Cognitive Flexibility and Emotion Regulation in Eating Disorder Patients with Comorbid Generalized Anxiety and Posttraumatic Stress Symptoms.

Research has found that difficulties in emotion regulation negatively impact mental health, whereas cognitive flexibility may promote stress resilience and positive mental health. Little is known about cognitive flexibility and emotion regulation in people with comorbid eating disorder (ED) and anxiety and stress disorders. A transdiagnostic ED population (N=227) at an outpatient ED treatment facility completed several self-report instruments that measured cognitive flexibility, emotion regulation difficulties, posttraumatic stress disorder (PTSD) symptoms, and generalized anxiety disorder (GAD) symptoms upon admission. We investigated cognitive flexibility and emotion regulation differences for those with an ED without comorbidity and those with various combinations of comorbidity. In a one-way between-groups ANOVA, we investigated differences in cognitive flexibility for those with GAD, PTSD, neither, and both comorbidities. We found a statistically significant difference between these groups, with mean cognitive flexibility inventory scores being the lowest in the group with both comorbidities. However, when controlling for emotion regulation, a one-way between-groups ANCOVA indicated no significant differences in cognitive flexibility between comorbidity groups F(3,222)=1.20,p=.31 Partial η2=.02. Though self-reported cognitive flexibility levels differ among ED patients with and without comorbidities, it appears that these differences are better explained by emotion regulation. Therefore, addressing emotion regulation early in treatment for all individuals with EDs, regardless of comorbidity. Further research is needed to understand the impact of treating emotion regulation on ED treatment engagement, dropout, and effectiveness.

Does Terminology Matter When Measuring Stigmatizing Attitudes About Weight? Validation of a Modified Attitudes Toward Obese Persons Scale.

Commonly used medical terms like "obesity" and "overweight" have been identified as stigmatizing. Thus, this study sought to revise a commonly used measure of weight stigmatizing attitudes, the Attitudes Toward Obese Persons (ATOP) scale. We compared the original terminology in the ATOP (e.g., "obese")to a Modified version using neutral terms (e.g., "higher weight"). We randomized participants (N = 599) to either receive the original or Modified ATOP and compared their scores. There was no significant difference between the scores of participants who received the original ATOP and the Modified ATOP, t(597) = -2.46, p = .550. Through principal component analysis, we found the Modified ATOP is best used as a 13-item unidimensional measure. Findings suggest a Modified version of the ATOP with neutral language is suitable for assessing negative attitudes about higher-weight people without sacrificing psychometric properties. Further examination of the terminology used in weight stigma measures is needed to determine how to best assess weight stigma without reinforcing stigmatizing attitudes. The findings of the present study suggest that the use of neutral terms in measures of anti-fat bias is a promising solution that warrants further investigation.

Nanomolar anti-SARS-CoV-2 Omicron activity of the host-directed TMPRSS2 inhibitor N-0385 and synergistic action with direct-acting antivirals.

SARS-CoV-2 Omicron subvariants with increased transmissibility and immune evasion are spreading globally with alarming persistence. Whether the mutations and evolution of spike (S) Omicron subvariants alter the viral hijacking of human TMPRSS2 for viral entry remains to be elucidated. This is particularly important to investigate because of the large number and diversity of mutations of S Omicron subvariants reported since the emergence of BA.1. Here we report that human TMPRSS2 is a molecular determinant of viral entry for all the Omicron clinical isolates tested in human lung cells, including ancestral Omicron subvariants (BA.1, BA.2, BA.5), contemporary Omicron subvariants (BQ.1.1, XBB.1.5, EG.5.1) and currently circulating Omicron BA.2.86. First, we used a co-transfection assay to demonstrate the endoproteolytic cleavage by TMPRSS2 of spike Omicron subvariants. Second, we found that N-0385, a highly potent TMPRSS2 inhibitor, is a robust entry inhibitor of virus-like particles harbouring the S protein of Omicron subvariants. Third, we show that N-0385 exhibits nanomolar broad-spectrum antiviral activity against live Omicron subvariants in human Calu-3 lung cells and primary patient-derived bronchial epithelial cells. Interestingly, we found that N-0385 is 10-20 times more potent than the repositioned TMPRSS2 inhibitor, camostat, against BA.5, EG.5.1, and BA.2.86. We further found that N-0385 shows broad synergistic activity with clinically approved direct-acting antivirals (DAAs), i.e., remdesivir and nirmatrelvir, against Omicron subvariants, demonstrating the potential therapeutic benefits of a multi-targeted treatment based on N-0385 and DAAs.

