Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Enzymatic isolation and microfluidic electrophoresis analysis of residual dsRNA impurities in mRNA vaccines and therapeutics.

The versatility, rapid development, and ease of production scalability of mRNA therapeutics have placed them at the forefront of biopharmaceutical research. However, despite their vast potential to treat diseases, their novelty comes with unsolved analytical challenges. A key challenge in ensuring sample purity has been monitoring residual, immunostimulatory dsRNA impurities generated during the in vitro transcription of mRNA. Here, we present a method that combines an enzyme, S1 nuclease, to identify and isolate dsRNA from an mRNA sample with a microfluidic electrophoresis analytical platform to characterize the impurity. After the method was developed and optimized, it was tested with clinically relevant, pseudouridine-modified 700 and 1800 bp dsRNA and 818-4451 nt mRNA samples. While the treatment impacted the magnitude of the fluorescent signal used to analyze the samples due to the interference of the buffer with the labeling of the sample, this signal loss was mitigated by 8.8× via treatment optimization. In addition, despite the mRNA concentration being up to 400× greater than that of the dsRNA, under every condition, there was a complete disappearance of the main mRNA peak. While the mRNA peak was digested, the dsRNA fragments remained physically unaffected by the treatment, with no change to their migration time. Using these samples, we detected 0.25% dsRNA impurities in mRNA samples using 15 µL with an analytical runtime of 1 min per sample after digestion and were able to predict their size within 8% of the expected length. The short runtime, sample consumption, and high throughput compatibility make it suitable to support the purity assessment of mRNA during purification and downstream.

Cyclophilin A promotes cell migration via the Abl-Crk signaling pathway.

Cyclophilin A (CypA) is overexpressed in a number of human cancer types, but the mechanisms by which the protein promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds the CrkII adaptor protein and prevents it from switching to the inhibited state. CrkII influences cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binding site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly upregulated.

Nanotechnology-Based Approaches for Guiding Neural Regeneration.

The mammalian brain is a phenomenal piece of "organic machinery" that has fascinated scientists and clinicians for centuries. The intricate network of tens of billions of neurons dispersed in a mixture of chemical and biochemical constituents gives rise to thoughts, feelings, memories, and life as we know it. In turn, subtle imbalances or damage to this system can cause severe complications in physical, motor, psychological, and cognitive function. Moreover, the inevitable loss of nerve tissue caused by degenerative diseases and traumatic injuries is particularly devastating because of the limited regenerative capabilities of the central nervous system (i.e., the brain and spinal cord). Among current approaches, stem-cell-based regenerative medicine has shown the greatest promise toward repairing and regenerating destroyed neural tissue. However, establishing controlled and reliable methodologies to guide stem cell differentiation into specialized neural cells of interest (e.g., neurons and oligodendrocytes) has been a prevailing challenge in the field. In this Account, we summarize the nanotechnology-based approaches our group has recently developed to guide stem-cell-based neural regeneration. We focus on three overarching strategies that were adopted to selectively control this process. First, soluble microenvironmental factors play a critical role in directing the fate of stem cells. Multiple factors have been developed in the form of small-molecule drugs, biochemical analogues, and DNA/RNA-based vectors to direct neural differentiation. However, the delivery of these factors with high transfection efficiency and minimal cytotoxicity has been challenging, especially to sensitive cell lines such as stem cells. In our first approach, we designed nanoparticle-based systems for the efficient delivery of such soluble factors to control neural differentiation. Our nanoparticles, comprising either organic or inorganic elements, were biocompatible and offered multifunctional capabilities such as imaging and delivery. Moving from the soluble microenvironment in which cells are immersed to the underlying surface, cells can sense and consequently respond to the physical microenvironment in which they reside. For instance, changes in cell adhesion, shape, and spreading are key cellular responses to surface properties of the underlying substrate. In our second approach, we modulated the surface chemistry of two-dimensional substrates to control neural stem cell morphology and the resulting differentiation process. Patterned surfaces consisting of immobilized extracellular matrix (ECM) proteins and/or nanomaterials were generated and utilized to guide neuronal differentiation and polarization. In our third approach, building on the above-mentioned approaches, we further tuned the cell-ECM interactions by introducing nanotopographical features in the form of nanoparticle films or nanofiber scaffolds. Besides providing a three-dimensional surface topography, our unique nanoscaffolds were observed to enhance gene delivery, facilitate axonal alignment, and selectively control differentiation into neural cell lines of interest. Overall, nanotechnology-based approaches offer the precise physicochemical control required to generate tools suitable for applications in neuroscience.

