Journal of Phycology

Marine diatoms contribute to about 20% of global primary production. We present the first basin-scale, multiyear assessment of diatom communities in the North Pacific, combining taxonomically high-resolution RuBisCO large subunit gene (rbcL) metabarcoding with concurrent environmental measurements. Using a nine-year time series of daily samples resolved at the species level via ~500 bp rbcL fragments, we performed multivariate analyses across biogeographic provinces, identifying significant correlations between community structure and environmental drivers such as temperature and macronutrient availability. We report the prevalence of a previously overlooked centric diatom species in the North Pacific, Eunotogramma lunatum, which appears to be near-dominant even in subarctic high-nitrate, low-chlorophyll waters where pennate diatoms are typically favored. These results demonstrate the power of rbcL for large-scale ocean monitoring and provide a critical baseline for future studies of diatom population dynamics, climate change impacts, and ecosystem resilience in a key marine region.

Intertidal mudflats are low hydrodynamic energy environments hosting microphytobenthic communities that experience strong spatiotemporal variability in light regimes, including changes in spectral quality and light intensity that can lead to cellular photooxidative stress. To cope with these fluctuations, autotrophs exhibit diverse and highly plastic adaptations that are often species-dependent and shaped by their ecological niches. This study investigates photophysiological responses and metabolic remodeling in a diatom assemblage originating from a natural winter microphytobenthic biofilm under contrasting red and blue light intensities. To this end, photosynthetic parameters were monitored alongside changes in lipophilic metabolites, including untargeted lipids and lipophilic pigments. While few metabolites showed temporal remodeling, rapid and contrasting changes were observed within 30 minutes in response to both spectral quality and light intensity. Red light treatments induced broader remodeling of lipophilic metabolites than blue light, whereas blue light appeared to have a greater impact on photosynthetic parameters. Moreover, red light induced xanthophyll-cycle responses comparable to those observed under blue light at equivalent incident intensity. We discuss these metabolic responses in relation to diatom photoadaptive strategies, placing these findings within the intertidal environmental framework. This work further underlines the importance of understanding rapid metabolic plasticity in coping with light fluctuations, providing new insights into the photoregulatory strategies of natural microphytobenthic communities.

High-phenolic plant genera present a major technical limitation in genomic research. Standard extraction approaches that perform reliably across diverse flora often perform poorly when applied to recalcitrant taxa, producing low DNA yield and integrity incompatible with sequencing requirements. The genus Anigozanthos (Kangaroo paws) from the family Haemodoraceae exemplifies this problem. We identified key physicochemical factors governing extraction failure in this genus and resolved them through targeted modifications to lysis chemistry and contaminant management. The resulting protocol achieved a near threefold improvement in DNA purity, substantially reducing contaminant carry over and consistently yielded high-integrity, long DNA fragments (DIN > 7) across a diverse sample set spanning cultivated and wild material across four diverse genera of Haemodoraceae. We also tested a straightforward purity assessment framework that can be implemented in any standard molecular laboratory, enabling rapid pre-submission quality assessment without the need for specialised equipment. Together these advances open a practical path to genomic characterisation of Anigozanthos that establishes a transferable model for genomic research across Australia ' s chemically complex native flora.

Myxococcus xanthus bacteria form aggregates when starved on solid surfaces and some cells differentiate into spores. Studies of mutants in monoculture have advanced knowledge of this multi-cellular developmental process, but our understanding of the genetic determinants is incomplete. To assess gene function genomewide, we generated a pool of barcoded transposon insertion mutants, subjected it to starvation, and separated developmental samples into non-aggregated cells, aggregated cells, and spores. We also subjected our pool to chemically-induced unicellular sporulation. Evaluation of changes in the abundance of mutants in samples allowed identification of 200 genes in which insertions reproducibly caused distinct patterns of depletion and/or accumulation over time. Many of these genes have well-established roles in development, validating our approach, while many others have not previously been associated with development. Genes involved in type IV pili (T4P)-dependent motility were more important than gliding motility genes for aggregation and sporulation in the mixed population. Although exopolysaccharide (EPS) synthesis genes are required for aggregation in monoculture, most were dispensable for aggregation in our pool, consistent with EPS sharing between cells, yet these genes were required cell-autonomously for efficient sporulation. Genes for positive regulators of EPS synthesis were important for aggregation as well as sporulation, suggesting functions beyond EPS production. Insertions in several novel genes impaired both starvation- and chemically-induced sporulation. Many genes increased the efficiency of starvation-induced sporulation. Some of these mutants, which we call "developmental winners", are novel cheaters. Our results demonstrate the power of using the newly-created mutant library to elucidate M. xanthus biology.

