High-throughput kinetics in drug discovery.

The importance of a drug's kinetic profile and interplay of structure-kinetic activity with PK/PD has long been appreciated in drug discovery. However, technical challenges have often limited detailed kinetic characterization of compounds to the latter stages of projects. This review highlights the advances that have been made in recent years in techniques, instrumentation, and data analysis to increase the throughput of detailed kinetic and mechanistic characterization, enabling its application earlier in the drug discovery process.

Aspartate ß-Hydroxylase Is Upregulated in Head and Neck Squamous Cell Carcinoma and Regulates Invasiveness in Cancer Cell Models.

Aspartate ß-hydroxylase (ASPH) is a protein associated with malignancy in a wide range of tumors. We hypothesize that inhibition of ASPH activity could have anti-tumor properties in patients with head and neck cancer. In this study, we screened tumor tissues of 155 head and neck squamous cell carcinoma (HNSCC) patients for the expression of ASPH using immunohistochemistry. We used an ASPH inhibitor, MO-I-1151, known to inhibit the catalytic activity of ASPH in the endoplasmic reticulum, to show its inhibitory effect on the migration of SCC35 head and neck cancer cells in cell monolayers and in matrix-embedded spheroid co-cultures with primary cancer-associated fibroblast (CAF) CAF 61137 of head and neck origin. We also studied a combined effect of MO-I-1151 and HfFucCS, an inhibitor of invasion-blocking heparan 6-O-endosulfatase activity. We found ASPH was upregulated in HNSCC tumors compared to the adjacent normal tissues. ASPH was uniformly high in expression, irrespective of tumor stage. High expression of ASPH in tumors led us to consider it as a therapeutic target in cell line models. ASPH inhibitor MO-I-1151 had significant effects on reducing migration and invasion of head and neck cancer cells, both in monolayers and matrix-embedded spheroids. The combination of the two enzyme inhibitors showed an additive effect on restricting invasion in the HNSCC cell monolayers and in the CAF-containing co-culture spheroids. We identify ASPH as an abundant protein in HNSCC tumors. Targeting ASPH with inhibitor MO-I-1151 effectively reduces CAF-mediated cellular invasion in cancer cell models. We propose that the additive effect of MO-I-1151 with HfFucCS, an inhibitor of heparan 6-O-endosulfatases, on HNSCC cells could improve interventions and needs to be further explored.

The Variability of MCID, SCB, PASS, and MOI Thresholds for PROMs in the Reverse Total Shoulder Arthroplasty Literature: A Systematic Review.

BACKGROUND: Reverse total shoulder arthroplasty (RTSA) is a common procedure utilized to address degenerative pathologies of the glenohumeral joint and rotator cuff. Increased reliance on patient-reported outcome measures (PROMs) has placed emphasis on the utilization of the minimum clinically important difference (MCID), substantial clinical benefit (SCB), patient acceptable symptom state (PASS), and maximal outcome improvement (MOI) thresholds to assess the clinical efficacy of RTSA. In this study, we systematically reviewed the MCID, SCB, PASS, and MOI thresholds reported for PROMs following RTSA. METHODS: PubMed, Embase, MEDLINE, Cochrane Library, and Google Scholar were queried for articles from Jan. 1, 2000-Aug. 31, 2023 reporting MCID, SCB, PASS, or MOI values for PROMs following RTSA. Patient demographic data, study characteristics, MCID/SCB/PASS/MOI thresholds, and threshold calculation methods were extracted. RESULTS: 141 articles were screened with 39 ultimately included, comprising 11,984 total patients that underwent RTSA. 34 (87%) studies reported MCID thresholds, 20 (51%) reported SCB, 5 (13%) reported PASS, and 2 (5%) reported MOI. 25/39 (64%) studies referenced a previous study when reporting MCID, SCB, PASS, or MOI values, 11 (28%) used an anchor-based method to calculate threshold values, 1 (3%) used a distribution-based method, and 2 (5%) used both anchor and distribution methods. There were 19 newly calculated MCID (11), SCB (5), PASS (1), and MOI (2) thresholds. For five of the six most utilized PROMs (ASES, SST, Constant, UCLA, SPADI), the range of reported MCID values exceeded 50% of the most common threshold. For three of the six, the range of SCB values exceeded 25% of the most common threshold. CONCLUSION: There is substantial variability in the MCID and SCB threshold values reported in the RTSA literature. Standardizing the methodologic calculation and utilization of MCID, SCB, PASS, and MOI thresholds for RTSA may allow for improved assessment of PROMs.

