Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications.

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.

Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems.

The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.

Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system.

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.

The MKK3 MAPK cascade integrates temperature and after-ripening signals to modulate seed germination.

The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.

CNS autoimmune response in the MAM/pilocarpine rat model of epileptogenic cortical malformation.

The development of seizures in epilepsy syndromes associated with malformations of cortical development (MCDs) has traditionally been attributed to intrinsic cortical alterations resulting from abnormal network excitability. However, recent analyses at single-cell resolution of human brain samples from MCD patients have indicated the possible involvement of adaptive immunity in the pathogenesis of these disorders. By exploiting the MethylAzoxyMethanol (MAM)/pilocarpine (MP) rat model of drug-resistant epilepsy associated with MCD, we show here that the occurrence of status epilepticus and subsequent spontaneous recurrent seizures in the malformed, but not in the normal brain, are associated with the outbreak of a destructive autoimmune response with encephalitis-like features, involving components of both cell-mediated and humoral immune responses. The MP brain is characterized by blood-brain barrier dysfunction, marked and persisting CD8+ T cell invasion of the brain parenchyma, meningeal B cell accumulation, and complement-dependent cytotoxicity mediated by antineuronal antibodies. Furthermore, the therapeutic treatment of MP rats with the immunomodulatory drug fingolimod promotes both antiepileptogenic and neuroprotective effects. Collectively, these data show that the MP rat could serve as a translational model of epileptogenic cortical malformations associated with a central nervous system autoimmune response. This work indicates that a preexisting brain maldevelopment predisposes to a secondary autoimmune response, which acts as a precipitating factor for epilepsy and suggests immune intervention as a therapeutic option to be further explored in epileptic syndromes associated with MCDs.

Epigenetic alterations fuel brain metastasis via regulating inflammatory cascade.

Brain metastasis (BrM) is a major threat to the survival of melanoma, breast, and lung cancer patients. Circulating tumor cells (CTCs) cross the blood-brain barrier (BBB) and sustain in the brain microenvironment. Genetic mutations and epigenetic modifications have been found to be critical in controlling key aspects of cancer metastasis. Metastasizing cells confront inflammation and gradually adapt in the unique brain microenvironment. Currently, it is one of the major areas that has gained momentum. Researchers are interested in the factors that modulate neuroinflammation during BrM. We review here various epigenetic factors and mechanisms modulating neuroinflammation and how this helps CTCs to adapt and survive in the brain microenvironment. Since epigenetic changes could be modulated by targeting enzymes such as histone/DNA methyltransferase, deacetylases, acetyltransferases, and demethylases, we also summarize our current understanding of potential drugs targeting various aspects of epigenetic regulation in BrM.

Unity among the diverse RNA-guided CRISPR-Cas interference mechanisms.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements (MGEs). The Cas protein-CRISPR RNA (crRNA) complex uses complementarity of the crRNA "guide" region to specifically recognize the invader genome. CRISPR effectors that perform targeted destruction of the foreign genome have emerged independently as multi-subunit protein complexes (Class 1 systems) and as single multi-domain proteins (Class 2). These different CRISPR-Cas systems can cleave RNA, DNA, and protein in an RNA-guided manner to eliminate the invader, and in some cases, they initiate programmed cell death/dormancy. The versatile mechanisms of the different CRISPR-Cas systems to target and destroy nucleic acids have been adapted to develop various programmable-RNA-guided tools and have revolutionized the development of fast, accurate, and accessible genomic applications. In this review, we present the structure and interference mechanisms of different CRISPR-Cas systems and an analysis of their unified features. The three types of Class 1 systems (I, III, and IV) have a conserved right-handed helical filamentous structure that provides a backbone for sequence-specific targeting while using unique proteins with distinct mechanisms to destroy the invader. Similarly, all three Class 2 types (II, V, and VI) have a bilobed architecture that binds the RNA-DNA/RNA hybrid and uses different nuclease domains to cleave invading MGEs. Additionally, we highlight the mechanistic similarities of CRISPR-Cas enzymes with other RNA-cleaving enzymes and briefly present the evolutionary routes of the different CRISPR-Cas systems.

