Structural and functional basis of VLDLR usage by Eastern equine encephalitis virus.

The very-low-density lipoprotein receptor (VLDLR) comprises eight LDLR type A (LA) domains and supports entry of distantly related alphaviruses, including Eastern equine encephalitis virus (EEEV) and Semliki Forest virus (SFV). Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage more than one LA domain simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection. Whereas all EEEV strains show conservation of two VLDLR-binding sites, the EEEV PE-6 strain and a few other EEE complex members feature a single amino acid substitution that enables binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.

Vaginal Lactobacillus fatty acid response mechanisms reveal a metabolite-targeted strategy for bacterial vaginosis treatment.

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related lactobacilli, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the vaginal microbiota and enhances bacterial fitness by biochemically sequestering OA in a derivative form only ohyA-harboring organisms can exploit. OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro BV model, suggesting a metabolite-based treatment approach.

SnapShot: Tissue Clearing.

Tissue clearing has become an important tool for the investigation of biological systems in three dimensions. However, many pioneering techniques were based on serendipitous discoveries. Next-generation clearing methods have been (re)designed with a better understanding of the chemistry and physics required to equalize the refractive index throughout a sample which prevents the random bending of light that clouds biological tissues.

Physiology, biomechanics, and biomimetics of hagfish slime.

Hagfishes thwart attacks by fish predators by producing liters of defensive slime. The slime is produced when slime gland exudate is released into the predator's mouth, where it deploys in a fraction of a second and clogs the gills. Slime exudate is composed mainly of secretory products from two cell types, gland mucous cells and gland thread cells, which produce the mucous and fibrous components of the slime, respectively. Here, we review what is known about the composition of the slime, morphology of the slime gland, and physiology of the cells that produce the slime. We also discuss several of the mechanisms involved in the deployment of both mucous and thread cells during the transition from thick glandular exudate to ultradilute material. We review biomechanical aspects of the slime, along with recent efforts to produce biomimetic slime thread analogs, and end with a discussion of how hagfish slime may have evolved.

Clarifying Tissue Clearing.

Biological specimens are intrinsically three dimensional; however, because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by "clearing" the tissue have been ongoing for over a century, there have been a large number of recent innovations. This Review introduces the physical basis for light scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Rare coding variants in ten genes confer substantial risk for schizophrenia.

Rare coding variation has historically provided the most direct connections between gene function and disease pathogenesis. By meta-analysing the whole exomes of 24,248 schizophrenia cases and 97,322 controls, we implicate ultra-rare coding variants (URVs) in 10 genes as conferring substantial risk for schizophrenia (odds ratios of 3-50, P < 2.14 × 10-6) and 32 genes at a false discovery rate of <5%. These genes have the greatest expression in central nervous system neurons and have diverse molecular functions that include the formation, structure and function of the synapse. The associations of the NMDA (N-methyl-D-aspartate) receptor subunit GRIN2A and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GRIA3 provide support for dysfunction of the glutamatergic system as a mechanistic hypothesis in the pathogenesis of schizophrenia. We observe an overlap of rare variant risk among schizophrenia, autism spectrum disorders1, epilepsy and severe neurodevelopmental disorders2, although different mutation types are implicated in some shared genes. Most genes described here, however, are not implicated in neurodevelopment. We demonstrate that genes prioritized from common variant analyses of schizophrenia are enriched in rare variant risk3, suggesting that common and rare genetic risk factors converge at least partially on the same underlying pathogenic biological processes. Even after excluding significantly associated genes, schizophrenia cases still carry a substantial excess of URVs, which indicates that more risk genes await discovery using this approach.

Prevotella are major contributors of sialidases in the human vaginal microbiome.