Diatoms are major contributors to marine primary production, yet current approaches for monitoring their abundance and function rely on coarse satellite chlorophyll estimates or sparse cell count and carbon fixation measurements. Molecular markers are a promising approach for high-resolution measurement of both abundance and metabolic activity through analysis of environmental DNA (eDNA) and RNA (eRNA). We present an isothermal quantitative recombinase polymerase amplification (qRPA) assay targeting rbcL gene copies and transcripts of marine diatoms, operating at low temperature and producing results in less than 15 min. We demonstrate specificity and calibration across diverse diatom taxa, then apply the assay to eDNA and eRNA samples from the Mare Chiara Long-Term Ecological Research site in the Bay of Naples, Italy, alongside microscopy, chlorophyll, physicochemical, and carbon-fixation data. Diatom rbcL DNA tracked abundance across five orders of magnitude despite seasonal shifts in community composition. Combining molecular and optical data revealed increased cellular rbcL copies and chlorophyll in low-light winter populations, suggesting enhanced photosynthetic capacity despite lower abundance. Furthermore, rbcL RNA reflected total carbon fixation rates and identified populations with differing carbon fixation activity. These results support rapid, RPA-based rbcL quantification as a robust approach for biomolecular ocean observing.

The ocean tides influence key Earth system processes at a range of spatial and temporal scales. It is known that the geometry of ocean basins is the leading controller of tidal energetics, so well-constrained palaeogeographic reconstructions and tidal properties for Earths past are imperative when investigating other Earth system processes. Here, we present a novel way to constrain both deep-time tidal model results and reconstructions, by combining palaeoecology with sedimentology. We compare new palaeo-tidal model simulations for the Cambrian period, significant for the early origin and radiation of major animal fauna, to tidal proxies. One of the most abundant soft-bodied organisms preserved during this time are cnidarian medusae (jellyfish). A total of 17 cnidarian medusae localities were obtained through the literature, which had an adequate global distribution and occurred at regular intervals throughout the period of study. In some locations there were also estimates of palaeo-tidal range. Our results show a good agreement between the simulations and proxy data. In the few locations where there is disagreement, it is proposed that the palaeogeographic reconstructions are missing details, e.g., island chains, and our results allow for the palaeogeographic reconstructions to be improved. The proxy method presented is promising and can be applied to other time-periods with different marine fossils, particularly at evolutionary and extinction periods where the marginal marine environment is of importance.

Three new species of sun orchid (Thelymitra) endemic to Aotearoa New Zealand are here described. These are T. palustris, T. scabrifolia and T. semaphora. The morphological distinctiveness of these three species has been acknowledged for decades; however, their taxonomic status has remained unresolved. Evidence from existing karyological data, recently generated DNA sequence data (LFY and ycf1) and morphological studies from historical and fresh collections are used here to support their formal description. Both, T. palustris and T. semaphora are restricted to wet habitats north of Auckland (North Island). Thelymitra scabrifolia inhabits mostly scrub, and it has a similar northern North Island distribution, but is has been found also in Manawat[a]whi / Three Kings Islands and historically in Otago (South Island). All three species are polyploids and are of conservation concern.

