title: Digital data repository and automatic analysis framework for FDOPA PET neuroimaging

abstract:
IntroductionFDOPA PET has been used extensively to image the human brain in many clinical disorders and has the potential to be used for patient stratification and individualized treatment. However, to reach its full and effective clinical translation, FDOPA PET requires both a robust data infrastructure and analytical protocol that are capable of ensuring high quality data and metadata, accurate biological quantification, and replicable results. In this study we evaluate a digital data repository and automated analytical framework for FDOPA PET neuroimaging that can produce an individualised quantification of dopamine synthesis capacity in the living human brain.

MethodsThe imaging platform XNAT was used to store the Kings College London institutional brain FDOPA PET imaging archive, alongside individual demographics and clinical information. A fully automated analysis pipeline for imaging processing and data quantification was developed in Python and integrated in XNAT using the Docker technology. Reproducibility was assessed in test-retest datasets both in controls and patients with psychosis. The agreement between the automated analysis estimates and the results derived by the manual analysis were compared. Finally, using a sample of healthy controls (N=115), a sensitivity analysis was performed to explore the impact of experimental and demographic variables on the FDOPA PET measures.

ResultsThe final data repository includes 892 FDOPA PET scans organized from 23 different studies, collected at five different imaging sites. After removing commercials studies, the infrastructure consisted of 792 FDOPA PET scans from 666 individuals (female 33.9%, healthy controls 29.1%) collected from four different imaging sites between 2004-2021. The automated analysis pipeline provided results that were in agreement with the results from the manual analysis, with a Pearsons correlation that ranged from 0.64 to 0.99 for Kicer, and from 0.79 to 1.00 for SUVR. The mean absolute difference between the two pipelines ranges from 3.4% to 9.4% for Kicer, and from 2.5% to 12.4% for SUVR. Moreover, we found good reproducibility of the data analysis by the automated pipeline (in the whole striatum for the Kicer: ICC for the controls = 0.71, ICC for the psychotic patients = 0.88). From the demographic and experimental variables assessed, gender was found to most influence striatal dopamine synthesis capacity (F = 10.7, p <0.001), with women showing greater dopamine synthesis capacity than men, while the effects of weight, age, injected radioactivity, and scanner, varied by brain region and parameter of interest.

ConclusionsCombining information from different neuroimaging studies has allowed us to test comprehensively the automated pipeline for quantification of dopamine synthesis capacity using FDOPA PET data and to validate its replicability and reproducibility performances on a large sample size. This validation process is a necessary methodological step for the development of the clinical application of FDOPA PET as precision medicine biomarker. The proposed infrastructure is generalisable behind the FDOPA radiotracer.

category: bioengineering


title: MMR Deficiency Defines Distinct Molecular Subtype of Breast Cancer with Unique Proteomic Networks and Variable Clinical Significance

abstract: Mismatch repair (MMR) alterations are important prognostic and predictive biomarkers in a variety of cancer subtypes including colorectal and endometrial. However, in breast cancer (BC), the distinction and clinical significance of MMR is largely unknown. This may be due in part to the fact that genetic alterations in MMR genes are rare, and only seen to occur in around 3% of BCs. In the present study we analyzed TCGA data using a multi-sample protein-protein interactions (PPI) analysis tool, Proteinarium, and showed a distinct separation in the MMR deficient and intact specific networks. MMR deficient tumor specific networks have a highly connected cluster of histone genes, identified by unique PPI. We also found that distribution of MMR deficient breast cancer is more prevalent in HER2-enriched and triple-negative (TN) BC subtypes compared to luminal BCs. Poorer survival was seen in patients with HER2-enriched BCs with MMR deficiency, whereas an improved survival was seen in TNBCs with MMR deficiency. We recommend defining MMR deficient breast cancer by next generation sequencing (NGS) when any somatic mutation is detected in one of the 7 MMR genes found in our study. Our recommendations include labeling patients with variants of undetermined significance (VUS) as MMR deficient supported by findings from distinct clusters of patients based on our network analysis. MMR may have a role in guiding the use of immunotherapy for both TN as well as HER2-enriched BC.

category: bioinformatics


title: Generative power of a protein language model trained on multiple sequence alignments

abstract: Computational models starting from large ensembles of evolutionarily related protein sequences capture a representation of protein families and learn constraints associated to protein structure and function. They thus open the possibility for generating novel sequences belonging to protein families. Protein language models trained on multiple sequence alignments, such as MSA Transformer, are highly attractive candidates to this end. We propose and test an iterative method that directly uses the masked language modeling objective to generate sequences using MSA Transformer. We demonstrate that the resulting sequences generally score better than those generated by Potts models, and even than natural sequences, for homology, coevolution and structure-based measures. Moreover, MSA Transformer better reproduces the higher-order statistics and the distribution of sequences in sequence space of natural data than Potts models, although Potts models better reproduce first- and second-order statistics. MSA Transformer is thus a strong candidate for protein sequence generation and protein design.


title: ScisorWiz: Visualizing Differential Isoform Expression in Single-Cell Long-Read Data

abstract: RNA isoforms contribute to the diverse functionality of the proteins they encode within the cell. Visualizing how isoform expression differs across cell types and brain regions can inform our understanding of disease and gain or loss of functionality caused by alternative splicing with potential negative impacts. However, the extent to which this occurs in specific cell types and brain regions is largely unknown. This is the kind of information that ScisorWiz plots can provide in an informative and easily communicable manner. ScisorWiz affords its user the opportunity to visualize specific genes across any number of cell types, and provides various sorting options for the user to gain different ways to understand their data. ScisorWiz provides a clear picture of differential isoform expression through various clustering methods and highlights features such as alternative exons and Single Nucleotide Variants (SNVs). Tools like Scisor-Wiz are key for interpreting single-cell isoform sequencing data. This tool applies to any single-cell long-read RNA sequencing data in any cell type, tissue, or species.


title: The Decision of the Optimal Rank of a Non-negative Matrix Factorization Model for Gene Expression Datasets Utilizing Unit Invariant Knee Method

abstract:
BackgroundThere is a great need to develop a computational approach to analyze and exploit the information contained in gene expression data. Recent utilization of non-negative matrix factorization (NMF) in computational biology has served its capability to derive essential details from a high amount of data in particular gene expression microarrays.

ObjectiveA common problem in NMF is finding the proper number rank (r) of factors. Thus, various techniques have been suggested to select the optimal value of rank factorization (r).

MethodThis study focused on the unit invariant knee (UIK) method to calculate factorization rank (basis vector) of the non-negative matrix factorization (NMF) of gene expression data sets is employed. Because the UIK method requires an extremum distance estimator (EDE) that is eventually employed for inflection and identification of a knee point, this study finds the first inflection point of curvature of RSS of the proposed algorithms using the UIK method on gene expression datasets as a target matrix.

ResultsComputation was conducted for the UIK task using the esGolub data set of R studio, and consequently, the distinct results of NMF was subjected to compare on different algorithms. The proposed UIK method is easy to perform, free of a priori rank value input, and does not require initial parameters that significantly influence the models functionality.

ConclusionThis study demonstrates that the UIK method provides a credible prediction for both gene expression data and precisely estimating of simulated mutational processes data with known dimensions.


title: Neural network approach to somatic SNP calling in WGS samples without a matched control.

abstract:
Somatic variants are usually called by analysing the DNA sequences of a tumor sample in conjunction with a matched normal. However, a matched normal is not always available for instance in diagnostic settings. To unlock such data for basic research single-sample somatic variant calling is required. Previous approaches can not easily be applied in the case of typical whole genome sequencing (WGS) samples.We present a neural network-based approach for calling somatic single nucleotide polymorphism (SNP) variants in tumor WGS samples without a matched normal. The method does not require any manual tuning of filtering parameters and can be applied under the conditions of a typical WGS experiment. We demonstrate the effectiveness of the proposed approach by reporting its performance on 5 SNP datasets corresponding to 5 different cancer types.

The proposed method is implemented in Python 3.6 and available as a GitHub repository at https://github.com/heiniglab/deepSNP.


title: A comprehensive study of mRNA and long noncoding RNAs in Indian Breast cancer patients using transcriptomics approach

abstract:
BackgroundBreast cancer (BC) is one of the leading causes of cancer-associated death in women. Despite the progress in therapeutic regimen, resistance and recurrence of Breast cancer have impacted Overall Survival. Transcriptomic profiling of tumour samples has led to identifying subtype-specific differences, identifying biomarkers, and designing therapeutic strategies. Although there are multiple transcriptomic studies on breast cancer patients from different geographical regions, a comprehensive study on long noncoding RNA (lncRNA) and mRNA in Indian Breast cancer patients in multiple subtypes are very limited. This study aims to understand the subtype-specific alterations and mRNA-lncRNA gene sets.

MethodWe have performed transcriptome analysis of 17 Indian breast cancer patients and matched normal belonging to 6 different subtypes, i.e., four patients in triple positive, three patients in estrogen receptor-positive (ER+ve), three patients in estrogen and progesterone receptor-positive (ER+ve, PR+ve), two patients in Human epidermal growth factor receptor (Her2+ve), three patients in triple-negative and one patient in ER+ve and Her2 +ve subtypes. Hierarchical clustering and principal component analysis were performed using R packages to derive gene sets. Univariate and multivariate Cox analyses were performed for survival analysis.

