Glycosylation Modulation Dictates Trafficking and Interaction of SARS-CoV-2 S1 Subunit and ACE2 in Intestinal Epithelial Caco-2 Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mainly targets the upper respiratory tract. It gains entry by interacting with the host cell receptor angiotensin-converting enzyme 2 (ACE2) via its heavily glycosylated spike glycoprotein. SARS-CoV-2 can also affect the gastrointestinal tract. Given the significant role of glycosylation in the life cycle of proteins and the multisystem target of SARS-CoV-2, the role of glycosylation in the interaction of S1 with ACE2 in Caco-2 cells was investigated after modulation of their glycosylation patterns using N-butyldeoxynojirimycin (NB-DNJ) and 1-deoxymannojirimycin (dMM), in addition to mutant CHO cells harboring mutations at different stages of glycosylation. The data show a substantial reduction in the interactions between the altered glycosylation forms of S1 and ACE2 in the presence of NB-DNJ, while varied outcomes resulted from dMM treatment. These results highlight the promising effects of NB-DNJ and its potential use as an off-label drug to treat SARS-CoV-2 infections.

The Antioxidant and Proapoptotic Effects of Sternbergia clusiana Bulb Ethanolic Extract on Triple-Negative and Estrogen-Dependent Breast Cancer Cells In Vitro.

BACKGROUND: Sternbergia clusiana belongs to the Amaryllidaceae family and is recognized for the valuable biological activity of its major bioactive compounds. The aim of the current is to evaluate the anticancer effects of the ethanolic bulb extract of Sternbergia clusiana (ScBEE) on breast cancer cells in vitro and to further reveal the underlying cellular mechanism. METHODS: An MTS cell viability assay was performed on MDA-MB-231 and MCF-7 cells, along with cell cycle analysis, cell death ELISA, Western blot analysis and an ROS production assay to decipher the mechanism of death. LC-MS/MS was also performed to identify the chemical composition of this ethanolic extract. RESULTS: The results show a selective antiproliferative effect on both cell lines with no effect on normal mesenchymal stem cells. Further analysis suggested the activation of the apoptotic pathway as reflected by the increase in cellular and DNA fragmentation and alterations in apoptotic proteins such as Bax, Bcl-2 and c-PARP. ScBEE was also found to exhibit antioxidant effect, as shown by a decrease in ROS production. The underlying mechanism of action was explained by the presence of several bioactive compounds identified by LC-MS/MS, including alkaloids, terpenoids and phenols, which are elaborated in the manuscript. CONCLUSION: This study highlights the antioxidant and anticancerous properties of S.clusiana for breast cancer treatment.

Lipid rafts disruption by statins negatively impacts the interaction between SARS-CoV-2 S1 subunit and ACE2 in intestinal epithelial cells.

The causative agent of the COVID-19 pandemic, SARS-CoV-2, is a virus that targets mainly the upper respiratory tract. However, it can affect other systems such as the gastrointestinal (GI) tract. Therapeutic strategies for this virus are still inconclusive and understanding its entry mechanism is important for finding effective treatments. Cholesterol is an important constituent in the structure of cellular membranes that plays a crucial role in a variety of cellular events. In addition, it is important for the infectivity and pathogenicity of several viruses. ACE2, the main receptor of SARS-CoV-2, is associated with lipid rafts which are microdomains composed of cholesterol and sphingolipids. In this study, we investigate the role of statins, lipid-lowering drugs, on the trafficking of ACE2 and the impact of cholesterol modulation on the interaction of this receptor with S1 in Caco-2 cells. The data show that fluvastatin and simvastatin reduce the expression of ACE2 to variable extents, impair its association with lipid rafts and sorting to the brush border membrane resulting in substantial reduction of its interaction with the S1 subunit of the spike protein. By virtue of the substantial effects of statins demonstrated in our study, these molecules, particularly fluvastatin, represent a promising therapeutic intervention that can be used off-label to treat SARS-CoV-2.