Squid abundance has been reported to increase globally between 1970 and 2010. This increase has been hypothesized to result from two primary factors: the loss of top predators due to overfishing and rising ocean temperatures. The decline in apex predators may lead to the expansion of squid populations either through reduced predation pressure or diminished competition with juvenile predators. Concurrently, increased temperatures could enhance the somatic growth rates of squid, thereby accelerating their population growth. However, empirically disentangling the impacts of predator loss and temperature on squid biomass remains challenging, especially in a food-web context. In this study, we used a size- and trait-based model of upper trophic levels that resolves the ecosystem structure -- biomass and trophic interactions of fish and squid -- for varying depth, temperature, and secondary production, to investigate two hypotheses of the historical expansion of squid, i.e., the effects of predator depletion from fishing and rising temperatures on squid biomass. Our model reveals that intensified fishing of squid predators -- specifically large demersal fish in shelf systems and large pelagic fish in open oceans -- leads to a slight increase in squid biomass. Conversely, elevated temperatures are associated with a decline in squid biomass. This temperature-driven reduction in biomass is attributed to an increased metabolism of squids beyond the available food supply. If historic overfishing on large marine predators continues to be curtailed, we expect a corresponding reduction in global squid biomass and fisheries potential, which could be further exacerbated by rising temperatures.

Protists play important roles in food chains and symbioses in soil and aquatic environments, displaying an enormous morphological and functional diversity. While most commonly found protist species are well known to science, our global-scale environmental DNA survey across soil, water, and sediments reveals dozens of novel, phylum-level phylogenetic lineages that remain to be characterized for basic morphology and function. A vast majority of these undescribed taxa occur in marine water and sediments, but some are common in soil. Most of these novel taxa have distinct substrate and habitat preferences and biogeographic patterns. To accord these lineages scientific agency and enable unambiguous scientific communication, we propose formal names for 150 species to phylum-level taxa from 25 deep lineages based on eDNA and rRNA gene long-read sequence information.

Hydrozoa is a group of relatively simple animals with a well-developed nervous system. The nervous system in all hydrozoan medusae is highly conserved and includes outer and inner nerve rings at the bell margin, a neural network in the manubrium, and radial neural pathways that connect them. However, one element of the nervous system shows substantial variability among species: the subumbrella neural network. We examined the structure of the nervous and muscular systems in the subumbrella of 14 species of hydrozoan medusae. The main conclusion of this study is that the distribution of neural networks in the subumbrella strongly correlates with the distribution of smooth radial muscles. This correlation suggests that smooth radial muscles are the primary target of the subumbrella nervous system. Most species in the order Anthoathecata show a trend toward secondary loss of the neural networks and radial smooth muscle fibers in the subumbrella region, concentrating neural elements and smooth muscles only in the radial pathways along the radial canals. By contrast, all studied species in the order Leptothecata have neural networks in the subumbrella area, as well as radial smooth muscle fibers spread throughout the entire subumbrella region. The correlation between radial smooth muscles and the nervous system is also observed in the radial pathways along the radial canals. All species with thick bundles of smooth radial muscles along the radial canals have clearly defined, dense neural pathways running along or even embedded within the smooth muscle bundles.

Heritable bacterial symbionts are pervasive in terrestrial arthropods, often imposing reproductive manipulations to promote their own spread within host populations. Co-infections are common, potentially allowing symbiont co-infectors to hitchhike through a host population. However, adverse thermal conditions can disrupt these communities, particularly when co-infectors vary in their thermal sensitivity. We used a multi-generation experiment to test whether warm (29 {degrees}C) conditions disrupted spread of heritable symbionts through uninfected populations of the spider, Mermessus fradeorum. We tested two common infection combinations: a single infection with a cytoplasmic incompatibility (CI) inducing Rickettsiella or a feminizing co-infection that included a feminizing Wolbachia, the same Rickettsiella, and up to three apparent hitchhikers (two additional Wolbachia strains and Tisiphia). We initiated replicate populations with 1/3 of one infection type and 2/3 uninfected spiders, evaluating population infection rate over 5 spider generations under different temperature regimes. Under cool (21{degrees}C) conditions, Wolbachia feminization drove co-infection to 88% and Rickettsiella CI drove single infection to 83% of host populations. Vertical transmission for all symbionts was high (97-99%) and hitchhiking symbionts also spread effectively. Under warm conditions, feminization and CI efficacy were reduced, and symbionts suffered variably reduced vertical transmission. Warm conditions ultimately destroyed the co-infecting symbiont consortium and impeded symbiont spread. On its own, though, Rickettsiella was still able to increase, despite reduced strength of CI. We hypothesize that contrasting tensions between feminizing spread of the symbiont consortium versus environmentally driven loss of function and transmission may explain observed patterns of mixed infections in field populations of this spider.