Master-slave locking of optomechanical oscillators over a long distance.

Frequency locking and other phenomena emerging from nonlinear interactions between mechanical oscillators are of scientific and technological importance. However, existing schemes to observe such behavior are not scalable over distance. We demonstrate a scheme to couple two independent mechanical oscillators, separated in frequency by 80 kHz and situated far from each other (3.2 km), via light. Using light as the coupling medium enables this scheme to have low loss and be extended over long distances. This scheme is reversible and can be generalized for arbitrary network configurations.

Synchronization and Phase Noise Reduction in Micromechanical Oscillator Arrays Coupled through Light.

Synchronization of many coupled oscillators is widely found in nature and has the potential to revolutionize timing technologies. Here, we demonstrate synchronization in arrays of silicon nitride micromechanical oscillators coupled in an all-to-all configuration purely through an optical radiation field. We show that the phase noise of the synchronized oscillators can be improved by almost 10 dB below the phase noise limit for each individual oscillator. These results open a practical route towards synchronized oscillator networks.

Smokeless Tobacco-associated Lesions: A Mobile Health Approach.

AIM: Globally, India accounts for the highest number of oral cancer cases. The survival rates are about 30% lower than those in developing countries. The main reason for these dismal figures is the late presentation of patients. In order to downstage oral cancer in such a scenario, screening and diagnosis at an early stage is warranted. A pragmatic approach is needed for an oral cancer screening program, hence a mobile health (mHealth) approach was used. In this approach, health workers were empowered with mobile phones with decision-based algorithm. Risk stratification of tobacco habit enables us to identify lesions associated with particular habits. MATERIALS AND METHODS: A specific cohort of factory employees who predominantly had pure tobacco chewing habit was chosen to examine the effect of pure tobacco on oral mucosa. One thousand three hundred and fifty-seven subjects were screened in two phases. In the first phase, habits and oral lesions were identified and photographed. The photographs were remotely diagnosed by an oral medicine specialist and those subjects requiring biopsy were recalled for phase II. Cytology and biopsy were performed in phase II. RESULTS: The predominant habit was smokeless tobacco (SLT), in 582 subjects. The most commonly encountered lesion was tobacco pouch keratosis seen in 397 subjects. Biopsy was performed for 71 subjects, most cases showed hyperkeratosis and mild dysplasia. One subject had moderate dysplasia. CONCLUSION: There was minimal alteration of tissues in our study subjects, which can be considered as low-risk. Use of mHealth empowered frontline healthcare workers to identify subjects with lesions and enabled remote diagnosis by specialist in resource-constrained settings. CLINICAL SIGNIFICANCE: Use of mHealth enabled us have an electronic record of subject details. This data shall be used for a planned follow-up of the same cohort after 3 years.

Unveiling an Uncommon Scenario of Co-occurrence of Multiple Odontomes With Impacted Maxillary Lateral Incisor and Canine in a 17-Year-Old Girl: A Unique and Rare Case Report.

This case report presents the enigma of multiple odontomes with overretained deciduous teeth leading to the impaction of permanent successors (22, 23) in an abnormal position in a 17-year-old female patient who reported the chief complaint of maligned teeth. Permanent maxillary canines and lateral incisors are the most common teeth to face the brunt of impaction due to a wide range of etiological factors. It is imperative for a clinician to diagnose cases at an early stage to accelerate the rate of eruption of such teeth. This is especially important in cases where initially the etiology seems to be simple but on careful and judicious evaluation of the case, numerous other etiologies are found to map together for the underlying pathology. This case discusses how the presence of multiple odontomes with delayed exfoliation of deciduous teeth leads to the impaction of a permanent successor. Understanding the underlying pathology is seemingly important to devise intricate treatment mechanics for traction of impacted teeth without taxing anchorage from dental units and taking cognizance of the amount of alveolar bone loss post-removal of multiple odontomes. The appropriate thickness of alveolar bone scaffolding is required for the canine to extrude down, with an adequate band of marginal gingiva encircling the cement-enamel junction of the impacted canine, preventing any kind of fenestration and dehiscence. Hence, meticulous care was taken during surgical exposure and removal of odontomes to preserve an adequate labial cortical plate intact for traction. These excavated tooth-like structures were later subjected to histopathological evaluation, which confirmed the diagnosis of compound odontomes.

Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

Achieving a controlled and reproducible means to direct stem cell differentiation is the single most critical concern scientists have been trying to address since the discovery of stem cells. In this regard, the use of small molecules and RNA interference offers unique advantages by targeting different cellular mechanisms. Our cyclodextrin-modified dendritic polyamine construct (termed DexAM) combines the unique properties of two distinct chemical moieties in a single delivery vehicle. DexAM is a single vehicle that not only solubilizes hydrophobic small molecules in physiological solutions but also forms complexes with siRNA molecules, making it an attractive delivery system for controlling stem cell differentiation. Herein, we report the synthesis and application of DexAM to simultaneously deliver hydrophobic small molecules and siRNA into neural stem cells to significantly enhance their neuronal differentiation.

High Q SiC microresonators.

We demonstrate photonic devices based on standard 3C SiC epitaxially grown on silicon. We achieve high optical confinement by taking advantage of the high stiffness of SiC and undercutting the underlying silicon substrate. We demonstrate a 20 µm radius suspended microring resonator with Q=14,100 fabricated on commercially available SiC-on-silicon substrates.

Co occurrance of Macular Telangiectasia type 2 with hemorrhagic polypoidal choroidal vasculopathy and response to anti-vascular endothelial growth factor.

We report the case of a 57-year-old female patient who presented with defective vision in the right eye due to large hemorrhagic pigment epithelial detachment with subretinal hemorrhage near the arcades with scattered retinal pigment epithelium (RPE) alteration at the macula and minimal vitreous hemorrhage inferiorly. The left eye showed right angle perifoveal dipping venule with RPE alteration temporal to the fovea, intraretinal RPE stellate plaque at the macula area. Multimodal imaging features including color fundus photo, red-free photograph, optical coherence tomography (OCT), enhanced depth imaging OCT, fundus fluorescein angiography, indocyanine green angiography, and OCT angiography were studied along with treatment response. One eye showed features of macular telangiectasia Type 2 (MacTel 2) with polypoidal choroidal vasculopathy (PCV) (hemorrhagic type) and the other eye showed features of MacTel 2 with pachychoroid pigment epitheliopathy. This report highlights the hitherto undescribed co-occurrence of MacTel 2 with bilateral pachychoroid and with unilateral hemorrhagic PCV in one eye and its favorable outcome with anti-vascular endothelial growth factor therapy.

Antimicrobial Efficacy of Chlorhexidine and Herbal Mouth Rinse on Salivary Streptococcus mutans in Children with Mixed Dentition: A Randomized Crossover Study.

Aim and objective: Streptococcus mutans (S. mutans) shows increased resistance to currently available antibiotics and chemotherapeutics. The present study compares the effectiveness of chlorhexidine and Herbal mouth rinse against salivary S. mutans in children with mixed dentition. Materials and methods: Subjects (n = 60) with mixed dentition were selected for the study. Caries status was recorded using Nyvard's criteria. Baseline saliva samples were collected and assessed for quantifying S. mutans. Subjects were instructed to rinse their mouths with 0.2 % w/v chlorhexidine and herbal mouth rinse for 7 days. Saliva samples were collected after 7 days and assessed for S. mutans. After a run-in period of 21 days, both the mouth rinses were crossed over according to the Latin square design, and a similar procedure was carried out. Later, determination of mean colony-forming units (CFU/mL) from the saliva samples was done. For statistical analysis, Kolmogorov and Mann-Whitney U tests were applied. Results: Both the groups showed a significant reduction in S. mutans count, at baseline and 7 days (p = 0.0001), and the reduction of S. mutans count in herbal mouth rinse as compared to chlorhexidine mouth rinse (p = 0.0209) was statistically significant. Conclusion: Herbal mouth rinse proved to have better antimicrobial efficacy than Chlorhexidine mouth rinse. How to cite this article: Shah SV, Badakar CM, Hugar SM, et al. Antimicrobial Efficacy of Chlorhexidine and Herbal Mouth Rinse on Salivary Streptococcus mutans in Children with Mixed Dentition: A Randomized Crossover Study. Int J Clin Pediatr Dent 2022;15(1):99-103.

Sequential multimodal imaging of isolated necrotic full-thickness macular hole secondary to toxoplasma retinochoroiditis.

