A collaborative approach to improving representation in viral genomic surveillance.

The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building 'next generation' genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness.

A collaborative approach to improve representation in viral genomic surveillance.

The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building microbial genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness. Author summary: Genomic surveillance involves decoding a pathogen’s genetic code to track its spread and evolution. During the pandemic, genomic surveillance programs around the world provided valuable data to scientists, doctors, and public health officials. Knowing the complete SARS-CoV-2 genome has helped detect the emergence of new variants, including ones that are more transmissible or cause more severe disease, and has supported the development of diagnostics, vaccines, and therapeutics. The impact of genomic surveillance on public health depends on representative sampling that accurately reflects the diversity and distribution of populations, as well as rapid turnaround time from sampling to data sharing. After a slow start, SARS-CoV-2 genomic surveillance in the United States grew exponentially. Despite this, many rural regions and ethnic minorities remain poorly represented, leaving significant gaps in the data that informs public health responses. To address this problem, we formed a network of universities and clinics in Louisiana, Georgia, and Mississippi with the goal of increasing SARS-CoV-2 sequencing volume, representation, and equity. Our results demonstrate the advantages of rapidly sequencing pathogens in the same communities where the cases occur and present a model that leverages existing academic and clinical infrastructure for a powerful decentralized genomic surveillance system.

Azithromycin Reduces Markers of Vascular Damage in Pediatric Patients With Sickle Cell Disease.

Background: Immunomodulatory effects of macrolides in chronic inflammation are well known. In this study, we tested our hypothesis that azithromycin (AZT) can decrease inflammation in pediatric patients with sickle cell disease (SCD). Methods: The use of AZT as an anti-inflammatory agent was evaluated in double-blind, placebo-controlled, cross-over study for 8 weeks of treatment with 8 weeks of washout. Blood samples were collected before (PRE) and after (POST) each 8-week treatment period. Repeated measures analysis of variance (ANOVA) with post hoc multiple comparison procedures and Chi-square test were used for statistical analysis of the data. Complete blood count, distribution of the lymphocyte subsets, and plasma levels of markers of vascular damage were analyzed. Results: A significant decrease in the number of leucocytes and granulocytes was observed in AZT group following treatment. An opposite dynamic was observed in placebo group; numbers of granulocytes significantly increased at POST interval. All markers of vascular damage were reduced in AZT group at POST interval with overall significance (P = 0.026). The most prominent significant changes were observed in levels of myeloid-related protein 8/14 (MRP8/14), lipocalin A (NGAL), matrix metalloproteinases (MMP) 9, and insulin-like growth factor-binding protein (IGFBP) 4. Plasma level of C-reactive protein (CRP) was significantly decreased in AZT group as well. Conclusions: Data suggested that AZT may be beneficial in management of microvascular injury in SCD.

Reaction of Cyanide with Hg0-Contaminated Gold Mining Tailings Produces Soluble Mercuric Cyanide Complexes.

Elemental mercury (Hg0) contamination in artisanal and small-scale gold mining (ASGM) communities is widespread, and Hg0-contaminated tailings are often reprocessed with cyanide (-CN) to extract residual gold remaining after amalgamation. Hg0 reacts with -CN under aerobic conditions to produce Hg(CN)42- and other Hg(CN)nn-2 complexes. The production of solvated Hg(CN)nn-2 complexes increases upon agitation in the presence of synthetic and authentic Hg0-contaminated tailings that aid in dispersing the Hg0, increasing its reactive surface area. Adult rats were exposed to various concentrations of Hg(CN)2, and accumulation in organs and tissues was quantified using direct mercury analysis. The primary site of Hg(CN)2 accumulation was the kidney, although accumulation was also detected in the liver, spleen, and blood. Little accumulation was observed in the brain, suggesting that Hg(CN)2 complexes do not cross the blood-brain barrier. Renal tissue was particularly sensitive to the effects of Hg(CN)2, with pathological changes observed at low concentrations. Hg(CN)2 complexes are handled by mammalian systems in a manner similar to other inorganic species of Hg, yet appear to be more toxic to organ systems. The findings from this study are the first to show that Hg(CN)2 complexes are highly stable complexes that can lead to cellular injury and death in mammalian organ systems.

The cannabinoid receptor 2 agonist, ß-caryophyllene, improves working memory and reduces circulating levels of specific proinflammatory cytokines in aged male mice.

