DNAJC9 prevents CENP-A mislocalization and chromosomal instability by maintaining the fidelity of histone supply chains.

The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.

The histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability.

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.

Flavonifractor plautii Protects Against Elevated Arterial Stiffness.

BACKGROUND: Dysbiosis of gut microbiota plays a pivotal role in vascular dysfunction and microbial diversity was reported to be inversely correlated with arterial stiffness. However, the causal role of gut microbiota in the progression of arterial stiffness and the specific species along with the molecular mechanisms underlying this change remain largely unknown. METHODS: Participants with elevated arterial stiffness and normal controls free of medication were matched for age and sex. The microbial composition and metabolic capacities between the 2 groups were compared with the integration of metagenomics and metabolomics. Subsequently, Ang II (angiotensin II)-induced and humanized mouse model were employed to evaluate the protective effect of Flavonifractor plautii (F plautii) and its main effector cis-aconitic acid. RESULTS: Human fecal metagenomic sequencing revealed a significantly high abundance and centrality of F plautii in normal controls, which was absent in the microbial community of subjects with elevated arterial stiffness. Moreover, blood pressure only mediated part of the effect of F plautii on lower arterial stiffness. The microbiome of normal controls exhibited an enhanced capacity for glycolysis and polysaccharide degradation, whereas, those of subjects with increased arterial stiffness were characterized by increased biosynthesis of fatty acids and aromatic amino acids. Integrative analysis with metabolomics profiling further suggested that increased cis-aconitic acid served as the main effector for the protective effect of F plautii against arterial stiffness. Replenishment with F plautii and cis-aconitic acid improved elastic fiber network and reversed increased pulse wave velocity through the suppression of MMP-2 (matrix metalloproteinase-2) and inhibition of MCP-1 (monocyte chemoattractant protein-1) and NF-κB (nuclear factor kappa-B) activation in both Ang II-induced and humanized model of arterial stiffness. CONCLUSIONS: Our translational study identifies a novel link between F plautii and arterial function and raises the possibility of sustaining vascular health by targeting gut microbiota.

Unpacking the navigation toolbox: insights from comparative cognition.

The study of navigation is informed by ethological data from many species, laboratory investigation at behavioural and neurobiological levels, and computational modelling. However, the data are often species-specific, making it challenging to develop general models of how biology supports behaviour. Wiener et al. outlined a framework for organizing the results across taxa, called the 'navigation toolbox' (Wiener et al. In Animal thinking: contemporary issues in comparative cognition (eds R Menzel, J Fischer), pp. 51-76). This framework proposes that spatial cognition is a hierarchical process in which sensory inputs at the lowest level are successively combined into ever-more complex representations, culminating in a metric or quasi-metric internal model of the world (cognitive map). Some animals, notably humans, also use symbolic representations to produce an external representation, such as a verbal description, signpost or map that allows communication of spatial information or instructions between individuals. Recently, new discoveries have extended our understanding of how spatial representations are constructed, highlighting that the hierarchical relationships are bidirectional, with higher levels feeding back to influence lower levels. In the light of these new developments, we revisit the navigation toolbox, elaborate it and incorporate new findings. The toolbox provides a common framework within which the results from different taxa can be described and compared, yielding a more detailed, mechanistic and generalized understanding of navigation.

Nest excavators' learning walks in the Australian desert ant Melophorus bagoti.

The Australian red honey ant, Melophorus bagoti, stands out as the most thermophilic ant in Australia, engaging in all outdoor activities during the hottest periods of the day during summer months. This species of desert ants often navigates by means of path integration and learning landmark cues around the nest. In our study, we observed the outdoor activities of M. bagoti workers engaged in nest excavation, the maintenance of the nest structure, primarily by taking excess sand out of the nest. Before undertaking nest excavation, the ants conducted a single exploratory walk. Following their initial learning expedition, these ants then engaged in nest excavation activities. Consistent with previous findings on pre-foraging learning walks, after just one learning walk, the desert ants in our study demonstrated the ability to return home from locations 2 m away from the nest, although not from locations 4 m away. These findings indicate that even for activities like dumping excavated sand within a range of 5-10 cm outside the nest, these ants learn and utilize the visual landmark panorama around the nest.