ResultsmRNA and lncRNA expression profiles segregated Indian Breast cancer subtypes with minimum overlap. We have identified a 25mRNA-27 lncRNA gene set, which displayed proper segregation of the subtypes in our data. The same gene set also segregated premenopausal women samples in The Cancer Genome Atlas (TCGA) data. Pathway analysis of the differentially expressed genes revealed unique pathways for premenopausal and postmenopausal women. Kaplan-Meier survival analysis revealed menopausal status, grade of the tumour, and hormonal status displayed statistically significant effects (p < 0.05) on the risk of mortality due to breast cancer. Her2+ve patients showed low overall survival

ConclusionThis is the first study describing subtype-specific mRNA and lncRNA gene expression in Indian Breast Cancer patients with unique pathway signatures for premenopausal and postmenopausal breast cancer patients. Additionally, our data identified an mRNA-lncRNA gene set that could segregate pre and postmenopausal women with Breast Cancer. Although the sample size is small, results from this study could be a foundation that could be validated further in a larger dataset to establish an mRNA-lncRNA signature specific to the Indian population which might, in turn, improve therapeutic decisions.


title: Proliferative advantage of specific aneuploid cells drives evolution of tumor karyotypes

abstract: Most tumors have abnormal karyotypes, which arise from mistakes during mitotic division of healthy euploid cells and evolve through numerous complex mechanisms. In a recent mouse model with high levels of chromosome missegregation, chromosome gains dominate over losses both in pretumor and tumor tissues, whereas tumors are characterized by gains of chromosomes 14 and 15. However, the mechanisms driving clonal selection leading to tumor karyotype evolution remain unclear. Here we show, by introducing a mathematical model based on a concept of a macro-karyotype, that tumor karyotypes can be explained by proliferation-driven evolution of aneuploid cells. In pretumor cells, increased apoptosis and slower proliferation of cells with monosomies lead to predominant chromosome gains over losses. Tumor karyotypes with gain of one chromosome can be explained by karyotype-dependent proliferation, while for those with two chromosomes an interplay with karyotype-dependent apoptosis is an additional possible pathway. Thus, evolution of tumor-specific karyotypes requires proliferative advantage of specific aneuploid karyotypes.

category: biophysics


title: Effect of Chlonisol (2--1,3-propanediol) on Overall Survival in Laboratory Rodents with Intracranial Tumors: A Meta-analysis of Preclinical Studies

abstract:
BackgroundThe need for new, effective, and affordable drugs for the treatment of primary and metastatic brain tumors remains unsatisfactory. Preclinical studies of chlonisol (2-[3-(2-chloroethyl)-3-nitrosoureido]-1,3-propanediol) showed promising results in the treatment of experimental intracranial tumors.

ObjectivesTo apply a meta-analytical approach to estimate the combined effect size of chlonisol on overall survival (OS) in rodents with brain tumor transplants.

Search strategyData for the meta-analysis were obtained from the laboratorys internal database from reports of preclinical studies of chlonisol.

Selection criteriaEligible studies were parallel preclinical trials in rodents (mice, rats) with intracranially transplanted tumors. Chlonisol was compared with active control treatment (lomustine or temozolomide). All cytostatics were administered at the maximum tolerated dose (MTD). The duration of the studies was at least 90 days. The main outcome was OS-HR (hazard ratio).

Data analysisWe applied the inverse variance technique for the meta-analysis of HRs. In HR analysis we adopted a random-effect model.

ResultsThe analysis included seven trials with 132 rodents. Studies were conducted between 2016 and 2022. As a murine intracranial grafts we used Ehrlichs carcinoma, Sarcoma 180 and the HER2-positive mammary tumor derived from a female FVB/N HER-2/neu transgenic mouse. Glioma 35 was transplanted into rats. Compared with active control, oral or intraperitoneal administration of chlonisol at MTD of 20 mg/kg, significantly reduced the risk of death by 63% (HR=0.37; 95% CI: 0.24-0.56; P<0.00001) in animals with intracranial tumors. The direction in favor of chlonisol was stable across studies despite the use of different animals and transplants, the routes of administration of chlonisol, and control treatment. No significant heterogeneity was observed between the studies (Tau2 = 0.03; Chi2 = 6.52; df = 6; P = 0.33; I2 = 8%).

ConclusionCompared with lomustine and temozolomide, chlonisol treatment in MTD provides an important advantage in OS in animals with intracranial tumors. Our results may serve as a basis for further study of chlonisol as a chemotherapy agent for primary and metastatic brain tumors.

category: pharmacology and toxicology


title: Top-down modulation and cortical-AMG/HPC interaction in familiar face processing

abstract:
Humans can accurately recognize familiar faces in only a few hundred milliseconds, but the underlying neural mechanism remains unclear. Here we recorded intracranial electrophysiological signals from ventral temporal cortex (VTC), superior/middle temporal cortex (STC/MTC), medial parietal cortex (MPC) and amygdala/hippocampus (AMG/HPC) in 20 epilepsy patients while they viewed faces of famous people and strangers as well as common objects. In posterior VTC and MPC, familiarity-sensitive responses emerged significantly later than initial face-selective responses, suggesting that familiarity enhances face representations after they are first being extracted. Moreover, viewing famous faces increased the coupling between cortical areas and AMG/HPC in multiple frequency bands. These findings imply that the top-down modulation in local face-selective response and interactions between cortical face areas and AMG/HPC contribute to the superior recognition of familiar faces.

TeaserTop-down modulation and cortical-AMG/HPC interactions contribute to the superior processing of familiar faces.

category: neuroscience


title: Tumor necrosis factor α modulates excitatory and inhibitory neurotransmission in a concentration-dependent manner

abstract: Microglia, the brains resident immune cells, have been implicated in important brain functions, such as synaptic transmission and plasticity. The pro-inflammatory cytokine tumor necrosis factor  (TNF), which is produced and secreted by microglia, has been linked to the expression of synaptic plasticity in neurons. However, the role of TNF-mediated activation of microglia has not been addressed in this context. Here, we assessed concentration-dependent effects of TNF on the balance of synaptic excitation/inhibition and the activation of microglia using mouse organotypic entorhino-hippocampal tissue cultures. We found that low concentrations of TNF enhanced excitatory synaptic strength while not affecting inhibitory neurotransmission. At higher concentrations, TNF increased inhibitory neurotransmission without affecting excitatory synaptic strength. Both low and high concentrations of TNF induced the synaptic accumulation of GluA1-containing AMPA receptors, suggesting that a high concentration of TNF exerts a homeostatic effect on excitatory neurotransmission that prevents synaptic strengthening. Consistent with this, high, but not low, concentrations of TNF activated microglia. Moreover, high concentrations of TNF enhanced excitatory neurotransmission in microglia-depleted tissue cultures. These findings extend our knowledge on the role of TNF on synaptic plasticity by demonstrating concentration-dependent effects on excitatory and inhibitory neurotransmission. They reveal a TNF-mediated negative feedback mechanism on excitatory neurotransmission that is dependent on the activation of microglia, thereby emphasizing their role as gatekeepers of TNF-mediated plasticity and homeostasis.


title: Limited Evidence for a Triple Dissociation in the Medial Temporal Lobe: an fMRI Recognition Memory Replication Study

abstract:
Common research practices in neuroimaging studies using functional magnetic resonance imaging may produce outcomes that are difficult to replicate. Results that cannot be replicated have contributed to a replication crisis in psychology, neuroscience, and other disciplines over the years. Here we replicate two previous papers in which the authors present two analysis paths for a dataset in which participants underwent fMRI while performing a recognition memory test for old and new words. Both studies found activation in the hippocampus, with the first demonstrating a distinction in hippocampal activation corresponding to true and perceived oldness of stimuli and the second demonstrating that hippocampal activation reflects the subjective experience of the participant. We replicated the behavioral and MRI acquisition parameters reported in the two target articles (Daselaar, Fleck, & Cabeza, 2006; Daselaar, Fleck, Prince, et al., 2006) with N=53 participants. We focused fMRI analyses on regions of interest reported in the target articles examining fMRI activation for differences corresponding with true and perceived oldness and those associated with the subjective memory experiences of recollection, familiarity, and novelty. Comparisons between true and perceived oldness revealed main effects not only for true, but also perceived oldness along with significant interaction effects. We replicate the findings of recollection and familiarity signals in the hippocampus and medial temporal lobe cortex, respectively, but failed to replicate a novelty signal in the anterior medial temporal lobe. These results remained when we analyzed only correct trials, indicating that the effects were not due to selectively averaging correct and incorrect trials. Taken together, we replicate the previous finding that activation in the hippocampus corresponds to the subjective experience associated with correct recognition memory retrieval.

