Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.

CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.

Chemogenetic approaches reveal dual functions of microglia in seizures.

Microglia are key players in maintaining brain homeostasis and exhibit phenotypic alterations in response to epileptic stimuli. However, it is still relatively unknown if these alterations are pro- or anti-epileptic. To unravel this dilemma, we employed chemogenetic manipulation of microglia using the artificial Gi-Dreadd receptor within a kainic acid (KA) induced murine seizure model. Our results indicate that acute Gi-Dreadd activation with Clozapine-N-Oxide can reduce seizure severity. Additionally, we observed increased interaction between microglia and neuronal soma, which correlated with reduced neuronal hyperactivity. Interestingly, prolonged activation of microglial Gi-Dreadds by repeated doses of CNO over 3 days, arrested microglia in a less active, homeostatic-like state, which associated with increased neuronal loss after KA induced seizures. RNAseq analysis revealed that prolonged activation of Gi-Dreadd interferes with interferon ß signaling and microglia proliferation. Thus, our findings highlight the importance of microglial Gi signaling not only during status epilepticus (SE) but also within later seizure induced pathology.

Optogenetic activation of spinal microglia triggers chronic pain in mice.

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1ß production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.

Mechanistic basis of 5'-3' translocation in SF1B helicases.

Superfamily 1B (SF1B) helicases translocate in a 5'-3' direction and are required for a range of cellular activities across all domains of life. However, structural analyses to date have focused on how SF1A helicases achieve 3'-5' movement along nucleic acids. We present crystal structures of the complex between the SF1B helicase RecD2 from Deinococcus radiodurans and ssDNA in the presence and absence of an ATP analog. These snapshots of the reaction pathway reveal a nucleotide binding-induced conformational change of the two motor domains that is broadly reminiscent of changes observed in other SF1 and SF2 helicases. Together with biochemical data, the structures point to a step size for translocation of one base per ATP hydrolyzed. Moreover, the structures also reveal a mechanism for nucleic acid translocation in the 5'-3' direction by SF1B helicases that is surprisingly different from that of 3'-5' translocation by SF1A enzymes, and explains the molecular basis of directionality.

Structure and regulation of the human INO80-nucleosome complex.

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines 1 . Biochemical studies2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2-a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)-in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding 8 , histone exchange 9 and nucleosome spacing10-12 remain poorly understood. Although some remodellers work as monomers 13 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain-unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.

When Change Chooses You: My Journey With Paralysis.

Life sometimes provides unannounced challenges that threaten the structure of our existence. This personal account of an ob-gyn's encounter with paralysis will illustrate the impact of one such experience.

Validity of the Hawkin Dynamics Wireless Dual Force Platform System Against a Piezoelectric Laboratory Grade System for Vertical Countermovement Jump Variables.

ABSTRACT: Dos'Santos, T, Evans, DT, and Read, DB. Validity of the Hawkin dynamics wireless dual force platform system against a piezoelectric laboratory grade system for vertical countermovement jump variables. J Strength Cond Res 38(6): 1144-1148, 2024-The aim of this study was to determine the criterion validity of the Hawkin Dynamics (HD) wireless dual force platform system for assessing vertical countermovement jump (CMJ) variables, compared with those derived from a Kistler piezoelectric laboratory grade force platform system. During a single testing session, HD force platforms were placed directly on top of 2 adjacent Kistler force platforms to simultaneously collect vertical ground reaction forces produced by 2 male recreational soccer players (age: 29.0 ± 2.8 years, height: 1.79 ± 0.01 m, mass: 85.6 ± 4.7 kg) that performed 25 vertical CMJs each. Sixteen vertical CMJ variables pertaining to jump height (JH), flight time (FT), time-to-take off (TTT), countermovement depth, body weight (BW), propulsive and braking mean, and peak powers, forces, and impulses were compared between systems. Fixed bias was observed for 6 of 16 variables (peak and mean braking power, mean propulsion force, TTT, FT, and BW), while proportional bias was present for 10 of 16 variables (peak and mean propulsive and braking force, TTT, FT, peak and mean braking power, mean propulsive power, and BW). For all variables regardless of fixed or proportional bias, percentage differences were ≤3.4% between force platform systems, with near perfect to perfect correlations (r or ρ = 0.977-1.000) observed for 15 of 16 variables. The HD dual wireless force platform system can be considered a valid alternative to a piezoelectric laboratory grade force platform system for the collection of vertical CMJ variables, particularly outcome (i.e., JH, reactive strength index modified) and strategy variables (countermovement depth).

