ABSTRACT
SARS-CoV-2 is a positive-sense RNA virus that contains open reading frame 1ab (ORF1ab) to produce 16 nonstructural proteins (nsps). Five stem-loops (SL) are found in the 5' UTR of the RNA that are involved in myriad viral functions and are labeled SL1 through SL5. SL1 is crucial to viral replication. Upon viral infection, nsp1 binds the ribosomal 40S subunit to inhibit all host mRNA translation. Upon SL1 binding to nsp1, viral mRNA can be processed by the ribosome, allowing viral proteins to be produced. In this study, we are examining small DNA oligonucleotides that bind to SL1-mimetic DNA in order to block SL1-nsp1 interactions. We designed a DNA analog of the SL1 hairpin and two small DNA oligonucleotides that are complementary to either the helical stem or the loop region of SL1. The binding of these oligonucleotides to the SL1 hairpin should allow the formation of either an alternate duplex or a triplex structure. Isothermal titration calorimetry (ITC) and circular dichroism (CD) techniques were performed in 1 MKCl and 10 mM MgCl2 at two different pH (5.5 and 7.0) to examine structural and thermodynamics of binding. ITC of the two oligonucleotides showed modest binding. Results from DNA binding experiments, thermal denaturation, and CD show the hairpin structure is thermodynamically more favored and mostly remains intact under the conditions examined.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
The COVID-19 outbreak has fashioned to severe threat to each and every individual in social and economic aspects in the country. This required improved wisdom to know how it is different and dominant, to diagnose and determine effective vaccines to avoid the transmission of these deadly causative agents. From this review, the probable property of these deadly transmissible viruses is related to that of SARS-CoV-2 as a fright zone of viruses. It also provides some sparks about effective and accurate diagnosis and treatment strategies. The effective management and control of panic zone of virus (PZV) and SARS-CoV-2 are more important to reduce the pandemic situation.Copyright © 2023 Inderscience Enterprises Ltd.
ABSTRACT
The genomic material of SARS-CoV-2 is a positive-sense single-stranded RNA. SARS-CoV-2 produces non-structural protein 1 (NSP1), which inhibits host cell translation by binding its' N-terminal to the host's 40S ribosomal subunit. Once NSP1 is bound its C-terminal domain folds and binds to the mRNA entry channel. Stem loop 1 (SL1) in the 5'-UTR of the viral mRNA binds to NSP1 to abrogate translation inhibition leading to the expression of viral proteins. SL1 contains a 1 x 2 internal loop that is not seen in other coronaviruses and may be involved in conformational changes that influence SL1-NSP1 interactions. The 1 x 2 internal loop of SL1 contains a putative A*C non-canonical base pair. The U6 snRNA also contains a 1 x 2 internal loop known to undergo conformation changes in response to pH and magnesium ion binding. Here we examine the thermodynamic properties and magnesium binding of the 1 x 2 internal loop of SL1 in varying helical contexts. Thermal denaturation experiments were performed on various DNA and RNA constructs in the presence of 1 M KCl or 10 mM magnesium chloride at a pH of 5.5 and 7. We show that formation of the A+*C base pair and the construct stability in the presence of magnesium ions is dependent on the helical context.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Raman spectroscopy probes the vibrational modes of a molecule. In recent years, surface-enhanced Raman spectra (SERS) of oligonucleotides on gold or silver nanoparticles have yielded significantly stronger signals. Raman spectra of DNA are high throughput, quantitative, and label-free and show distinct features created by vibrational modes such as ring deformation, backbone bending, and hydrogen bond stretching. Here we are using gold nanoparticles to probe various structural changes in a short helical DNA designed to mimic SL1 of coronavirus and SL of U6 snRNA. Phosphate buffers containing 1 M KCl or 10 mM magnesium chloride were utilized at two different pH (5.5 and 7). Differences in peak intensity are being observed between canonically paired helical DNA and DNA of similar composition with modifications containing non-canonical A*C base pair. We are comparing ion binding, pH-related, and temperature-variable conditions to observe changes in DNA structures.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Background & Aim: Background: Traditionally, ‘fresh’ Hematopoietic progenitors cell (HPC) infusions have been preferred over cryopreserved HPC in Allo-HCT because cryopreservation and thawing leads to cell loss, besides DMSO-related adverse reactions in patients. Emergence of COVID-19 pandemic has severely affected fresh HPC infusions and most professional bodies recommend cryopreservation of HPC products before initiating conditioning chemotherapy. Although some western studies suggest no significant impact of graft manipulation on patient outcome, there is no available data from the developing world.Aim: We compare neutrophil and platelet engraftment in patients undergoing Allo-HCT with fresh and cryopreserved HPC products. Methods, Results & Conclusion: Material and Method: Allo-HCT data from October 2018 to October 2021 were analyzed. Cryopreservation was performed by controlled-rate freezing using 10% DMSO, plasmalyte- A and human albumin ( 1:2:1) as cryoprotectant. Cryopreserved products were stored in vapour-phase of Liquid nitrogen tank. CD34+ enumeration and viablity( by 7-AAD) was done on Flow-cytometry on fresh and post-thaw HPC samples. Neutrophil engraftment was defined as absolute neutrophil count >0.5 ×109/L for 3 days. Platelet engraftment was defined as independence from platelet transfusion for at least 7 days with a platelet count >20 × 109/L. Statistical analysis using Wilcoxon Rank Sum test. Results: Ninety-six patients underwent allo-HCT (46 received fresh and 50 received cryopreserved HPC products) (Table 1). There was no significant difference in neutrophil engraftment with fresh and cryopreserved grafts (p>0.05) in different types of transplants( Matched related/unrelated and haploidentical). 