Canalization of Phenotypes-When the Transcriptome is Constantly but Weakly Perturbed.

Recent studies have increasingly pointed to microRNAs (miRNAs) as the agent of gene regulatory network (GRN) stabilization as well as developmental canalization against constant but small environmental perturbations. To analyze mild perturbations, we construct a Dicer-1 knockdown line (dcr-1 KD) in Drosophila that modestly reduces all miRNAs by, on average, ∼20%. The defining characteristic of stabilizers is that, when their capacity is compromised, GRNs do not change their short-term behaviors. Indeed, even with such broad reductions across all miRNAs, the changes in the transcriptome are very modest during development in stable environment. By comparison, broad knockdowns of other regulatory genes (esp. transcription factors) by the same method should lead to drastic changes in the GRNs. The consequence of destabilization may thus be in long-term development as postulated by the theory of canalization. Flies with modest miRNA reductions may gradually deviate from the developmental norm, resulting in late-stage failures such as shortened longevity. In the optimal culture condition, the survival to adulthood is indeed normal in the dcr-1 KD line but, importantly, adult longevity is reduced by ∼90%. When flies are stressed by high temperature, dcr-1 KD induces lethality earlier in late pupation and, as the perturbations are shifted earlier, the affected stages are shifted correspondingly. Hence, in late stages of development with deviations piling up, GRN would be increasingly in need of stabilization. In conclusion, miRNAs appear to be a solution to weak but constant environmental perturbations.

Deciphering clonal dynamics and metastatic routines in a rare patient of synchronous triple-primary tumors and multiple metastases with MPTevol.

Multiple primary tumor (MPT) is a special and rare cancer type, defined as more than two primary tumors presenting at the diagnosis in a single patient. The molecular characteristics and tumorigenesis of MPT remain unclear due to insufficient approaches. Here, we present MPTevol, a practical computational framework for comprehensively exploring the MPT from multiregion sequencing (MRS) experiments. To verify the utility of MPTevol, we performed whole-exome MRS for 33 samples of a rare patient with triple-primary tumors and three metastatic sites and systematically investigated clonal dynamics and metastatic routines. MPTevol assists in comparing genomic profiles across samples, detecting clonal evolutionary history and metastatic routines and quantifying the metastatic history. All triple-primary tumors were independent origins and their genomic characteristics were consistent with corresponding sporadic tumors, strongly supporting their independent tumorigenesis. We further showed two independent early monoclonal seeding events for the metastases in the ovary and uterus. We revealed that two ovarian metastases were disseminated from the same subclone of the primary tumor through undergoing whole-genome doubling processes, suggesting metastases-to-metastases seeding occurred when tumors had similar microenvironments. Surprisingly, according to the metastasis timing model of MPTevol, we found that primary tumors of about 0.058-0.124 cm diameter have been disseminating to distant organs, which is much earlier than conventional clinical views. We developed MPT-specialized analysis framework MPTevol and demonstrated its utility in explicitly resolving clonal evolutionary history and metastatic seeding routines with a rare MPT case. MPTevol is implemented in R and is available at https://github.com/qingjian1991/MPTevol under the GPL v3 license.

Controlled generation of self-sustained oscillations in complex artificial neural networks.

Spatially distinct, self-sustained oscillations in artificial neural networks are fundamental to information encoding, storage, and processing in these systems. Here, we develop a method to induce a large variety of self-sustained oscillatory patterns in artificial neural networks and a controlling strategy to switch between different patterns. The basic principle is that, given a complex network, one can find a set of nodes-the minimum feedback vertex set (mFVS), whose removal or inhibition will result in a tree-like network without any loop structure. Reintroducing a few or even a single mFVS node into the tree-like artificial neural network can recover one or a few of the loops and lead to self-sustained oscillation patterns based on these loops. Reactivating various mFVS nodes or their combinations can then generate a large number of distinct neuronal firing patterns with a broad distribution of the oscillation period. When the system is near a critical state, chaos can arise, providing a natural platform for pattern switching with remarkable flexibility. With mFVS guided control, complex networks of artificial neurons can thus be exploited as potential prototypes for local, analog type of processing paradigms.

Tumorigenesis as the Paradigm of Quasi-neutral Molecular Evolution.