The complement C3-C3aR pathway mediates microglia-astrocyte interaction following status epilepticus.

Gliosis is a histopathological characteristic of epilepsy that comprises activated microglia and astrocytes. It is unclear whether or how crosstalk occurs between microglia and astrocytes in the evolution of epilepsy. Here, we report in a mouse model of status epilepticus, induced by intracerebroventricular injection of kainic acid (KA), sequential activation of microglia and astrocytes and their close spatial interaction in the hippocampal CA3 region. Microglial ablation reduced astrocyte activation and their upregulation of complement C3. When compared to wild-type mice, both C3-/- and C3aR-/- mice had significantly less microglia-astrocyte interaction in response to KA-induced status epilepticus. Additionally, KA-injected C3-/- mice had significantly less histochemical evidence of neurodegeneration. The results suggest that the C3-C3aR pathway contributes to KA-induced neurodegeneration by mediating microglia-astrocyte communication. The C3-C3aR pathway may prove to be a potential therapeutic target for epilepsy treatment.

Microglia reprogram metabolic profiles for phenotype and function changes in central nervous system.

In response to various types of environmental and cellular stress, microglia rapidly activate and exhibit either pro- or anti-inflammatory phenotypes to maintain tissue homeostasis. Activation of microglia can result in changes in morphology, phagocytosis capacity, and secretion of cytokines. Furthermore, microglial activation also induces changes to cellular energy demand, which is dependent on the metabolism of various metabolic substrates including glucose, fatty acids, and amino acids. Accumulating evidence demonstrates metabolic reprogramming acts as a key driver of microglial immune response. For instance, microglia in pro-inflammatory states preferentially use glycolysis for energy production, whereas, cells in anti-inflammatory states are mainly powered by oxidative phosphorylation and fatty acid oxidation. In this review, we summarize recent findings regarding microglial metabolic pathways under physiological and pathological circumtances. We will then discuss how metabolic reprogramming can orchestrate microglial response to a variety of central nervous system pathologies. Finally, we highlight how manipulating metabolic pathways can reprogram microglia towards beneficial functions, and illustrate the therapeutic potential for inflammation-related neurological diseases.

Regulatory T cells protect against brain damage by alleviating inflammatory response in neuromyelitis optica spectrum disorder.

BACKGROUND AND PURPOSE: Neuromyelitis optica spectrum disorder (NMOSD) is mainly an anti-aquaporin 4 (anti-AQP4) autoantibodies-mediated idiopathic inflammatory demyelinating disease of the central nervous system. Systemic and local inflammatory responses play a key role in the pathophysiology of NMOSD. However, the role of the crucial immunomodulators CD4+CD25+ forkhead box P3+ (Foxp3) regulatory T cells (Tregs) has not been investigated in NMOSD. METHODS: Twenty-five patients with anti-AQP4-postive NMOSD undergoing an attack and 21 healthy controls (HCs) were enrolled. Frequencies of T cell subsets and Tregs in the peripheral blood were assessed by flow cytometry. Additionally, a model of NMOSD using purified immunoglobulin G from anti-AQP4-antibodies-positive patients with NMOSD and human complement injected into brain of female adult C57BL/6J mice was established. Infiltrated Tregs into NMOSD mouse brain lesions were analyzed by flow cytometry, histological sections, and real-time quantitative Polymerase Chain Reaction. Astrocyte loss, demyelination, and inflammatory response were also evaluated in our NMOSD mouse model. Finally, we examined the effects of both depletion and adoptive transfer of Tregs. RESULTS: The percentage of Tregs, especially naïve Tregs, among total T cells in peripheral blood was significantly decreased in NMOSD patients at acute stage when compared to HCs. Within our animal model, the number and proportion of Tregs among CD4+ T cells were increased in the lesion of mice with NMOSD. Depletion of Tregs profoundly enhanced astrocyte loss and demyelination in these mice, while adoptive transfer of Tregs attenuated brain damage. Mechanistically, the absence of Tregs induced more macrophage infiltration, microglial activation, and T cells invasion, and modulated macrophages/microglia toward a classical activation phenotype, releasing more chemokines and pro-inflammatory cytokines. In contrast, Tregs transfer ameliorated immune cell infiltration in NMOSD mice, including macrophages, neutrophils, and T cells, and skewed macrophages and microglia towards an alternative activation phenotype, thereby decreasing the level of chemokines and pro-inflammatory cytokines. CONCLUSION: Tregs may be key immunomodulators ameliorating brain damage via dampening inflammatory response after NMOSD.

