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Background: Diarrhoea remains one of the leading causes of childhood mortality globally. Recent epidemiological studies conducted in low-middle income countries (LMICs) identified Shigella spp. as the first and second most predominant agent of dysentery and moderate diarrhoea, respectively. Antimicrobial therapy is often necessary for Shigella infections; however, we are reaching a crisis point with efficacious antimicrobials. The rapid emergence of resistance against existing antimicrobials in Shigella spp. poses a serious global health problem. Methods: Aiming to identify alternative antimicrobial chemicals with activity against antimicrobial resistant Shigella, we initiated a collaborative academia-industry drug discovery project, applying high-throughput phenotypic screening across broad chemical diversity and followed a lead compound through in vitro and in vivo characterisation. Results: We identified several known antimicrobial compound classes with antibacterial activity against Shigella. These compounds included the oral carbapenem Tebipenem, which was found to be highly potent against broadly susceptible Shigella and contemporary MDR variants for which we perform detailed pre-clinical testing. Additional in vitro screening demonstrated that Tebipenem had activity against a wide range of other non-Shigella enteric bacteria. Cognisant of the risk for the development of resistance against monotherapy, we identified synergistic behaviour of two different drug combinations incorporating Tebipenem. We found the orally bioavailable prodrug (Tebipenem pivoxil) had ideal pharmacokinetic properties for treating enteric pathogens and was effective in clearing the gut of infecting organisms when administered to Shigella-infected mice and gnotobiotic piglets. Conclusions: Our data highlight the emerging antimicrobial resistance crisis and shows that Tebipenem pivoxil (licenced for paediatric respiratory tract infections in Japan) should be accelerated into human trials and could be repurposed as an effective treatment for severe diarrhoea caused by MDR Shigella and other enteric pathogens in LMICs. Funding: Tres Cantos Open Lab Foundation (projects TC239 and TC246), the Bill and Melinda Gates Foundation (grant OPP1172483) and Wellcome (215515/Z/19/Z).

The genome of the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals the presence of a rich repertoire of esterases and lipases highlighting their important role in cellular metabolism. Among them is the carboxylesterase LpEst1 a bacterial enzyme related to the mammalian hormone-sensitive lipase, which is known to play a central role in energy homeostasis. In this study, the crystal structure of LpEst1 has been determined at 2.05 Å resolution; it exhibits an αβ-hydrolase fold, consisting of a central β-sheet surrounded by α-helices, endowed with novel topological features. The structure reveals a dimeric assembly not comparable with any other enzyme from the bacterial hormone-sensitive lipase family, probably echoing the specific structural features of the participating subunits. Biophysical studies including analytical gel filtration and ultracentrifugation support the dimeric nature of LpEst1. Structural and mutational analyses of the substrate-binding pocket and active site together with biochemical studies provided insights for understanding the substrate profile of LpEst1 and suggested for the first time the conserved Asp173, which is adjacent to the nucleophile, as a key element in the stabilization of the loop where the oxyanion hole resides.

Human noroviruses (HuNoVs) are a major cause of diarrheal disease, yet critical aspects of their biology, including cellular tropism, remain unclear. Although research has traditionally focused on the intestinal epithelium, the hypothesis that HuNoV infects macrophages has been recurrently discussed and is investigated here using a zebrafish larval model. Through single-cell RNA sequencing of dissected zebrafish intestines, we unbiasedly identified macrophages as host cells for HuNoV replication, with all three open reading frames mapped to individual macrophages. Notably, HuNoV preferentially infects actively phagocytosing inflammatory macrophages. HuNoV capsid proteins and double-stranded RNA colocalized within intestinal macrophages of infected zebrafish larvae, and the negative-strand RNA intermediate was detected within FACS-sorted macrophages. Flow cytometry confirmed viral replication within these macrophages, constituting approximately 23% of HuNoV’s host cells. Identifying macrophages as host cells prompts a reevaluation of their role in HuNoV pathogenesis, offering new directions for understanding and controlling this infection.

Mental health issues among Ph.D. students are prevalent and on the rise, with multiple studies showing that Ph.D. students are more likely to experience symptoms of mental health-related issues than the general population. However, the data is still sparse. This study aims to investigate the mental health of 589 Ph.D. students at a public university in Germany using a mixed quantitative and qualitative approach. We administered a web-based self-report questionnaire to gather data on the mental health status, investigated mental illnesses such as depression and anxiety, and potential areas for improvement of the mental health and well-being of Ph.D. students. Our results revealed that one-third of the participants were above the cut-off for depression and that factors such as perceived stress and self-doubt were prominent predictors of the mental health status of Ph.D. students. Additionally, we found job insecurity and low job satisfaction to be predictors of stress and anxiety. Many participants in our study reported working more than full-time while being employed part-time. Importantly, deficient supervision was found to have a negative effect on Ph.D. students’ mental health. The study’s results are in line with those of earlier investigations of mental health in academia, which likewise reveal significant levels of depression and anxiety among Ph.D. students. Overall, the findings provide a greater knowledge of the underlying reasons and potential interventions required for advancing the mental health problems experienced by Ph.D. students. The results of this research can guide the development of effective strategies to support the mental health of Ph.D. students.

