Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-based Reporter Assays.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli. Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence. These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Using the Zebrafish Larval Model of Infection to Investigate Antibiotic Efficacy and Combination Treatments Against Staphylococcus aureus.

There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist pathogen that has developed resistance to a range of antibiotics. The zebrafish larval model of systemic disease has been increasingly utilized to elucidate S. aureus virulence mechanisms and host-pathogen interactions. Here, we outline how this model can be used to investigate the effects of different antibiotics alone and in combination against S. aureus.

Three-Dimensional Rotary Culture to Model Mycobacterial Biofilms in Low-Shear Detergent-Free Liquid Suspension.

In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.

Single-Crystal Cage Framework with High Selectivity and Reversibility in Fullerene Binding.

Host-guest chemistry, a pivotal branch of supramolecular chemistry, plays an essential role in understanding and constructing complex structures through non-covalent interactions. Organic molecular cages, characterized by their intrinsic confined cavities, can selectively bind a variety of guest molecules. Their host-guest chemistry has been well studied in the solution phase, and several attempts have been made to encode well-defined molecular architectures into solid-state polymeric materials. However, only limited studies have explored their potential in the solid state, where their lack of robustness and less ordered networks significantly hinder practical applications. Herein, we report the synthesis of a single-crystal cage framework and a systematic study of its host-guest chemistry, spanning from the solution state to the solid state. Our studies reveal that the host-guest interactions inherent to the cage are successfully maintained in the solid-state polymeric material. Furthermore, the framework's robustness allows for the guest molecules (fullerene) to be released triggered by an organic acid (trifluoracetic acid), with subsequent regeneration of the framework through an organic base (triethylamine) treatment. Our findings represent the first synthesis of a robust, single-crystal cage framework exhibiting highly selective and reversible host-guest chemistry, thus showing great potential towards molecular separation application.

The relationship between sustained hamstring pain and reorganisation of somatosensory representations: a randomised, controlled study.

ABSTRACT: Recurrent hamstring injuries are highly prevalent amongst sporting populations. It has been hypothesised that pain from an initial hamstring injury may induce reorganisation of somatosensory representations that could contribute to reinjury. However, because of the cross-sectional nature of existing research, it remains unknown whether somatosensory changes are a cause or effect of pain or if they are driven by other potentially confounding factors. Here, we explored the effect of experimentally induced sustained hamstring pain on tasks that interrogate somatosensory and spatial representations. Fifty healthy participants were randomly allocated to an experimental group that performed an eccentric exercise protocol on the right hamstring to induce delayed onset muscle soreness or a control group performing a repetition-matched concentric exercise protocol. The tactile cortical representation was assessed using two-point discrimination and tactile localisation, whereas the proprioceptive representation was assessed using a left-right judgement task. Peripersonal spatial representations were assessed using an auditory localisation task. Assessments were performed at baseline and day 2. No between-group differences in tactile acuity were observed. However, improvements in left-right judgments and worsening of auditory localisation occurred in the experimental group compared with the control group. This study provides preliminary evidence showing that somatosensory changes occur in response to sustained hamstring pain. Experimentally induced, sustained hamstring pain elicited enhancements in proprioceptive processing and deficits in peripersonal spatial processing, suggesting a shift in the allocation of attentional resources from the external (peripersonal) to internal (body) environment. These findings may hold important implications for reinjury risk and rehabilitation following hamstring pain.

The impact of biomarkers of malignancy (IMWG SLiM criteria) in myeloma in a real-world population: Clinical characteristics, therapy and outcomes from the Australian and New Zealand Myeloma and Related Diseases Registry (ANZ MRDR).

A decade after International Myeloma Working Group (IMWG) biomarkers (SLiM criteria) were introduced, this real-world study examined their impact on diagnosis, therapy and outcomes in myeloma. Using the ANZ MRDR, 3489 newly diagnosed patients from 2013 to 2023, comprising 3232 diagnosed by CRAB ('CRAB patients', including 1758 who also satisfied ≥1 SLiM criteria) and 257 by SLiM ('SLiM patients') criteria were analysed. CRAB patients had higher R-ISS and lower performance status, with no difference in cytogenetic risk. SLiM patients had improved progression-free survival (PFS, 37.5 vs. 32.2 months, hazard ratio [HR] 1.31 [1.08-1.59], p = 0.003), overall survival (80.9 vs. 73.2 months, HR 1.64 [1.26-2.13], p < 0.001) and PFS2 (54.6 vs. 40.3 months, HR 1.51 [1.22-1.86], p < 0.001) compared with CRAB patients, partially explained by earlier diagnosis, with no differential impact between the plasma cell and light-chain criteria on PFS. However, 34% of CRAB patients did not manifest SLiM characteristics, raising the possibility that SLiM features are associated with different biological behaviours contributing to a better prognosis, for example, improved PFS2 in SLiM patients suggested less disease resistance at first relapse. These data support earlier initiation of therapy by SLiM. The superior survival outcomes of SLiM versus CRAB patients highlight the importance of defining these subgroups when interpreting therapeutic outcomes at induction and first relapse.

Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53.

Purpose: Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN). Methods: After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS). Results: In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53. Conclusion: The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and -9.1 °C.

Bayesian phylogenetic analysis of Philippine languages supports a rapid migration of Malayo-Polynesian languages.

The Philippines are central to understanding the expansion of the Austronesian language family from its homeland in Taiwan. It remains unknown to what extent the distribution of Malayo-Polynesian languages has been shaped by back migrations and language leveling events following the initial Out-of-Taiwan expansion. Other aspects of language history, including the effect of language switching from non-Austronesian languages, also remain poorly understood. Here we apply Bayesian phylogenetic methods to a core-vocabulary dataset of Philippine languages. Our analysis strongly supports a sister group relationship between the Sangiric and Minahasan groups of northern Sulawesi on one hand, and the rest of the Philippine languages on the other, which is incompatible with a simple North-to-South dispersal from Taiwan. We find a pervasive geographical signal in our results, suggesting a dominant role for cultural diffusion in the evolution of Philippine languages. However, we do find some support for a later migration of Gorontalo-Mongondow languages to northern Sulawesi from the Philippines. Subsequent diffusion processes between languages in Sulawesi appear to have led to conflicting data and a highly unstable phylogenetic position for Gorontalo-Mongondow. In the Philippines, language switching to Austronesian in 'Negrito' groups appears to have occurred at different time-points throughout the Philippines, and based on our analysis, there is no discernible effect of language switching on the basic vocabulary.

Novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives and their antitrypanosomal activities against T.brucei.

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense and is invariably fatal unless treated. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work, informed by previous findings, presents novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives with promising antitrypanosomal activity. In particular, 32 exhibits an in vitro EC50 value of 0.5 µM against Trypanosoma brucei rhodesiense, and analogues 29, 30 and 33 show antitrypanosomal activities in the <1 µM range. We have demonstrated that substituted 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidines present promising antitrypanosomal hit molecules with potential for further preclinical development.

Prediction of motor and non-motor Parkinson's disease symptoms using serum lipidomics and machine learning: a 2-year study.

Identifying biological factors which contribute to the clinical progression of heterogeneous motor and non-motor phenotypes in Parkinson's disease may help to better understand the disease process. Several lipid-related genetic risk factors for Parkinson's disease have been identified, and the serum lipid signature of Parkinson's disease patients is significantly distinguishable from controls. However, the extent to which lipid profiles are associated with clinical outcomes remains unclear. Untargeted high-performance liquid chromatography-tandem mass spectrometry identified >900 serum lipids in Parkinson's disease subjects at baseline (n = 122), and the potential for machine learning models using these lipids to predict motor and non-motor clinical scores after 2 years (n = 67) was assessed. Machine learning models performed best when baseline serum lipids were used to predict the 2-year future Unified Parkinson's disease rating scale part three (UPDRS III) and Geriatric Depression Scale scores (both normalised root mean square error = 0.7). Feature analysis of machine learning models indicated that species of lysophosphatidylethanolamine, phosphatidylcholine, platelet-activating factor, sphingomyelin, diacylglycerol and triacylglycerol were top predictors of both motor and non-motor scores. Serum lipids were overall more important predictors of clinical outcomes than subject sex, age and mutation status of the Parkinson's disease risk gene LRRK2. Furthermore, lipids were found to better predict clinical scales than a panel of 27 serum cytokines previously measured in this cohort (The Michael J. Fox Foundation LRRK2 Clinical Cohort Consortium). These results suggest that lipid changes may be associated with clinical phenotypes in Parkinson's disease.

Arthrospira platensis nanoparticles dietary supplementation improves growth performance, steroid hormone balance, and reproductive productivity of Nile tilapia (Oreochromis niloticus) broodstock.