Chemogenetic manipulation of microglia inhibits neuroinflammation and neuropathic pain in mice.

Microglia play an important role in the central sensitization and chronic pain. However, a direct connection between microglial function and pain development in vivo remains incompletely understood. To address this issue, we applied chemogenetic approach by using CX3CR1creER/+:R26LSL-hM4Di/+ transgenic mice to enable expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (Gi DREADD) in microglia. We found that microglial Gi DREADD activation inhibited spinal nerve transection (SNT)-induced microglial reactivity as well as chronic pain in both male and female mice. Gi DREADD activation downregulated the transcription factor interferon regulatory factor 8 (IRF8) and its downstream target pro-inflammatory cytokine interleukin 1 beta (IL-1ß). Using in vivo spinal cord recording, we found that activation of microglial Gi DREADD attenuated synaptic transmission following SNT. Our results demonstrate that microglial Gi DREADD reduces neuroinflammation, synaptic function and neuropathic pain after SNT. Thus, chemogenetic approaches provide a potential opportunity for interrogating microglial function and neuropathic pain treatment.

Jump Training in Rugby Union Players: Barbell or Hexagonal Bar?

ABSTRACT: Weakley, JJS, Till, K, Read, DB, Leduc, C, Roe, GAB, Phibbs, PJ, Darrall-Jones, J, and Jones, B. Jump training in rugby union players: barbell or hexagonal bar?. J Strength Cond Res 35(3): 754-761, 2021-The countermovement jump (CMJ) is an exercise that can develop athletic performance. Using the conventional barbell (BAR) and hexagonal barbell (HEX) while jumping, the intensity can be increased. However, the bar that provides greater adaptations is unknown. Therefore, this study aimed to assess changes in loaded and unloaded CMJ with either a BAR or HEX across a 4-week mesocycle in rugby union players. Twenty-nine subjects were strength-matched and randomized into 2 groups. Subjects completed 3 sets of CMJ at 20% of 1 repetition maximum back squat, 3 times per week for 4 weeks, using either a BAR or HEX. Subjects completed an unloaded CMJ on a force plate before and after, whereas the highest peak concentric velocity during the jump squat was recorded in the first and last training sessions using a linear position transducer. Magnitude-based inferences assessed meaningful changes within- and between-groups. Possibly greater improvements in unloaded CMJ were found in the HEX group in jump height (effect size ± 90% confidence intervals: 0.27 ± 0.27), relative peak (0.21 ± 0.23), and mean power (0.32 ± 0.36). In addition, likely to very likely greater improvements were observed in the HEX group in peak velocity (0.33 ± 0.27), relative mean power (0.53 ± 0.30), mean force (0.47 ± 0.27), and 100-ms impulse (0.60 ± 0.48). Similar raw changes in jump squat peak velocity occurred (0.20-0.25 m·s-1), despite the likely greater ES occurring with the BAR (0.32 ± 0.26). These results indicate that training with the HEX leads to superior unloaded CMJ adaptations. In addition, practitioners should use either the HEX or BAR when aiming to enhance loaded jump ability.

Flaxseed Increases Animal Lifespan and Reduces Ovarian Cancer Severity by Toxically Augmenting One-Carbon Metabolism.

We used an LC-MS/MS metabolomics approach to investigate one-carbon metabolism in the plasma of flaxseed-fed White Leghorn laying hens (aged 3.5 years). In our study, dietary flaxseed (via the activity of a vitamin B6 antagonist known as "1-amino d-proline") induced at least 15-fold elevated plasma cystathionine. Surprisingly, plasma homocysteine (Hcy) was stable in flaxseed-fed hens despite such highly elevated cystathionine. To explain stable Hcy, our data suggest accelerated Hcy remethylation via BHMT and MS-B12. Also supporting accelerated Hcy remethylation, we observed elevated S-adenosylmethionine (SAM), an elevated SAM:SAH ratio, and elevated methylthioadenosine (MTA), in flaxseed-fed hens. These results suggest that flaxseed increases SAM biosynthesis and possibly increases polyamine biosynthesis. The following endpoint phenotypes were observed in hens consuming flaxseed: decreased physiological aging, increased empirical lifespan, 9-14% reduced body mass, and improved liver function. Overall, we suggest that flaxseed can protect women from ovarian tumor metastasis by decreasing omental adiposity. We also propose that flaxseed protects cancer patients from cancer-associated cachexia by enhancing liver function.