Quantitative and Standardized Pseudovirus Neutralization Assay for COVID-19.

COVID-19 is a global pandemic caused by the highly infectious SARS-CoV-2 virus. Efforts to combat SARS-CoV-2 infection include mass vaccination and development of monoclonal and convalescent plasma therapeutics that require precise measurements of correlative, functional neutralizing antibodies that prevent virus infection. Developing rapid, safe, easy-to-use, and high-quality neutralization assays are essential for the success of the massive effort. Here, we developed a vesicular stomatitis virus-based neutralization assay that was capable of quantifying varying degrees of neutralization in patient serum samples. This assay has two detection readouts, flow cytometry and live cell imaging. The two readout methods produced consistent values of all 50% neutralization titers, further enhancing measurement confidence on the assay. Moreover, the use of available reference standards such as the World Health Organization International Standard (NIBSC code 20/136) enables quantification and standardization of the pseudovirus neutralization assay with neutralizing antibody titers measured in International Units/mL. Quantitative and standardized neutralization assays are critical for reliable efficacy evaluation and comparison of numerous vaccines and therapeutics.

A Comparative Study of Genetic Diversity and Multiplicity of Infection in Uncomplicated Plasmodium falciparum Infections in Selected Regions of Pre-Elimination and High Transmission Settings Using MSP1 and MSP2 Genes.

BACKGROUND: Understanding the genetic structure of P. falciparum population in different regions is pivotal to malaria elimination. Genetic diversity and the multiplicity of infection are indicators used for measuring malaria endemicity across different transmission settings. Therefore, this study characterized P. falciparum infections from selected areas constituting pre-elimination and high transmission settings in South Africa and Nigeria, respectively. METHODS: Parasite genomic DNA was extracted from 129 participants with uncomplicated P. falciparum infections. Isolates were collected from 78 participants in South Africa (southern Africa) and 51 in Nigeria (western Africa). Allelic typing of the msp1 and msp2 genes was carried out using nested PCR. RESULTS: In msp1, the K1 allele (39.7%) was the most common allele among the South African isolates, while the RO33 allele (90.2%) was the most common allele among the Nigerian isolates. In the msp2 gene, FC27 and IC3D7 showed almost the same percentage distribution (44.9% and 43.6%) in the South African isolates, whereas FC27 had the highest percentage distribution (60.8%) in the Nigerian isolates. The msp2 gene showed highly distinctive genotypes, indicating high genetic diversity in the South African isolates, whereas msp1 showed high genetic diversity in the Nigerian isolates. The RO33 allelic family displayed an inverse relationship with participants' age in the Nigerian isolates. The overall multiplicity of infection (MOI) was significantly higher in Nigeria (2.87) than in South Africa (2.44) (p < 0.000 *). In addition, heterozygosity was moderately higher in South Africa (1.46) than in Nigeria (1.13). CONCLUSIONS: The high genetic diversity and MOI in P. falciparum that were observed in this study could provide surveillance data, on the basis of which appropriate control strategies should be adopted.

Automating Predictive Phage Therapy Pharmacology.

Viruses that infect as well as often kill bacteria are called bacteriophages, or phages. Because of their ability to act bactericidally, phages increasingly are being employed clinically as antibacterial agents, an infection-fighting strategy that has been in practice now for over one hundred years. As with antibacterial agents generally, the development as well as practice of this phage therapy can be aided via the application of various quantitative frameworks. Therefore, reviewed here are considerations of phage multiplicity of infection, bacterial likelihood of becoming adsorbed as a function of phage titers, bacterial susceptibility to phages also as a function of phage titers, and the use of Poisson distributions to predict phage impacts on bacteria. Considered in addition is the use of simulations that can take into account both phage and bacterial replication. These various approaches can be automated, i.e., by employing a number of online-available apps provided by the author, the use of which this review emphasizes. In short, the practice of phage therapy can be aided by various mathematical approaches whose implementation can be eased via online automation.

Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

Carry-Over Effects of Climate Variability at Breeding and Non-Breeding Grounds on Spring Migration in the European Wren Troglodytes troglodytes at the Baltic Coast.

