A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis in vivo.

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wildtype. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels compared to the wildtype virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection and showed no signs of lung pathology. Our data validate the SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a promising target to develop antivirals.

Tumor microenvironment signaling and therapeutics in cancer progression.

Tumor development and metastasis are facilitated by the complex interactions between cancer cells and their microenvironment, which comprises stromal cells and extracellular matrix (ECM) components, among other factors. Stromal cells can adopt new phenotypes to promote tumor cell invasion. A deep understanding of the signaling pathways involved in cell-to-cell and cell-to-ECM interactions is needed to design effective intervention strategies that might interrupt these interactions. In this review, we describe the tumor microenvironment (TME) components and associated therapeutics. We discuss the clinical advances in the prevalent and newly discovered signaling pathways in the TME, the immune checkpoints and immunosuppressive chemokines, and currently used inhibitors targeting these pathways. These include both intrinsic and non-autonomous tumor cell signaling pathways in the TME: protein kinase C (PKC) signaling, Notch, and transforming growth factor (TGF-ß) signaling, Endoplasmic Reticulum (ER) stress response, lactate signaling, Metabolic reprogramming, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and Siglec signaling pathways. We also discuss the recent advances in Programmed Cell Death Protein 1 (PD-1), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), T-cell immunoglobulin mucin-3 (TIM-3) and Lymphocyte Activating Gene 3 (LAG3) immune checkpoint inhibitors along with the C-C chemokine receptor 4 (CCR4)- C-C class chemokines 22 (CCL22)/ and 17 (CCL17), C-C chemokine receptor type 2 (CCR2)- chemokine (C-C motif) ligand 2 (CCL2), C-C chemokine receptor type 5 (CCR5)- chemokine (C-C motif) ligand 3 (CCL3) chemokine signaling axis in the TME. In addition, this review provides a holistic understanding of the TME as we discuss the three-dimensional and microfluidic models of the TME, which are believed to recapitulate the original characteristics of the patient tumor and hence may be used as a platform to study new mechanisms and screen for various anti-cancer therapies. We further discuss the systemic influences of gut microbiota in TME reprogramming and treatment response. Overall, this review provides a comprehensive analysis of the diverse and most critical signaling pathways in the TME, highlighting the associated newest and critical preclinical and clinical studies along with their underlying biology. We highlight the importance of the most recent technologies of microfluidics and lab-on-chip models for TME research and also present an overview of extrinsic factors, such as the inhabitant human microbiome, which have the potential to modulate TME biology and drug responses.

CAR T-Cell-Based gene therapy for cancers: new perspectives, challenges, and clinical developments.

Chimeric antigen receptor (CAR)-T cell therapy is a progressive new pillar in immune cell therapy for cancer. It has yielded remarkable clinical responses in patients with B-cell leukemia or lymphoma. Unfortunately, many challenges remain to be addressed to overcome its ineffectiveness in the treatment of other hematological and solidtumor malignancies. The major hurdles of CAR T-cell therapy are the associated severe life-threatening toxicities such as cytokine release syndrome and limited anti-tumor efficacy. In this review, we briefly discuss cancer immunotherapy and the genetic engineering of T cells and, In detail, the current innovations in CAR T-cell strategies to improve efficacy in treating solid tumors and hematologic malignancies. Furthermore, we also discuss the current challenges in CAR T-cell therapy and new CAR T-cell-derived nanovesicle therapy. Finally, strategies to overcome the current clinical challenges associated with CAR T-cell therapy are included as well.

COVID-19 and diabetes: What do we know so far?

Coronavirus disease 2019 (COVID-19) management has been challenging for patients with comorbidities. Patients with diabetes and COVID-19, in particular, have shown severe symptoms and rapid progression of the disease. They also have a high mortality rate compared to the non-diabetic population. The high mortality rate is caused in people with diabetes who are in a pro-inflammatory condition; this could worsen COVID-19. In addition, people with diabetes have circulatory issues and COVID-19 infection can lead to further clotting problems. It is critical to understand the mechanisms underlying the adverse clinical outcomes in patients with diabetes and COVID-19. This review discusses various disease conditions contributing to poor prognosis in diabetic COVID-19 patients such as hyperglycemia, insulin resistance, impaired pancreatic function, and production of advanced glycation end products.

Plasma Biomarkers as Risk Factors for Incident CKD.