Ploidy variation induces butterfly effect on chromatin topology in wheat.

Polyploidy is a prominent driver of plant diversification, accompanied with dramatic chromosomal rearrangement and epigenetic changes that affect gene expression. How chromatin interactions within and between subgenomes adapt to ploidy transition remains poorly understood. We generate open chromatin interaction maps for natural hexaploid wheat (AABBDD), extracted tetraploid wheat (AABB), diploid wheat progenitor Aegilops tauschii (DD) and resynthesized hexaploid wheat (RHW, AABBDD). Thousands of intra- and interchromosomal loops are de novo established or disappeared in AB subgenomes after separation of D subgenome, in which 37-95% of novel loops are lost again in RHW after merger of D genome. Interestingly, more than half of novel loops are formed by cascade reactions that are triggered by disruption of chromatin interaction between AB and D subgenomes. The interaction repressed genes in RHW relative to DD are expression suppressed, resulting in more balanced expression of the three homoeologs in RHW. The interaction levels of cascade anchors are decreased step-by-step. Leading single nucleotide polymorphisms of yield- and plant architecture-related quantitative trait locus are significantly enriched in cascade anchors. The expression of 116 genes interacted with these anchors are significantly correlated with the corresponding traits. Our findings reveal trans-regulation of intrachromosomal loops by interchromosomal interactions during genome merger and separation in polyploid species.

A Randomized Trial of Two Remote Health Care Delivery Models on the Uptake of Genetic Testing and Impact on Patient-Reported Psychological Outcomes in Families With Pancreatic Cancer: The Genetic Education, Risk Assessment, and Testing (GENERATE) Study.

BACKGROUND & AIMS: Genetic testing uptake for cancer susceptibility in family members of patients with cancer is suboptimal. Among relatives of patients with pancreatic ductal adenocarcinoma (PDAC), The GENetic Education, Risk Assessment, and TEsting (GENERATE) study evaluated 2 online genetic education/testing delivery models and their impact on patient-reported psychological outcomes. METHODS: Eligible participants had ≥1 first-degree relative with PDAC, or ≥1 first-/second-degree relative with PDAC with a known pathogenic germline variant in 1 of 13 PDAC predisposition genes. Participants were randomized by family, between May 8, 2019, and June 1, 2021. Arm 1 participants underwent a remote interactive telemedicine session and online genetic education. Arm 2 participants were offered online genetic education only. All participants were offered germline testing. The primary outcome was genetic testing uptake, compared by permutation tests and mixed-effects logistic regression models. We hypothesized that Arm 1 participants would have a higher genetic testing uptake than Arm 2. Validated surveys were administered to assess patient-reported anxiety, depression, and cancer worry at baseline and 3 months postintervention. RESULTS: A total of 424 families were randomized, including 601 participants (n = 296 Arm 1; n = 305 Arm 2), 90% of whom completed genetic testing (Arm 1 [87%]; Arm 2 [93%], P = .014). Arm 1 participants were significantly less likely to complete genetic testing compared with Arm 2 participants (adjusted ratio [Arm1/Arm2] 0.90, 95% confidence interval 0.78-0.98). Among participants who completed patient-reported psychological outcomes questionnaires (Arm 1 [n = 194]; Arm 2 [n = 206]), the intervention did not affect mean anxiety, depression, or cancer worry scores. CONCLUSIONS: Remote genetic education and testing can be a successful and complementary option for delivering genetics care. (Clinicaltrials.gov, number NCT03762590).

Cucumber JASMONATE ZIM-DOMAIN 6 interaction with transcription factor MYB6 impairs waterlogging-triggered adventitious rooting.