22% (11/50) of cryopreserved graft infusions were associated with Grade-1 DMSO-related adverse reactions, which were managed with symptomatic treatment. Cryopreservation increased the cost of related allogeneic transplants by USD1100. No cryopreserved HPC product was culture positive on microbiological assessment. Conclusion: In our experience, the engraftment kinetics were similar with fresh and cryopreserved HPC products as CD34+cell dose administered was almost the same. Cryopreserved grafts had a median 7% CD34+cell loss, associated with mild DMSO-related adverse reactions and cost increment. Even though, graft cryopreservation is a feasible alternative during the pandemic, it is crucial to ensure graft quality and promptly manage DMSO-related adverse reactions.(Table Presented) Table 1 Comparison of Fresh and cryopreserved HPC products in Allo-HCT
ABSTRACT
Background & Aim: Hematopoietic progenitor cells (HPCs) are infused for hematopoietic reconstitution in the setting of malignancy and inherited or acquired hematological deficiencies. Given the global COVID-19 pandemic, the recommendation was made to cryopreserve all allogeneic HPCs to protect recipients by allowing for subclinical cases of infection to present prior to infusion. As such, consideration of HPC stability programs (SP) and their rigor has risen. The goal of a SP is to prove the rigor of a transplant program’s cryopreservation and storage standard operating procedures so that sufficient HPC viability and potency are maintained for engraftment. SPs are also required by accreditation agencies such as AABB and FACT. Many HPC SP have validated product expirations out to 10 years. Here we share our SP to 20 years with ongoing validation for 30-year expiration. Methods, Results & Conclusion: Program Design: Current testing frequency of our SP is within the first year, and then at three-year intervals (3, 6, 9, 12, 15, 19, 21). Our rolling SP includes 2 additional (Figure Presented) Fig. 1.Current vs proposed HPC product testing and cryopreseveration schema. samples tested at 0, 5, & 9 years, then at 3-year intervals (12, 15, 19, 21, 24, 27, 30). SP samples are collected from donors requiring additional days to reach their goal but are in excess at the conclusion of collection (e.g., Day 1 collection 4.5e6 CD34+ cells/Kg, goal 5e6 CD34+ cells/ Kg). Samples are collected on a quarterly basis with ten 1mL cryovials being drawn (Figure 1). CD3+ and CD34+ viabilities are tested after cryopreservation with an acceptable threshold set at ≥75% for both. Conclusion: We are validating our SP up to 20 years with intention to validate to 30 years. Thus far, our SP reveals product age has no to low correlation with engraftment, suggesting maintenance of potency over time in a cryopreservative of 10% DMSO, 10% plasma, and 30% PlasmaLyte-A with a final cell concentration of ≤3×108 NC/mL (Figure 2). Successful engraftment has been seen in all recipients. Transplant programs should modify testing frequency, acceptance criteria, and product expiration to meet individual need while working towards standardization in the field. Given the frequency of DLIs and 2nd/3rd transplants at the Mayo Clinic, a 30-year SP reflects the need of our transplant program.(Figure Presented)Fig. 2 . ANC and platelet engraftment dates for ≥10-year-old HPC products
ABSTRACT
Dry air alters salt and water balance in the upper airways and increases the risks of COVID-19 among other respiratory diseases. We explored whether such upper airway variations in salt and water balance might alter respiratory droplet generation and potentially contribute to observed impacts of airway hydration on respiratory disease. In a randomized 4-arm study of 21 healthy human subjects we found that the breathing of humid air, the wearing of cotton masks, and the delivery of (sodium, calcium, and magnesium chloride) salt droplets sized to deposit in the nose, trachea, and main bronchi similarly reduce the exhalation of respiratory droplets by approximately 50% (P < 0.05) within 10 minutes following hydration. Respiratory droplet generation returns to relatively high baseline levels within 60–90 minutes on return to dry air in all cases other than on exposure to divalent (calcium and magnesium) salts, where suppression continues for 4–5 hours. We also found via a preliminary ecological regression analysis of COVID-19 cases in the United States between January 2020 and March 2021 that exposure to elevated airborne salt on (Gulf and Pacific) US coastlines appears to suppress by approximately 25%–30% (P < 0.05) COVID-19 incidence and deaths per capita relative to inland counties — accounting for ten potential confounding environmental, physiological, and behavioral variables including humidity. We conclude that the hydration of the upper airways by exposure to humidity, the wearing of masks, or the breathing of airborne salts that deposit in the upper airways diminish respiratory droplet generation and may reduce the risks of COVID-19 incidence and symptoms.
ABSTRACT
Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.