Organosulfur compounds are the predominant sulfur source in terrestrial environments, requiring bacteria to use enzymes for their assimilation. Most described organosulfur-assimilating enzymes require oxygen, and enzymes that function under anoxic conditions remain poorly understood. Recently, methylthio-alkane reductase (Mar), a nitrogenase-like enzyme that reduces the volatile organic sulfur compounds (VOSCs) methylthio ethanol (MT-EtOH), dimethyl sulfide (DMS), and ethyl methyl sulfide (EMS) under anoxic conditions, was identified in the purple nonsulfur bacterium Rhodospirillum rubrum. However, another purple nonsulfur bacterium, Rhodopseudomonas palustris, has three loci of nitrogen fixation-like (NFL) genes with high sequence similarity to Mar, suggesting additional Mar-like enzymes with distinct roles. Here, we tested whether these NFL genes are required for VOSC assimilation in R. palustris. RNA-seq analysis revealed that all three NFL loci are upregulated under sulfur limitation, supporting a role in sulfur assimilation. Only disruption of the NFL genes encoded by RPA2634-37, renamed marBHDK1, caused fitness defects with EMS, DMS, and dimethylsulfoniopropionate (DMSP) as sulfur sources, indicating a functional Mar enzyme. The NFL genes RPA2347-48 and RPA2353-54, renamed marKD2 and marHB2, were required for activity with MT-EtOH or ethanethiol but not DMS, EMS, or DMSP. No activity was observed for the third locus, RPA2363-64, renamed nflDK. Overall, two Mar homologs in R. palustris are capable of VOSC reduction, one specialized for simple VOSCs and the other preferring a substrate with an additional functional group.

Urbanization is reshaping freshwater ecosystems, with well-documented effects across gradients of land-use change, hydrologic alteration, and habitat degradation. However, how biodiversity is organized among neighboring urban aquatic habitats that differ in hydrologic connectivity, disturbance transmission, residence time, management history, and opportunities for species movement is often less clear. This creates a challenge for interpreting urban fish communities at local scales as species occurrence may reflect both contemporary habitat filtering and historical contingencies including native persistence, interbasin transfer, stocking, and nonindigenous introductions. Here we use eDNA detections, historical records, phylogenetic information, and species trait data to investigate the fish assemblages of the Charlotte metropolitan region. We detect a highly mixed fauna that also depicts a strong signature of structured biodiversity profiles across taxonomic, phylogenetic, functional, and life-history dimensions between habitat types. In particular, bounded habitats contained assemblages with larger-bodied species that are fecund and faster to reproduce relative to free-flowing habitats. Species-level occurrence models did not support a simple trait-by-habitat rule. Instead our results demonstrate that urban aquatic habitats can sort historically mixed regional species pools into predictable assemblage-level life-history profiles while simultaneously retaining signatures of evolutionary and historical biogeographic contingency.

Purpose: To evaluate the safety, procedural performance, sample recovery, and surgeon preference of an ophthalmic needle designed specifically for anterior chamber (AC) paracentesis. Methods: In this multicenter study, AC paracentesis was performed in clinic and operating-room settings using a 32-gauge x 4-mm needle with low dead space. The procedure was evaluated using a standardized physician survey. Prespecified outcomes included procedure-related adverse events (primary outcome), needle entry and handling, aspiration and sample recovery, comparative performance versus a 30-gauge needle, and physician preference for future use. Results: A total of 110 needle uses by eight surgeons were included. No ocular complications occurred, including lens or iris injury, hyphema, AC collapse, wound leak, hypotony, infection, or retinal complication, and no procedure required needle exchange or conversion to another device. Two technical events without ocular sequelae were noted, in which needle entry was partial thickness and did not reach the AC (1.8%; exact 95% CI, 0.2%-6.4%). Physicians rated needle entry, handling and sample recovery as good or excellent. Compared with a 30-gauge needle, the study needle was rated as at least comparable across all assessed domains. All surgeons rated it better or much better for intra-procedural safety and preferred it for future AC taps. Conclusions and Relevance: This short, 32-gauge low-dead-space ophthalmic needle demonstrated a favorable safety profile and was preferred over a 30-gauge needle by all surgeons. As aqueous humor liquid biopsy expands in clinical diagnostics and trials, an ophthalmic-specific needle design may help improve the consistency and safety of aqueous humor collection for molecular analysis and broader clinical use. Keywords: Anterior chamber paracentesis; Aqueous humor; Liquid biopsy; Low dead space; Ophthalmic needle