Waterlogging is a serious abiotic stress that drastically decreases crop productivity by damaging the root system. Jasmonic acid (JA) inhibits waterlogging-induced adventitious root (AR) formation in cucumber (Cucumis sativus L.). However, we still lack a profound mechanistic understanding of how JA governs AR formation under waterlogging stress. JAZ (JASMONATE ZIM-DOMAIN) proteins are responsible for repressing JA signaling in a transcriptional manner. In this study, we showed that overexpressing CsJAZ8 inhibited the formation of ARs triggered by waterlogging. Molecular analyses revealed that CsJAZ8 inhibited the activation of the R2R3-MYB transcription factor CsMYB6 via direct interaction. Additionally, silencing of CsMYB6 negatively impacted AR formation under waterlogging stress, as CsMYB6 could directly bind to the promoters of 1-aminocyclopropane-1-carboxylate oxidase2 gene CsACO2 and gibberellin 20-oxidases gene CsGA20ox2, facilitating the transcription of these genes. The overexpression of CsACO2 and CsGA20ox2 led to increased levels of ethylene and gibberellin, which facilitated AR formation under waterlogging conditions. On the contrary, silencing these genes resulted in contrasting phenotypes of AR formation. These results highlight that the transcriptional cascade of CsJAZ8 and CsMYB6 plays a critical role in regulating hormonal-mediated cucumber waterlogging-triggered AR formation by inhibiting ethylene and gibberellin accumulation. We anticipate that our findings will provide insights into the molecular mechanisms that drive the emergence of AR in cucumber plants under waterlogging stress.

Genetic testing in early-onset atrial fibrillation.

Atrial fibrillation (AF) is a globally prevalent cardiac arrhythmia with significant genetic underpinnings, as highlighted by recent large-scale genetic studies. A prominent clinical and genetic overlap exists between AF, heritable ventricular cardiomyopathies, and arrhythmia syndromes, underlining the potential of AF as an early indicator of severe ventricular disease in younger individuals. Indeed, several recent studies have demonstrated meaningful yields of rare pathogenic variants among early-onset AF patients (∼4%-11%), most notably for cardiomyopathy genes in which rare variants are considered clinically actionable. Genetic testing thus presents a promising opportunity to identify monogenetic defects linked to AF and inherited cardiac conditions, such as cardiomyopathy, and may contribute to prognosis and management in early-onset AF patients. A first step towards recognizing this monogenic contribution was taken with the Class IIb recommendation for genetic testing in AF patients aged 45 years or younger by the 2023 American College of Cardiology/American Heart Association guidelines for AF. By identifying pathogenic genetic variants known to underlie inherited cardiomyopathies and arrhythmia syndromes, a personalized care pathway can be developed, encompassing more tailored screening, cascade testing, and potentially genotype-informed prognosis and preventive measures. However, this can only be ensured by frameworks that are developed and supported by all stakeholders. Ambiguity in test results such as variants of uncertain significance remain a major challenge and as many as ∼60% of people with early-onset AF might carry such variants. Patient education (including pretest counselling), training of genetic teams, selection of high-confidence genes, and careful reporting are strategies to mitigate this. Further challenges to implementation include financial barriers, insurability issues, workforce limitations, and the need for standardized definitions in a fast-moving field. Moreover, the prevailing genetic evidence largely rests on European descent populations, underscoring the need for diverse research cohorts and international collaboration. Embracing these challenges and the potential of genetic testing may improve AF care. However, further research-mechanistic, translational, and clinical-is urgently needed.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) as a compensatory protease inhibitor in hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE. OBJECTIVE: We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways. METHODS: Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls. RESULTS: In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks. CONCLUSION: The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.

Temporal (Dis)Assembly of Peptide Nanostructures Dictated by Native Multistep Catalytic Transformations.