Elevated bacterial sialidase activity in the female genital tract is strongly associated with poor health outcomes including preterm birth and bacterial vaginosis (BV). These negative effects may arise from sialidase-mediated degradation of the protective mucus layer in the cervicovaginal environment. Prior biochemical studies of vaginal bacterial sialidases have focused solely on the BV-associated organism Gardnerella vaginalis. Despite their implications for sexual and reproductive health, sialidases from other vaginal bacteria have not been characterized. Here, we show that vaginal Prevotella species produce sialidases that possess variable activity toward mucin substrates. The sequences of sialidase genes and their presence are largely conserved across clades of Prevotella from different geographies, hinting at their importance globally. Finally, we find that Prevotella sialidase genes and transcripts, including those encoding mucin-degrading sialidases from Prevotella timonensis, are highly prevalent and abundant in human vaginal genomes and transcriptomes. Together, our results identify Prevotella as a critical source of sialidases in the vaginal microbiome, improving our understanding of this detrimental bacterial activity.

Trial of an Intervention to Improve Acute Heart Failure Outcomes.

BACKGROUND: Patients with acute heart failure are frequently or systematically hospitalized, often because the risk of adverse events is uncertain and the options for rapid follow-up are inadequate. Whether the use of a strategy to support clinicians in making decisions about discharging or admitting patients, coupled with rapid follow-up in an outpatient clinic, would affect outcomes remains uncertain. METHODS: In a stepped-wedge, cluster-randomized trial conducted in Ontario, Canada, we randomly assigned 10 hospitals to staggered start dates for one-way crossover from the control phase (usual care) to the intervention phase, which involved the use of a point-of-care algorithm to stratify patients with acute heart failure according to the risk of death. During the intervention phase, low-risk patients were discharged early (in ≤3 days) and received standardized outpatient care, and high-risk patients were admitted to the hospital. The coprimary outcomes were a composite of death from any cause or hospitalization for cardiovascular causes within 30 days after presentation and the composite outcome within 20 months. RESULTS: A total of 5452 patients were enrolled in the trial (2972 during the control phase and 2480 during the intervention phase). Within 30 days, death from any cause or hospitalization for cardiovascular causes occurred in 301 patients (12.1%) who were enrolled during the intervention phase and in 430 patients (14.5%) who were enrolled during the control phase (adjusted hazard ratio, 0.88; 95% confidence interval [CI], 0.78 to 0.99; P = 0.04). Within 20 months, the cumulative incidence of primary-outcome events was 54.4% (95% CI, 48.6 to 59.9) among patients who were enrolled during the intervention phase and 56.2% (95% CI, 54.2 to 58.1) among patients who were enrolled during the control phase (adjusted hazard ratio, 0.95; 95% CI, 0.92 to 0.99). Fewer than six deaths or hospitalizations for any cause occurred in low- or intermediate-risk patients before the first outpatient visit within 30 days after discharge. CONCLUSIONS: Among patients with acute heart failure who were seeking emergency care, the use of a hospital-based strategy to support clinical decision making and rapid follow-up led to a lower risk of the composite of death from any cause or hospitalization for cardiovascular causes within 30 days than usual care. (Funded by the Ontario SPOR Support Unit and others; COACH ClinicalTrials.gov number, NCT02674438.).

Lactobacillus-Deficient Cervicovaginal Bacterial Communities Are Associated with Increased HIV Acquisition in Young South African Women.

Elevated inflammation in the female genital tract is associated with increased HIV risk. Cervicovaginal bacteria modulate genital inflammation; however, their role in HIV susceptibility has not been elucidated. In a prospective cohort of young, healthy South African women, we found that individuals with diverse genital bacterial communities dominated by anaerobes other than Gardnerella were at over 4-fold higher risk of acquiring HIV and had increased numbers of activated mucosal CD4+ T cells compared to those with Lactobacillus crispatus-dominant communities. We identified specific bacterial taxa linked with reduced (L. crispatus) or elevated (Prevotella, Sneathia, and other anaerobes) inflammation and HIV infection and found that high-risk bacteria increased numbers of activated genital CD4+ T cells in a murine model. Our results suggest that highly prevalent genital bacteria increase HIV risk by inducing mucosal HIV target cells. These findings might be leveraged to reduce HIV acquisition in women living in sub-Saharan Africa.

The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.

Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-ß is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-ß. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-ß. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.

Two C. elegans DM domain proteins, DMD-3 and MAB-3, function in late stages of male somatic gonad development.