Many studies have linked changes in avian phenology in Europe to the North Atlantic Oscillation (NAO), which serves as a proxy for conditions in western Europe. However, the effects of climate variation in other regions of Europe on the phenology of short-distance migrants with large non-breeding grounds remain unclear. We determined the combined influence of large-scale climate indices, NAO, the Mediterranean Oscillation Index (MOI), and the Scandinavian Pattern (SCAND), during the preceding year on spring migration timing of European wren at the southern Baltic coast during 1982-2021. We modelled the effects of these climate variables on the entire passage and subsequent percentiles of the wren's passage at Bukowo-Kopan and Hel ringing stations. Over 1982-2021, the start and median of migration shifted earlier at Hel, but the end of passage shifted later at both stations. In effect, the duration of passage at Hel was extended by 7.6 days. Early passage at Hel was related with high MOI in spring and the preceding autumn. Spring passage at Bukowo-Kopan was delayed after high NAO in the previous breeding season, and high winter and spring NAO. Late spring passage occurred at both stations following a high SCAND in the previous summer. At both locations, an early start or median of passage followed high local temperatures. We conclude that phenology of the wren's spring migration at the Baltic coast was shaped by conditions encountered at wintering quarters in western Europe, where NAO operates, and in the south-eastern Europe, where the MOI operates, in conjunction with conditions in Scandinavia during the previous breeding season. We demonstrated that climate variability in various parts of the migrants' range has combined carry-over effects on in migrants' phenology in Europe.

Human IGF2 Gene Epigenetic and Transcriptional Regulation: At the Core of Developmental Growth and Tumorigenic Behavior.

Regulation of the human IGF2 gene displays multiple layers of control, which secures a genetically and epigenetically predetermined gene expression pattern throughout embryonal growth and postnatal life. These predominantly nuclear regulatory mechanisms converge on the function of the IGF2-H19 gene cluster on Chromosome 11 and ultimately affect IGF2 gene expression. Deregulation of such control checkpoints leads to the enhancement of IGF2 gene transcription and/or transcript stabilization, ultimately leading to IGF-II peptide overproduction. This type of anomaly is responsible for the effects observed in terms of both abnormal fetal growth and increased cell proliferation, typically observed in pediatric overgrowth syndromes and cancer. We performed a review of relevant experimental work on the mechanisms affecting the human IGF2 gene at the epigenetic, transcriptional and transcript regulatory levels. The result of our work, indeed, provides a wider and diversified scenario for IGF2 gene activation than previously envisioned by shedding new light on its extended regulation. Overall, we focused on the functional integration between the epigenetic and genetic machinery driving its overexpression in overgrowth syndromes and malignancy, independently of the underlying presence of loss of imprinting (LOI). The molecular landscape provided at last strengthens the role of IGF2 in cancer initiation, progression and malignant phenotype maintenance. Finally, this review suggests potential actionable targets for IGF2 gene- and regulatory protein target-degradation therapies.

Uma leitura psicanalítica sobre o consumo de manipulações corporais na contemporaneidade / Une lecture psychanalytique sur la consommation des manipulations corporelles à l'époque contemporaine / A psychoanalytical reading on the consumption of body manipulations in contemporary times

O presente artigo visa abordar o fenômeno contemporâneo do consumo de manipulações corporais do tipo estético a partir da teoria e da clínica psicanalíticas. Nossa hipótese de pesquisa é que a menor potência do ideal do eu em sua vertente de interdito afeta a consistência da imagem corporal, culminando em um sofrimento psíquico provocado pelo desregramento pulsional. Indagamos se a intervenção no corpo poderia apaziguar a relação imaginária através de uma idealização do corpo vendida como mercadoria

Cet article vise à aborder le phénomène contemporain de consommation de manipulations corporelles de type esthétique en s'appuyant sur la théorie et la clinique psychanalytiques. Notre hypothèse de recherche est que la puissance inférieure de l'Idéal du Moi dans son aspect d'interdit affecte la cohérence de l'imagecorporelle, culminant dans la souffrance psychique causée par le trouble instinctif. Nous nous sommes demandé si l'intervention sur le corps pouvait apaiser la relation imaginaire par une idéalisation du corps vendu comme une marchandise

This article aims to address the contemporary phenomenon of consumption of body manipulations of the aesthetic type based on psychoanalytic theory and clinics. Our research hypothesis is that the lower power of the ego ideal in its interdict aspect affects the consistency of the body image, culminating in psychic suffering caused by instinctual disorder. We asked whether intervention in the body could appease the imaginary relationship through an idealization of the body sold as a merchandise.