Emergence of complex catalytic machinery via simple building blocks under non-equilibrium conditions can contribute toward the system level understanding of the extant biocatalytic reaction network that fuels metabolism. Herein, we report temporal (dis)assembly of peptide nanostructures in presence of a cofactor dictated by native multistep cascade transformations. The short peptide can form a dynamic covalent bond with the thermodynamically activated substrate and recruit cofactor hemin to access non-equilibrium catalytic nanostructures (positive feedback). The neighboring imidazole and hemin moieties in the assembled state rapidly converted the substrate to product(s) via a two-step cascade reaction (hydrolase-peroxidase like) that subsequently triggered the disassembly of the catalytic nanostructures (negative feedback). The feedback coupled reaction cycle involving intrinsic catalytic prowess of short peptides to realize the advanced trait of two-stage cascade degradation of a thermodynamically activated substrate foreshadows the complex non-equilibrium protometabolic networks that might have preceded the chemical emergence of life.

Tandem Integration of Biological and Electrochemical Catalysis for Efficient Polyester Upcycling under Ambient Conditions.

Excessive production of waste polyethylene terephthalate (PET) poses an ecological challenge, which necessitates developing technologies to extract the values from end-of-life PET. Upcycling has proven effective in addressing the low profitability of current recycling strategies, yet existing upcycling technologies operate under energy-intensive conditions. Here we report a cascade strategy to steer the transformation of PET waste into glycolate in an overall yield of 92.6% under ambient conditions. The cascade approach involves setting up a robust hydrolase with 95.6% PET depolymerization into ethylene glycol (EG) monomer within 12 h, followed by an electrochemical process initiated by a CO-tolerant Pd/Ni(OH)2 catalyst to convert the EG intermediate into glycolate with high Faradaic efficiency of 97.5%. Techno-economic analysis and life cycle assessment indicate that, compared with the widely adopted electrochemical technology that heavily relies on alkaline pretreatment for PET depolymerization, our designed enzymatic-electrochemical approach offers a cost-effective and low-carbon pathway to upgrade PET.

DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

A Guanidinobenzol-Rich Polymer Overcoming Cascade Delivery Barriers for CRISPR-Cas9 Genome Editing.

The efficient cytosolic delivery of the CRISPR-Cas9 machinery remains a challenge for genome editing. Herein, we performed ligand screening and identified a guanidinobenzol-rich polymer to overcome the cascade delivery barriers of CRISPR-Cas9 ribonucleoproteins (RNPs) for genome editing. RNPs were stably loaded into the polymeric nanoparticles (PGBA NPs) by their inherent affinity. The polymer facilitated rapid endosomal escape of RNPs via a dynamic multiple-step cascade process. Importantly, the incorporation of fluorescence in the polymer helps to identify the correlation between cellular uptake and editing efficiency, increasing the efficiency up to 70% from the initial 30% for the enrichment of edited cells. The PGBA NPs efficiently deliver RNPs for in vivo gene editing via both local and systemic injections and dramatically reduce PCSK9 level. These results indicate that PGBA NPs enable the cascade delivery of RNPs for genome editing, showing great promise in broadening the therapeutic potential of the CRISPR-Cas9 technique.

Polyvinylpyrrolidone-Coated Cubic Hollow Nanocages of PdPt3 and PdIr3 as Highly Efficient Self-Cascade Uricase/Peroxidase Mimics.

Uricase-catalyzed uric acid (UA) degradation has been applied for hyperuricemia therapy, but this medication is limited by H2O2 accumulation, which can cause oxidative stress of cells, resulting in many other health issues. Herein, we report a robust cubic hollow nanocage (HNC) system based on polyvinylpyrrolidone-coated PdPt3 and PdIr3 to serve as highly efficient self-cascade uricase/peroxidase mimics to achieve the desired dual catalysis for both UA degradation and H2O2 elimination. These HNCs have hollow cubic shape with average wall thickness of 1.5 nm, providing desired synergy to enhance catalyst's activity and stability. Density functional theory calculations suggest the PdIr3 HNC surface tend to promote OH*/O* desorption for better peroxidase-like catalysis, while the PdPt3 HNC surface accelerates the UA oxidation by facilitating O2-to-H2O2 conversion. The dual catalysis power demonstrated by these HNCs in cell studies suggests their great potential as a new type of nanozyme for treating hyperuricemia.