Regulation of MBK-2/DYRK by CDK-1 and the pseudophosphatases EGG-4 and EGG-5 during the oocyte-to-embryo transition.

DYRKs are kinases that self-activate in vitro by autophosphorylation of a YTY motif in the kinase domain, but their regulation in vivo is not well understood. In C. elegans zygotes, MBK-2/DYRK phosphorylates oocyte proteins at the end of the meiotic divisions to promote the oocyte-to-embryo transition. Here we demonstrate that MBK-2 is under both positive and negative regulation during the transition. MBK-2 is activated during oocyte maturation by CDK-1-dependent phosphorylation of serine 68, a residue outside of the kinase domain required for full activity in vivo. The pseudotyrosine phosphatases EGG-4 and EGG-5 sequester activated MBK-2 until the meiotic divisions by binding to the YTY motif and inhibiting MBK-2's kinase activity directly, using a mixed-inhibition mechanism that does not involve tyrosine dephosphorylation. Our findings link cell-cycle progression to MBK-2/DYRK activation and the oocyte-to-embryo transition.

Effect of repetition of vertical and horizontal routes on navigation performance in Australian bull ants.

Solitarily foraging ant species differ in their reliance on their two primary navigational systems- path integration and visual learning. Despite many species of Australian bull ants spending most of their foraging time on their foraging tree, little is known about the use of these systems while climbing. "Rewinding" displacements are commonly used to understand navigational system usage, and work by introducing a mismatch between these navigational systems, by displacing foragers after they have run-down their path integration vector. We used rewinding to test the role of path integration on the arboreal and terrestrial navigation of M. midas. We rewound foragers along either the vertical portion, the ground surface portion, or across both portions of their homing trip. Since rewinding involves repeatedly capturing and releasing foragers, we included a nondisplacement, capture-and-release control, in which the path integration vector is unchanged. We found that rewound foragers do not seem to accumulate path integration vector, although a limited effect of vertical rewinding was found, suggesting a potential higher sensitivity while descending the foraging tree. However, the decrease in navigational efficiency due to capture was larger than the vertical rewinding effect, which along with the negative impact of the vertical surface, and an interaction between capture and rewinding, may suggest aversion rather than path integration caused the vertical rewinding response. Together these results add to the evidence that M. midas makes minimal use of path integration while foraging, and the growing evidence that they are capable of quickly learning from aversive stimulus.

Landmarks, beacons, or panoramic views: What do pigeons attend to for guidance in familiar environments?

Birds and social insects represent excellent systems for understanding visually guided navigation. Both animal groups use surrounding visual cues for homing and foraging. Ants extract sufficient spatial information from panoramic views, which naturally embed all near and far spatial information, for successful homing. Although egocentric panoramic views allow for parsimonious explanations of navigational behaviors, this potential source of spatial information has been mostly neglected during studies of vertebrates. Here we investigate how distinct landmarks, a beacon, and panoramic views influence the reorientation behavior in pigeons (Columba livia). Pigeons were trained to search for a location characterized by a beacon and several distinct landmarks. Transformation tests manipulated aspects of the landmark configuration, allowing for a dissociation among navigational strategies. Quantitative image and path analyses provided support that the panoramic view was used by the pigeons. Although the results from some individuals support the use of beaconing, overall the pigeons relied predominantly on the panoramic view when spatial cues provided conflicting information regarding the goal location. Reorientation based on vector and bearing information derived from distinct landmarks as well as environmental geometry failed to account fully for the results. Thus, the results of our study support that pigeons can use panoramic views for reorientation in familiar environments. Given that the current model for landmark use by pigeons posits the use of different vectors from an object, a global panorama-matching strategy suggests a fundamental change in the theory of how pigeons use surrounding visual cues for localization.

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors.

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1-CUL4CDT2 ubiquitin ligase.

Whole exome sequencing improves genetic diagnosis of fetal clubfoot.