Enforced Disappearances and Torture Today: A View from Analytical Psychology 2. Torture Survivors and the Unthinkable: A Hyper-Present Body in the Therapeutic Process: 2. Torture Survivors and the Unthinkable: A Hyper-Present Body in the Therapeutic Process1.

In very rare cases, individuals survive the atrocities of abduction, imprisonment and torture that are part of the hallmark of enforced disappearances. Cases of people who survive torture and seek asylum in a third country help us understand some important aspects related to the crime of enforced disappearance. In the psychotherapy of torture survivors, at an early stage and for a long time, words often do not convey the core of the patient's experience. Survivors usually have tormented bodies in which individual and collective violence, hatred, anger, guilt and shame are painfully inscribed. Corporeal countertransference becomes the only possible way for a therapist to get in touch with a survivor's experience through a kind of body-to-body communication. The centrality of the body in these therapies suggests that the body is the involuntary recipient and container of mass political atrocities and, for this reason, the place where, in the case of horrific social violence, the possibility of social "knowing" is stored and can be retrieved. Thus, when it comes to forced disappearance, the determination of the relatives to get to the truth through the discovery of the remains of their disappeared demonstrates the importance of the body as the final witness of what happened, beyond any possible manipulation.

Dans de très rares cas, des personnes survivent aux atrocités de l'enlèvement, de l'emprisonnement et de la torture qui caractérisent les disparitions forcées. Les cas de personnes qui survivent à la torture et demandent l'asile dans un pays tiers nous aident à comprendre certains aspects importants liés au crime de disparition forcée. Dans la psychothérapie des survivants de la torture, à un stade précoce et pendant longtemps, les mots ne transmettent souvent pas le cœur de l'expérience du patient. Les survivants ont généralement des corps tourmentés dans lesquels la violence individuelle et collective, la haine, la colère, le sentiment de culpabilité et la honte sont douloureusement inscrites. Le contre-transfert corporel devient le seul moyen possible pour un thérapeute d'entrer en contact avec l'expérience d'un survivant par une sorte de communication de corps à corps. La centralité du corps dans ces thérapies suggère que le corps est le destinataire involontaire et le conteneur des atrocités politiques de masse et, pour cette raison, le lieu où, dans le cas d'une violence sociale horrible, la possibilité d'un "savoir" social est stockée et peut être récupérée. Ainsi, lorsqu'il s'agit de disparitions forcées, la détermination des des membres de la famille à accéder à la vérité en retrouvant les restes de la personne disparue montre l'importance du corps en tant que dernier témoin de ce qui a eu lieu, au-delà de toute manipulation.

En casos muy raros, las personas sobreviven a las atrocidades del secuestro, el encarcelamiento y la tortura que forman parte del sello distintivo de las desapariciones forzadas. Los casos de personas que sobreviven a la tortura y buscan asilo en un tercer país nos ayudan a comprender algunos aspectos importantes relacionados con el delito de desaparición forzada. En la psicoterapia de supervivientes de tortura, en una fase temprana y durante mucho tiempo, las palabras no suelen transmitir el núcleo de la experiencia del paciente. Los supervivientes suelen tener cuerpos atormentados en los que se inscriben dolorosamente la violencia individual y colectiva, el odio, la ira, el sentimiento de culpa y la vergüenza. La contratransferencia corporal se convierte en la única vía posible para que un terapeuta entre en contacto con la experiencia de un superviviente a través de una especie de comunicación cuerpo a cuerpo. La centralidad del cuerpo en estas terapias sugiere que el cuerpo es el receptor involuntario y el contenedor de las atrocidades políticas masivas y, por esta razón, el lugar donde, en el caso de la violencia social horrorosa, se almacena y puede recuperarse la posibilidad del "conocimiento" social. Así, cuando se trata de desapariciones forzadas, la determinación de los familiares por llegar a la verdad a través del hallazgo de los restos de sus desaparecidos demuestra la importancia del cuerpo como testigo final de lo sucedido, más allá de cualquier posible manipulación.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Application of the PHENotype SIMulator for rapid identification of potential candidates in effective COVID-19 drug repurposing.