Acute and early symptoms of forest dieback linked to climate warming and drought episodes have been reported for relict Abies pinsapo Boiss. fir forests from Southern Spain, particularly at their lower ecotone. Satellite, orthoimages, and field data were used to assess forest decline, tree mortality, and gap formation and recolonization in the lower half of the altitudinal range of A. pinsapo forests (850-1550 m) for the last 36 years (1985-2020). Field surveys were carried out in 2003 and in 2020 to characterize changes in stand canopy structure and mortality rates across the altitudinal range. Time series of the Normalized Difference Vegetation Index (NDVI) at the end of the dry season (derived from Landsat 5 and 7 imagery) were used for a Dynamic Factor Analysis to detect common trends across altitudinal bands and topographic solar incidence gradients (SI). Historical canopy cover changes were analyzed through aerial orthoimages classification. Here we show that extensive decline and mortality contrast to the almost steady alive basal area for 17 years, as well as the rising photosynthetic activity derived from NDVI since the mid-2000s and an increase in the forest canopy cover in the late years at mid and high altitudes. We hypothesized that these results suggest an unexpected resilience in A. pinsapo forests to climate change-induced dieback, that might be promoted by compensation mechanisms such as (i) recruitment of new A. pinsapo individuals; (ii) facilitative effects on such recruitment mediated by revegetation with other species; and (iii) a ‘release effect’ in which surviving trees can thrive with fewer resource competition. Future research is needed to understand these compensation mechanisms and their scope in future climate change scenarios.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to date is incurable. The major cause of death is dilated cardiomyopathy however, its pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived induced pluripotent stem cells (DMD-COs) and isogenic-corrected controls (DMD-Iso-COs) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-COs lack initial proliferative capacity, displayed a progressive loss of sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, cardiomyocyte deterioration, fibrosis and aberrant adipogenesis were observed in DMD-COs over time. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-COs which was associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies.

This paper examines multiple sluicing constructions in Mandarin Chinese (henceforth, MC) experimentally. The acceptability status of such constructions in MC is controversial, and the judgments reported in the previous literature vary. Obtaining experimental evidence on the acceptability status is, therefore, important to advance the research on multiple sluicing in MC. Consequently, the present study conducts two sets of experiments to investigate factors affecting the acceptability of multiple sluicing sentences and the influence of the distribution of shi preceding wh-remnants on acceptability ratings. The results show that multiple sluicing in MC is generally a marked construction. Nevertheless, factors including prepositionhood and specificity have ameliorating effects on the acceptability of such constructions. Moreover, the influence of the distribution of shi on the acceptability ratings is related to the nature of wh-remnants; that is, its presence significantly improves the acceptability of cases of multiple sluicing when it precedes bare wh-arguments. We argue that the observed ameliorating effects on multiple sluicing can be explained by a cue-based retrieval approach to cross-linguistic elliptical constructions. Compared to bare wh-arguments, prepositional and discourse-linked wh-phrases provide cues to facilitate the retrieval of information from antecedent clauses.

In this study we combine information from aerial LIDAR and hemispherical images taken in the field with ForeStereo—a forest inventory device—to assess the vulnerability and to design conservation strategies for endangered Mediterranean fir forests based on the mapping of fire risk and canopy structure spatial variability. We focused on the largest continuous remnant population of the endangered tree species Abies pinsapo Boiss. spanning 252 ha in Sierra de las Nieves National Park (South Andalusia, Spain). We established 49 sampling plots over the study area. Stand structure variables were derived from ForeStereo device, a proximal sensing technology for tree diameter, height and crown dimensions and stand crown cover and basal area retrieval from stereoscopic hemispherical images photogrammetry. With this information, we developed regression models with airborne LIDAR data (spatial resolution of 0.5 points∙m−2). Thereafter, six fuel models were fitted to the plots according to the UCO40 classification criteria, and then the entire area was classified using the Nearest Neighbor algorithm on Sentinel imagery (overall accuracy of 0.56 and a KIA-Kappa Coefficient of 0.46). FlamMap software was used for fire simulation scenarios based on fuel models, stand structure, and terrain data. Besides the fire simulation, we analyzed canopy structure to assess the status and vulnerability of this fir population. The assessment shows a secondary growth forest that has an increasing presence of fuel models with the potential for high fire spread rate fire and burn probability. Our methodological approach has the potential to be integrated as a support tool for the adaptive management and conservation of A. pinsapo across its whole distribution area (

Sexual forms of malaria parasites are responsible for transmission to the mosquito. Anti-malarial drug resistance remains a serious problem and requires advent of new drug therapies. Here, the authors present a high-throughput screen of potential antimalarial compounds, identifying seventeen drug-like molecules specifically targeting transmission.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.

Mesodermal iPSC-derived progenitors (MiPs) can regenerate both skeletal and cardiac muscle. Here, the authors show that a microRNA cocktail stimulates skeletal muscle differentiation and that human MiPs can engraft into striated muscle in mice.