PURPOSE: To describe the sequential multimodal imaging features of an isolated necrotic macular hole secondary to Toxoplasma retinochoroiditis. OBSERVATION: A 23-year-old male was referred for surgical management of an idiopathic macular hole following sudden decrease in vision in his right eye. Right eye examination showed best-corrected visual acuity of 20/200, mild anterior segment inflammation, and a full thickness non operculated macular hole (MH) with ill-defined ragged margins and surrounding strip of pallid edema. Further multimodal imaging including optical coherence tomography (OCT), fundus autofluorescence (FAF), fluorescein angiography (FFA), and OCT-angiography confirmed the atypical configuration and inflammatory nature of MH. Serological tests showed elevated level of Toxoplasma gondii-specific antibodies. A diagnosis of necrotic isolated full-thickness MH secondary to toxoplasma retinochoroiditis was made. Patient was treated medically with anti-toxoplasma medication for 6 months. Sequential multimodal imaging highlighted the healing process of necrotic MH with vision improving to 20/80 at 6 months after presentation. CONCLUSION AND IMPORTANCE: A high level of suspicion and multimodal imaging plays an important role in accurate etiological diagnosis and management of atypical macular hole as in our case. Sequential multimodal imaging may provide an insight into the pathogenesis and healing pattern of such lesion.

Microparticle-Based Biochemical Sensing Using Optical Coherence Tomography and Deep Learning.

Advancing continuous health monitoring beyond vital signs to biochemistry will revolutionize personalized medicine. Herein, we report a biosensing platform to achieve remote biochemical monitoring using microparticle-based biosensors and optical coherence tomography (OCT). Stimuli-responsive, polymeric microparticles were designed to serve as freely dispersible biorecognition units, wherein binding with a target biochemical induces volumetric changes of the microparticle. Analytical approaches to detect these submicron changes in 3D using OCT were devised by modeling the microparticle as an optical cavity, enabling estimations far below the resolution of the OCT system. As a proof of concept, we demonstrated the 3D spatiotemporal monitoring of glucose-responsive microparticles distributed throughout a tissue mimic in response to dynamically fluctuating levels of glucose. Deep learning was further implemented using 3D convolutional neural networks to automate the vast processing of the continuous stream of three-dimensional time series data, resulting in a robust end-to-end pipeline with immense potential for continuous in vivo biochemical monitoring.

Comparative cytopathologic evaluation using acridine orange with Papanicolaou stain in psychoactive substances abusers with potentially malignant and malignant disorders.

INTRODUCTION: Oral cancer is a major health problem and its early detection is advantageous for therapeutic purposes. According to available evidence, the risks of oral malignancies increase with the usage of tobacco and other psychoactive substances (PSs). The present study showed expression pattern of nuclear and cytoplasmic changes from normal individuals without habit to oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) in PS abusers with the help of fluorescence acridine orange (AO) stain and Papanicolaou (PAP) stain. AIM AND OBJECTIVES: This study aimed to investigate and compare diagnostic efficacy of fluorescence microscopic evaluation of AO stain in cytological smears with PAP staining under light microscopy in PS abusers having oral potentially malignant and malignant lesions. MATERIALS AND METHODS: Oral smears from 120 individuals among which 40 from potentially malignant disorders, 40 from oral malignancy and 40 normal buccal mucosa smears were prepared. One set of smears was stained by AO staining and the other by PAP staining and examined under fluorescence and light microscope, respectively. The results of both the stainings were evaluated by grading cytology smears in class-I to class-V cytology. RESULTS: The AO fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence. The efficacy of AO fluorescence stain was higher than PAP stain in screening of oral lesions suspicious of malignancy. The sensitivity of PAP staining and AO staining is 57.50% and 61.25%, respectively. CONCLUSION: As compared to PAP staining method, fluorescent AO method is more effective in screening of OPMD and OSCC in PS abusers.

Castleman's disease.

Castleman's disease, a type of lymph node hyperplasia, usually occurs in the mediastinum and rarely presents in the cervical region as an asymptomatic solitary mass. Clinically, they are of two types-solitary and multi-centric. Most of the solitary types are asymptomatic with no associated symptoms, whereas the multi-centric type is associated with systemic symptoms and has a poor prognosis. Histologically, they can be classified as-Hyaline vascular, plasma cell, transitional and stromal rich type. We report a case of Castleman's disease involving the submandibular lymph node in a 75-year-old male patient whose definitive diagnosis was made only on histological examination. Isolated Castleman's disease of the submandibular node is rare and a thorough clinical and histological examination is necessary to rule out the systemic form of the disease and other diseases with manifestations as a cervical lymph node enlargement.

Effect of tobacco in human oral leukoplakia: a cytomorphometric analysis.