A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants.

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

Synthetic Analogs of the Sponge Sesterterpenoid Alotaketal C are Potent Inhibitors of SARS-CoV-2 Omicron BA.1 and BA.5 Infections of Human Lung Cells.

The protein kinase C-activating sponge natural product alotaketal C (1) potently inhibits the infection of human Calu-3 lung cells by SARS-CoV-2 Omicron BA.1 and BA.5 variants. Simplified analogs of 1 have been synthesized and tested for anti-SARS-CoV-2 activity providing SAR data for the antiviral pharmacophore of 1. Analogs 19 and 23, which are missing the C-11 substituents in 1 and have modified C-13 appendages, are ∼2- to 7-fold more potent than 1 and have equal or larger selectivity indices.

Berbamine suppresses intestinal SARS-CoV-2 infection via a BNIP3-dependent autophagy blockade.

SARS-CoV-2, the causative virus of COVID-19, continues to threaten global public health. COVID-19 is a multi-organ disease, causing not only respiratory distress, but also extrapulmonary manifestations, including gastrointestinal symptoms with SARS-CoV-2 RNA shedding in stool long after respiratory clearance. Despite global vaccination and existing antiviral treatments, variants of concern are still emerging and circulating. Of note, new Omicron BA.5 sublineages both increasingly evade neutralizing antibodies and demonstrate an increased preference for entry via the endocytic entry route. Alternative to direct-acting antivirals, host-directed therapies interfere with host mechanisms hijacked by viruses, and enhance cell-mediated resistance with a reduced likelihood of drug resistance development. Here, we demonstrate that the autophagy-blocking therapeutic berbamine dihydrochloride robustly prevents SARS-CoV-2 acquisition by human intestinal epithelial cells via an autophagy-mediated BNIP3 mechanism. Strikingly, berbamine dihydrochloride exhibited pan-antiviral activity against Omicron subvariants BA.2 and BA.5 at nanomolar potency, providing a proof of concept for the potential for targeting autophagy machinery to thwart infection of current circulating SARS-CoV-2 subvariants. Furthermore, we show that autophagy-blocking therapies limited virus-induced damage to intestinal barrier function, affirming the therapeutic relevance of autophagy manipulation to avert the intestinal permeability associated with acute COVID-19 and post-COVID-19 syndrome. Our findings underscore that SARS-CoV-2 exploits host autophagy machinery for intestinal dissemination and indicate that repurposed autophagy-based antivirals represent a pertinent therapeutic option to boost protection and ameliorate disease pathogenesis against current and future SARS-CoV-2 variants of concern.

Discovery of lead natural products for developing pan-SARS-CoV-2 therapeutics.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC50) below 50 µM against mNG-SARS-CoV-2; 16 of these had EC50 values below 10 µM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants.

Dolutegravir-containing HIV therapy reversibly alters mitochondrial health and morphology in cultured human fibroblasts and peripheral blood mononuclear cells.

OBJECTIVES: Given the success of combination antiretroviral therapy (cART) in treating HIV viremia, drug toxicity remains an area of interest in HIV research. Despite newer integrase strand transfer inhibitors (InSTIs), such as dolutegravir (DTG) and raltegravir (RAL), having excellent clinical tolerance, there is emerging evidence of off-target effects and toxicities. Although limited in number, recent reports have highlighted the vulnerability of mitochondria to these toxicities. The aim of the present study is to quantify changes in cellular and mitochondrial health following exposure to current cART regimens at pharmacological concentrations. A secondary objective is to determine whether any cART-associated toxicities would be modulated by human telomerase reverse transcriptase (hTERT). METHODS: We longitudinally evaluated markers of cellular (cell count, apoptosis), and mitochondrial health [mitochondrial reactive oxygen species (mtROS), membrane potential (MMP) and mass (mtMass)] by flow cytometry in WI-38 human fibroblast with differing hTERT expression/localization and peripheral blood mononuclear cells. This was done after 9 days of exposure to, and 6 days following the removal of, seven current cART regimens, including three that contained DTG. Mitochondrial morphology was assessed by florescence microscopy and quantified using a recently developed deep learning-based pipeline. RESULTS: Exposure to DTG-containing regimens increased apoptosis, mtROS, mtMass, induced fragmented mitochondrial networks compared with non-DTG-containing regimens, including a RAL-based combination. These effects were unmodulated by telomerase expression. All effects were fully reversible following removal of drug pressure. CONCLUSION: Taken together, our observations indicate that DTG-containing regimens negatively impact cellular and mitochondrial health and may be more toxic to mitochondria, even among the generally well tolerated InSTI-based cART.