The pro-apoptotic properties of a phytonutrient rich infusion of A. cherimola leaf extract on AML cells.

Annonaceae family has broad uses in herbal medicine for treatment of several diseases, whether through seeds' or leaves' extracts. The present study investigates the antiproliferative and antitumor activity of Annona cherimola aqueous leaf (AAL) extract/infusion in acute myeloid leukemia (AML) cell lines in vitro. High-resolution LC-MS was first used to analyze the composition of the aqueous extract. Cell proliferation assay, Annexin V staining, cell cycle analysis, dual Annexin V/PI staining, cell death quantification by ELISA, ROS level detection and Western Blotting were then performed to elucidate the therapeutic effects of AAL extract. The results obtained revealed a potent antioxidant activity of AAL extract. Moreover, the extract exhibited dose- and time-dependent antiproliferative effects on AML cell lines by decreasing cell viability with an IC50 of 5.03% (v/v) at 24 h of treatment of KG-1 cells. This decrease in viability was accompanied with a significant increase in apoptotic cell death with cell cycle arrest and flipping of the phosphatidylserine from the inner to the outer leaflet of the cell membrane. The respective overexpression and downregulation of proapoptotic proteins like cleaved caspase-8, cleaved PARP-1 and Bax and antiapoptotic proteins like Bcl-2 further validated the apoptotic pathway induced by AAL on AML cells. Finally, LC-MS revealed the presence of several compounds like fatty acids, terpenes, phenolics, cinnamic acids and flavonoids that could contribute to the antioxidant and anti-cancer effects of this herbal infusion. In addition to the generally known nutritional effects of the Annona cherimola fruit and leaves, the presented data validates the antioxidant and anti-cancerous effects of the leaf infusion on AML cell lines, proposing its potential therapeutic use against acute myeloid leukemia with future in vivo and clinical trials.

Malva pseudolavatera Leaf Extract Promotes ROS Induction Leading to Apoptosis in Acute Myeloid Leukemia Cells In Vitro.

Malva pseudolavatera Webb & Berthel. is a plant from the Malvaceae family that has long been included in the human diet due to its various curative effects. Many plant leaf extracts from the various species of Malva genus have been reported to possess anti-cancer properties, however, studies on M. pseudolavatera Webb & Berthel. leaves have documented anti-inflammatory and anti-oxidant effects with no emphasis on their possible anti-cancer potential. The present study explores the anti-cancer properties of Malva pseudolavatera Webb & Berthel. leaf extract on acute myeloid leukemia (AML) cell lines in vitro and deciphers the underlying molecular mechanism. Treatment of AML cell lines with M. pseudolavatera methanolic leaf extract showed a dose- and time-dependent inhibition of proliferation and a dose-dependent increase in apoptotic hallmarks such as an increase in phosphatidylserine on the outer membrane leaflet and membrane leakage in addition to DNA fragmentation. The pro-apoptotic effect was induced by reactive oxygen species (ROS) as well as an upregulation of cleaved poly(ADP-ribose) polymerase (PARP), increase in Bax/Bcl-2 ratio, andrelease of cytochrome-c from the mitochondria. Major compounds of the extract included methyl linolenate, phytol, γ-sitosterol, and stigmasterol as revealed by gas chromatography coupled with mass spectrometry, and amino acids, amino acid derivatives, tiliroside, 13-hydroxyperoxyoctadecadienoic, and quercitrin as detected by liquid chromatography coupled to mass spectrometry.

The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines.