Age-related cognitive decline has been associated with proinflammatory cytokines, yet the precise relationship between cognitive decline and cytokine load remains to be elucidated. ß-caryophyllene (BCP) is a cannabinoid receptor 2 (CB2) agonist with established anti-inflammatory effects that is known to improve memory and increase lifespan. It is of interest to explore the potential of BCP to reduce age-related cognitive decline and proinflammatory cytokine load. In this study, we assessed changes in circulating cytokines across the lifespan, memory performance in young and aged mice, and the effects of BCP on memory function and cytokine load. The plasma levels of 12 cytokines were assessed in male Swiss-Webster mice at 3, 12, and 18 months of age using multiplexed flow cytometry. Working memory was compared in 3 and 12 month-old mice using spontaneous alternations. A dose-response function (100-300 mg/kg, subchronic administration) for BCP-induced memory restoration was determined in 3- and 12- month-old mice. Finally, the effects on cytokine levels of the peak memory enhancing dose of BCP were assessed in 18- month-old mice. Circulating levels of several cytokines significantly increased with age. Multilinear regression analysis showed that IL-23 levels were most strongly associated with age. Aged mice showed deficits in working memory and higher levels of IL-23, both of which were reversed by BCP treatment. BCP appears to reverse age-associated impairments in memory and modulates cytokine production. IL-23 may play a significant role in the aging process, and future research should determine whether it has utility as a biomarker for novel anti-aging therapeutics.

Using glycyrrhizic acid to target sumoylation processes during Epstein-Barr virus latency.

Cellular sumoylation processes are proposed targets for anti-viral and anti-cancer therapies. We reported that Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) dysregulates cellular sumoylation processes, contributing to its oncogenic potential in EBV-associated malignancies. Ginkgolic acid and anacardic acid, known inhibitors of sumoylation, inhibit LMP1-induced protein sumoylation; however, both drugs have adverse effects in hosts. Here we test the effects of glycyrrhizic acid, a medicinal botanical extract with anti-inflammatory, anti-carcinogenic, and anti-viral properties, on cellular sumoylation processes. While glycyrrhizic acid is known to inhibit EBV penetration, its affect on cellular sumoylation processes remains to be documented. We hypothesized that glycyrrhizic acid inhibits cellular sumoylation processes and may be a viable treatment for Epstein-Barr virus-associated malignancies. Results showed that glycyrrhizic acid inhibited sumoylation processes (without affecting ubiquitination processes), limited cell growth, and induced apoptosis in multiple cell lines. Similar to ginkgolic acid; glycyrrhizic acid targeted the first step of the sumoylation process and resulted in low levels of spontaneous EBV reactivation. Glycyrrhizic acid did not affect induced reactivation of the virus, but the presence of the extract did reduce the ability of the produced virus to infect additional cells. Therefore, we propose that glycyrrhizic acid may be a potential therapeutic drug to augment the treatment of EBV-associated lymphoid malignancies.

Treatment of Hematological Malignancies with Glycyrrhizic Acid.

The current study examined the effectiveness of glycyrrhizic acid (GA) in reducing cell viability and inducing apoptosis in human chronic myeloid leukemia (CML) in vitro and a mouse lymphoma in vivo. Additionally, we assessed GA as a candidate for combinational therapy in CML along with the current frontline treatment, imatinib (IM). Treatment of K562 CML cells with GA alone resulted in significant induction of apoptosis and loss of cell viability. GA was well tolerated by peripheral blood mononuclear cells (PBMCs) up to 2 mM doses which were subsequently used in combination with IM. Co-treatment of CML with GA and IM greatly enhanced the levels of apoptosis in human CML. The effectiveness of GA was not limited to in vitro studies as treatment of EL-4 lymphoma-bearing mice with GA (50 or 500 mg/kg/day) led to significant dose-related decrease in tumor burden that correlated with a significant increase in the level of apoptotic tumors in vivo. The broad activity of GA against different tumor cell types, its tolerance by PBMCs and synergistic effects when combined with IM suggests that GA may be a viable candidate for combinational treatment strategies in CML and other hematological malignancies.

Inhibition of hyaluronic acid formation sensitizes chronic myelogenous leukemia to treatment with doxorubicin.

In the current study we examined the ability of 4-methylumbelliferone (4-MU), which can inhibit hyaluronic acid synthesis, to sensitize K562 chronic myelogenous leukemia (CML) cells to doxorubicin therapy. Exposure of K562 cells to doxorubicin led to increased hyaluronic acid synthase (HAS) gene expression and increased levels of cell surface hyaluronic acid. Furthermore, exposure of K562 cells to exogenous HA caused resistance to doxorubicin-induced cell death. The combination of low dose 4-MU and doxorubicin led to increased apoptosis when compared to higher doses of any agent alone. Additionally, treatment with 4-MU led to a significant reduction in doxorubicin-induced increase in HA cell surface expression. Mechanistically, 4-MU treatment led to an increase in p38 activation and PARP cleavage. The role of p38 in 4-MU/doxorubicin-treated K562 cells was confirmed when p38 inhibitors led to protection from 4-MU/doxorubicin-induced apoptosis. Together, results from this study suggest that treatment with 4-MU increases the sensitivity of CML to chemotherapeutics by decreasing their HA-mediated resistance to apoptosis.