Background. Videolaryngoscopy still requires adjuncts or hyperangulated rescue in a clinically important minority, and bedside screening discriminates modestly. Point-of-care ultrasound (POCUS) of the anterior airway is a promising alternative, but existing prediction models are opaque or assume a pre-specified functional form. We developed and internally validated a parsimonious, fully disclosed POCUS risk equation whose form is recovered from data and whose structural properties are machine-checked by formal proof - to our knowledge the first formally verified clinical risk predictor - following TRIPOD+AI 2024. Methods. In a prospective single-centre, single-operator cohort of 259 adults undergoing elective videolaryngoscopy (no-Easy airway 68/259, 26.3%), Sequentially Thresholded Least Squares with bootstrap stability selection (B=300) screened a 71-term library of nine POCUS features and retained a seven-term logistic equation; a two-term bootstrap-stable model was pre-specified as robustness analysis. Internal validation used 5x10 repeated cross-validation plus temporal and device hold-outs, with pre-specified overfitting and optimism assessments. Five behavioural properties of the deployed equation were machine-checked in Lean 4. Results. Two interactions met the |c|/sigma_c>2 stability criterion: skin-to-epiglottis x skin-to-hyoid-bone distance and tongue volume x sagittal tongue area. The seven-term equation reached a 5x10 cross-validated C-statistic of 0.966 (optimism-corrected 0.968) and held across temporal and device hold-outs (0.94-0.97). Calibration-in-the-large matched prevalence, with cross-validated slope 0.90 attenuating to 0.625 out-of-time; standard recalibration restored 0.92 without loss of discrimination. The pre-specified two-term robustness model reproduced this performance (C-statistic 0.964-0.968; events-per-parameter 34; shrinkage 0.99), confirming the result is not an artefact of the screening stage. Net benefit over a clinical baseline was positive across 10-50% thresholds. All five Lean 4 theorems compiled without sorry. Conclusions. A sparse, formally verified POCUS equation predicts difficult videolaryngoscopy with high internally validated discrimination and quantified, modest overfitting. Because the equation was developed in a single-operator cohort and its inputs are operator-dependent, external validation requires prior harmonisation of the measurement protocol and operator credentialing.

Introduction: Polyendocrine metabolic ovarian syndrome (PMOS), formerly known as polycystic ovary syndrome (PCOS), is a common endocrine disorder affecting women of reproductive age. Besides reproductive and metabolic disturbances, PMOS negatively impacts psychological well-being and quality of life. Despite available treatment options, there remains a need for safe and effective therapies that improve both clinical symptoms and fertility outcomes. Aim: To compare the efficacy of VAMHA and MYRHA tablet combination therapy with standard non-hormonal therapy in restoring regular menstruation. Secondary objectives included assessment of ovulation, menstrual symptoms, polycystic ovarian morphology, hormonal and metabolic parameters, anthropometric measures, and skin manifestations. Study Design: Open-label, randomized, multicentre, prospective comparative clinical study. Methods: Seventy-one women with PMOS were randomized to Group A (n=37) or Group B (n=34). Group A received VAMHA and MYRHA tablets (2 tablets each), while Group B received Metformin 500 mg plus Myoinositol 600 mg (1 tablet), twice daily for 180 days. Data were recorded in Case Report Forms. Statistical Analysis: Continuous variables were summarized using mean and standard deviation, while categorical variables were expressed as frequencies and percentages. Appropriate statistical tests, including Chi-square, were used. A p-value [&le;]0.05 was considered significant. Results: Significantly more participants in Group A achieved regular menstrual cycles than Group B (31 vs. 22; p<0.05). Ovulation occurred in 16 participants in Group A compared with 6 in Group B (p<0.05). Both groups showed significant improvement in menstrual irregularity and related symptoms. Significant reductions in Anti-Mullerian Hormone (AMH), fasting insulin, and body mass index (BMI) were observed in both groups (p<0.05). Resolution of polycystic ovarian morphology occurred in 13 participants (38.23%) in Group A and 10 (33.33%) in Group B. Both treatments were well tolerated with no major safety concerns. Conclusions: VAMHA and MYRHA combination therapy was superior to standard non-hormonal therapy in improving menstrual regularity and ovulation. It also produced favourable metabolic, hormonal, and ultrasonographic outcomes, suggesting its potential as a safe and effective option for comprehensive PMOS management and fertility enhancement.