BACKGROUND: The edible fruit Annona cherimola has previously shown many nutritional and medicinal properties. The current study evaluates the anti-cancer and anti-proliferative properties of Annona cherimola ethanolic leaf extract (AELE) on Acute Myeloid Leukemia (AML) cell lines cultured in vitro (Monomac-1 and KG-1). METHODS: The anti-proliferative effect of A. cherimola ethanolic leaf extract was evaluated via cell viability assay. Its pro-apoptotic effect was assessed through Cell Death ELISA and dual Annexin V/PI staining. To further investigate the molecular mechanism by which the extract promoted apoptosis and inhibited the proliferation of the AML cells used, apoptotic protein expression was determined through western blots. Extract composition was elucidated by Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: Our results showed that the treatment with A. cherimola ethanolic leaf extract exhibited an inhibitory effect on the proliferation of both cancer cell lines used in a dose- and time-dependent manner, with no toxic effects on normal mononuclear cells (MNCs) isolated from human bone marrow. This effect was mediated by DNA fragmentation and apoptosis, as revealed by Cell Death ELISA and dual Annexin V/PI staining. Western blot analysis revealed a Bax/Bcl2 dependent mechanism of apoptosis, as well as PARP cleavage, confirming the apoptotic results observed previously. These effects may be attributed to the presence of terpenes which constitute a large component of the leafy extract, as revealed via GC-MS. CONCLUSION: All the data presented in our study show that the terpene-rich A. cherimola ethanolic leaf extract exhibits an anti-proliferative and pro-apoptotic effect on the AML cell lines used.

The high efficacy of muscarinic M4 receptor in D1 medium spiny neurons reverses striatal hyperdopaminergia.

The opposing action of dopamine and acetylcholine has long been known to play an important role in basal ganglia physiology. However, the quantitative analysis of dopamine and acetylcholine signal interaction has been difficult to perform in the native context because the striatum comprises mainly two subtypes of medium-sized spiny neurons (MSNs) on which these neuromodulators exert different actions. We used biosensor imaging in live brain slices of dorsomedial striatum to monitor changes in intracellular cAMP at the level of individual MSNs. We observed that the muscarinic agonist oxotremorine decreases cAMP selectively in the MSN subpopulation that also expresses D1 dopamine receptors, an action mediated by the M4 muscarinic receptor. This receptor has a high efficacy on cAMP signaling and can shut down the positive cAMP response induced by dopamine, at acetylcholine concentrations which are consistent with physiological levels. This supports our prediction based on theoretical modeling that acetylcholine could exert a tonic inhibition on striatal cAMP signaling, thus supporting the possibility that a pause in acetylcholine release is required for phasic dopamine to transduce a cAMP signal in D1 MSNs. In vivo experiments with acetylcholinesterase inhibitors donepezil and tacrine, as well as with the positive allosteric modulators of M4 receptor VU0152100 and VU0010010 show that this effect is sufficient to reverse the increased locomotor activity of DAT-knockout mice. This suggests that M4 receptors could be a novel therapeutic target to treat hyperactivity disorders.

Counseling Women and Couples on Family Planning: A Randomized Study in Jordan.

This article evaluates the effects of involving men in family planning counseling in Jordan using a randomized experiment. We randomly assigned a sample of 1,247 married women to receive women-only counseling, couples counseling, or no counseling. We measured the effects of each type of counseling on family planning use, knowledge, attitudes, and spousal communication about family planning. Compared to no counseling, couples counseling led to a 54 percent increase in uptake of modern methods. This effect is not significantly different from the 46 percent increase in modern method uptake as a result of women-only counseling. This outcome may be due, in part, to lower rates of compliance with the intervention among those assigned to couples counseling compared to women-only counseling. To realize the possible added benefits of involving men, more tailored approaches may be needed to increase men's participation.

Improved Childhood Diarrhea Treatment Practices in Ghana: A Pre-Post Evaluation of a Comprehensive Private-Sector Program.