DPI_CDF: druggable protein identifier using cascade deep forest.

BACKGROUND: Drug targets in living beings perform pivotal roles in the discovery of potential drugs. Conventional wet-lab characterization of drug targets is although accurate but generally expensive, slow, and resource intensive. Therefore, computational methods are highly desirable as an alternative to expedite the large-scale identification of druggable proteins (DPs); however, the existing in silico predictor's performance is still not satisfactory. METHODS: In this study, we developed a novel deep learning-based model DPI_CDF for predicting DPs based on protein sequence only. DPI_CDF utilizes evolutionary-based (i.e., histograms of oriented gradients for position-specific scoring matrix), physiochemical-based (i.e., component protein sequence representation), and compositional-based (i.e., normalized qualitative characteristic) properties of protein sequence to generate features. Then a hierarchical deep forest model fuses these three encoding schemes to build the proposed model DPI_CDF. RESULTS: The empirical outcomes on 10-fold cross-validation demonstrate that the proposed model achieved 99.13 % accuracy and 0.982 of Matthew's-correlation-coefficient (MCC) on the training dataset. The generalization power of the trained model is further examined on an independent dataset and achieved 95.01% of maximum accuracy and 0.900 MCC. When compared to current state-of-the-art methods, DPI_CDF improves in terms of accuracy by 4.27% and 4.31% on training and testing datasets, respectively. We believe, DPI_CDF will support the research community to identify druggable proteins and escalate the drug discovery process. AVAILABILITY: The benchmark datasets and source codes are available in GitHub: http://github.com/Muhammad-Arif-NUST/DPI_CDF .

Sensational site: the sodium pump ouabain-binding site and its ligands.

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and ß subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology.

Rapid Initiation of Antiretroviral Therapy With Coformulated Bictegravir, Emtricitabine, and Tenofovir Alafenamide Versus Efavirenz, Lamivudine, and Tenofovir Disoproxil Fumarate in HIV-Positive Men Who Have Sex With Men in China: Week 48 Results of the Multicenter, Randomized Clinical Trial.

BACKGROUND: Most international treatment guidelines recommend rapid initiation of antiretroviral therapy (ART) for people newly diagnosed with human immunodeficiency virus (HIV)-1 infection, but experiences with rapid ART initiation remain limited in China. We aimed to evaluate the efficacy and safety of efavirenz (400 mg) plus lamivudine and tenofovir disoproxil fumarate (EFV + 3TC + TDF) versus coformulated bictegravir, emtricitabine, and tenofovir alafenamide (BIC/FTC/TAF) in rapid ART initiation among men who have sex with men (MSM) who have been diagnosed with HIV. METHODS: This multicenter, open-label, randomized clinical trial enrolled MSM aged ≥18 years to start ART within 14 days of confirmed HIV diagnosis. The participants were randomly assigned in a 1:1 ratio to receive EFV (400 mg) + 3TC + TDF or BIC/FTC/TAF. The primary end point was viral suppression (<50 copies/mL) at 48 weeks per US Food and Drug Administration Snapshot analysis. RESULTS: Between March 2021 and July 2022, 300 participants were enrolled; 154 were assigned to receive EFV + 3TC + TDF (EFV group) and 146 BIC/FTC/TAF (BIC group). At week 48, 118 (79.2%) and 140 (95.9%) participants in the EFV and BIC group, respectively, were retained in care with viral suppression, and 24 (16.1%) and 1 (0.7%) participant in the EFV and BIC group (P < .001), respectively, discontinued treatment because of adverse effects, death, or lost to follow-up. The median increase of CD4 count was 181 and 223 cells/µL (P = .020), respectively, for the EFV and BIC group, at week 48. The overall incidence of adverse effects was significantly higher for the EFV group (65.8% vs 37.7%, P < .001). CONCLUSIONS: BIC/FTC/TAF was more efficacious and safer than EFV (400 mg) + 3TC + TDF for rapid ART initiation among HIV-positive MSM in China.