Background & Aims: Fatigue is a central symptom of chronic liver disease (CLD), substantially impacting health-related quality of life (HRQoL). This study aimed to further understand CLD symptomatology, including fatigue, and its impact on HRQoL from a patient perspective. Methods: Abbott Global Assessment of Patients unmet needs (aGAP) was a multinational, cross-sectional survey in adults with compensated CLD in China, India and Mexico, conducted between July and November 2024. Adult participants who self-reported that they had physician-diagnosed CLD and were experiencing fatigue completed a quantitative survey to assess symptom burden and included three HRQoL patient-reported outcome (PRO) questionnaires (Patient-Reported Outcomes Measurement Information System [PROMIS]-29+2, Work Productivity and Activity Impairment - Specific Health Problem version 2.0 [WPAI: SHP], Multidimensional Fatigue Inventory [MFI]). Results: Overall, 505 participants (China: 200; Mexico: 105; India: 200) completed the study. Participants reported that their CLD-related fatigue sometimes, often or always affected their self-esteem/confidence (45.1%) and ability to maintain or acquire new employment (38.6%). Most participants reported moderate (51.3%) or serious (26.9%) fatigue, with 33.5% experiencing fatigue every day or almost every day. Many participants felt their social life was negatively impacted by their fatigue (47.3%) and that there were related financial difficulties (53.9%). Use of validated PRO tools demonstrated severe fatigue (MFI: overall mean [SD] 13.9 [3.4] general fatigue and 13.4 [3.6] physical fatigue) as well as substantial levels of work and activity impairment (WPAI: SHP overall mean [SD] 53.0 [26.4]) and high levels of anxiety, pain interference, depression and sleep interference (PROMIS T-scores [&ge;]54). Conclusions: Fatigue has a substantial impact on HRQoL among adults with CLD across several countries, highlighting a global unmet need for targeted interventions to effectively identify and manage the condition.

Importance: Evidence on GLP-1/GIP therapies is largely derived from trials enrolling selected populations or medical records that miss utilization outside healthcare channels. No nationally representative cohort has characterized real-world uptake, indications, and access. Objective: To characterize GLP-1/GIP prevalence, indication, clinical profile, and access. Design: Prospective cohort study with three GLP-1/GIP surveillance waves (March 2024, December 2024, October 2025). Setting: The Understanding America Study, an address-based, nationally representative panel of approximately 15,000 US adults aged 18+ years initiated in 2014. Participants: UAS participants responding to at least one surveillance wave (n=9150). Exposures: GLP-1/GIP use status (never vs any use, comprising current and former use), self-reported primary indication (diabetes, weight loss, or other), and access pathway (traditional vs non-traditional). Main Outcomes and Measures: Survey-weighted prevalence of GLP-1/GIP use, overall and by indication and access pathway; sociodemographic, cardiometabolic, treatment, and access characteristics; and smartwatch-derived resting heart rate, heart rate variability, maximum activity heart rate, step count, and sleep duration and variability. Results: Among n=9150 adults (1274 with any use; 60.9% female; median age 53 years), weighted prevalence increased 46%, from 8.2% (March 2024) to 12.0% (October 2025) representing 32 million. Weight-loss indications grew, reaching nearly half of use (4.1% to 5.6%); diabetes-indicated use was stable (5.3% to 5.4%). Users carried high cardiometabolic burden (obesity, 68.2%; diabetes, 53.6%) but diverged by indication: diabetes-indicated users were older (median, 59 vs 49 years), whereas weight-loss-indicated users were more often female (69.9% vs 51.3%) and healthier. One in three users (~9 million) had non-traditional access, especially in weight-loss-indicated users, of whom 33% had no conventional prescription; 41% used compounding, online, or foreign pharmacies; and, 43% lacked coverage. Non-traditional users were five times as likely to report an unlisted, likely compounded formulation (19.8% vs 4.1%). All p<0.05. Conclusions and Relevance: Real-world GLP-1/GIP use has grown rapidly and diversified substantially in indication, access, and population profile. One in 3 users obtained treatment through nontraditional channels largely invisible to claims data, raising long-term safety, efficacy, and coverage questions. GLIMMER provides a public, nationally representative longitudinal evidence base for future payer and provider decisions.