A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Enhanced Facilitated Diffusion of Membrane-Associating Proteins under Symmetric Confinement.

The facilitated surface diffusion of transiently adsorbing molecules in a planar confined microenvironment (i.e., slit-like confinement) is highly relevant to biological phenomena, such as extracellular signaling, as well as numerous biotechnology systems. Here, we studied the surface diffusion of individual proteins confined between two symmetric lipid bilayer membranes, under a continuum of confinement heights, using single-molecule tracking and convex lens-induced confinement as well as hybrid, kinetic Monte Carlo simulations of a generalized continuous time random walk process. Surface diffusion was observed to vary non-monotonically with confinement height, exhibiting a maximum at a height of ∼750 nm, where diffusion was nearly 40% greater than that for a semi-infinite system. This demonstrated that planar confinement can, in fact, increase surface diffusion, qualitatively validating previous theoretical predictions. Simulations reproduced the experimental results and suggested that confinement enhancement of surface diffusion for symmetric systems is limited to cases where the adsorbate exhibits weak surface sticking.

Minimal Cycle Representatives in Persistent Homology Using Linear Programming: An Empirical Study With User's Guide.

Cycle representatives of persistent homology classes can be used to provide descriptions of topological features in data. However, the non-uniqueness of these representatives creates ambiguity and can lead to many different interpretations of the same set of classes. One approach to solving this problem is to optimize the choice of representative against some measure that is meaningful in the context of the data. In this work, we provide a study of the effectiveness and computational cost of several ℓ 1 minimization optimization procedures for constructing homological cycle bases for persistent homology with rational coefficients in dimension one, including uniform-weighted and length-weighted edge-loss algorithms as well as uniform-weighted and area-weighted triangle-loss algorithms. We conduct these optimizations via standard linear programming methods, applying general-purpose solvers to optimize over column bases of simplicial boundary matrices. Our key findings are: 1) optimization is effective in reducing the size of cycle representatives, though the extent of the reduction varies according to the dimension and distribution of the underlying data, 2) the computational cost of optimizing a basis of cycle representatives exceeds the cost of computing such a basis, in most data sets we consider, 3) the choice of linear solvers matters a lot to the computation time of optimizing cycles, 4) the computation time of solving an integer program is not significantly longer than the computation time of solving a linear program for most of the cycle representatives, using the Gurobi linear solver, 5) strikingly, whether requiring integer solutions or not, we almost always obtain a solution with the same cost and almost all solutions found have entries in { - 1,0,1 } and therefore, are also solutions to a restricted ℓ 0 optimization problem, and 6) we obtain qualitatively different results for generators in Erdos-Rényi random clique complexes than in real-world and synthetic point cloud data.

Cadherin cis and trans interactions are mutually cooperative.

Cadherin transmembrane proteins are responsible for intercellular adhesion in all biological tissues and modulate tissue morphogenesis, cell motility, force transduction, and macromolecular transport. The protein-mediated adhesions consist of adhesive trans interactions and lateral cis interactions. Although theory suggests cooperativity between cis and trans bonds, direct experimental evidence of such cooperativity has not been demonstrated. Here, the use of superresolution microscopy, in conjunction with intermolecular single-molecule Förster resonance energy transfer, demonstrated the mutual cooperativity of cis and trans interactions. Results further demonstrate the consequent assembly of large intermembrane junctions, using a biomimetic lipid bilayer cell adhesion model. Notably, the presence of cis interactions resulted in a nearly 30-fold increase in trans-binding lifetimes between epithelial-cadherin extracellular domains. In turn, the presence of trans interactions increased the lifetime of cis bonds. Importantly, comparison of trans-binding lifetimes of small and large cadherin clusters suggests that this cooperativity is primarily due to allostery. The direct quantitative demonstration of strong mutual cooperativity between cis and trans interactions at intermembrane adhesions provides insights into the long-standing controversy of how weak cis and trans interactions act in concert to create strong macroscopic cell adhesions.