Diarrhea is the fourth leading cause of child mortality in Ghana. In 2010, Ghana endorsed guidelines from the World Health Organization and the United Nations Children's Fund for use of zinc with low-osmolarity oral rehydration salts (ORS) for the treatment of acute childhood diarrhea. From late 2011 through 2014, the Strengthening Health Outcomes through the Private Sector (SHOPS) project implemented a comprehensive program in 3 regions of Ghana to increase the availability and use of ORS and zinc and to decrease incorrect use of antibiotics and antidiarrheals. The program included (1) partnering with local pharmaceutical firms to introduce and market locally produced zinc products, (2) collaborating with the Ghanaian Pharmacy Council to provide training and supportive supervision of private-sector providers on diarrhea management, and (3) conducting mass media campaigns to raise caregiver awareness. We evaluated the effect of this program using a baseline survey of 754 caregivers of children under 5 with diarrhea at the start of the intervention in 2012 and a follow-up survey of 751 caregivers in 2014. Regression analysis showed that use of ORS with zinc increased from 0.8% in 2012 to 29.2% in 2014 (P<.001), and antibiotic use declined from 66.2% to 38.2% (P<.001) during the same period. The magnitude and statistical significance of these results remained the same after including potential confounding factors as covariates. Inappropriate antibiotic use, however, remained high at follow-up. We conclude that similar programs applied in other settings have the potential to rapidly scale up use of ORS and zinc. Additional efforts are required to reduce persistent incorrect antibiotic use.

Effectiveness of evidence-based medicine on knowledge, attitudes, and practices of family planning providers: a randomized experiment in Jordan.

BACKGROUND: Provider misconceptions and biases about contraceptive methods are major barriers to family planning access for women in low- and middle-income countries, including Jordan. Evidence-Based Medicine (EBM) programs aim to reduce biases and misconceptions by providing doctors with the most up-to-date scientific evidence on contraceptive methods. METHODS: We evaluate the effects of an EBM program conducted in Jordan on private providers' knowledge, attitudes, and practices. Family planning providers randomly assigned to a treatment group were invited to attend a roundtable seminar on the injectable contraceptive Depot Medroxy Progesterone Acetate (DMPA), and to receive two 15 min one-on-one educational visits that reinforce the messages from the seminar. RESULTS: There was low compliance with the EBM program. The study fails to detect an impact on providers' knowledge of DMPA's side effects or on reported clinical practices. There is suggestive evidence of a positive impact on providers' attitudes toward and confidence in prescribing the contraceptive to their patients. There is also evidence of positive selection into program participation. CONCLUSIONS: We conclude that EBM may not be effective as a stand-alone program targeting a family planning method with a high level of provider and consumer bias. Evidence of positive selection into program participation underscores the importance of randomization to avoid overestimating the true effects of interventions. TRIAL REGISTRATION: AEA RCT Registry, AEARCTR0000539 , 11/3/2014.

User fee exemptions and equity in access to caesarean sections: an analysis of patient survey data in Mali.

INTRODUCTION: Little rigorous evidence exists on how health service utilization varies across socioeconomic groups after a user fee exemption policy has been implemented, and the evidence that does exist is mixed. In this paper, we estimate the distribution of caesarean section deliveries across socioeconomic groups following Mali's implementation of a fee exemption policy for caesareans in 2005. METHODS: We conducted a patient survey in 2010 to collect socioeconomic data from 2,477 women who had caesareans in a representative sample of 25 facilities across all regions of Mali. We used these data along with data from the most recent Demographic and Health Survey to construct a wealth index and classify women into population-based wealth groupings. We compared the wealth distribution of women delivering via caesarean section to that of a nationally representative sample of women giving birth. RESULTS: We found that wealthier women make up a disproportionate share of those having free caesareans, five years after implementation of the fee exemption policy. Women in the richest two quintiles accounted for 58 percent of all caesareans, while women in the poorest two quintiles accounted for 27 percent of all caesareans. Fewer women in the poorest two-fifths of the population are receiving caesareans than what we would expect given their share in the population of women giving birth. CONCLUSIONS: While fee exemptions remove important financial barriers to accessing priority maternal health services, they are insufficient to ensure equal access among wealth groups.