Introduction: Earlier identification of individuals at high risk of chronic kidney disease (CKD) may facilitate improved risk factor mitigation. Methods: We evaluated the association of novel plasma biomarkers with incident CKD using a case-cohort design in participants without diabetes and with baseline estimated glomerular filtration rate (eGFR) ≥ 60 ml/min per 1.73 m2 in the Multi-Ethnic Study of Atherosclerosis (MESA) and Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohorts. Incident CKD was defined as development of eGFR < 60 ml/min per 1.73 m2 and ≥40% decline in eGFR from baseline. We measured plasma markers of inflammation/fibrosis-soluble tumor necrosis factor receptors (TNFRs) 1 and 2 (TNFR-1 and TNFR-2), monocyte chemotactic protein-1 (MCP-1), chitinase 3-like protein 1 (YKL-40), and soluble urokinase-type plasminogen activator receptor (suPAR)-and tubular injury (kidney injury molecule 1 [KIM-1]). Cox regression models weighted for the case-cohort design were used to estimate hazard ratios (HRs) of incident CKD after adjustment for CKD risk factors, eGFR, and albuminuria. Results: In MESA (median follow-up of 9.2 years), there were 497 individuals in the random subcohort and 163 incident CKD cases. In REGARDS (median follow-up of 9.4 years), there were 497 individuals in the random subcohort and 497 incident CKD cases. Each 2-fold higher plasma KIM-1 (adjusted HR 1.38 [95% CI 1.05-1.81]), suPAR (1.96 [1.10-3.49]), TNFR-1 (1.65 [1.04-2.62]), TNFR-2 (2.02 [1.21-3.38]), and YKL-40 (1.38 [1.09-1.75]) concentrations were associated with incident CKD in MESA. In REGARDS, TNFR-1 (1.99 [1.43-2.76]) and TNFR-2 (1.76 [1.22-2.54]) were associated with incident CKD. Conclusion: Plasma concentrations of soluble TNFR-1 and TNFR-2 are consistently associated with incident CKD in nondiabetic community-living individuals in MESA and REGARDS.

Biomarkers of Kidney Tubule Disease and Risk of End-Stage Kidney Disease in Persons With Diabetes and CKD.

Introduction: Tubulointerstitial damage in diabetes and chronic kidney disease (CKD) is poorly captured by estimated glomerular filtration rate (eGFR) and albuminuria. Urine biomarkers of kidney health may better elucidate disease progression in persons with diabetes and CKD. Methods: Per case-cohort design, we randomly selected a subcohort of 560 study participants of the REasons for Geographic And Racial Differences in Stroke (REGARDS) study from 1092 adults with diabetes and baseline eGFR <60 ml/min per 1.73 m2 and registered a total of 161 end-stage kidney disease (ESKD) cases (n = 93 from the subcohort; n = 68 from outside the subcohort) during 4.3 ± 2.7 years mean follow-up. We measured urine biomarkers of kidney tubule injury (kidney injury molecule-1 [KIM-1]), inflammation and fibrosis (monocyte chemoattractant protein-1 [MCP-1]), repair (chitinase-3-like protein 1 [YKL-40]), and tubule function, including reabsorption (alpha-1-microglobulin [α1m]) and synthetic capacity (epidermal growth factor [EGF] and uromodulin [UMOD]). Weighted Cox regression models estimated ESKD risk adjusting for demographics, ESKD risk factors, and baseline eGFR and urine albumin. Least absolute shrinkage and selection operator (LASSO) regression identified a subset of biomarkers most strongly associated with ESKD. Results: At baseline, subcohort participants had mean age of 70 ± 9 years, mean eGFR of 40 ±13 ml/min per 1.73 m2, and median urine albumin-to-creatinine ratio of 33 (interquartile range 10-213) mg/g. Adjusting for baseline eGFR and albuminuria, each 2-fold higher urine KIM-1 (hazard ratio = 1.43 [95% CI: 1.17-1.75]), α1m (hazard ratio = 1.47 [1.19-1.82]), and MCP-1 (hazard ratio = 1.27 [1.06-1.53]) were independently associated with ESKD. LASSO retained KIM-1 and α1m for associations with ESKD. Conclusion: Among adults with diabetes and eGFR <60 ml/min per 1.73 m2, higher urine KIM-1, α1m, and MCP-1 are independently associated with incident ESKD, providing insight into kidney disease progression in persons with diabetes and CKD.