To bring about sexual dimorphism in form, information from the sex determination pathway must trigger sex-specific modifications in developmental programs. DM-domain encoding genes have been found to be involved in sex determination in a multitude of animals, often at the level of male somatic gonad formation. Here we report our findings that the DM-domain transcription factors MAB-3 and DMD-3 function together in multiple steps during the late stages of C. elegans male somatic gonad development. Both mab-3 and dmd-3 are expressed in the linker cell and hindgut of L4 males and dmd-3 is also expressed in presumptive vas deferens cells. Furthermore, dmd-3, but not mab-3, expression in the linker cell is downstream of nhr-67, a nuclear hormone receptor that was previously shown to control late stages of linker cell migration. In mab-3; dmd-3 double mutant males, the last stage of linker cell migration is partially defective, resulting in aberrant linker cell shapes and often a failure of the linker cell to complete its migration to the hindgut. When mab-3; dmd-3 double mutant linker cells do complete their migration, they fail to be engulfed by the hindgut, indicating that dmd-3 and mab-3 activity are essential for this process. Furthermore, linker cell death and clearance are delayed in mab-3; dmd-3 double mutants, resulting in the linker cell persisting into adulthood. Finally, DMD-3 and MAB-3 function to activate expression of the bZIP transcription factor encoding gene zip-5 and downregulate the expression of the zinc metalloprotease ZMP-1 in the linker cell. Taken together, these results demonstrate a requirement for DM-domain transcription factors in controlling C. elegans male gonad formation, supporting the notion that the earliest DM-domain genes were involved in male somatic gonad development in the last common ancestor of the bilaterians.

International Initiatives of the American Heart Association: Original Concepts, Present Programs, and Future Focus: A Science Advisory From the American Heart Association.

The American Heart Association (AHA), founded in 1924, is anchored in the core belief that scientific research can lead the way to better prevention, treatment, recovery, and ultimately a cure for cardiovascular disease. Historically, the association's involvement in international efforts centered on scientific cooperation. Activities mostly involved AHA leadership presenting at international scientific meetings and leaders from other countries sharing scientific and medical information at AHA meetings. Although the AHA's and American Stroke Association's international efforts have expanded substantially since those early days, global knowledge exchange remains the bedrock of its international endeavors. As the AHA turns 100, we reflect on the successful global efforts in prevention, resuscitation, global advocacy, quality improvement, and health equity that have guided the organization to a place of readiness for "advancing health and hope, for everyone, everywhere." Motivated by the enormous potential for population health gains in an aging world, the AHA is entering its second century with redoubled commitment to improving global cardiovascular and cerebrovascular health for all.

Safety and Efficacy of a Third Dose of BNT162b2 Covid-19 Vaccine.

BACKGROUND: Active immunization with the BNT162b2 vaccine (Pfizer-BioNTech) has been a critical mitigation tool against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the coronavirus disease 2019 (Covid-19) pandemic. In light of reports of waning protection occurring 6 months after the primary two-dose vaccine series, data are needed on the safety and efficacy of offering a third (booster) dose in persons 16 years of age or older. METHODS: In this ongoing, placebo-controlled, randomized, phase 3 trial, we assigned participants who had received two 30-µg doses of the BNT162b2 vaccine at least 6 months earlier to be injected with a third dose of the BNT162b2 vaccine or with placebo. We assessed vaccine safety and efficacy against Covid-19 starting 7 days after the third dose. RESULTS: A total of 5081 participants received a third BNT162b2 dose and 5044 received placebo. The median interval between dose 2 and dose 3 was 10.8 months in the vaccine group and 10.7 months in the placebo group; the median follow-up was 2.5 months. Local and systemic reactogenicity events from the third dose were generally of low grade. No new safety signals were identified, and no cases of myocarditis or pericarditis were reported. Among the participants without evidence of previous SARS-CoV-2 infection who could be evaluated, Covid-19 with onset at least 7 days after dose 3 was observed in 6 participants in the vaccine group and in 123 participants in the placebo group, which corresponded to a relative vaccine efficacy of 95.3% (95% confidence interval, 89.5 to 98.3). CONCLUSIONS: A third dose of the BNT162b2 vaccine administered a median of 10.8 months after the second dose provided 95.3% efficacy against Covid-19 as compared with two doses of the BNT162b2 vaccine during a median follow-up of 2.5 months. (Funded by BioNTech and Pfizer; C4591031 ClinicalTrials.gov number, NCT04955626.).