Cadherin clusters stabilized by a combination of specific and nonspecific cis-interactions.

We demonstrate a combined experimental and computational approach for the quantitative characterization of lateral interactions between membrane-associated proteins. In particular, weak, lateral (cis) interactions between E-cadherin extracellular domains tethered to supported lipid bilayers, were studied using a combination of dynamic single-molecule Förster Resonance Energy Transfer (FRET) and kinetic Monte Carlo (kMC) simulations. Cadherins are intercellular adhesion proteins that assemble into clusters at cell-cell contacts through cis- and trans- (adhesive) interactions. A detailed and quantitative understanding of cis-clustering has been hindered by a lack of experimental approaches capable of detecting and quantifying lateral interactions between proteins on membranes. Here single-molecule intermolecular FRET measurements of wild-type E-cadherin and cis-interaction mutants combined with simulations demonstrate that both nonspecific and specific cis-interactions contribute to lateral clustering on lipid bilayers. Moreover, the intermolecular binding and dissociation rate constants are quantitatively and independently determined, demonstrating an approach that is generalizable for other interacting proteins.

Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance.

Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.

Cadherin Extracellular Domain Clustering in the Absence of Trans-Interactions.

While both cis and trans (adhesive)-interactions cooperate in the assembly of intercellular adhesions, computational simulations have predicted that two-dimensional confinement may promote cis-oligomerization, in the absence of trans-interactions. Here, single-molecule tracking of cadherin extracellular domains on supported lipid bilayers revealed the density-dependent formation of oligomers and cis-clusters in the absence of trans-interactions. Lateral oligomers were virtually eliminated by mutating a putative cis (lateral) binding interface. At low cadherin surface coverage, wild-type and mutant cadherin diffused rapidly, consistent with the motion of a lipid molecule within a cadherin-free supported bilayer and with cadherins diffusing as monomers. Although the diffusion of mutant cadherin did not change appreciably with increasing surface coverage, the average short-time diffusion coefficient of wild-type cadherin slowed significantly above a fractional surface coverage of ∼0.01 (∼1100 molecules/µm2). A detailed analysis of molecular trajectories suggested the presence of a broad size distribution of cis-cadherin oligomers. These findings verify predictions that two-dimensional confinement promotes cis-oligomerization, in the absence of trans-interactions.

Transient Telomerase Inhibition with Imetelstat Impacts DNA Damage Signals and Cell-Cycle Kinetics.

Telomerase is the ribonucleoprotein reverse transcriptase that catalyzes the synthesis of telomeres at the ends of linear chromosomes and contributes to proper telomere-loop (T-loop) formation. Formation of the T-loop, an obligate step before cell division can proceed, requires the generation of a 3'-overhang on the G-rich strand of telomeric DNA via telomerase or C-strand specific nucleases. Here, it is discovered that telomerase activity is critical for efficient cell-cycle progression using transient chemical inhibition by the telomerase inhibitor, imetelstat. Telomerase inhibition changed cell cycle kinetics and increased the proportion of cells in G2-phase, suggesting delayed clearance through this checkpoint. Investigating the possible contribution of unstructured telomere ends to these cell-cycle distribution changes, it was observed that imetelstat treatment induced γH2AX DNA damage foci in a subset of telomerase-positive cells but not telomerase-negative primary human fibroblasts. Chromatin-immunoprecipitation with γH2AX antibodies demonstrated imetelstat treatment-dependent enrichment of this DNA damage marker at telomeres. Notably, the effects of telomerase inhibition on cell cycle profile alterations were abrogated by pharmacological inhibition of the DNA-damage-repair transducer, ATM. Also, imetelstat potentiation of etoposide, a DNA-damaging drug that acts preferentially during S-G2 phases of the cell cycle, depends on functional ATM signaling. Thus, telomerase inhibition delays the removal of ATM-dependent DNA damage signals from telomeres in telomerase-positive cancer cells and interferes with cell cycle progression through G2Implications: This study demonstrates that telomerase activity directly facilitates the progression of the cell cycle through modulation of transient telomere dysfunction signals. Mol Cancer Res; 16(8); 1215-25. ©2018 AACR.

Formation of a [2]pseudorotaxane based on a pillar[5]arene and a rigid guest in solution and in the solid state.

A pillar[5]arene with mono(ethylene oxide) substituents could form a highly stable [2]pseudorotaxane with a rigid guest both in solution and in the solid state.