Microglial P2Y12 Receptor Regulates Seizure-Induced Neurogenesis and Immature Neuronal Projections.

Seizures are common in humans with various etiologies ranging from congenital aberrations to acute injuries that alter the normal balance of brain excitation and inhibition. A notable consequence of seizures is the induction of aberrant neurogenesis and increased immature neuronal projections. However, regulatory mechanisms governing these features during epilepsy development are not fully understood. Recent studies show that microglia, the brain's resident immune cell, contribute to normal neurogenesis and regulate seizure phenotypes. However, the role of microglia in aberrant neurogenic seizure contexts has not been adequately investigated. To address this question, we coupled the intracerebroventricular kainic acid model with current pharmacogenetic approaches to eliminate microglia in male mice. We show that microglia promote seizure-induced neurogenesis and subsequent seizure-induced immature neuronal projections above and below the pyramidal neurons between the DG and the CA3 regions. Furthermore, we identify microglial P2Y12 receptors (P2Y12R) as a participant in this neurogenic process. Together, our results implicate microglial P2Y12R signaling in epileptogenesis and provide further evidence for targeting microglia in general and microglial P2Y12R in specific to ameliorate proepileptogenic processes.SIGNIFICANCE STATEMENT Epileptogenesis is a process by which the brain develops epilepsy. Several processes have been identified that confer the brain with such epileptic characteristics, including aberrant neurogenesis and increased immature neuronal projections. Understanding the mechanisms that promote such changes is critical in developing therapies to adequately restrain epileptogenesis. We investigated the role of purinergic P2Y12 receptors selectively expressed by microglia, the resident brain immune cells. We report, for the first time, that microglia in general and microglial P2Y12 receptors in specific promote both aberrant neurogenesis and increased immature neuronal projections. These results indicate that microglia enhance epileptogenesis by promoting these processes and suggest that targeting this immune axis could be a novel therapeutic strategy in the clinic.

Deficiency of microglial Hv1 channel is associated with activation of autophagic pathway and ROS production in LPC-induced demyelination mouse model.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated demyelinated disease of the central nervous system. Activation of microglia is involved in the pathogenesis of myelin loss. OBJECTIVE: This study is focused on the role of Hv1 in regulating demyelination and microglial activation through reactive oxygen species (ROS) production after lysophosphatidylcholine (LPC)-mediated demyelination. We also explored autophagy in this process. METHODS: A model of demyelination using two-point LPC injection into the corpus callosum was established. LFB staining, immunofluorescence, Western blot, and electron microscopy were used to study the severity of demyelination. Microglial phenotype and autophagy were detected by immunofluorescence and Western blot. Morris water maze was used to test spatial learning and memory ability. RESULTS: We have identified that LPC-mediated myelin damage was reduced by Hv1 deficiency. Furthermore, we found that ROS and autophagy of microglia increased in the demyelination region, which was also inhibited by Hv1 knockout. CONCLUSION: These results suggested that microglial Hv1 deficiency ameliorates demyelination through inhibition of ROS-mediated autophagy and microglial phenotypic transformation.

Microglial depletion aggravates the severity of acute and chronic seizures in mice.

Microglia are the resident immune cells of the center nervous system and participate in various neurological diseases. Here we determined the function of microglia in epileptogenesis using microglial ablation approaches. Three different microglia-specific genetic tools were used, CX3CR1CreER/+:R26iDTA/+, CX3CR1CreER/+:R26iDTR/+, and CX3CR1CreER/+:Csf1rFlox/Flox mice. We found that microglial depletion led to worse kainic acid (KA)-induced status epilepticus, higher mortality rate, and increased neuronal degeneration in the hippocampus. In KA-induced chronic spontaneous recurrent seizures, microglial depletion increased seizure frequency, interictal spiking, and seizure duration. Therefore, microglial depletion aggravates the severity of KA-induced acute and chronic seizures. Interestingly, microglial repopulation reversed the effects of depletion upon KA-induced status epilepticus. Our results demonstrate a beneficial role of microglia in suppressing both acute and chronic seizures, suggesting that microglia are a potential therapeutic target for the management of epilepsy.

Structural basis for translocation by AddAB helicase-nuclease and its arrest at χ sites.

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi) and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence, whereupon they produce a 3' single-stranded DNA tail onto which they initiate loading of the RecA protein. Consequently, regulation of the AddAB/RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments.

The Effects of Superset Configuration on Kinetic, Kinematic, and Perceived Exertion in the Barbell Bench Press.