OBJECTIVE: This retrospective study aimed to investigate the value of whole exome sequencing (WES) for clubfoot (CF) fetuses with or without other structural abnormalities and to further explore the genetic causes of fetal CF. METHODS: this study included 83 singleton pregnancies diagnosed with fetal CF referred to our center between January 2016 and March 2022; cases were divided into two groups: isolated CF and non-isolated CF. After excluding cases with positive karyotyping and chromosomal microarray analysis results, WES was performed for the eligible fetuses and parents. Monogenic variants detected by WES and perinatal outcomes were recorded and evaluated at postnatal follow-up. RESULTS: overall, clinically significant variations were identified in 12.0% (10/83) of fetuses, and the detection rate was significantly higher in the non-isolated than in the isolated CF group (8/36, 22.2% vs. 2/47, 4.3%, p = 0.031). We additionally detected eight (9.6%) fetuses harboring variants of unknown significance. We identified 11 clinically significant variations correlating with clinical phenotypes in nine genes from ten fetuses, with KLHL40 being the most frequent (n = 2). Furthermore, we observed a significant difference in termination and survival rates between isolated and non-isolated CF cases (27.6 vs. 77.8% and 59.6 vs. 19.4%, p < 0.001 for both). CONCLUSION: our data indicate that WES has a high additional diagnostic yield for the molecular diagnosis of fetal CF, markedly enhancing existing prenatal diagnostic capabilities and expanding our understanding of intrauterine genetic disorders, thus assisting us to better interpret fetal phenotype in the future.

Prenatal diagnosis in the fetal hyperechogenic kidneys: assessment using chromosomal microarray analysis and exome sequencing.

Fetal hyperechogenic kidneys (HEK) is etiologically a heterogeneous disorder. The aim of this study was to identify the genetic causes of HEK using prenatal chromosomal microarray analysis (CMA) and exome sequencing (ES). From June 2014 to September 2022, we identified 92 HEK fetuses detected by ultrasound. We reviewed and documented other ultrasound anomalies, microscopic and submicroscopic chromosomal abnormalities, and single gene disorders. We also analyzed the diagnostic yield of CMA and ES and the clinical impact the diagnosis had on pregnancy management. In our cohort, CMA detected 27 pathogenic copy number variations (CNVs) in 25 (25/92, 27.2%) fetuses, with the most common CNV being 17q12 microdeletion syndrome. Among the 26 fetuses who underwent further ES testing, we identified 7 pathogenic/likely pathogenic variants and 8 variants of uncertain significance in 9 genes in 12 fetuses. Four novel variants were first reported herein, expanding the mutational spectra for HEK-related genes. Following counseling, 52 families chose to continue the pregnancy, and in 23 of them, postnatal ultrasound showed no detectable renal abnormalities. Of these 23 cases, 15 had isolated HEK on prenatal ultrasound. Taken together, our study showed a high rate of detectable genetic etiologies in cases with fetal HEK at the levels of chromosomal (aneuploidy), sub-chromosomal (microdeletions/microduplications), and single gene (point mutations). Therefore, we speculate that combined CMA and ES testing for fetal HEK is feasible and has good clinical utility. When no genetic abnormalities are identified, the findings can be transient, especially in the isolated HEK group.

Scanning behaviour in ants: an interplay between random-rate processes and oscillators.

At the start of a journey home or to a foraging site, ants often stop, interrupting their forward movement, turn on the spot a number of times, and fixate in different directions. These scanning bouts are thought to provide visual information for choosing a path to travel. The temporal organization of such scanning bouts has implications about the neural organisation of navigational behaviour. We examined (1) the temporal distribution of the start of such scanning bouts and (2) the dynamics of saccadic body turns and fixations that compose a scanning bout in Australian desert ants, Melophorus bagoti, as they came out of a walled channel onto open field at the start of their homeward journey. Ants were caught when they neared their nest and displaced to different locations to start their journey home again. The observed parameters were mostly similar across familiar and unfamiliar locations. The turning angles of saccadic body turning to the right or left showed some stereotypy, with a peak just under 45°. The direction of such saccades appears to be determined by a slow oscillatory process as described in other insect species. In timing, however, both the distribution of inter-scanning-bout intervals and individual fixation durations showed exponential characteristics, the signature for a random-rate or Poisson process. Neurobiologically, therefore, there must be some process that switches behaviour (starting a scanning bout or ending a fixation) with equal probability at every moment in time. We discuss how chance events in the ant brain that occasionally reach a threshold for triggering such behaviours can generate the results.