Prebiotic Potential and Value-Added Products Derived from Spirulina laxissima SV001-A Step towards Healthy Living.

Lately, microalgae-based value-added products have been gaining market value because they moderate the dependency on fossil fuel and high-value chemical products. To this end, the purpose of this study was to develop prebiotic products from the microalgae Spirulina sp. The microalgae were isolated from the fresh water and characterized at the molecular level. The dry biomass, chlorophyll content, phycocyanin, cytotoxicity and antimicrobial and antioxidant properties of the isolated strains were analyzed. Moreover, value-added products like Spirulina cake, chocolate, tea, vermicelli and Spirulina juice were made for a vulnerable population due to high nutritive value.

Convalescent serum-derived exosomes: Attractive niche as COVID-19 diagnostic tool and vehicle for mRNA delivery.

The spread of SARS-CoV-2 over the entire world is more commonly known as COVID-19. COVID-19 has impacted society in every aspect of routine life. SARS-CoV-2 infection is often misdiagnosed as influenza or seasonal upper respiratory tract viral infections. General diagnostic tools can detect the viral antigen or isotypes of antibodies. However, inter- and intraindividual variations in antibody levels can cause false negatives in antibody immunoassays. On the contrary, the false-positive test results can also occur due to either cross-reactivity of the viral antigens or some other patient-related autoimmune factors. There is need for a cogent diagnostic tool with more specificity, selectivity, and reliability. Here, we have described the potential of convalescent serum-derived exosome as a diagnostic tool for the detection of SARS-CoV-2, even in asymptomatic patients, which is a limitation for currently practiced diagnostic tests throughout the globe. In addition, its potential as a vehicle for messenger RNA (mRNA) delivery is also emphasized.

Application of Orthobiologics in Achilles Tendinopathy: A Review.

Orthobiologics are biological materials that are intended for the regeneration of bone, cartilage, and soft tissues. In this review, we discuss the application of orthobiologics in Achilles tendinopathy, more specifically. We explain the concepts and definitions of each orthobiologic and the literature regarding its use in tendon disorders. The biological potential of these materials can be harnessed and administered into injured tissues, particularly in areas where standard healing is disrupted, a typical feature of Achilles tendinopathy. These products contain a wide variety of cell populations, cytokines, and growth factors, which have been shown to modulate many other cells at local and distal sites in the body. Collectively, they can shift the state of escalated inflammation and degeneration to reestablish tissue homeostasis. The typical features of Achilles tendinopathy are failed healing responses, persistent inflammation, and predominant catabolic reactions. Therefore, the application of orthobiologic tools represents a viable solution, considering their demonstrated efficacy, safety, and relatively easy manipulation. Perhaps a synergistic approach regarding the combination of these orthobiologics may promote more significant clinical outcomes rather than individual application. Although numerous optimistic results have been registered in the literature, additional studies and clinical trials are still highly desired to further illuminate the clinical utility and efficacy of these therapeutic strategies in the management of tendinopathies.

An Update on the Effectiveness of Probiotics in the Prevention and Treatment of Cancer.

Probiotics are living microbes that play a significant role in protecting the host in various ways. Gut microbiota is one of the key players in maintaining homeostasis. Cancer is considered one of the most significant causes of death worldwide. Although cancer treatment has received much attention in recent years, the number of people suffering from neoplastic syndrome continues to increase. Despite notable improvements in the field of cancer therapy, tackling cancer has been challenging due to the multiple properties of cancer cells and their ability to evade the immune system. Probiotics alter the immunological and cellular responses by enhancing the epithelial barrier and stimulating the production of anti-inflammatory, antioxidant, and anticarcinogenic compounds, thereby reducing cancer burden and growth. The present review focuses on the various mechanisms underlying the role of probiotics in the prevention and treatment of cancer.

Urine Biomarkers of Kidney Tubule Health, Injury, and Inflammation are Associated with Progression of CKD in Children.