HighlightsO_LIWe replicated the methods of two influential papers using fMRI to examine recognition memory.
C_LIO_LIThe hippocampus consistently signals the subjective experience of remembering but not the objective old/new status of stimuli.
C_LIO_LIHippocampal response was consistent with recollection while the medial temporal lobe (MTL) cortex response was consistent with familiarity.
C_LIO_LIWe did not observe any regions in the MTL that signaled novelty, indicating recognition memory does not involve a triple dissociation of recollection, familiarity, and novelty in the MTL.
C_LI


title: 3D pose estimation enables virtual head-fixation in freely moving rats

abstract: The impact of spontaneous movements on neuronal activity has created the need to quantify behavior. We present a versatile framework to directly capture the 3D motion of freely definable body points in a marker-free manner with high precision and reliability. Combining the tracking with neural recordings revealed multiplexing of information in the motor cortex neurons of freely moving rats. By integrating multiple behavioral variables into a model of the neural response, we derived a virtual head-fixation for which the influence of specific body movements was removed. This strategy enabled us to analyze the behavior of interest (e.g., front paw movements). Thus, we unveiled an unexpectedly large fraction of neurons in the motor cortex with tuning to the paw movements, which was previously masked by body posture tuning. Once established, our framework can be efficiently applied to large datasets while minimizing the experimental workload caused by animal training and manual labeling.


title: Somatosensory Cortical Signature of Facial Nociception and Vibrotactile Touch Induced Analgesia

abstract:
Pain relief by vibrotactile touch is a common human experience. Previous neurophysiological investigations in animals focused on spinal mechanisms while human studies suggested the involvement of supraspinal mechanisms. Here we asked whether and how the primary somatosensory cortex (S1) is involved in touch induced analgesia. We discovered that in mice, vibrotactile reafferent signals from self-generated whisking significantly reduce facial nociception, which is abolished by specifically blocking touch transmission from thalamus to the barrel cortex (S1B). The presence of whisking altered nociceptive signal processing in S1B neurons. Intrinsic manifold analysis of S1B population activity revealed that whisking pushes the transition of neural state induced by noxious stimuli towards the state encoding non-nocifensive actions. Thus, S1B integrates facial tactile and noxious signals to enable touch mediated analgesia.

TeaserVibrotactile signals modulate barrel cortex population dynamics during touch mediated facial analgesia


title: Impact of HIV infection and integrase strand transfer inhibitors-based treatment on gut virome

abstract:
Viruses are the most abundant components of the microbiome in human beings with a significant impact on health and disease. However, the impact of human immunodeficiency virus (HIV) infection on gut virome has been scarcely analyzed. On the other hand, several studies suggested that not all antiretrovirals for treating HIV infection exert similar effects on the gut bacteriome, being the integrase strand transfers inhibitors (INSTIs) --first-choice treatment of naive HIV-infected patients nowadays-- those associated with a healthier gut. Thus, the aim of this study was to evaluate the effects of HIV infection and INSTIs in first line of treatment on gut virome composition. To accomplish this objective, 26 non-HIV-infected volunteers, 15 naive HIV-infected patients and 15 INSTIs-treated HIV-infected patients were recruited and gut virome composition was analysed using shotgun sequencing. The results showed that bacteriophages are the most abundant and diverse viruses in the gut independent from the HIV-status and the use of treatment. HIV infection was accompanied by a decrease in phage richness which was reverted after INSTIs-based treatment (p<0.01 naive vs. control Richness index and p<0.05 naive vs. control Fishers alpha index). {beta}-diversity of phages revealed that samples from HIV-infected samples clustered separately from those belonging to the control group (padj<0.01 naive vs. control and padj<0.05 INSTIs vs. control). However, it is worth mentioning that samples coming from INSTIs-treated patients were more grouped than those from naive patients. Differential abundant analysis of phages showed an increase of Caudoviricetes class in the naive group compared to control the group (padj<0.05) and a decrease of Malgrandaviricetes class in the INSTIs-treated group compared to the control group (padj<0.001). Besides, it was observed that INSTIs-based treatment was not able to reverse the increase of lysogenic phages associated with HIV infection (p<0.05 vs. control) or to modify the decrease observed on the relative abundance of Proteobacteria-infecting phages (p<0.05 vs. control). To sum up, our study describes for the first time the impact of HIV and INSTIs on gut virome and demonstrates that INSTIs-based treatments are able to partially restore gut dysbiosis not only at bacterial but also at viral level, which opens several opportunities for new studies focused on microbiota-based therapies.

Author summaryThe impact of human immunodeficiency virus (HIV) infection and the effects of integrase strand transfer inhibitors (INSTIs)-based treatments --first-choice treatment of naive HIV-infected patients nowadays-- on gut virome are unknown. In this study, we have confirmed that phages are the most abundant viral component of the human gut virome. Besides, we have described for the first time that INSTIs-based treatments are able to partially restore gut dysbiosis induced by HIV infection not only at bacteria but also at viral level. This fact opens new opportunities for future studies and approaches focused on microbiota-based therapies in the context of HIV infection and treatment.

category: microbiology


title: Genetic admixture history and forensic characteristics of Tibeto-Burman-speaking Qiang people explored via the newly developed Y-STR panel and genome-wide SNP data

abstract: Fine-scale patterns of population genetic structure and diversity of ethnolinguistically diverse populations are important for biogeographical ancestry inference, kinship testing and also for the development and validation of new kits focused on forensic personal identification. Analyses focused on forensic markers and genome-wide SNP data can provide new insights into the origin, admixture processes and forensic characteristics of targeted populations. Qiang people with a large sample size among Tibeto-Burman-speaking populations widely reside in the middle latitude of the Tibetan Plateau. However, their genetic structure and forensic features have remained uncharacterized due to the paucity of comprehensive genetic analyses. Here, we first developed and validated the AGCU-Y30 Y-STR panel, which contains slowly and moderately mutating Y-STRs, and then we conducted comprehensive population genetic analyses based on Y-STRs and genome-wide SNPs to explore the admixture history of Qiang people and their neighbours. The validated results of this panel showed that the new Y-STR kit was sensitive and robust enough for forensic applications. Haplotype diversity (HD) ranging from 0.9932 to 0.9996 and allelic frequencies ranging from 0.001946 to 0.8326 in 514 Qiang people demonstrated that all included markers were highly polymorphic in Tibeto-Burman people. Population genetic analyses based on Y-STRs (RST, FST, MDS, NJ, PCA and MJNs) revealed that the Qiang people harboured a paternally close relationship with lowland Tibetan-Yi corridor populations. Furthermore, we made a comprehensive population admixture analysis among Eurasian modern and ancient populations based on the shared alleles. We determined that the Qiang people were a genetically admixed population and showed the closest relationship with Tibetan and Neolithic Yellow River farmers. Admixture modelling showed that Qiang people shared the primary ancestry with Tibetan and was derived from North China, supporting the hypothesis of common origin between Tibetan and Qiang people.

category: genetics


title: Optimal gap-affine alignment in O(s) space

abstract:
MotivationPairwise sequence alignment remains a fundamental problem in computational biology and bioinformatics. Recent advances in genomics and sequencing technologies demand for faster and scalable algorithms that can cope with the ever-increasing sequence lengths. Classical pairwise alignment algorithms based on dynamic programming are strongly limited by quadratic requirements in time and memory. The recently proposed wavefront alignment (WFA) algorithm introduced an efficient algorithm to perform exact alignment in O(ns) time where s is the optimal score and n is the sequence length. Notwithstanding these bounds, WFAs O(s2) memory requirements become computationally impractical for genome-scale alignments, leading to a need for further improvement.

ResultsIn this paper, we present the bidirectional WFA algorithm (BiWFA), the first gap-affine algorithm capable of computing optimal alignments in O(s) memory while retaining the WFAs time complexity of O(ns). In practice, our implementation never requires more than 183 MB aligning long and noisy sequences up to 1 Mbp long, while maintaining competitive execution times.

AvailabilityAll code is publicly available at https://github.com/smarco/BiWFA-paper

Contactsantiagomsola@gmail.com


title: A spectral algorithm for polynomial-time graph isomorphism testing

abstract: Polynomial time solutions to the graph isomorphism problem have remained elusive to date, presenting one of the most important issues in computer science. This study describes a spectral algorithm to infer quadratic permutations mapping tuples of isomorphic graphs in [Formula] time. Robustness to degeneracy and multiple isomorphisms are achieved through low dimensional eigenspace projections and iterative perturbations respectively. An analytic proof is provided and experimental performance is demonstrated in synthetic and biological data.


title: resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

abstract: Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a  reservoir of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.


title: How does target lesion selection affect RECIST? A computer simulation study

abstract: RECIST is grounded on the assumption that target lesion selection is objective and representative of the total tumor burdens response to therapy. A computer simulation model was designed to study target lesion selections impact on response assessment. Readers disagreement as a function of the total number of lesions, affected organs, and lesion growth was analyzed. Disagreement aggravates when the number of lesions increases, when lesions are concentrated on few organs, and when lesion growth borders the thresholds of progressive disease and partial response. In a metastatic setting, RECIST displays a non-linear, unpredictable behavior, not reliable for response assessment due to target lesion selection variability. Including more (if not all) lesions in the quantitative analysis of tumor burden is desirable.

category: cancer biology


title: Coupling and Heterogeneity Modulate Pacemaking Capability in Healthy and Diseased Two-Dimensional Sinoatrial Node Tissue Models

abstract: Both experimental and modeling studies have attempted to determine mechanisms by which a small anatomical region, such as the sinoatrial node (SAN), can robustly drive electrical conduction in the human heart. However, despite important advances from prior research, important questions remain unanswered. This study aimed to investigate, through mathematical modeling, the role of intercellular coupling and cellular heterogeneity in synchronization and pacemaking within the healthy and diseased SAN. In a multicellular computational model of a monolayer of either human or rabbit SAN cells, simulations revealed that heterogenous cells synchronize their discharge frequency into a unique beating rhythm across a wide range of cellular heterogeneity and intercellular coupling. However, an insightful and unanticipated behavior appears in pathological conditions precipitated by perturbations of certain ionic currents (gCaL = 0.5): an intermediate range of intercellular coupling (900-4000 M{Omega}) is beneficial to the human SAN automaticity, enabling a very small portion of tissue (3.4%) to drive propagation, which fails with both lower and higher resistances. This protective effect of intercellular coupling and heterogeneity, seen in both human and rabbit tissues, highlights the remarkable resilience of the SAN. Overall, the model here developed allowed insight into the mechanisms of automaticity of the human sinoatrial node. The simulations suggest that certain degrees of gap junctional expressions protect the SAN from ionic perturbations that might be caused by drugs or mutations.

category: physiology


title: Combined effects of 12-week yoga warm-up on athletic performance in male high school track and field athletes

abstract:
AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSPracticing yoga could improve balance and flexibility, but its positive significance as a long-term warm-up for formal training was uncertain. We hypothesized that practicing yoga during warm-up might positively affect balance, flexibility, and speed performance in male high school track and field athletes.