The current, rapidly diversifying pandemic has accelerated the need for efficient and effective identification of potential drug candidates for COVID-19. Knowledge on host-immune response to SARS-CoV-2 infection, however, remains limited with few drugs approved to date. Viable strategies and tools are rapidly arising to address this, especially with repurposing of existing drugs offering significant promise. Here we introduce a systems biology tool, the PHENotype SIMulator, which -by leveraging available transcriptomic and proteomic databases-allows modeling of SARS-CoV-2 infection in host cells in silico to i) determine with high sensitivity and specificity (both>96%) the viral effects on cellular host-immune response, resulting in specific cellular SARS-CoV-2 signatures and ii) utilize these cell-specific signatures to identify promising repurposable therapeutics. Powered by this tool, coupled with domain expertise, we identify several potential COVID-19 drugs including methylprednisolone and metformin, and further discern key cellular SARS-CoV-2-affected pathways as potential druggable targets in COVID-19 pathogenesis.

Hepatitis C virus replication requires integrity of mitochondria-associated ER membranes.

Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

Human Cardiac Pericytes Are Susceptible to SARS-CoV-2 Infection.

COVID-19 is associated with serious cardiovascular complications, with incompletely understood mechanism(s). Pericytes have key functions in supporting endothelial cells and maintaining vascular integrity. We demonstrate that human cardiac pericytes are permissive to SARS-CoV-2 infection in organotypic slice and primary cell cultures. Viral entry into pericytes is mediated by endosomal proteases, and infection leads to up-regulation of inflammatory markers, vasoactive mediators, and nuclear factor kappa-B-dependent cell death. Furthermore, we present evidence of cardiac pericyte infection in COVID-19 myocarditis patients. These data demonstrate that human cardiac pericytes are susceptible to SARS-CoV-2 infection and suggest a role for pericyte infection in COVID-19.

Low genetic diversity of Plasmodium falciparum merozoite surface protein 1 and 2 and multiplicity of infections in western Ethiopia following effective malaria interventions.

BACKGROUND: Genetic diversity of malaria parasites can inform the intensity of transmission and poses a major threat to malaria control and elimination interventions. Characterization of the genetic diversity would provide essential information about the ongoing control efforts. This study aimed to explore allelic polymorphism of merozoite surface protein 1 (msp1) and merozoite surface protein 2 (msp2) to determine the genetic diversity and multiplicity of Plasmodium falciparum infections circulating in high and low transmission sites in western Ethiopia. METHODS: Parasite genomic DNA was extracted from a total of 225 dried blood spots collected from confirmed uncomplicated P. falciparum malaria-infected patients in western Ethiopia. Of these, 72.4% (163/225) and 27.6% (62/225) of the samples were collected in high and low transmission areas, respectively. Polymorphic msp1 and msp2 genes were used to explore the genetic diversity and multiplicity of falciparum malaria infections. Genotyping of msp1 was successful in 86.5% (141/163) and 88.7% (55/62) samples collected from high and low transmission areas, respectively. Genotyping of msp2 was carried out among 85.3% (139/163) and 96.8% (60/62) of the samples collected in high and low transmission sites, respectively. Plasmodium falciparum msp1 and msp2 genes were amplified by nested PCR and the PCR products were analysed by QIAxcel ScreenGel Software. A P-value of less or equal to 0.05 was considered significant. RESULTS: High prevalence of falciparum malaria was identified in children less than 15 years as compared with those ≥ 15 years old (AOR = 2.438, P = 0.005). The three allelic families of msp1 (K1, MAD20, and RO33) and the two allelic families of msp2 (FC27 and 3D7), were observed in samples collected in high and low transmission areas. However, MAD 20 and FC 27 alleles were the predominant allelic families in both settings. Plasmodium falciparum isolates circulating in western Ethiopia had low genetic diversity and mean MOI. No difference in mean MOI between high transmission sites (mean MOI 1.104) compared with low transmission area (mean MOI 1.08) (p > 0.05). The expected heterozygosity of msp1 was slightly higher in isolates collected from high transmission sites (He = 0.17) than in those isolates from low transmission (He = 0.12). However, the heterozygosity of msp2 was not different in both settings (Pfmsp2: 0.04 in high transmission; pfmsp2: 0.03 in low transmission). CONCLUSION: Plasmodium falciparum from clinical malaria cases in western Ethiopia has low genetic diversity and multiplicity of infection irrespective of the intensity of transmission at the site of sampling. These may be signaling the effectiveness of malaria control strategies in Ethiopia; although further studies are required to determine how specific intervention strategies and other parameters that drive the pattern.