BACKGROUND: Novel urine biomarkers may improve identification of children at greater risk of rapid kidney function decline, and elucidate the pathophysiology of CKD progression. METHODS: We investigated the relationship between urine biomarkers of kidney tubular health (EGF and α-1 microglobulin), tubular injury (kidney injury molecule-1; KIM-1), and inflammation (monocyte chemoattractant protein-1 [MCP-1] and YKL-40) and CKD progression. The prospective CKD in Children Study enrolled children aged 6 months to 16 years with an eGFR of 30-90ml/min per 1.73m2. Urine biomarkers were assayed a median of 5 months [IQR: 4-7] after study enrollment. We indexed the biomarker to urine creatinine by dividing the urine biomarker concentration by the urine creatinine concentration to account for the concentration of the urine. The primary outcome was CKD progression (a composite of a 50% decline in eGFR or kidney failure) during the follow-up period. RESULTS: Overall, 252 of 665 children (38%) reached the composite outcome over a median follow-up of 6.5 years. After adjustment for covariates, children with urine EGF concentrations in the lowest quartile were at a seven-fold higher risk of CKD progression versus those with concentrations in the highest quartile (fully adjusted hazard ratio [aHR], 7.1; 95% confidence interval [95% CI], 3.9 to 20.0). Children with urine KIM-1, MCP-1, and α-1 microglobulin concentrations in the highest quartile were also at significantly higher risk of CKD progression versus those with biomarker concentrations in the lowest quartile. Addition of the five biomarkers to a clinical model increased the discrimination and reclassification for CKD progression. CONCLUSIONS: After multivariable adjustment, a lower urine EGF concentration and higher urine KIM-1, MCP-1, and α-1 microglobulin concentrations were each associated with CKD progression in children.

NP-ALT, a Liposomal:Peptide Drug, Blocks p27Kip1 Phosphorylation to Induce Oxidative Stress, Necroptosis, and Regression in Therapy-Resistant Breast Cancer Cells.

Resistance to cyclin D-CDK4/6 inhibitors (CDK4/6i) represents an unmet clinical need and is frequently caused by compensatory CDK2 activity. Here we describe a novel strategy to prevent CDK4i resistance by using a therapeutic liposomal:peptide formulation, NP-ALT, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. We find that NP-ALT blocks proliferation in HR+ breast cancer cells, as well as CDK4i-resistant cell types, including triple negative breast cancer (TNBC). The peptide ALT is not as stable in primary mammary epithelium, suggesting that NP-ALT has little effect in nontumor tissues. In HR+ breast cancer cells specifically, NP-ALT treatment induces ROS and RIPK1-dependent necroptosis. Estrogen signaling and ERα appear required. Significantly, NP-ALT induces necroptosis in MCF7 ESRY537S cells, which contain an ER gain of function mutation frequently detected in metastatic patients, which renders them resistant to endocrine therapy. Here we show that NP-ALT causes necroptosis and tumor regression in treatment naïve, palbociclib-resistant, and endocrine-resistant BC cells and xenograft models, demonstrating that p27 is a viable therapeutic target to combat drug resistance. IMPLICATIONS: This study reveals that blocking p27 tyrosine phosphorylation inhibits CDK4 and CDK2 activity and induces ROS-dependent necroptosis, suggesting a novel therapeutic option for endocrine and CDK4 inhibitor-resistant HR+ tumors.

SARS-CoV-2 variants: A double-edged sword?

Since the worldwide emergence of the COVID-19 outbreak, there have been international concerns about the possible viral evolution into variants with underlying mutations that may contribute to their increased transmissibility, disease severity, risk of death, and their potential escape from the immune response or may even lead to its extinction. Rigorous surveillance has revealed the variants harboring mutations in the spike protein, the main target of neutralizing antibodies generated through vaccination or herd immunity. In this review, we have highlighted major SARS-CoV-2 variants as well as other local strains along with their specific mutations, suspected changes in their characteristics, and their impact on the current pandemic and vaccine efficacy. We have also emphasized the need to develop widely protective interventions to curb further transmission of variants.

COVID-19: Antiviral agents and enzyme inhibitors/receptor blockers in development.

Novel 2019 coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) and coronavirus disease 2019 (COVID-19), the respiratory syndrome it causes, have shaken the world to its core by infecting and claiming the lives of many people since originating in December 2019 in Wuhan, China. World Health Organization and several states have declared a pandemic situation and state of emergency, respectively. As there is no treatment for COVID-19, several research institutes and pharmaceutical companies are racing to find a cure. Advances in computational approaches have allowed the screening of massive antiviral compound libraries to identify those that may potentially work against SARS-CoV-2. Antiviral agents developed in the past to combat other viruses are being repurposed. At the same time, new vaccine candidates are being developed and tested in preclinical/clinical settings. This review provides a detailed overview of select repurposed drugs, their mechanism of action, associated toxicities, and major clinical trials involving these agents.