MethodsOver a 12-week period, athletes in a yoga group (YG) (n=10) practiced yoga for 15 min 4 times a week during warm-up, while athletes in a dynamic stretching group (DSG) (n=10) practiced 15 min of dynamic stretching. Except for the warm-up activities, the training content of the two groups of students was the same. we tested performance indicators immediately before and after the intervention, including lower extremity flexibility test (right hip active flexion range), lower extremity balance test [using surface electromyography (sEMG) to measure right leg tibialis anterior (TA) activation during one-leg stance (OLS) with eyes closed], and speed performance test (100-meter and 800-meter tests).

ResultsWe performed between-group and within-group comparisons for indicators of two groups by using SPSS (version 26.0). Within-group comparisons showed a significant improvement in flexibility (P=0.002) and balance (P=0.003) in YG, but no significant change in DSG, after the 12-week intervention. In addition, speed performance of both YG (100m, P=0.026; 800m, P=0.045) and DSG (100m, P=0.029; 800m, P=0.006) was significantly improved. Between-group comparison showed that YG had a significant advantage in 800m (P=0.045) and flexibility (P=0.031).

ConclusionsThese data suggested that practicing yoga as a long-term warm-up could help male high school track and field athletes improve lower body flexibility and 800m speed. In addition, yoga had a certain positive effect on balance, but it was not significant overall.


title: PvC3H29 interacts with and inhibits DNA binding of PvNAPs to finetune leaf senescence in switchgrass (Panicum virgatum)

abstract:
Finetuning the progression of leaf senescence is important for plants fitness in nature, while  staygreen with delayed leaf senescence has been considered as a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass CCCH-type Zinc finger gene, PvC3H29, was characterized as a suppressor of leaf senescence that over-expressing or suppressing the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll catabolic pathway genes were involved in the PvC3H29-regulated leaf senescence. PvC3H29 was a nucleus-localized protein with no transcriptional activity. By Y2H screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1&2. Over-expressing the PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating chlorophyll catabolic genes to promote chlorophyll degradation. PvC3H29, through protein-protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioned as a  brake in the progression of leaf senescence. Moreover, over-expressing PvC3H29 resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops.

One-sentence summaryPvC3H29 interacts with transcription factors, PvNAP1&2, to inhibit their transactivation on chlorophyll catabolism and leaf senescence in switchgrass.

category: plant biology


title: The Main Themes and Experiences Shared with the Hashtag #BlackintheIvory: A Qualitative Analysis

abstract:
BackgroundThe movement #BlackintheIvory gave Black academics an opportunity to connect through a social media platform allowing them to share common experiences in the pursuit of higher education. Through the analysis of Twitter posts, using the hashtag #BlackintheIvory, this study investigates the main themes identified among Black scholars in academia and their shared experiences with teaching, mentoring, collegiality, identity, service, and racism.

MethodsUsing the Twitter API, we isolated all publicly available tweets, which can include text, images, and links to websites, posted with the hashtag #BlackintheIvory on the Twitter website (www.twitter.com) from the inception of the hashtag in June 2020 to the end of December 2020. To evaluate the tweets, we categorized the tweets inductively. Based on the content of the posts, we identified 6 themes: Teaching, Mentoring, Collegiality, Identity, Service, and Racism.

ResultsOur search yielded a total of 12,538 original posts, including tweets between inception in June 2020 to December 2020 from profiles made public (excluding modified tweets and duplicate tweets). We selected and analyzed the top retweeted 2500 tweets, which is 20% (2500/12538) of the total number of downloaded tweets. The greatest percentage of posts were about Teaching (881; 35%), followed by Service (441; 18%) and Racism (414; 17%). The remaining tweets were categorized as Collegiality (388; 15%), Identity (210; 8%), and Mentoring (166; 7%) of the total number of tweets from June-December 2020.

ConclusionThe experiences, perspectives, and narratives among the Black diaspora within #BlackintheIvory are not uniform. The commonality exists within the structural systemic racism which impacts Black academics within the ivory tower, this study is a resounding wake-up call for action.

FundingThis study was not subject to any funding

category: scientific communication and education


title: Multi-Omics Regulatory Network Inference in the Presence of Missing Data

abstract:
MotivationA key problem in systems biology is the discovery of regulatory mechanisms that drive phenotypic behavior of complex biological systems in the form of multi-level networks. Modern multiomics profiling techniques probe these fundamental regulatory networks but are often hampered by experimental restrictions leading to missing data or partially measured omics types for subsets of individuals due to cost restrictions. In such scenarios, in which missing data is present, classical computational approaches to infer regulatory networks are limited. In recent years, approaches have been proposed to infer sparse regression models in the presence of missing information. Nevertheless, these methods have not been adopted for regulatory network inference yet.

ResultsIn this study, we integrated regression-based methods that can handle missingness into KiMONo, a Knowledge guIded Multi-Omics Network inference approach, and benchmark their performance on commonly encountered missing data scenarios in single- and multi-omics studies. Overall, two-step approaches that explicitly handle missingness performed best for a wide range of random- and block-missingness and noise levels, while methods implicitly handling missingness performed worst and were generally unstable. Our results show that robust multi-omics network inference with KiMONo is feasible and thus allows users to leverage available multi-omics data to its full extent.

Availabilityhttps://github.com/cellmapslab/kimono

Contactbenjamin.schubert@helmholtz-muenchen.de

Supplementary informationSupplementary data are available at Bioinformatics online.

category: systems biology


title: Systems Biology Approach of Understanding Insulin Resistance: Linkage between Type 2 Diabetes and Alzheimer's Disease

abstract: Insulin resistance (IR) is a physiological condition in which cells in the body become resistant to insulin. It is a known risk factor associated to type 2 diabetes (T2D). Recently, the idea that IR plays an important role in the progression of Alzheimers disease (AD) has been gaining a lot of attention. Comparing the components of the insulin signalling pathway in relation to T2D and AD, there seems to be a lot of commonality. However, on what role IR plays in linking T2D and AD remains unknown. Through systems biology approach, we extended an existing mathematical model (i.e. ODE based) to study and understand the role IR plays in linking T2D and AD. The simulations, together with the experimental data collected from the literature, show that the common components in T2D and AD express the same dynamical behaviors. This result provides the bases for further modelling of the insulin signaling pathway in determining the link between T2D and AD.


title: Bidirectional alterations in brain temperature profoundly modulate spatiotemporal neurovascular responses in-vivo: Implications for theragnostics

abstract: Neurovascular coupling (NVC) is a mechanism that, amongst other known and latent critical functions, ensures activated brain regions are adequately supplied with oxygen and glucose. This biological phenomenon underpins non-invasive perfusion-related neuroimaging techniques and recent reports have implicated NVC impairment in several neurodegenerative disorders. Yet, much remains unknown regarding NVC in health and disease, and only recently has there been burgeoning recognition of a close interplay with brain thermodynamics. Accordingly, we developed a novel multi-modal approach to systematically modulate cortical temperature and interrogate the spatiotemporal dynamics of sensory-evoked NVC. We show that changes in cortical temperature profoundly and intricately modulate NVC, with low temperatures associated with diminished oxygen delivery, and high temperatures inducing a distinct vascular oscillation. These observations provide novel insights into the relationship between NVC and brain thermodynamics, with important implications for brain-temperature related therapies, functional biomarkers of elevated brain temperature, and in-vivo methods to study neurovascular coupling.


title: A bug's tale: revealing the history, biogeography and ecological patterns of 500 years of insect introductions

abstract: The arrival of Europeans to the Americas triggered a massive exchange of organisms on a continental scale. This exchange was accelerated by the amazing increase in the movement of people and goods during the 20th century. In Chile, scientific and technical literature contains hundreds of records of exotic insect species in different parts of the territory, from the hyperarid Atacama Desert to the Magallanes Region. Here, we analyze the biological patterns obtained from the database of introductions of exotic insects to Chile from the European arrival to the present. Our task includes a review of old records in museums, libraries, collections, expedition records and catalogs. Almost 600 species of exotic insects have been reported in Chile. Introductions started at the very arrival of Europeans to the central valley of Chile and underwent a huge acceleration in the second half of the 20th century. The order Hemiptera was the most prevalent among introduced insects. Most species are linked to agriculture and forestry. Species are of Palearctic origin in more than 50% of the records. In terms of temporal trends, the rate of introduced species shows an abrupt increase at the beginning of the 1950s. This change may be associated with the strong development in agriculture and forestry in Chile after World War II and the increase in intercontinental air traffic. We believe that the understanding of past patterns of introductions is an important component in the design of current policies to minimize the impact of invasive insects.