Muscle architecture shapes muscle function and changes with age, growth, training and disease, yet quantifying three-dimensional (3D) muscle architecture in vivo remains challenging. We introduce a hybrid fascicle tractography approach for freehand 3D ultrasound data that accurately reconstructs 3D muscle fascicles with respect to an objective, anatomically relevant coordinate system defined by the muscle's central aponeurosis. The hybrid approach combines Hessian-based fascicle detection with wavelet-based refinement to generate volumetric fascicle orientations. In a synthetic dataset with known ground truth, fascicle orientations and lengths were estimated with errors of [&le;]2{degrees} and ~1.5%, respectively. In vivo, the approach detected physiologically plausible fascicle lengthening in the human tibialis anterior following a passive plantar flexion rotation, whereas diffusion tensor imaging of the same muscle did not. The proposed method enables anatomically relevant, objective and non-invasive quantification of 3D muscle architecture in vivo, providing a practical framework for applications in clinical and applied muscle physiology.

Background. In Europe, first-trimester combined screening with the Fetal Medicine Foundation (FMF) algorithm identifies women at increased risk of preeclampsia who may benefit from personalized aspirin prophylaxis. However, a substantial proportion of early-onset preeclampsia (EOPE) remains undetected at clinically acceptable specificity. Objective. To evaluate the first-trimester performance of MaiRa for early-onset preeclampsia (EOPE) risk stratification by benchmarking it against FMF screening in the same women, characterizing discordant patient-level classification profiles and exploring potential implementation strategies. Study Design. This secondary case-control analysis was nested within the prospective, multicentre PREMOM cohort [NCT04990141], which enrolled women with singleton pregnancies across 14 tertiary hospitals in Spain. First-trimester MaiRa and FMF risk estimates were evaluated in the same 126 pregnant women, comprising 99 uncomplicated controls and 27 EOPE cases, defined by disease onset before 34 weeks. Discrimination was compared using a stratified paired bootstrap analysis of the areas under the receiver-operating-characteristic curves. Performance was assessed at prespecified clinical thresholds, and detection rates were evaluated at fixed false-positive rates. Universal and contingent MaiRa implementation strategies were also evaluated. Results. MaiRa showed greater first-trimester discrimination for EOPE than FMF combined screening (AUC, 0.974 vs 0.900; P=.040) and consistently achieved higher detection rates across fixed false-positive rates. At false-positive rates of 5% and 10%, MaiRa detected 85.2% and 92.6% of EOPE cases, compared with 44.4% and 70.4% for FMF, respectively. Patient-level analysis demonstrated that MaiRa identified 12 of 27 EOPE cases (44.4%) classified as low risk by FMF; these pregnancies generally exhibited less abnormal conventional first-trimester profiles, including fewer maternal risk factors, lower mean arterial pressure and lower uterine artery pulsatility index, yet 8 of 12 (66.7%) subsequently developed severe EOPE. Exploratory implementation analyses showed that universal MaiRa screening achieved the highest EOPE detection, whereas a contingent strategy using FMF for triage and reflex MaiRa testing reduced molecular testing to 35.7% of pregnancies while maintaining 77.8% sensitivity and 97.0% specificity. Conclusion. MaiRa provided greater first-trimester discrimination for EOPE than conventional combined screening and detected additional pregnancies that later developed severe disease despite less abnormal conventional screening profiles. The findings suggest that maternal plasma cfRNA profiling captures biological alterations not fully reflected by combined first-trimester screening and support further prospective evaluation in an independent, unselected obstetric population. Key words: early-onset preeclampsia; first-trimester screening; cell-free RNA; liquid biopsy; Fetal Medicine Foundation algorithm; combined screening; risk stratification; aspirin prophylaxis.