Delayed Lysis Time at High Multiplicities of Particles in a Chlorovirus-Chlorella Interaction.

When viruses infect microbial cells, their phenotypes depend on the host's genotype and on the environmental conditions. Here we describe such an effect in laboratory strains of the chlorovirus PBCV-1 and its algal host Chlorella variabilis. We studied the growth of six virus isolates, and found that the mean lysis time was 1.34±0.05 times longer at multiplicity of particles (MOP) 10 than at MOP 1. We could not detect any associated changes in burst size. This is a novel plastic trait for chloroviruses, and we hypothesize that it is caused by our specific laboratory algae.

Plasticity and therapeutic potential of cAMP and cGMP-specific phosphodiesterases in Toxoplasma gondii.

Toxoplasma gondii is a common zoonotic protozoan pathogen adapted to intracellular parasitism in many host cells of diverse organisms. Our previous work has identified 18 cyclic nucleotide phosphodiesterase (PDE) proteins encoded by the parasite genome, of which 11 are expressed during the lytic cycle of its acutely-infectious tachyzoite stage in human cells. Here, we show that ten of these enzymes are promiscuous dual-specific phosphodiesterases, hydrolyzing cAMP and cGMP. TgPDE1 and TgPDE9, with a Km of 18 µM and 31 µM, respectively, are primed to hydrolyze cGMP, whereas TgPDE2 is highly specific to cAMP (Km, 14 µM). Immuno-electron microscopy revealed various subcellular distributions of TgPDE1, 2, and 9, including in the inner membrane complex, apical pole, plasma membrane, cytosol, dense granule, and rhoptry, indicating spatial control of signaling within tachyzoites. Notably, despite shared apical location and dual-catalysis, TgPDE8 and TgPDE9 are fully dispensable for the lytic cycle and show no functional redundancy. In contrast, TgPDE1 and TgPDE2 are individually required for optimal growth, and their collective loss is lethal to the parasite. In vitro phenotyping of these mutants revealed the roles of TgPDE1 and TgPDE2 in proliferation, gliding motility, invasion and egress of tachyzoites. Moreover, our enzyme inhibition assays in conjunction with chemogenetic phenotyping underpin TgPDE1 as a target of commonly-used PDE inhibitors, BIPPO and zaprinast. Finally, we identified a retinue of TgPDE1 and TgPDE2-interacting kinases and phosphatases, possibly regulating the enzymatic activity. In conclusion, our datasets on the catalytic function, physiological relevance, subcellular localization and drug inhibition of key phosphodiesterases highlight the previously-unanticipated plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii.

Evolutionary genetics of malaria.

Many standard-textbook population-genetic results apply to a wide range of species. Sometimes, however, population-genetic models and principles need to be tailored to a particular species. This is particularly true for malaria, which next to tuberculosis and HIV/AIDS ranks among the economically most relevant infectious diseases. Importantly, malaria is not one disease-five human-pathogenic species of Plasmodium exist. P. falciparum is not only the most severe form of human malaria, but it also causes the majority of infections. The second most relevant species, P. vivax, is already considered a neglected disease in several endemic areas. All human-pathogenic species have distinct characteristics that are not only crucial for control and eradication efforts, but also for the population-genetics of the disease. This is particularly true in the context of selection. Namely, fitness is determined by so-called fitness components, which are determined by the parasites live-history, which differs between malaria species. The presence of hypnozoites, i.e., dormant liver-stage parasites, which can cause disease relapses, is a distinct feature of P. vivax and P. ovale sp. In P. malariae inactivated blood-stage parasites can cause a recrudescence years after the infection was clinically cured. To properly describe population-genetic processes, such as the spread of anti-malarial drug resistance, these features must be accounted for appropriately. Here, we introduce and extend a population-genetic framework for the evolutionary dynamics of malaria, which applies to all human-pathogenic malaria species. The model focuses on, but is not limited to, the spread of drug resistance. The framework elucidates how the presence of dormant liver stage or inactivated blood stage parasites that act like seed banks delay evolutionary processes. It is shown that, contrary to standard population-genetic theory, the process of selection and recombination cannot be decoupled in malaria. Furthermore, we discuss the connection between haplotype frequencies, haplotype prevalence, transmission dynamics, and relapses or recrudescence in malaria.