category: ecology


title: Differential methylation, epigenetic clocks, and island-mainland divergence in an insectivorous small mammal

abstract: Geographically isolated populations, including island-mainland populations, tend to exhibit phenotypic variation in many species. The so-called island syndrome occurs when different environmental pressures lead to insular divergence from mainland populations. This phenomenon can be seen in an island population of Nova Scotia masked shrews (Sorex cinereus), which have developed a specialized feeding habit and digestive enzyme compared to their mainland counterparts. Epigenetic modifications, such as DNA methylation (DNAm), can impact phenotypes by altering gene expression without changing the DNA sequence. Here, we used a de novo masked shrew genome assembly and epigenome-wide assay to investigate morphological and DNA methylation patterns between island and mainland populations. Island shrews were morphologically and epigenetically different than their mainland counterparts, exhibiting a smaller body size; remarkably, the gene ontology analyses mirrored these phenotypes including genes for the digestive enzyme phenotypes. Moreover, island shrews appeared to age faster than their mainland counterparts. This study provides an example counter to the prevailing island syndrome morphological patterns, along with novel epigenomic insight into the drivers of island-mainland divergence in mammals.

category: genomics


title: BANKSY: A Spatial Omics Algorithm that Unifies Cell Type Clustering and Tissue Domain Segmentation

abstract: Each cell type in a solid tissue has a characteristic transcriptome and spatial arrangement, both of which are observable using modern spatial omics assays. However, the common practice is still to ignore spatial information when clustering cells to identify cell types. In fact, spatial location is typically considered only when solving the related, but distinct, problem of demarcating tissue domains (which could include multiple cell types). We present BANKSY, an algorithm that unifies cell type clustering and domain segmentation by constructing a product space of cell and neighbourhood transcriptomes, representing cell state and microenvironment, respectively. BANKSYs spatial kernel-based feature augmentation strategy improves per-formance and scalability on both tasks when tested on FISH-based and sequencing-based spatial omics data. Uniquely, BANKSY identified hitherto undetected niche-dependent cell states in two mouse brain regions. Lastly, we show that quality control of spatial omics data can be formulated as a domain identification problem and solved using BANKSY. BANKSY represents a biologically motivated, scalable, and versatile framework for analyzing spatial omics data.


title: Roseoflavin, a natural riboflavin analogue, possesses in vitro and in vivo antiplasmodial activity

abstract: The ability of the human malaria parasite Plasmodium falciparum to access and utilise vital nutrients is critical to its growth and proliferation. Molecules that interfere with these process could potentially serve as antimalarials. We found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin metabolism and/or the utilisation of the riboflavin metabolites flavin mononucleotide and flavin adenine dinucleotide. An additional eight riboflavin analogues were evaluated, but none were found to be more potent than roseoflavin, nor was their activity on target. Focussing on roseoflavin, we tested its antimalarial activity in vivo against Plasmodium vinckei vinckei in mice. We found that roseoflavin decreased the parasitemia by 46-fold following a 4 day suppression test and, on average, increased the survival of mice by 4-5 days. Our data are consistent with riboflavin metabolism and/or the utilisation of riboflavin-derived cofactors being viable drug targets for the development of new antimalarials and that roseoflavin could serve as a potential starting point.


title: Cooperative ectodomain interaction among TCRαβ, CD3γε, and CD3δε enhances TCR mechanotransduction

abstract: The T-cell receptor (TCR) complex comprises TCR{beta}, CD3{gamma}{varepsilon}, CD3{delta}{varepsilon}, and CD3{zeta}{zeta}. TCR{beta} engagement with peptide-bound major histocompatibility complex (pMHC) triggers CD3 phosphorylation, which is regulated by mechanical force. However, the inner workings of the TCR mechanotransduction machinery remains unclear. TCR ectodomain (ECD) interactions have been inferred from structural and mutagenesis studies. Due to their extreme weakness, however, direct measurements of affinity had failed and of force regulation have never been attempted. Here we measured two-dimensional affinities and force-dependent lifetimes of interactions among TCR{beta}, CD3{gamma}{varepsilon}, and CD3{delta}{varepsilon} ECDs, showing a cooperative CD3{delta}{varepsilon}-TCR{beta}-CD3{gamma}{varepsilon} catch bond with longer- lasting lifetime that exceeds the TCR-pMHC bond lifetime. Molecular dynamics stimulations revealed a central interacting region surrounded by TCR ECDs and identified critical interacting residues at their interfaces. Interfering TCR ECD interactions by antibodies impaired TCR-pMHC interaction and T cell function. Mutating residues that mediate TCR ECD cis-interactions with CD3s altered the catch bond of TCR-pMHC trans-interaction, which correlates with changed T cell cytokine production. Thus, TCR mechanotransduction is supported by cooperative TCR ECD interactions, which regulates T cell function. Our results provide a missing link between pMHC ligation and CD3 signaling and may guide future TCR engineering design for immunotherapies.

category: immunology


title: A Genome-Scale Metabolic Model of Marine Heterotroph Vibrio splendidus sp. 1A01

abstract: While the Vibrio splendidus species is best known as an opportunistic pathogen in oysters, the Vibrio splendidus sp. 1A01 strain was first identified as an early colonizer of synthetic chitin particles incubated in seawater. To gain a better understanding of its metabolism, a genome-scale metabolic model (GSMM) of V. splendidus sp. 1A01 was reconstructed. GSMMs enable us to simulate all metabolic reactions in a bacterial cell using Flux Balance Analysis. A draft model was built using an automated pipeline from BioCyc. Manual curation was then performed based on experimental data, in part by gap-filling metabolic pathways and tailoring the models biomass reaction to V. splendidus sp. 1A01. The challenges of building a metabolic model for a marine microorganism like V. splendidus sp. 1A01 are described.


title: Infected I. scapularis nymphs maintained in prolonged questing under optimal environmental conditions for one year can transmit B. burgdorferi to uninfected hosts

abstract:
In recent decades, Lyme disease has been expanding to previous non-endemic areas. We hypothesized that infected I. scapularis nymphs that retain host-seeking behavior under optimal environmental conditions are fit to fulfil their transmission role in the enzootic cycle of B. burgdorferi. To test this hypothesis, we produced nymphal ticks in the laboratory under controlled temperature (22-25{degrees}C), humidity (80-90%) and natural daylight cycle conditions to allow them to retain host-seeking/questing behavior for 1 year. We then analyzed differences in B. burgdorferi infection prevalence in questing and diapause nymphs at 6 weeks post-molting (prime questing) as well as differences in infection prevalence of questing nymphs maintained under prolonged environmental induced questing over 12 months (prolonged questing). Lastly, we analyzed the fitness of nymphal ticks subjected to prolonged questing in transmission of B. burgdorferi to naive mice over the course of the year. Our study shows that B. burgdorferi infected unfed I. scapularis nymphal ticks maintained under optimal environmental conditions in the laboratory not only survived for a year in a developmental state of prolonged questing (host-seeking), as they retained an infection prevalence sufficient to effectively fulfil transmission of B. burgdorferi to uninfected mice after tick challenge. Our study is important for understanding and possibly modeling Lyme disease expansion into former non-endemic regions due to global warming.

IMPORTANCELyme disease is rapidly spreading from its usual endemic areas in the Northeast, Midwest and Midatlantic states into neighboring areas which could be due to changing climate patterns. Our study shows that unfed I. scapularis nymphal ticks kept under optimal environmental conditions in the laboratory survived for a year while exhibiting aggressive host-seeking behavior and they maintained a B. burgdorferi infection prevalence which was sufficient to infect naive reservoir hosts after tick challenge. Our study raises important questions regarding prolonged survival of B. burgdorferi infected host-seeking nymphal I. scapularis ticks that can potentially increase the risk of Lyme disease incidence, if conditions of temperature and humidity become amenable to the enzootic cycle of B. burgdorferi in regions currently classified as non-endemic.


title: A landscape of differentiated biological processes involved in the initiation of sex differentiation in zebrafish

abstract: Zebrafish (Danio rerio) has been used as a promising animal model to study gonadal development and gametogenesis. Although previous studies have identified critical molecules participating in zebrafish gonad differentiation, a landscape view of the biological processes involved in this process is still lacking. Here we isolated intact zebrafish differentiating gonads, at 25 days post-fertilization (dpf) and 30 dpf, and conducted RNA-seq analysis between the juvenile gonads that tended to develop into ovaries or testes. Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the level of biological process. During ovary differentiation, the biological processes related to metabolic activities in production of energy and maternal substances, RNA degradation, and DNA repair were enriched. During testis differentiation, the biological processes related to cell proliferation, differentiation, and morphogenesis were enriched, with a total of 15 signaling pathways. Notably, we reveal that the immune-related processes are extensively involved in the regulation of testis development. Overall, this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.

category: developmental biology


title: The E3/E4 ubiquitin ligase UFD-2 mediates negative feedback on Raf protein stability

abstract:
Signaling by the kinase cascade comprised of Raf, MEK, and ERK is critical for animal development; moreover, its inappropriate activation is commonly found in human malignancies. In a genetic screen for factors that control signaling by the Caenorhabditis elegans Raf ortholog LIN-45, we found that it is negatively regulated by the E3/E4 ubiquitin ligase UFD-2. Both UFD-2 and its partner, the ATP-dependent unfoldase CDC-48, were required for degradation of LIN-45 protein. Our structure-function studies showed that disruption of LIN-45 domains that mediate protein interactions and complex formation, including the Ras binding domain, cysteine-rich domain, or C-terminus, allow for UFD-2-independent degradation. We propose a model whereby UFD-2 mediates a novel step of Raf degradation, by acting with the CDC-48 unfoldase machinery to extract Raf from multiprotein complexes.