Treatment with the hyaluronic Acid synthesis inhibitor 4-methylumbelliferone suppresses LPS-induced lung inflammation.

Exposure to bacterial endotoxins, such as lipopolysaccharide (LPS), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for LPS-induced inflammation. In the current study, we investigated the potential use of the hyaluronic acid (HA) synthesis inhibitor 4-methylumbelliferone (4-MU) on LPS-induced acute lung inflammation. Culturing LPS-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production, and an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from LPS-induced lung injury. Specifically, 4-MU treatment led to a reduction in LPS-induced hyaluronic acid synthase (HAS) messenger RNA (mRNA) levels, reduction in lung permeability, and reduction in proinflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target HA production may be an effective treatment for the inflammatory response following exposure to LPS.

Treatment with the hyaluronic acid synthesis inhibitor 4-methylumbelliferone suppresses SEB-induced lung inflammation.

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for SEB-induced inflammation. In the current study we investigated the potential use of the hyaluronic acid synthase inhibitor 4-methylumbelliferone (4-MU) on staphylococcal enterotoxin B (SEB) induced acute lung inflammation. Culturing SEB-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production as well as an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from SEB-induced lung injury. Specifically, 4-MU treatment led to a reduction in SEB-induced HA levels, reduction in lung permeability, and reduced pro-inflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target hyaluronic acid production may be an effective treatment for the inflammatory response following exposure to SEB.

Development of a novel treatment for leukemia directed at tumor-associated mRNA splicing.

This report describes a novel approach to cancer therapy that targets genes that are preferentially alternatively spliced and expressed in leukemia. We developed CD44v6 and CD44v8 splicing constructs fused with GFP or a humanized fragment of Pseudomonas aeruginosa exotoxin A (hPE24). Transfection of K562 leukemia cells with the GFP-linked splicing constructs led to subsequent production of detectable levels of GFP. Transfection of K562 cells with the hPE24-linked splicing constructs led to significant reduction of cell viability and an increase in the induction of apoptosis. Normal human PBMCs were unaffected by following transfection with these constructs.

Targeting hyaluronic acid production for the treatment of leukemia: treatment with 4-methylumbelliferone leads to induction of MAPK-mediated apoptosis in K562 leukemia.

The current study examined the effect of modulation of hyaluronic acid (HA) synthesis on leukemia cell survival using the hyaluronic acid synthesis inhibitor 4-methylumbelliferone (4-MU). Treatment of CML cells with 4-MU led to caspase-dependent apoptosis characterized by decreased HA production, PARP cleavage, and increased phosphorylation of p38. Addition of exogenous HA, the pan caspase inhibitor Z-VAD-FMK or the p38 inhibitor SB203580 to 4-MU treated cells was able to protect cells from apoptosis. Treatment of tumor-bearing mice with 4-MU led to a significant reduction in tumor load which was mediated through the induction of apoptosis.

The role of hyaluronic acid in SEB-induced acute lung inflammation.

We investigated the role of the extracellular matrix component, hyaluronic acid (HA) in SEB-induced ALI/ARDS. Intranasal exposure of mice to SEB led to a significant increase in the level of soluble hyaluronic acid in the lungs. Similarly, in an endothelial cell/spleen cell co-culture, SEB exposure led to significant increases in soluble levels of hyaluronic acid, cellular proliferation, and cytokine production compared with SEB-exposed spleen cells or endothelial cells alone. Exposure of SEB-activated spleen cells to hyaluronic acid led to increased cellular proliferation and increased cytokine production. SEB-induced cytokine production and proliferation in vitro were significantly reduced by the hyaluronic acid blocking peptide, Pep-1. Finally, treatment of SEB-exposed mice with Pep-1 significantly reduced SEB-induced ALI/ARDS, through reduction of cytokine production and numbers of lung inflammatory cells, compared to mice treated with a control peptide. Together, these results suggest the possibility of targeting HA for the treatment of SEB-induced ALI/ARDS.

Fatigue in medical residents leads to reactivation of herpes virus latency.

The main objective of this study was to detect fatigue-induced clinical symptoms of immune suppression in medical residents. Samples were collected from the subjects at rest, following the first night (low-stress), and the last night (high-stress) of night float. Computerized reaction tests, Epworth Sleepiness Scale, and Wellness Profile questionnaires were used to quantify fatigue level. DNA of human herpes viruses HSV-1, VZV, EBV, as well as cortisol and melatonin concentrations, were measured in saliva. Residents at the high-stress interval reported being sleepier compared to the rest interval. EBV DNA level increased significantly at both stress intervals, while VZV DNA level increased only at low-stress. DNA levels of HSV-1 decreased at low-stress but increased at high-stress. Combined assessment of the viral DNA showed significant effect of stress on herpes virus reactivation at both stress intervals. Cortisol concentrations at both stress intervals were significantly higher than those at rest.