From representations to servomechanisms to oscillators: my journey in the study of cognition.

The study of comparative cognition bloomed in the 1970s and 1980s with a focus on representations in the heads of animals that undergird what animals can achieve. Even in action-packed domains such as navigation and spatial cognition, a focus on representations prevailed. In the 1990s, I suggested a conception of navigation in terms of navigational servomechanisms. A servomechanism can be said to aim for a goal, with deviations from the goal-directed path registering as an error. The error drives action to reduce the error in a negative-feedback loop. This loop, with the action reducing the very signal that drove action in the first place, is key to defining a servomechanism. Even though actions are crucial components of servomechanisms, my focus was on the representational component that encodes signals and evaluates errors. Recently, I modified and amplified this view in claiming that, in navigation, servomechanisms operate by modulating the performance of oscillators, endogenous units that produce periodic action. The pattern is found from bacteria travelling micrometres to sea turtles travelling thousands of kilometres. This pattern of servomechanisms working with oscillators is found in other realms of cognition and of life. I think that oscillators provide an effective way to organise an organism's own activities while servomechanisms provide an effective means to adjust to the organism's environment, including that of its own body.

The Basis of Navigation Across Species.

Animals navigate a wide range of distances, from a few millimeters to globe-spanning journeys of thousands of kilometers. Despite this array of navigational challenges, similar principles underlie these behaviors across species. Here, we focus on the navigational strategies and supporting mechanisms in four well-known systems: the large-scale migratory behaviors of sea turtles and lepidopterans as well as navigation on a smaller scale by rats and solitarily foraging ants. In lepidopterans, rats, and ants we also discuss the current understanding of the neural architecture which supports navigation. The orientation and navigational behaviors of these animals are defined in terms of behavioral error-reduction strategies reliant on multiple goal-directed servomechanisms. We conclude by proposing to incorporate an additional component into this system: the observation that servomechanisms operate on oscillatory systems of cycling behavior. These oscillators and servomechanisms comprise the basis for directed orientation and navigational behaviors.

Ants on featureless saltpans build tall nest mounds.

North African desert ants Cataglyphis fortis living on a featureless saltpan far from the shoreline build a mound at their nest entrance. Experimental manipulations show that they do this purposefully to make it easier for returning foragers to find their nest.

Resolving conflict between aversive and appetitive learning of views: how ants shift to a new route during navigation.

Ants store and recall views associated with foraging success, facilitating future foraging journeys. Negative views are also learned, but instead prompt avoidance behaviors such as turning away. However, little is known about the aversive view's role in navigation, the effect of cue conflict, or the contextual relationship between learning and recalling. In this study, we tested Myrmecia midas' capacity for aversive learning of views either independently of or in conflict with appetitive events. We either captured and released foragers when reaching a location or let them pass unhindered. After a few journeys, captured foragers exhibited aversive learning by circumventing the capture location and increasing both meandering and scanning. Ants that experienced foraging-appetitive and homing-aversive events on their journeys exhibited lower rates of avoidance behavior and scans than those experiencing aversive events in both outbound and homebound journeys. The foraging-aversive and homing-aversive ants exhibited similar levels of avoidance and scanning as those that experienced the foraging-aversive and homing-appetitive. We found that foragers showed evidence of context specificity in their scanning behavior, but not in other measures of aversive learning. The foragers did not increase their meandering and scans while approaching the views associated with aversive events. In addition to shedding light on the role of aversive views in navigation, our finding has important implications for understanding the learning mechanisms triggered by handling animals.

Oscillators and servomechanisms in orientation and navigation, and sometimes in cognition.