One-Sentence SummaryRaf kinase complexes are degraded by the UFD-2 ubiquitin ligase and CDC-48 unfoldase during Raf-MEK-ERK signal transduction.


title: High genetic diversity in the pelagic deep-sea fauna of the Atacama Trench revealed by environmental DNA

abstract: A current paradigm in marine biodiversity states that faunal richness decreases with depth. However, the deep-ocean ecosystem has been significantly under-sampled, hindering a complete view of its biodiversity. This situation is accentuated in ultra-deep waters, where the remote and extreme conditions unfit the traditional sampling methods. Using environmental DNA, we assessed the pelagic metazoan diversity of the Atacama Trench from the high-productive near-surface level down to ~8000 m depth. Our results show that waters deeper than 4000 m contributed up to 50% of the overall genetic diversity. These findings contrast with similar observations in the less-productive Kermadec Trench, where the diversity in deep waters was lower than in shallower waters. Moreover, both deep pelagic ecosystems exhibited some unknown phylogenetic clades within the dominant taxonomic groups: hydrozoans and copepods. The deep-ocean biota may thus contribute to global biodiversity far more than hitherto suggested, especially in zones influenced by high primary production. Our results underline the need for increased effort to study these remote ecosystems and improve our understanding of their contribution to the ecology and biogeochemistry of the deep-sea pelagic and benthic realms.


title: Performance Comparison Of Agilent New SureSelect All Exon v8 Probes With v7 Probes For Exome Sequencing

abstract: Exome sequencing may become routine in health care it increases the chance of pinpointing the genetic cause of an individual patients condition and thus making an accurate diagnosis. It is important for facilities providing genetic services to keep track of changes in the technology of exome capture in order to maximize throughput while reducing cost per sample. In this study, we focused on comparing the newly released exome probe set Agilent SureSelect Human All Exon v8 and the previous probe set v7. In preparation for higher throughput of exome sequencing using the DNBSEQ-G400, we evaluated target design, coverage statistics, and variants across these two different exome capture products. Although the target size of the v8 design has not changed much compared to the v7 design (35.24 Mb vs 35.8 Mb), the v8 probe design allows you to call more of SNVs (+3.06%) and indels (+8.49%) with the same number of raw reads per sample on the common target regions (34.84 Mb). Our results suggest that the new Agilent v8 probe set for exome sequencing yields better data quality than the current Agilent v7 set.


title: Integrative chromatin domain annotation throughgraph embedding of Hi-C data

abstract:
MotivationThe organization of the genome into domains plays a central role in gene expression and other cellular activities. Researchers identify genomic domains mainly through two views: 1D functional assays such as ChIP-seq, and chromatin conformation assays such as Hi-C. Fully understanding domains requires integrative modeling that combines these two views. However, the predominant form of integrative modeling uses segmentation and genome annotation (SAGA) along with the rigid assumption that loci in contact are more likely to share the same domain type, which is not necessarily true for epigenomic domain types and genome-wide chromatin interactions.

ResultsHere, we present an integrative approach that annotates domains using both 1D functional genomic signals and Hi-C measurements of genome-wide 3D interactions without the use of a pairwise prior. We do so by using a graph embedding to learn structural features corresponding to each genomic region, then inputting learned structural features along with functional genomic signals to a SAGA algorithm. We show that our domain types recapitulate well-known subcompartments with an additional granularity that distinguishes a combination of the spatial and functional states of the genomic regions. In particular, we identified a division of the previously-identified A2 subcompartment such that the divided domain types have significantly varying expression levels.

Availabilityhttps://github.com/nedashokraneh/genome-structure-function-aggregation

Contactmaxwl@sfu.ca

Supplementary information


title: Rare Monoclonal Antibody Discovery Based on Indirect Competitive Screening of Single Hapten-specific Rabbit Antibody Secreting Cell

abstract: Rare antibody that is able to tolerate physio-chemical factors is preferred and highly demanded in diagnosis and therapy. Rabbit monoclonal antibodies (RmAbs) are distinguished owing to their high affinity and stability. However, the efficiency and availability of traditional methods for RmAb discovery are limited, especially for small molecules. Here, we present an indirect competitive screening method in nanowells, named CSMN, for single rabbit antibody secreting cells (ASCs) selection with 20.6 h and proposed an efficient platform for RmAb production against small molecule with 5.8 days for the first time. Chloramphenicol (CAP) as an antibacterial agent has the great threats for public health. We applied the CSMN to select CAP-specific ASCs and produced one high affinity RmAb, surprisingly showing extremely halophilic properties with an IC50 of 0.08 ng mL-1 in saturated salt solution which has as yet not been shown by other antibodies. Molecular dynamic simulation showed that the negatively charged surface improved the stability of the RmAb structure with additional disulfide bonds compared with mouse antibody. Moreover, the reduced solvent accessible surface area of the binding pocket increased the interactions of RmAb with CAP in a saturated salt solution. Furthermore, the RmAb was used to develop an immunoassay for the detection of CAP in real biological samples with simple pretreatment, shorter assay time, and higher sensitivity. The results demonstrated that the practical and efficient CSMN is suitable for rare RmAb discovery against small molecules.

category: biochemistry


title: Structural Characterization of L-Galactose Dehydrogenase: A Key Enzyme for Vitamin C Biosynthesis

abstract: In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a ({beta}/)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.


title: 3DPolyS-LE: an accessible simulation framework to model the interplay between chromatin and loop extrusion

abstract:
MotivationRecent studies suggest that the loop extrusion activity of Structural Maintenance of Chromosomes complexes is central to proper organization of genomes in vivo. Polymer physics-based modeling of chromosome structure has been instrumental to assess which structures such extrusion can create. Only few laboratories however have the technical and computational expertise to create in silico models combining dynamic features of chromatin and loop extruders.

ResultsHere we present 3DPolyS-LE, a self-contained, easy to use modeling and simulation framework allowing non-specialists to ask how specific properties of loop extruders and boundary elements impact on 3D chromosome structure. 3DPolyS-LE also provides algorithms to compare predictions with experimental Hi-C data.

Availability and implementationSoftware available at https://gitlab.com/togop/3DPolyS-LE ; implemented in Python and Fortran 2003 and supported on any Unix-based operating system (Linux, Mac OS).

Contactpeter.meister@unibe.ch and daniel.jost@ens-lyon.fr

Supplementary InformationSupplemental data are available at Bioinformatics online


title: Biological misinterpretation of transcriptional signatures in tumour samples can unknowingly undermine mechanistic understanding and faithful alignment with preclinical data

abstract:
Precise mechanism-based gene expression signatures (GESs) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signalling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumour samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumour stroma percentage can directly influence GESs, undermining the intended mechanistic signalling.

Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single cell RNAseq and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely-used GESs to be influenced, or confounded, by stromal content in tumour tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets.

Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumour models not fully recapitulating the stromal and immune microenvironment. As such, efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.


title: WNT5A-RHOA axis is a new vulnerability in small-cell lung cancer

abstract: WNT signaling presents an attractive target for cancer therapy due to its widespread oncogenic role. However, the molecular players involved in WNT signaling and the impact of their perturbation remain unknown for numerous recalcitrant cancers including small-cell lung cancer (SCLC). Here we show that beta-catenin, a master mediator of canonical WNT signaling, is not required for SCLC development in genetically engineered mouse models (GEMMs) and its transcriptional program is largely silenced during tumor development. Instead, inactivation of p130 in SCLC cells induces expression of WNT5A, a ligand for beta-catenin-independent WNT pathways. WNT5A is both sufficient and required for SCLC development and cell proliferation and selectively induces Rhoa transcription and activates RHOA protein to drive SCLC. Rhoa knockout suppresses SCLC development in vivo, and chemical perturbation of RHOA selectively inhibits SCLC cell proliferation. These findings suggest a novel requirement for the WNT5A-RHOA axis in SCLC that is distinct from other noncanonical WNT pathways. This vulnerability of p130-WNT5A-RHOA pathway provides critical insight into the development of novel therapeutic strategies for the recalcitrant cancer, as well as the stratification of patients who may benefit from them. This study also sheds new light on the heterogeneity of WNT signaling and the molecular determinants of its cell-type specificity.


title: The shape of aroma: measuring and modeling citrus oil gland distribution

abstract:
O_LICitrus come in diverse sizes and shapes, and play a key role in world culture and economy. Citrus oil glands in particular contain essential oils which include plant secondary metabolites associated with flavor and aroma. Capturing and analyzing nuanced information behind the citrus fruit shape and its oil gland distribution provides a morphology-driven path to further our insight into phenotype-genotype interactions.
C_LIO_LIWe investigated the shape of citrus fruit of 51 accessions based on 3D X-ray CT scan reconstructions. Accessions include all three ancestral citrus species, accessions from related genera, and several interspecific hybrids. We digitally separate and compare the size of fruit endocarp, mesocarp, exocarp, and oil gland tissue. Based on the centers of the oil glands, overall fruit shape is approximated with an ellipsoid. Possible oil gland distributions on this ellipsoid surface are explored using directional statistics.
C_LIO_LIThere is a strong allometry along fruit tissues; that is, we observe a strong linear relationship between the volume of any pair of major tissues. This suggests that the relative growth of fruit tissues with respect to each other follows a power law. We also observe that on average, glands distance themselves from their nearest neighbor following a square root relationship, which suggests normal diffusion dynamics at play.
C_LIO_LIThe observed allometry and square root models point to the existence of biophysical developmental constraints that govern novel relationships between fruit dimensions from both evolutionary and breeding perspectives. Understanding these biophysical interactions prompt an exciting research path on fruit development and breeding.
C_LI

