ABSTRACT
The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance and for determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for detection of SARS-CoV-2, with an additional advantage of enabling genetic epidemiology of SARS-CoV-2.
ABSTRACT
In the present study, we propose a platform for topical wound dressing material using a polydimethylsiloxane (PDMS) scaffold in order to enhance the skin healing process. In vitro co-culture assessment of epidermal-origin mouse B16-F10 melanocyte cells and mouse L929 fibroblast cells in three-dimensional polymeric scaffolds has been carried out towards developing bio-stable, interconnected, highly macroporous, PDMS based tissue-engineered scaffolds, using the salt leaching method. To determine a suitable ratio of salt to PDMS pre-polymer in the scaffold, two different samples with ratios 2:1 and 3:1 [w/w], were fabricated. Effective pore sizes of both scaffolds were observed to lie in the desirable range of 152-165 µm. In addition, scaffolds were pre-coated with collagen and investigated as a podium for culturing the chosen cells (fibroblast and melanocyte cells). Experimental results demonstrate not only a high proliferative potential of the skin tissue-specific cells within the fabricated PDMS based scaffolds but also confirm the presence of several other essential attributes such as high interconnectivity, optimum porosity, excellent mechanical strength, gaseous permeability, promising cell compatibility, water absorption capability and desired surface wettability. Therefore, scaffolds facilitate a high degree of cellular adhesion while providing a microenvironment necessary for optimal cellular infiltration and viability. Thus, the outcomes suggest that PDMS based macroporous scaffold can be used as a potential candidate for skin dressing material. In addition, the fabricated PDMS scaffolds may also be exploited for a plethora of other applications in tissue engineering and drug delivery.
ABSTRACT
Biodegradable poly(lactic acid) (PLA) is widely used to fabricate 3D scaffolds for tissue regeneration. However, PLA lacks cell adhering functional moieties, which limit its successful application in tissue engineering. Herein, we have tailored the cell adhesive properties of star shaped poly(d,l-lactide) (ss-PDLLA) by grafting gelatin to their 4 arms. Grafting of gelatin on PDLLA backbone was confirmed by 1H NMR and FTIR. The synthesized star shaped poly(d,l-lactide)-b-gelatin (ss-pLG) exhibited enhanced wettability and protein adsorption. The modification also facilitated better cell adhesion and proliferation on their respective polymer coated 2D substrates, compared to their respective unmodified ss-PDLLA. Further, 3D scaffolds were fabricated from gelatin grafted and unmodified polymers. The fabricated scaffolds were shown to be cytocompatible to 3T3-L1 cells and hemocompatible to red blood cells (RBCs). Cell proliferation was increased up to 2.5-fold in ss-pLG scaffolds compared to ss-PDLLA scaffolds. Furthermore, a significant increase in cell number reveals a high degree of infiltration of cells into the scaffolds, forming a viable and healthy 3D interconnected cell community. In addition to that, burst release of docetaxal (DTX) was observed from ss-pLG scaffolds. Hence, this new system of grafting polymers followed by fabricating 3D scaffolds could be utilized as a successful approach in a variety of applications where cell-containing depots are used.