Navigational mechanisms have been characterized as servomechanisms. A navigational servomechanism specifies a goal state to strive for. Discrepancies between the perceived current state and the goal state specify error. Servomechanisms adjust the course of travel to reduce the error. I now add that navigational servomechanisms work with oscillators, periodic movements of effectors that drive locomotion. I illustrate this concept selectively over a vast range of scales of travel from micrometres in bacteria to thousands of kilometres in sea turtles. The servomechanisms differ in sophistication, with some interrupting forward motion occasionally or changing travel speed in kineses and others adjusting the direction of travel in taxes. I suggest that in other realms of life as well, especially in cognition, servomechanisms work with oscillators.

What view information is most important in the homeward navigation of an Australian bull ant, Myrmecia midas?

Many insects orient by comparing current panoramic views of their environment to memorised views. We tested the navigational abilities of night-active Myrmecia midas foragers while we blocked segments of their visual panorama. Foragers failed to orient homewards when the front view, lower elevations, entire terrestrial surround, or the full panorama was blocked. Initial scanning increased whenever the visual panorama was blocked but scanning only increased along the rest of the route when the front, back, higher, or lower elevations were blocked. Ants meandered more when the front, the back, or the higher elevations were obscured. When everything except the canopy was blocked, the ants were quick and direct, but moved in random directions, as if to escape. We conclude that a clear front view, or a clear lower panorama is necessary for initial homeward headings. Furthermore, the canopy is neither necessary nor sufficient for homeward initial heading, and the back and upper segments of views, while not necessary, do make finding home easier. Discrepancies between image analysis and ant behaviour when the upper and lower views were blocked suggests that ants are selective in what portions of the scene they attend to or learn.

Effects of dual chelation therapy with deferasirox and deferoxamine in patients with beta thalassaemia major.

BACKGROUND AND OBJECTIVES: Patients with thalassaemia experience complications related to iron overload. In Australia currently, the two main options for iron chelation are deferasirox and deferoxamine. Optimal iron chelation using monotherapy can be limited due to toxicity or tolerability. Dual chelation therapy (DCT) may provide more aggressive iron chelation. MATERIAL AND METHODS: A retrospective, observational study was performed on a state-wide referral centre for patients receiving red cell transfusions for haemoglobinopathies (Monash Health, Australia). All patients prescribed DCT were identified using a local pharmacy dispensing database and were included in the study. Pre-DCT initiation and post-DCT completion were correlated with serum ferritin, cardiac iron loading (based on MRI T2* measurements) and liver iron content (LIC) using Wilcoxon signed-rank test. RESULTS: A total of 18 patients (12 adults, 6 children) were identified as receiving DCT. All patients received a combination of deferasirox and deferoxamine. The median duration of therapy was 23 months (range 2-73). Median serum ferritin reduced by 42% (p = 0.004) and there was a 76% reduction in LIC (p = 0.062). No significant changes were seen in cardiac iron loading. CONCLUSION: DCT over a prolonged period is effective at reducing serum ferritin and may contribute to improvement in liver iron loading.

Prenatal exome sequencing in fetuses with callosal anomalies.

OBJECTIVE: We aimed to investigate the value of exome sequencing (ES) in fetuses with callosal anomalies (CA) with or without other structural anomalies, but with normal findings by karyotyping and chromosome microarray analysis (CMA). METHODS: Cases with CA with or without other structural anomalies were screened for eligibility. Fetuses with abnormal karyotyping or CMA results were excluded. We performed ES on DNA samples from eligible fetus-parental trios and identified diagnostic genetic variants based on the ultrasonographic features. RESULTS: A total of 50 eligible fetus-parental trios were successfully analyzed by ES. We found 17 likely pathogenic or pathogenic variants in 14 genes from 17 fetuses, with a total proportion of diagnostic genetic variants equal to 34.0% (17/50). Of the 17 cases with a diagnosis, 10 (29.4%, 10/35) were isolated and 7 (43.8%, 7/15) were non-isolated. Pregnancy outcome data showed that 70.0% (7/10) of the surviving isolated CA fetuses with negative ES results had a good prognosis in early childhood. CONCLUSIONS: Our study used ES prenatally for CA and showed that ES can be used diagnostically to define the molecular defects that underlie unexplained CA. Most subjects with isolated CA with negative results for genetic causes will have a favorable prognosis in early childhood.