Societal Impact StatementCitrus are intrinsically connected to human health and culture, including preventing human diseases like scurvy, and inspiring sacred rituals. Citrus fruits come in a stunning number of different sizes and shapes, ranging from small clementines to oversized pummelos, and fruits display a vast diversity of flavors and aromas. These qualities are key in both traditional and modern medicine and the production of cleaning and perfume products. By quantifying and modeling overall fruit shape and oil gland distribution, we can gain further insight into citrus development and the impacts of domestication and improvement on multiple characteristics of the fruit.


title: Parallel lipid-carried epitope pillars enhance stimulation of chimeric antigen receptor T cells

abstract: Chimeric antigen receptor T cells are genetically engineered to express a specific T cell receptor of interest, such as to target a cancer-specific antigen. Signaling events in chimeric antigen receptor T cells are essential for their proliferation, survival, and function. To achieve optimal antitumor efficacy, signaling motifs as well as the structure of the transmembrane domain of the chimeric antigen receptor have to be carefully designed. However, it remains unclear whether the arrangement, and therefore the movement and elasticity, of tumor antigens influence their stimulation of chimeric antigen receptor T cells. Here we show that MUC1 molecules tethered to a glycosylphosphatidylinositol anchor drastically increased its stimulation of chimeric antigen receptor T cells. In particular, MUC1 molecules containing one tandem repeat sequence showed significantly greater stimulatory activity than five tandem repeat sequences. Thus glycosylphosphatidylinositol-anchored, tumor antigen epitope pillars in parallel are attractive targets for the design of chimeric antigen receptor T cells. These novel findings, which we propose as the parallel lipid-carried epitope model, emphasize the importance of epitope arrangement in selecting highly effective chimeric antigen receptor T cells, potentially revolutionizing the applicability of this therapy. Furthermore, our data have implications for the necessity of methodologies to measure the elasticity, movement, and density of antigen pillars in parallel, as key tools to guide future chimeric antigen receptor T cell therapy.


title: Light-inducible T cell engagers trigger, tune and shape the activation of primary T cells

abstract: Cells perceive overtime complex sequences of receptor stimulation that they integrate to mount an appropriate response. Yet, the influence of signal dynamics on cell responses has been poorly characterized due to technical limitations. Here, we present a generalizable approach to control receptor stimulation on unmodified primary cells. Indeed, for applications on primary murine T cells, we have engineered the LiTe system, a new recombinant optogenetics-based Light-inducible T cell engager which allows tunable and reversible spatiotemporal control of the T Cell Receptor (TCR) stimulation. We also provided in vitro evidence that this system enables efficient T cell activation with light, leading to cytokine secretion or tumor cell killing. Using specific time-gated stimulations, we have been able to orient the outcome of the activation of T cells. Overall, the LiTe system constitutes a versatile ON/OFF molecular switch allowing to decipher the cellular response to stimulation dynamics. Its original control over T cell activation opens new avenues for future precision cancer immunotherapy.


title: A new chemotype of chemically tractable nonsteroidal estrogens based on a thienopyrimidine core.

abstract:
Despite continued interest in development of nonsteroidal estrogens and antiestrogens, there are only a few chemotypes of estrogen receptor ligands. Using targeted screening in a ligand sensing assay we identified a phenolic thieno[2,3-d]pyrimidine with affinity for estrogen receptor . An efficient three-step synthesis of the heterocyclic core and structure-guided optimization of the substituents resulted in a series of potent nonsteroidal estrogens. The chemical tractability of the thieno[2,3-d]pyrimidine chemotype will support the design of new estrogen receptor ligands as therapeutic hormones and antihormones.

GRAPHICAL ABSTRACT

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category: cell biology


title: Comparative Analysis of rRNA removal methods for RNA-seq Differential Expression in Halophilic Archaea

abstract: Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high ratio rRNA:mRNA (80-90%: [~]10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA and therefore requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here we show that after the discontinuation of the previous version of RiboZero (Illumina) that was useful to partially deplete rRNA from halophilic archaea, archaeal transcriptomics studies have experienced a standstill. To overcome this limitation, here we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper we provide insights to the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.


title: Study on the interference of DicdsVg4 and DicdsVgR in egg formation in the ovary of Diaphorina citri (Kuwayama)

abstract: The ovary is the key organ in insect female oviposition. For the continuation of populations in nature, the whole life cycle of female adults is centred on reproduction. Vg and VgR genes are important in determining the synthesis and transport of vitellogenin. Therefore, Vg and VgR gene expression in insect females affects the formation of mature eggs in the ovary, which is very important. RNAi interference of Vg and VgR genes may become an important means to control D. citri populations. A study found that the expression of the DicVg4 and DicVgR genes was significantly affected by DicdsVg4 and DicdsVgR. DicVg4 and DicVgR gene expression interference could prevent the accumulation of vitellogenin in the ovaries of females, rendering ovaries unable to form mature eggs normally and leading to the production of abnormal eggs and nymphs. After knockdown of DicVg4 and DicVgR gene expression, the proportion of ovarian mature eggs was only 8.33 {+/-} 1.67% and 7.75 {+/-} 0.5% in the 10-d developmental stage, the proportion of negative control group ovarian mature eggs was 13.19 {+/-} 0.14% in the 10-d developmental stage, and the proportion of ovarian mature eggs in the interference group was 0.00 {+/-} 0.00% in the 5, 15, 20, 25, and 30-d developmental stages. These results provide a theoretical basis for the use of mixed polygene interference technology to control damage from D. citri populations.


title: Compendium-wide analysis of P. aeruginosa core and accessory genes reveal more nuanced transcriptional patterns

abstract:
Strains of Pseudomonas aeruginosa, an opportunistic pathogen that causes difficult to treat infections, have significant genomic heterogeneity including the presence of diverse accessory genes that are only present in some strains or clades. Both core genes, which are conserved across strains, and accessory genes have been associated with traits such as biofilm formation and virulence. Much of what we know about core and accessory gene content comes from genome analyses. Here, we use a newly assembled transcriptome compendium to analyze the transcriptional patterns of core and accessory gene expression in PAO1 and PA14 strains across thousands of samples from hundreds of distinct experiments. We found that a subset of core genes were stable, having consistent correlated expression patterns across samples regardless of strain background, with a focus on strains PAO1 and PA14. These stable core genes had fewer co-expressed neighbors that were accessory genes.

SignificancePseudomonas aeruginosa is a ubiquitous pathogen. There is a lot of diversity amongst P. aeruginosa strains, some which are clinically relevant. Understanding how these different strain-level traits manifest is important for identifying targets that regulate different traits of interest. With the availability of a PAO1-mapped and PA14-mapped RNA-seq compendium, which contain hundreds of strains, it is now possible to examine the effect of different strains on expression, which can mediate different traits. In this study we developed an approach to compare expression profiles across different P. aeruginosa gene groups - core and accessory genes. This approach revealed a subset of core genes with different transcriptional patterns across strains, which could contribute to trait differences.

category: bioinformatics


title: Ganglioside enriched phospholipid vesicles induce cooperative Aβ oligomerization and membrane disruption

abstract:
A major hallmark of Alzheimer disease (AD) is the accumulation of extracellular aggregates of amyloid-{beta} (A{beta}). Structural polymorphism observed among A{beta} fibrils in AD brains seem to correlate with the clinical sub-types suggesting a link between fibril polymorphism and pathology. Since fibrils emerge from a templated growth of low-molecular weight oligomers, understanding the factors affecting oligomer generation is important. The membrane lipids are key factors that influence early stages of A{beta} aggregation and oligomer generation, and cause membrane disruption. We have previously demonstrated that conformationally discrete A{beta} oligomers can be generated by modulating the charge, composition, chain length of lipids and surfactants. Here, we extend our studies into liposomal models by investigating A{beta} oligomerization on large unilamellar vesicles (LUVs) of total brain extracts (TBE), reconstituted lipid rafts (LRs) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Specifically, we varied the vesicle composition by varying the amount of GM1 gangliosides added as a constituent. We found that liposomes enriched in GM1 induce the formation of toxic, low-molecular weight oligomers that are isolable in a lipid-complexed form. Importantly, the data indicate that oligomer formation and membrane disruption are highly cooperative processes. Numerical simulations on the experimental data confirm cooperativity and reveal that GM1-enriched liposomes form twice as many numbers of pores as those without GM1. Overall, this study uncovers mechanisms of cooperativity between oligomerization and membrane disruption under controlled lipid compositional bias, and refocuses the significance of the early stages of A{beta} aggregation in polymorphism, propagation, and toxicity in AD.

TOC figure O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: biophysics


title: Functional testing of PI3K inhibitors stratifies responders to idelalisib and identifies treatment vulnerabilities in idelalisib-refractory/intolerant chronic lymphocytic leukemia

abstract:
PurposePhosphatidylinositol 3-kinase inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). While patients may show an initial response, development of treatment intolerance or resistance remains a clinically challenging. Prediction of individual treatment responses based on clinically actionable biomarkers is needed to overcome these challenges. Here, we investigated whether ex vivo functional responses to targeted therapies can stratify responders to idelalisib and guide precision medicine in CLL.