A mini-review on the effects of COVID-19 on younger individuals.

Coronavirus disease 2019 (COVID-19) pandemic has uprooted our lives like never before since its onset in the late December 2019. The world has seen mounting infections and deaths over the past few months despite the unprecedented measures countries are implementing, such as lockdowns, social distancing, mask-wearing, and banning gatherings in large groups. Interestingly, young individuals seem less likely to be impacted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19. While the rate of transmission, symptom presentation, and fatality is lower in children than people from other age groups, they have been disproportionately affected by strict lockdown measures needed to curb viral spread. In this review, we describe the association between patient age and COVID-19, epidemiology of SARS-CoV-2 infection in children, psychological effects associated with lockdowns and school closures, and possible mechanisms underlying lower transmission rate of COVID-19 in children.

Recent developments in autophagy-targeted therapies in cancer.

Autophagy plays a crucial role in cellular development and differentiation as well as in the maintenance of homeostasis in healthy cells. Autophagy is well documented in neurodegenerative disorders, aging, and infectious diseases. However, recognizing its significance in cancer has always been challenging due to its tumor-promoting and suppressive attributes. Various modulators targeting key components of autophagy machinery directly or indirectly have been developed over the years, and have shown promising results in preclinical models. Some of these compounds are even being tested in clinical trials for safety and efficacy. A detailed review of strategies used to target autophagy in cancer is presented including our opinion on developing better therapies and outstanding issues.

Novel 2019 coronavirus: Genome structure, clinical trials, and outstanding questions.

IMPACT STATEMENT: Early availability of the sequence, the genetic material of SARS-CoV-2 (the virus that causes COVID-19), has prompted efforts towards identifying a safe and effective vaccine in the current public health emergency. To that end, understanding the pathophysiology of disease is crucial for scientists around the world. Since conventional vaccine development and manufacturing may take several years, it is important to think about alternative strategies that we could use to mitigate imminent catastrophe. We hope that this article will open up new avenues and provide insights that could potentially save hundreds of lives affected by COVID-19.

Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line.

Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in in vitro culture systems have demonstrated that the use of three-dimensional (3D) culture models could be more relevant for this purpose as compared to monolayer or two-dimensional (2D) models due to their resemblance to in vivo cancer pathology. High-throughput techniques such as RNA sequencing, microarray analyses and cloning could provide useful insights into the relevance of these systems to the native tissue. Previous studies have reported RNA extraction protocols needed for such applications. We have modified these protocols to suit the isolation of total RNA from monolayer and hydrogel cultures of astrocytoma established using basement membrane matrix, Geltrex™. We have used this method to demonstrate the differences in the expression of genes involved in autophagy, a process deregulated in many cancer types, in monolayer and hydrogel cultures using quantitative polymerase chain reaction (qPCR). This protocol can be adopted by the researchers who wish to understand the molecular basis of gene expression in hydrogel cultures of normal as well as cancer cell lines.

Morphometric analysis of a triple negative breast cancer cell line in hydrogel and monolayer culture environments.

Triple negative breast cancer (TNBC) is a belligerent carcinoma that is unresponsive to targeted receptor therapies. Development of new treatment strategies would benefit from an expanded repertoire of in vitro cell culture systems, such as those that support tridimensional growth in the presence of hydrogel scaffolds. To this end, we established protocols for maintenance of the TNBC cell line HCC70 in monolayer culture and in a commercially available basement membrane matrix hydrogel. We evaluated the general morphology of cells grown in both conditions with light microscopy, and examined their subcellular organization using transmission electron microscopy (TEM). Phase contrast and confocal microscopy showed the prevalence of irregularly shaped flattened cells in monolayer cultures, while cells maintained in hydrogel organized into multi-layered spheroids. A quantitative ultrastructural analysis comparing cells from the two culture conditions revealed that cells that formed spheroids comprised a greater number of mitochondria, autophagic vacuoles and intercellular junctions than their monolayer counterparts, within the equivalent area of sampled tissue. These observations suggest that triple negative breast cancer cells in culture can alter their organelle content, as well as their morphology, in response to their microenvironment. Methods presented here may be useful for those who intend to image cell cultures with TEM, and for investigators who seek to implement diverse in vitro models in the search for therapeutic molecular targets for TNBC.