Targeting breast and pancreatic cancer metastasis using a dual-cadherin antibody.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

Stroke risk in women with atrial fibrillation.

BACKGROUND AND AIMS: Female sex is associated with higher rates of stroke in atrial fibrillation (AF) after adjustment for other CHA2DS2-VASc factors. This study aimed to describe sex differences in age and cardiovascular care to examine their relationship with stroke hazard in AF. METHODS: Population-based cohort study using administrative datasets of people aged ≥66 years diagnosed with AF in Ontario between 2007 and 2019. Cause-specific hazard regression was used to estimate the adjusted hazard ratio (HR) for stroke associated with female sex over a 2-year follow-up. Model 1 included CHA2DS2-VASc factors, with age modelled as 66-74 vs. ≥ 75 years. Model 2 treated age as a continuous variable and included an age-sex interaction term. Model 3 further accounted for multimorbidity and markers of cardiovascular care. RESULTS: The cohort consisted of 354 254 individuals with AF (median age 78 years, 49.2% female). Females were more likely to be diagnosed in emergency departments and less likely to receive cardiologist assessments, statins, or LDL-C testing, with higher LDL-C levels among females than males. In Model 1, the adjusted HR for stroke associated with female sex was 1.27 (95% confidence interval 1.21-1.32). Model 2 revealed a significant age-sex interaction, such that female sex was only associated with increased stroke hazard at age >70 years. Adjusting for markers of cardiovascular care and multimorbidity further decreased the HR, so that female sex was not associated with increased stroke hazard at age ≤80 years. CONCLUSION: Older age and inequities in cardiovascular care may partly explain higher stroke rates in females with AF.

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii.

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

A conduit to metastasis: circulating tumor cell biology.

Advances in the enrichment and analysis of rare cells from the bloodstream have allowed for detection and characterization of circulating tumor cells (CTCs) from patients with cancer. The analysis of CTCs has provided significant insight into the metastatic process. Studies on the biology of CTCs have begun to elucidate the molecular mechanisms of CTC generation, intravasation, survival, interactions with components of the blood, extravasation, and colonization of distant organs. Additionally, the study of CTCs has exposed dramatic intrapatient and interpatient heterogeneity and their evolution over time. In this review, we focus on the current knowledge of CTC biology and the potential clinical implications.

Addition of temsirolimus to chemotherapy in children, adolescents, and young adults with intermediate-risk rhabdomyosarcoma (ARST1431): a randomised, open-label, phase 3 trial from the Children's Oncology Group.