This study evaluates the impact of dietary supplementation of the blue-green alga Arthrospira platensis NIOF17/003 nanoparticles (AN) on the growth performance, whole-body biochemical compositions, blood biochemistry, steroid hormonal, and fry production efficiency of Nile tilapia (Oreochromis niloticus) broodstock, during the spawning season. After a 21-day preparation period to equip the females and ensure that their ovaries were filled with eggs, mating between the mature females and males took place in a 3:1 ratio during a 14-day spawning cycle. A total of 384 tilapia broodstock 288 females and 96 males with an initial body weight of 450.53±0.75, were divided into four groups; AN0: a basal diet as a control group with no supplementation of Arthrospira platensis, and the other three groups (AN2, AN4, and AN6) were diets supplemented with nanoparticles of A. platensis at levels of 2, 4, and 6 g kg─1 diet, respectively. The results found that fish-fed group AN6 showed the highest significant differences in weight gain (WG), final weight (FW), feed conversion ratio (FCR), protein efficiency ratio (PER), and feed efficiency ratio (FER). Females fed the AN6 diet showed the highest significant fat content. Compared to the AN0 group, fish fed on the supplemented diets showed significant improvement (p < 0.05) in triglyceride, glucose, and aspartate aminotransferase (AST). A gradual increase in AN inclusion level resulted in a gradual increase in the concentrations of luteinizing hormone (LH), and follicle-stimulating hormone (FSH), testosterone, progesterone, and prolactin. The rates (%) of increase in fry production for females fed supplemented diets were 10.5, 18.6, and 32.2% for AN2, AN4, and AN6, respectively, compared to the control group. This work concluded that the inclusion levels of 6 g kg─1 of A. platensis nanoparticles in the diet of Nile tilapia broodstock significantly improved the growth performances, steroid hormone concentrations, and increased the fry production efficiency by 32.2%, respectively. These findings revealed that A. platensis nanoparticles resulted in a significantly enhanced female' reproductive productivity of Nile tilapia broodstock.

Juvenile social play predicts adult reproductive success in male bottlenose dolphins.

For over a century, the evolution of animal play has sparked scientific curiosity. The prevalence of social play in juvenile mammals suggests that play is a beneficial behavior, potentially contributing to individual fitness. Yet evidence from wild animals supporting the long-hypothesized link between juvenile social play, adult behavior, and fitness remains limited. In Western Australia, adult male bottlenose dolphins (Tursiops aduncus) form multilevel alliances that are crucial for their reproductive success. A key adult mating behavior involves allied males using joint action to herd individual females. Juveniles of both sexes invest significant time in play that resembles adult herding-taking turns in mature male (actor) and female (receiver) roles. Using a 32-y dataset of individual-level association patterns, paternity success, and behavioral observations, we show that juvenile males with stronger social bonds are significantly more likely to engage in joint action when play-herding in actor roles. Juvenile males also monopolized the actor role and produced an adult male herding vocalization ("pops") when playing with females. Notably, males who spent more time playing in the actor role as juveniles achieved more paternities as adults. These findings not only reveal that play behavior provides male dolphins with mating skill practice years before they sexually mature but also demonstrate in a wild animal population that juvenile social play predicts adult reproductive success.

Resolving length-scale-dependent transient disorder through an ultrafast phase transition.

Material functionality can be strongly determined by structure extending only over nanoscale distances. The pair distribution function presents an opportunity for structural studies beyond idealized crystal models and to investigate structure over varying length scales. Applying this method with ultrafast time resolution has the potential to similarly disrupt the study of structural dynamics and phase transitions. Here we demonstrate such a measurement of CuIr2S4 optically pumped from its low-temperature Ir-dimerized phase. Dimers are optically suppressed without spatial correlation, generating a structure whose level of disorder strongly depends on the length scale. The redevelopment of structural ordering over tens of picoseconds is directly tracked over both space and time as a transient state is approached. This measurement demonstrates the crucial role of local structure and disorder in non-equilibrium processes as well as the feasibility of accessing this information with state-of-the-art XFEL facilities.

Engineering Lipophilic Aggregation of Adapalene and Adamantane-Based Cocrystals via van der Waals Forces and Hydrogen Bonding.

Lipophilic aggregation using adamantanes is a widely exploited molecular property in medicinal and materials chemistry. Adamantanes are traditionally installed to molecular units via covalent bonds. However, the noncovalent installation of adamantanes has been relatively underexplored and presents the potential to bring properties associated with adamantanes to molecules without affecting their intrinsic properties (e.g., pharmacophores). Here, we systematically study a series of adamantanecarboxylic acids with varying substitution levels of methyl groups and their cocrystals with bipyridines. Specifically, single-crystal X-ray diffraction shows that while the directionality of single-component adamantanes is notably sensitive to changes in methyl substitution, hydrogen-bonded cocrystals with bipyridines show consistent and robust packing due to π-stacking predominance. Our observations are supported by Hirshfeld surface and energy framework analyses. The applicability of cocrystal formation of adamantanes bearing carboxylic acids was used to generate the first cocrystals of adapalene, an adamantane-bearing retinoid used for treating acne vulgaris. We envisage our study to inspire noncovalent (i.e., cocrystal) installation of adamantanes to generate lipophilic aggregation in multicomponent systems.

From yield stress to elastic instabilities: Tuning the extensional behavior of elastoviscoplastic fluids.