Weakley, JJS, Till, K, Read, DB, Phibbs, PJ, Roe, G, Darrall-Jones, J, and Jones, BL. The effects of superset configuration on kinetic, kinematic, and perceived exertion in the barbell bench press. J Strength Cond Res 34(1): 65-72, 2020-Training that is efficient and effective is of great importance to an athlete. One method of improving efficiency is by incorporating supersets into resistance training routines. However, the structuring of supersets is still unexplored. Therefore, the purpose of this study was to assess the effects of agonist-antagonist (A-A), alternate peripheral (A-P), and similar biomechanical (SB) superset configurations on rate of perceived exertion (RPE), kinetic and kinematic changes during the bench press. Ten subjects performed resistance training protocols in a randomized-crossover design, with magnitude-based inferences assessing changes/differences within and between protocols. Changes in rate of perceived exertion were very likely and almost certainly greater in the A-P and SB protocols when compared with the A-A, whereas all superset protocols had very likely to almost certain reductions in mean velocity and power from baseline. Reductions in mean velocity and power were almost certainly greater in the SB protocol, with differences between the A-A and A-P protocols being unclear. Decreases in peak force were likely and almost certain in the A-A and SB protocols respectively, with changes in A-P being unclear. Differences between these protocols showed likely greater decreases in SB peak forces when compared to A-A, with all other superset comparisons being unclear. This study demonstrates the importance of exercise selection when incorporating supersets into a training routine. It is suggested that the practitioner uses A-A supersets when aiming to improve training efficiency and minimize reductions in kinetic and kinematic output of the agonist musculature while completing the barbell bench press.

Microglial proliferation and monocyte infiltration contribute to microgliosis following status epilepticus.

Microglial activation has been recognized as a major contributor to inflammation of the epileptic brain. Seizures are commonly accompanied by remarkable microgliosis and loss of neurons. In this study, we utilize the CX3CR1GFP/+ CCR2RFP/+ genetic mouse model, in which CX3CR1+ resident microglia and CCR2+ monocytes are labeled with GFP and RFP, respectively. Using a combination of time-lapse two-photon imaging and whole-cell patch clamp recording, we determined the distinct morphological, dynamic, and electrophysiological characteristics of infiltrated monocytes and resident microglia, and the evolution of their behavior at different time points following kainic acid-induced seizures. Seizure activated microglia presented enlarged somas with less ramified processes, whereas, infiltrated monocytes were smaller, highly motile cells that lacked processes. Moreover, resident microglia, but not infiltrated monocytes, proliferate locally in the hippocampus after seizure. Microglial proliferation was dependent on the colony-stimulating factor 1 receptor (CSF-1R) pathway. Pharmacological inhibition of CSF-1R reduced seizure-induced microglial proliferation, which correlated with attenuation of neuronal death without altering acute seizure behaviors. Taken together, we demonstrated that proliferation of activated resident microglia contributes to neuronal death in the hippocampus via CSF-1R after status epilepticus, providing potential therapeutic targets for neuroprotection in epilepsy.

POST: A framework for set-based association analysis in high-dimensional data.

Evaluating the differential expression of a set of genes belonging to a common biological process or ontology has proven to be a very useful tool for biological discovery. However, existing gene-set association methods are limited to applications that evaluate differential expression across k⩾2 treatment groups or biological categories. This limitation precludes researchers from most effectively evaluating the association with other phenotypes that may be more clinically meaningful, such as quantitative variables or censored survival time variables. Projection onto the Orthogonal Space Testing (POST) is proposed as a general procedure that can robustly evaluate the association of a gene-set with several different types of phenotypic data (categorical, ordinal, continuous, or censored). For each gene-set, POST transforms the gene profiles into a set of eigenvectors and then uses statistical modeling to compute a set of z-statistics that measure the association of each eigenvector with the phenotype. The overall gene-set statistic is the sum of squared z-statistics weighted by the corresponding eigenvalues. Finally, bootstrapping is used to compute a p-value. POST may evaluate associations with or without adjustment for covariates. In simulation studies, it is shown that the performance of POST in evaluating the association with a categorical phenotype is similar to or exceeds that of existing methods. In evaluating the association of 875 biological processes with the time to relapse of pediatric acute myeloid leukemia, POST identified the well-known oncogenic WNT signaling pathway as its top hit. These results indicate that POST can be a very useful tool for evaluating the association of a gene-set with a variety of different phenotypes. We have developed an R package named POST which is freely available in Bioconductor.