially, but as the cholesteatoma expands, erosion of the otic capsule, fallopian canal and tegmen can occur. Erosion of the tegmen tymapani or tegmen mastoideum may lead to development of a brain hernia or cerebrospinal fluid leakage. Invasion of jugular bulb, sigmoid sinus, internal carotid artery are noticed in extensive cholesteatoma and are quite challenging and requires expertise. Neurosurgical intervention should be considered along with the otological management in the same sitting in all possible cases. A retrospective review of 12 patients were carried out to assess the clinical presentation, complications, surgical management and postoperative outcomes of extensive cholesteatomas presenting at our centre between January 2017 and December 2019. CT or MRI findings, extent of cholesteatoma intra-operatively along with the status of major neurovascular structures and disease clearance, and the post-operative outcomes including morbidity and mortality were noted. All patients underwent canal wall down mastoidectomy with or without ossiculoplasty. Post operatively all patients were treated with intravenous antibiotics and if required intravenous steroids. Amongst the 12 patients of extensive cholesteatoma (EC), all of them (100%) presented with foul smelling, purulent ear discharge. 9 (75%) patients presented with otalgia. 4 (33.33%) patients had temporal headache. 10 (83.33%) patients complained of hard of hearing. 7 (58.33%) patients gives history of vertigo at the time of presentation. In 8 (66.66%) patients there was tegmen plate erosion noticed in CT scan. In 3 (25%) patients, the disease was invading the sigmoid sinus and in 1 (8.33%) patient jugular bulb was involved. In 3 (25%) cases of EC, blind sac closure was performed. In two patients who developed cerebellar abscess, drainage procedure was performed. 2 (16.66%) patients developed sigmoid sinus thrombosis, 1 (8.33%) patient had petrositis.

Microsurgical anatomy of the internal carotid artery at the skull base.

BACKGROUND: The aetiology and significance of internal carotid artery variations at the skull base remain controversial after decades, with limited available literature. Approximately 10-40 per cent of the general population has parapharyngeal internal carotid artery variations. METHOD: A prospective observational study was conducted on internal carotid artery variations in 36 cadavers, in a tertiary care hospital, between March 2019 to March 2020. RESULTS: The most common internal carotid artery variation observed in the specimens was tortuosity, in 30 per cent, followed by kinking in 18 per cent and coiling in 10 per cent. Thirty per cent of specimens had variations present bilaterally. A loop pattern of the internal carotid artery was identified. Coiling of the internal carotid artery may present as a node; hence, meticulous dissection is advocated near the skull base to avoid complications. These variations hold utmost importance for otorhinolaryngologists performing pharyngeal and nasopharyngeal surgical procedures. CONCLUSION: A detailed knowledge of anatomy, along with its variations, and surgical expertise, will help reduce the incidence of surgical complications.

Detection of a target protein (GroEl2) in Mycobacterium tuberculosis using a derivative of 1,2,4-triazolethiols.

Herein, we identified a potent lead compound RRA2, within a series of 54 derivatives of 1,2,4-triazolethiols (exhibit good potency as an anti-mycobacterial agents) against intracellular Mycobacterium tuberculosis (Mtb). Compound RRA2 showed significant mycobactericidal activity against active stage Mycobacterium bovis BCG and Mtb with minimum inhibitory concentration (MIC) values of 2.3 and 2.0 µg/mL, respectively. At MIC value, RRA2 compound yielded 0.82 log reduction of colony-forming unit (cfu) against non-replicating Mtb. Furthermore, RRA2 compound was selected for further target identification due to the presence of alkyne group, showing higher selectivity index (> 66.66 ± 0.22, in non-replicating stage). Using "click" chemistry, we synthesized the biotin linker-RRA2 conjugate, purified with HPLC method and confirmed the conjugation of biotin linker-RRA2 complex by HR-MS analysis. Furthermore, we successfully pulled down and identified a specific target protein GroEl2, from Mtb whole-cell extract. Furthermore, computational molecular modeling indicated RRA2 could interact with GroEl2, which explains the structure-activity relationship observed in this study. GroEL-2 identified a potent and specific target protein for RRA 2 compound in whole cell extract of Mtb H37Ra.

Probiotic properties of Lactobacillus casei - MYSRD 108 and Lactobacillus plantarum-MYSRD 71 with potential antimicrobial activity against Salmonella paratyphi.

A total of 130 isolates were screened, twelve isolates were characterized for probiotic attributes and two isolates with best probiotic features were evaluated in the study. Isolates MYSRD108 and MYSRD71 survived gastric conditions and were susceptible to tested antibiotics. Isolates showed more vital cell surface traits such as autoaggregation of 89.2 and 88.5% and cell surface hydrophobicity of 61 and 64%. PCR amplification followed by 16sRNA sequencing results confirmed that the isolates as Lactobacillus casei (MYSRD 108) and Lactobacillus plantarum (MYSRD 71). During this study, the Cells and their Cell Free Supernatant (CFS) were examined for antimicrobial activity. Both the isolates inhibited different bacterial pathogens in which the growth of S. paratyphi was significantly reduced. Further, their CFS also showed inhibitory effects against S. paratyphi with agar well diffusion and Minimum Inhibitory Concentration using Broth micro dilution method. The antimicrobial compounds in the CFS was characterized to different constraints such as pH neutralization, heat treatment, Hydrogen peroxide test and storage stability at -20> °C and represented that the antagonistic acitivity against Salmonella is due to the presence of organic acids in the supernatants that lowered the pH. These strains were further examined for the inhibition of S. paratyphi biofilm. The results indicated that CFS reduced S. paratyphi biofilm by more than 75% and the number of Salmonella biofilm was effectively reduced using 15% concentration of CFS. These strains may be used to produce antimicrobial compounds which can be a substitute for chemical preservatives in food industry.

Heterogeneity and Dynamics of Vasculature in the Endocrine System During Aging and Disease.

The endocrine system consists of several highly vascularized glands that produce and secrete hormones to maintain body homeostasis and regulate a range of bodily functions and processes, including growth, metabolism and development. The dense and highly vascularized capillary network functions as the main transport system for hormones and regulatory factors to enable efficient endocrine function. The specialized capillary types provide the microenvironments to support stem and progenitor cells, by regulating their survival, maintenance and differentiation. Moreover, the vasculature interacts with endocrine cells supporting their endocrine function. However, the structure and niche function of vasculature in endocrine tissues remain poorly understood. Aging and endocrine disorders are associated with vascular perturbations. Understanding the cellular and molecular cues driving the disease, and age-related vascular perturbations hold potential to manage or even treat endocrine disorders and comorbidities associated with aging. This review aims to describe the structure and niche functions of the vasculature in various endocrine glands and define the vascular changes in aging and endocrine disorders.