BACKGROUND: The Children's Oncology Group defines intermediate-risk rhabdomyosarcoma as unresected FOXO1 fusion-negative disease arising at an unfavourable site or non-metastatic FOXO1 fusion-positive disease. Temsirolimus in combination with chemotherapy has shown promising activity in patients with relapsed or refractory rhabdomyosarcoma. We aimed to compare event-free survival in patients with intermediate-risk rhabdomyosarcoma treated with vincristine, actinomycin, and cyclophosphamide alternating with vincristine and irinotecan (VAC/VI) combined with temsirolimus followed by maintenance therapy versus VAC/VI alone with maintenance therapy. METHODS: ARST1431 was a randomised, open-label, phase 3 trial conducted across 210 institutions in Australia, Canada, New Zealand, and the USA. Eligible patients were those aged 40 years or younger with non-metastatic FOXO1-positive rhabdomyosarcoma or unresected FOXO1-negative rhabdomyosarcoma disease from unfavourable sites. Two other groups of patients were also eligible: those who had FOXO1-negative disease at a favourable site (excluding orbit) that was unresected; and those who were aged younger than 10 years with stage IV FOXO1-negative disease with distant metastases. Eligible patients had to have a Lansky performance status score of 50 or higher if 16 years or younger and a Karnofsky performance status score of 50 or higher if older than 16 years; all patients were previously untreated. Patients were randomised (1:1) in blocks of four and stratified by histology, stage, and group. Patients received intravenous VAC/VI chemotherapy with a cyclophosphamide dose of 1·2 g/m2 per dose per cycle with or without a reducing dose of intravenous weekly temsirolimus starting at 15 mg/m2 or 0·5 mg/kg per dose for those who weighed less than 10 kg. The total duration of therapy was 42 weeks followed by 6 months of maintenance therapy with oral cyclophosphamide plus intravenous vinorelbine for all patients. Temsirolimus was withheld during radiotherapy and for 2 weeks before any major surgical procedure. The primary endpoint was 3-year event-free survival. Data were analysed with a revised intention-to-treat approach. The study is registered with ClinicalTrials.gov (NCT02567435) and is complete. FINDINGS: Between May 23, 2016, and Jan 1, 2022, 325 patients were enrolled. In 297 evaluable patients (148 assigned to VAC/VI alone and 149 assigned to VAC/VI with temsirolimus), the median age was 6·3 years (IQR 3·0-11·3); 33 (11%) patients were aged 18 years or older; 179 (60%) of 297 were male. 113 (77%) of 148 patients were FOXO1 negative in the VAC/VI group, and 108 (73%) of 149 were FOXO1 negative in the VAC/VI with temsirolimus group. With a median follow-up of 3·6 years (IQR 2·8-4·5), 3-year event-free survival did not differ significantly between the two groups (64·8% [95% CI 55·5-74·1] in the VAC/VI group vs 66·8% [57·5-76·2] in the VAC/VI plus temsirolimus group (hazard ratio 0·86 [95% CI 0·58-1·26]; log-rank p=0·44). The most common grade 3-4 adverse events were anaemia (62 events in 60 [41%] of 148 patients in the VAC/VI group vs 89 events in 87 [58%] of 149 patients in the VAC/VI with temsirolimus group), lymphopenia (83 events in 65 [44%] vs 99 events in 71 [48%]), neutropenia (160 events in 99 [67%] vs 164 events in 105 [70%]), and leukopenia (121 events in 86 [58%] vs 132 events in 93 [62%]). There was one treatment-related death in the VAC/VI with temsirolimus group, categorised as not otherwise specified. INTERPRETATION: Addition of temsirolimus to VAC/VI did not improve event-free survival in patients with intermediate-risk rhabdomyosarcoma defined by their FOXO1 translocation status and clinical factors. Novel biology-based strategies are needed to improve outcomes in this population. FUNDING: The Children's Oncology Group (supported by the US National Cancer Institute, US National Institutes of Health).

Directional change during active diffusion of viral ribonucleoprotein particles through cytoplasm.

A mesh of cytoskeletal fibers, consisting of microtubules, intermediate filaments, and fibrous actin, prevents the Brownian diffusion of particles with a diameter larger than 0.10 µm, such as vesicular stomatitis virus ribonucleoprotein (RNP) particles, in mammalian cells. Nevertheless, RNP particles do move in random directions but at a lower rate than Brownian diffusion, which is thermally driven. This nonthermal biological transport process is called "active diffusion" because it is driven by ATP. The ATP powers motor proteins such as myosin II. The motor proteins bend and cross-link actin fibers, causing the mesh to jiggle. Until recently, little was known about how RNP particles get through the mesh. It has been customary to analyze the tracks of particles like RNPs by computing the slope of the ensemble-averaged mean-squared displacement of the particles as a signature of mechanism. Although widely used, this approach "loses information" about the timing of the switches between physical mechanisms. It has been recently shown that machine learning composed of variational Bayesian analysis, Gaussian mixture models, and hidden Markov models can use "all the information" in a single track to reveal that that the positions of RNP particles are spatially clustered. Machine learning assigns a number, called a state, to each cluster. RNP particles remain in one state for 0.2-1.0 s before switching (hopping) to a different state. This earlier work is here extended to analyze the movements of a particle within a state and to determine particle directionality within and between states.