OBJECTIVES: Tobacco use is one of the most critical risk factors for different oral diseases. The aim of this study is to demonstrate the effect of tobacco on oral mucosa by cytomorphometric analysis of cells with the help of exfoliative cytology and to find out the improvement in diagnostic sensitivity of exfoliative cytology in the detection of dysplastic changes and early oral malignancy. METHODS: The nuclear area (NA) and cytoplasmic area (CA) of cells were measured within cytological smear obtained from leukoplakia lesions of buccal mucosa of 90 tobacco users, 30 smokers (TS), 30 chewers (TC) and 30 with combined habit of smoking and chewing (TSC) and from normal buccal mucosa of 30 non users (NU) of tobacco. Each habit group consisted of 30 tobacco users with oral leukoplakia lesion with mild epithelial dysplasia only. The 30 non-users of tobacco served as controls. The mean values of the CA and NA were obtained for each case, and the nuclear/cytoplasmic area (NA/CA) ratio was calculated. RESULTS: The results showed a statistically significant increase (P<0.001) in mean NA and a statistically significant decrease (P<0.001) in mean CA values of tobacco users with leukoplakia as compared to non-users, hence NA/CA ratio value was significantly higher in tobacco users with the lesion. CONCLUSION: The changes in cellular morphology caused by tobacco use can be visualized by use of exfoliative cytology with the help of cytomorphometric analysis. The evaluation of parameters (NA, CA and NA/CA ratio) may increase the sensitivity of exfoliative cytology for the early diagnosis of oral premalignant and malignant lesions.

Comparative Evaluation of Retention and Antibacterial Efficacy of Compomer and Glass Hybrid Bulk Fill Restorative Material as a Conservative Adhesive Restoration in Children with Mixed Dentition-An In Vivo Two-arm Parallel-group Double-blinded Randomized Controlled Study.

AIM AND OBJECTIVE: To compare and evaluate the retention along with antibacterial efficacy of colored compomer and glass hybrid bulk fill glass ionomer restorative material as a conservative adhesive restoration in children of age 6-12 years. MATERIALS AND METHODS: Sixty children were selected fulfilling the inclusion and exclusion criteria falling in the age group of 6-12 years with mixed dentition and two groups were formed: group I-colored compomer and group II-glass hybrid bulk fill material. Initially, oral prophylaxis was carried out and baseline collection of saliva was completed. Then, the restorative treatment was completed. Retention of the material and antibacterial count [colony-forming units (CFU)/mL of saliva] was estimated at 1, 3, and 6 months after the restorative procedure. RESULTS: It was seen that retention rate with glass hybrid bulk fill group was 100%, whereas with colored compomer group it was 90% at end of 6 months. Although good antibacterial activity was shown by both the group at 1, 3, and 6 months follow-up but statistically significant drop was seen in the glass hybrid bulk fill group at 3-month intervals than the colored compomer group with a p value of 0.0001 (p < 0.05). CONCLUSION: Among both the materials, glass hybrid bulk fill restorative material showed good retention compared to Colored compomer material but it was not statistically very significant. Also, both the materials have shown good antimicrobial activity at 1, 3, and 6 months follow-up. HOW TO CITE THIS ARTICLE: Mundada MV, Hugar SM, Hallikerimath S, e t a l. Comparative Evaluation of Retention and Antibacterial Efficacy of Compomer and Glass Hybrid Bulk Fill Restorative Material as a Conservative Adhesive Restoration in Children with Mixed Dentition-An In Vivo Two-arm Parallel-group Double-blinded Randomized Controlled Study. Int J Clin Pediatr Dent 2020;13(S-1):S45-S54.

Carbon fiber doped thermosetting elastomer for flexible sensors: physical properties and microfabrication.

We have developed conductive microstructures using micropatternable and conductive hybrid nanocomposite polymer. In this method carbon fibers (CFs) were blended into polydimethylsiloxane (PDMS). Electrical conductivities of different compositions were investigated with various fiber lengths (50-250 µm), and weight percentages (wt%) (10-60 wt%). Sample composites of 2 cm × 1 cm × 500 µm were fabricated for 4-point probe conductivity measurements. The measured percolation thresholds varied with length of the fibers: 50 wt% (307.7 S/m) for 50 µm, 40 wt% (851.1 S/m) for 150 µm, and 30 wt% (769.23 S/m) for 250 µm fibers. The conductive composites showed higher elastic modulus when compared to that of PDMS.