Experimental designCLL cells from treatment naive, idelalisib-responding, and idelalisib-refractory/intolerant patients (n=33 in total) were profiled against ten PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay.

ResultsAmong the ten PI3Ki studied, pan-PI3Ki were most effective at inhibiting PI3K signaling and cell viability, and they showed activity also in CLL cells from idelalisib-refractory/intolerant patients. The pan-PI3Ki copanlisib, but not the p110{delta} inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, while it did not significantly affect T cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Based on ex vivo drug sensitivity testing, a relapsed CLL patient was treated with idelalisib plus venetoclax, and the patient achieved a partial response. A more systematic analysis revealed that CLL cells from patients with a long-term response to idelalisib showed significantly higher drug sensitivities to 73 drug combinations at baseline compared to short-term responders.

ConclusionsOur findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and demonstrate that ex vivo functional profiling may guide precision medicine and predict treatment responses of individual CLL patients.

TRANSLATIONAL RELEVANCEThe phosphatidylinositol 3-kinase inhibitors (PI3Ki) idelalisib and duvelisib are approved for relapsed chronic lymphocytic leukemia (CLL), but their use has been limited by severe toxicity and acquired resistance. Identification of biomarkers that predict individual treatment responses, as well as alternative treatment vulnerabilities in PI3Ki refractory/intolerant patients, is needed to optimally tailor CLL therapy. We performed functional analyses of CLL cells from treatment naive, idelalisib-responding and idelalisib-refractory/intolerant patients to identify clinically actionable biomarkers. We show that CLL cells from idelalisib-refractory/intolerant patients remain sensitive to pan-PI3Ki and PI3Ki plus venetoclax combinations. Ex vivo drug sensitivity testing was used to guide treatment of a relapsed CLL patient who obtained a partial response after idelalisib plus venetoclax therapy. A systematic analysis of drug sensitivities to 73 drug combinations stratified responders to idelalisib using baseline samples from short-term and long-term responders to idelalisib. Our study demonstrates the power of functional precision medicine in relapsed CLL.


title: Berberine Bridge Enzyme-Like Oligosaccharide Oxidases Act As Enzymatic Transducers Between Microbial Glycoside Hydrolases And Plant Peroxidases

abstract: OG-oxidases (OGOXs) and CD-oxidase (CELLOX) are plant berberine bridge enzyme-like oligosaccharide oxidases that oxidize oligogalacturonides (OGs) and cellodextrins (CDs), cell wall fragments with nature of damage-associated molecular patterns (DAMPs). The oxidation of OGs and CDs attenuates their elicitor activity by concomitantly releasing H2O2. Here, we demonstrate that the H2O2 generated downstream of the combined action between a fungal polygalacturonase and OGOX1 or an endoglucanase and CELLOX can be directed by plant peroxidases (PODs) either towards a reaction possibly involved in plant defence such as the oxidation of monolignol or a reaction possibly involved in a developmental event such as the oxidation of auxin (IAA), pointing to OGOX1 and CELLOX as enzymatic transducers between microbial glycoside hydrolases and plant PODs.


title: Snake Fungal Disease Caused by the Fungal Pathogen Ophidiomyces ophidiicola in Texas

abstract: The pathogen Ophidiomyces ophidiicola (O.o.), widely known as the primary cause of snake fungal disease (SFD) has been detected in Texass naive snakes. Our team set out to begin to characterize O. ophidiicolas spread in east Texas. From July 2019 until October 2021, we sampled 176 snakes across east Texas and detected 27 positives cases (qPCR confirmed 27/176). From a ribbon snake with clear clinical display, we isolated and cultured what we believe to be the Texas isolate of O. ophidiicola. With over 1/10 snakes that may be infected in East Texas, gives credence to the onset of SFD in Texas.


title: The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

abstract:
The serine/threonine protein kinase AKT is a critical mediator of growth factor signaling that broadly impacts mRNA translation, metabolism, programmed cell death and stress responses. Human cytomegalovirus (HCMV), a large dsDNA virus in the herpesvirus family, dramatically remodels host cell signaling pathways during lytic infection. Here, we show that AKT accumulates in an inactive form during HCMV infection, as indicated by hypo-phosphorylation at Thr308 and Ser473, as well as nuclear localization of FOXO3a. Moreover, we find that expression of constitutively active myristoylated AKT (myr-AKT) causes a 1-log defect in viral replication, accompanied by defects in viral DNA synthesis and viral late gene expression. These findings suggest that HCMV shutoff of AKT is not merely incidental to viral factors such as pUL38 that maintain high levels of mTORC1 activity independently of extracellular growth factor signals. Short interfering RNA knockdown of FOXO3a, but not FOXO1, phenocopied the replication defects seen during expression of myr-AKT, corroborating a role for FOXO3a during HCMV infection. Accordingly, a chimeric FOXO3a-estrogen receptor fusion protein, whose nuclear localization is regulated by the small molecule ligand 4-hydroxytamoxifen instead of AKT activity, rescues virtually all the replication defects induced by myr-AKT expression. Taken together, our results argue that HCMV dampens AKT signaling to promote nuclear localization of FOXO transcription factors, which are required for efficient viral replication.

IMPORTANCEEvidence from a diverse herpesvirus infection models suggests that the PI3K / AKT signaling pathway suppresses reactivation from latency, and that inactivation of the pathway stimulates viral lytic replication. Here, we show that AKT accumulates in an inactive state during HCMV infection of lytically permissive cells while the presence of constitutive AKT activity causes substantial viral replication defects. Although AKT phosphorylates a diverse array of cellular substrates, we identify an important role for the Forkhead box class O transcription factors. Our findings show that when FoxO3a nuclear localization is decoupled from its negative regulation by AKT, the viral replication defects observed in the presence of constitutively active AKT are reversed. Collectively, our results reveal that HCMV inactivates AKT to promote the nuclear localization of FOXO transcription factors, which strongly implies that FOXOs play critical roles in transactivating cellular and/or viral genes during infection.


title: 1700029I15Rik orchestrates the biosynthesis of acrosomal membrane proteins required for sperm-egg fusion

abstract:
Sperm acrosomal membrane proteins, such as IZUMO1 and SPACA6, play an essential role in mammalian sperm-egg fusion. How their biosynthesis is regulated during spermiogenesis has largely remained unknown. Here, we show that the 1700029I15Rik knockout male mice are severely subfertile and their spermatozoa do not fuse with eggs. 1700029I15Rik encodes a type-II transmembrane protein that is expressed in early spermatids but not in mature spermatozoa. 1700029I15Rik is associated with proteins involved in N-glycosylation, disulfide isomerisation, and ER- Golgi trafficking, suggesting its involvement in nascent protein processing. 1700029I15Rik knockout testis has a normal level of sperm plasma membrane proteins, but decreased expression of multiple acrosomal membrane proteins. The knockout sperm exhibit elevated ubiquitinated proteins and upregulated ER-associated degradation; strikingly, SPACA6 becomes undetectable. Our results support for a specific, 1700029I15Rik-mediated pathway in spermiogenesis for the assembly of acrosomal membrane proteins.

Significance StatementIn sexually reproducing species, life begins with the fusion between a sperm and an egg. Multiple sperm acrosomal membrane proteins have been reported indispensable for sperm-egg fusion in mammals, yet the mechanism underlying their biosynthesis remains unknown. The present study demonstrates the existence of a 1700029I15Rik-mediated pathway specifically coordinating the processing and assembly of acrosomal membrane proteins. It represents an intriguing paradigm where the biosynthesis of proteins destined for various subcellular compartments might be orchestrated in a spatiotemporal manner. Given 1700029I15Rik is highly conserved in human, our findings provide potential insights into the aetiology of idiopathic male infertility and the development of a novel contraceptive approach involving molecular interventions in the maturation of gamete fusion-required acrosomal proteins.


title: Validation of the first PGT-whole genome sequencing approach including mitochondrial variants

abstract: Whole genome screening is currently not available in preimplantation genetic testing (PGT) due to poor performance of whole genome amplification methods. Here, we present the first validation study using our latest whole genome amplification (WGA) protocol, which yields amplified DNA with comparable quality to genomic DNA when perfoming whole genome sequencing assays. We started by validating PGT for aneuploidy (PGT-A) using our WGA protocol on cell lines and donated human embryos, where amplification success rates were >99.9% and 96.2% respectively. Accuracy on both was >99.9% on aneuploidy status. We next validated whole genome sequencing using amplified and genomic DNA from the Genome in the Bottle reference sample NA12878 to demonstrate accuracy, specificity, sensitivity, and precision of our WGA methods. Amplified DNA and genomic DNA are comparable in this case, with 99.99% accuracy, 99.99% specificity, 98.0% sensitivity and 98.1% precision with our WGA protocol. We additionally examined variant calls between biopsies and the remaining embryos from which they were derived. Again, 99.99% accuracy, 99.99% specificity, 98.1% sensitivity and 98.0% precision were observed. Mitochondrial heteroplasmy between biopsies and embryos was validated, and 99.9% accuracy, 100% specificity, 99.1% sensitivity and 100% precision were observed. Finally, we demonstrated the ability to improve the accuracy of certain types of mendelian variants in our data to close to 100% by leveraging parental and a born childs genome to perform linkage analysis.