In this study, we delve into the intricacies of elastoviscoplastic (EVP) fluids, particularly focusing on how polymer additives influence their extensional behavior. Our findings reveal that polymer additives significantly alter the extensional properties of the EVP fluids, such as relaxation time and extensional stresses while having negligible impact on the shear rheology. Interestingly, the modified fluids exhibit a transition from yield stress-like behavior to viscoelastic-like behavior under high extensional rates, ultimately leading to destabilization under extreme deformation. This research enhances the fundamental understanding of EVP fluids and highlights potential advancements in applications, especially in precision-demanding fields like 3D printing.

Enzyme-like Acyl Transfer Catalysis in a Bifunctional Organic Cage.

Amide-based organic cage cavities are, in principle, ideal enzyme active site mimics. Yet, cage-promoted organocatalysis has remained elusive, in large part due to synthetic accessibility of robust and functional scaffolds. Herein, we report the acyl transfer catalysis properties of robust, hexaamide cages in organic solvent. Cage structural variation reveals that esterification catalysis with an acyl anhydride acyl carrier occurs only in bifunctional cages featuring internal pyridine motifs and two crucial antipodal carboxylic acid groups. 1H NMR data and X-ray crystallography show that the acyl carrier is rapidly activated inside the cavity as a covalent mixed-anhydride intermediate with an internal hydrogen bond. Michaelis-Menten (saturation) kinetics suggest weak binding (KM = 0.16 M) of the alcohol pronucleophile close to the internal anhydride. Finally, activation and delivery of the alcohol to the internal anhydride by the second carboxylic acid group forms ester product and releases the cage catalyst. Eyring analysis indicates a strong enthalpic stabilization of the transition state (5.5 kcal/mol) corresponding to a rate acceleration of 104 over background acylation, and an ordered, associative rate-determining attack by the alcohol, supported by DFT calculations. We conclude that internal bifunctional organocatalysis specific to the cage structural design is responsible for the enhancement over the background reaction. These results pave the way for organic-phase enzyme mimicry in self-assembled cavities with the potential for cavity elaboration to enact selective acylations.

Statistical optimization of guest uptake in crystalline sponges: grading structural outcomes.

Investigation of the analyte soaking conditions on the crystalline sponge {[(ZnI2)3(tpt)2·x(solvent)]n} method using a statistical design of experiments model has provided fundamental insights into the influence of experimental variables. This approach focuses on a single analyte tested via 60 experiments (20 unique conditions) to identify the main effects for success and overall guest structure quality. This is employed as a basis for the development of a novel molecular structure grading system that enables the quantification of guest exchange quality.

Coronavirus Disease 2019 (COVID-19) Vaccination and Stillbirth in the Vaccine Safety Datalink.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) vaccination is recommended in pregnancy to reduce the risk of severe morbidity from COVID-19. However, vaccine hesitancy persists among pregnant people, with risk of stillbirth being a primary concern. Our objective was to examine the association between COVID-19 vaccination and stillbirth. METHODS: We performed a matched case-control study in the Vaccine Safety Datalink (VSD). Stillbirths and live births were selected from singleton pregnancies among persons aged 16-49 years with at least one prenatal, delivery, or postpartum visit at eight participating VSD sites. Stillbirths identified through diagnostic codes were adjudicated to confirm the outcome, date, and gestational age at fetal death. Confirmed antepartum stillbirths that occurred between February 14, 2021, and February 27, 2022, then were matched 1:3 to live births by pregnancy start date, VSD site, and maternal age at delivery. Associations among antepartum stillbirth and COVID-19 vaccination in pregnancy, vaccine manufacturer, number of vaccine doses received, and vaccination within 6 weeks before stillbirth (or index date in live births) were evaluated using conditional logistic regression. RESULTS: In the matched analysis of 276 confirmed antepartum stillbirths and 822 live births, we found no association between COVID-19 vaccination during pregnancy and stillbirth (38.4% stillbirths vs 39.3% live births in vaccinated individuals, adjusted odds ratio [aOR] 1.02, 95% CI, 0.76-1.37). Furthermore, no association between COVID-19 vaccination and stillbirth was detected by vaccine manufacturer (Moderna: aOR 1.00, 95% CI, 0.62-1.62; Pfizer-BioNTech: aOR 1.00, 95% CI, 0.69-1.43), number of vaccine doses received during pregnancy (1 vs 0: aOR 1.17, 95% CI, 0.75-1.83; 2 vs 0: aOR 0.98, 95% CI, 0.81-1.17), or COVID-19 vaccination within the 6 weeks before stillbirth or index date compared with no vaccination (aOR 1.16, 95% CI, 0.74-1.83). CONCLUSION: No association was found between COVID-19 vaccination and stillbirth. These findings further support recommendations for COVID-19 vaccination in pregnancy.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex.

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.