In the absence of both positive and negative selections, coding sequences evolve at a neutral rate (R = 1). Such a high genomic rate is generally not achievable due to the prevalence of negative selection against codon substitutions. Remarkably, somatic evolution exhibits the seemingly neutral rate R ∼ 1 across normal and cancerous tissues. Nevertheless, R ∼ 1 may also mean that positive and negative selections are both strong, but equal in intensity. We refer to this regime as quasi-neutral. Indeed, individual genes in cancer cells often evolve at a much higher, or lower, rate than R ∼ 1. Here, we show that 1) quasi-neutrality is much more likely when populations are small (N < 50); 2) stem-cell populations in single normal tissue niches, from which tumors likely emerge, have a small N (usually <50) but selection at this stage is measurable and strong; 3) when N dips below 50, selection efficacy decreases precipitously; and 4) notably, N is smaller in the stem-cell niche of the small intestine than in the colon. Hence, the ∼70-fold higher rate of phenotypic evolution (observed as cancer risk) in the latter can be explained by the greater efficacy of selection, which then leads to the fixation of more advantageous and fewer deleterious mutations in colon cancers. In conclusion, quasi-neutral evolution sheds a new light on a general evolutionary principle that helps to explain aspects of cancer evolution.

Molecular Evolution in Large Steps-Codon Substitutions under Positive Selection.

Molecular evolution is believed to proceed in small steps. The step size can be defined by a distance reflecting physico-chemical disparities between amino acid (AA) pairs that can be exchanged by single 1-bp mutations. We show that AA substitution rates are strongly and negatively correlated with this distance but only when positive selection is relatively weak. We use the McDonald and Kreitman test to separate the influences of positive and negative selection. While negative selection is indeed stronger on AA substitutions generating larger changes in chemical properties of AAs, positive selection operates by different rules. For 65 of the 75 possible pairs, positive selection is comparable in strength regardless of AA distance. However, the ten pairs under the strongest positive selection all exhibit large leaps in chemical properties. Five of the ten pairs are shared between Drosophila and Hominoids, thus hinting at a common but modest biochemical basis of adaptation across taxa. The hypothesis that adaptive changes often take large functional steps will need to be extensively tested. If validated, molecular models will need to better integrate positive and negative selection in the search for adaptive signal.

Benzo[a]pyrene promotes progression in tongue squamous cell carcinoma.

OBJECTIVES: Benzo[a]pyrene (B[a]P) is a member of the polycyclic aromatic hydrocarbon (PAH) family. Although the potent carcinogenicity of high-dose B[a]P has been extensively reported, the effects of long-term exposure to B[a]P on the progression of tongue squamous cell carcinoma (TSCC) are poorly understood. METHODS: In the present study, TSCC cells were treated with 5 or 50 nM of B[a]P for three months. The proliferation and chemoresistance of B[a]P-treated cells to 5-fluorouracil or cisplatin were detected by CCK8. The motility of the B[a]P-treated cells was evaluated with wound healing analysis, invasion assay, and three-dimensional culture in decellularized mouse tongue matrix. Xenograft assay was used to investigate the aggressiveness of B[a]P-treated cells. Immunofluorescence staining, terminal restriction fragment assay, and whole-genome sequence were used to determine the mutation spectrums. RESULTS: Long-term 50 nM B[a]P-treated cells exhibited increased aggressiveness and chemoresistance to 5-fluorouracil or cisplatin. In addition, data from whole-genome sequencing demonstrated that C:T to A:T transitions were the predominant nucleotide substitutions occurred in 50 nM B[a]P-treated CAL27 cells. Furthermore, 102 non-synonymous or stop-gain mutations were enriched in the extracellular-matrix-receptor interactive pathway. CONCLUSIONS: B[a]P exposure may contribute to genomic instability, and therefore, B[a]P may promote the progression of TSCC.

Direct measurement of pervasive weak repression by microRNAs and their role at the network level.

BACKGROUND: A gene regulatory network (GRN) comprises many weak links that are often regulated by microRNAs. Since miRNAs rarely repress their target genes by more than 30%, doubts have been expressed about the biological relevance of such weak effects. These doubts raise the possibility of under-estimation as miRNA repression is usually estimated indirectly from equilibrium expression levels. RESULTS: To measure miRNA repression directly, we inhibited transcript synthesis in Drosophila larvae and collected time-course data on mRNA abundance, the decline of which reflects transcript degradation. The rate of target degradation in the absence of miR310s, a moderately expressed miRNA family, was found to decrease by 5 to 15%. A conventional analysis that does not remove transcript synthesis yields an estimate of 6.5%, within the range of the new estimates. These data permit further examinations of the repression mechanisms by miRNAs including seed matching types, APA (alternative polyadenylation) sites, effects of other highly-expressed miRNAs and the length of 3'UTR. Our direct measurements suggest the latter two factors have a measurable effect on decay rate. CONCLUSION: The direct measurement confirms pervasive weak repression by miRNAs, supporting the conclusions based on indirect assays. The confirmation suggests that this weak repression may indeed be miRNAs' main function. In this context, we discuss the recent proposal that weak repression is "cumulatively powerful" in stabilizing GRNs.

Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution.

The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 "polymorphic" SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model, MALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors.

Proteomic Profiling of Serum Extracellular Vesicles Identifies Diagnostic Signatures and Therapeutic Targets in Breast Cancer.

Analysis of extracellular vesicles (EVs) is a promising noninvasive liquid biopsy approach for breast cancer (BC) detection, prognosis, and therapeutic monitoring. A comprehensive understanding of the characteristics and proteomic composition of BC-specific EVs from human samples is required to realize the potential of this strategy. In this study, we applied a mass spectrometry-based, data-independent acquisition (DIA) proteomic approach to characterize human serum EVs derived from patients with BC (n = 126) and healthy donors (HDs, n = 70) in a discovery cohort and validated the findings in five independent cohorts. Examination of the EV proteomes enabled construction of specific EV protein classifiers for diagnosing BC and distinguishing patients with metastatic disease. Of note, TALDO1 was found to be an EV biomarker of distant metastasis of BC. In vitro and in vivo analysis confirmed the role of TALDO1 in stimulating BC invasion and metastasis. Finally, high-throughput molecular docking and virtual screening of a library consisting of 271,380 small molecules identified a potent TALDO1 allosteric inhibitor, AO-022, which could inhibit BC migration in vitro and tumor progression in vivo. Together, this work elucidates the proteomic alterations in the serum EVs of BC patients to guide development of improved diagnosis, monitoring, and treatment strategies.

Precise microdissection of gastric mixed adeno-neuroendocrine carcinoma dissects its genomic landscape and evolutionary clonal origins.

Gastric mixed adenoneuroendocrine carcinoma (MANEC) is a clinically aggressive and heterogeneous tumor composed of adenocarcinoma (ACA) and neuroendocrine carcinoma (NEC). The genomic properties and evolutionary clonal origins of MANEC remain unclear. We conduct whole-exome and multiregional sequencing on 101 samples from 33 patients to elucidate their evolutionary paths. We identify four significantly mutated genes, TP53, RB1, APC, and CTNNB1. MANEC resembles chromosomal instability stomach adenocarcinoma in that whole-genome doubling in MANEC is predominant and occurs earlier than most copy-number losses. All tumors are of monoclonal origin, and NEC components show more aggressive genomic properties than their ACA counterparts. The phylogenetic trees show two tumor divergence patterns, including sequential and parallel divergence. Furthermore, ACA-to-NEC rather than NEC-to-ACA transition is confirmed by immunohistochemistry on 6 biomarkers in ACA- and NEC-dominant regions. These results provide insights into the clonal origin and tumor differentiation of MANEC.

Is the inverted surface landing more suitable in evaluating ankle braces and ankle inversion perturbation?

OBJECTIVE: To investigate biomechanical (kinematic) differences between 2 ankle brace testing protocols: landing on an inverted surface (IS) and inversion drop on an inversion platform. DESIGN: Five trials in each of 4 dynamic movement conditions were performed: inversion drop and drop landing from 0.45 m onto an IS without and with an ankle brace. A 7-camera motion analysis system was used to obtain the 3-dimensional kinematics. A 2 × 2 (brace × movement) repeated measures analysis of variance was used to evaluate selected variables for inversion drop and IS landing. SETTING: Research laboratory. PATIENTS: Eleven healthy subjects participated in the study. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Maximum ankle frontal plane and sagittal plane joint angles, range of motion, and maximum angular velocity. RESULTS: The IS landing resulted in significantly earlier maximum inversion, inversion velocities, dorsiflexion range of motion (ROM), contact dorsiflexion velocity, and maximum dorsiflexion velocity compared with the inversion drop. The ankle brace application during the IS landing reduced the contact plantarflexion angle, dorsiflexion ROM and maximum dorsiflexion velocity, and maximum inversion. CONCLUSIONS: The results from this study showed that the IS landing protocol produced significantly earlier maximum inversion angle and velocity and inversion velocities compared with the inversion drop protocol. These results showed that the IS landing is more demanding and should be considered in future investigations of ankle braces and lateral ankle performance/injury mechanisms.

Two decades of suspect evidence for adaptive molecular evolution-negative selection confounding positive-selection signals.

There has been a large literature in the last two decades affirming adaptive DNA sequence evolution between species. The main lines of evidence are from (i) the McDonald-Kreitman (MK) test, which compares divergence and polymorphism data, and (ii) the phylogenetic analysis by maximum likelihood (PAML) test, which analyzes multispecies divergence data. Here, we apply these two tests concurrently to genomic data of Drosophila and Arabidopsis. To our surprise, the >100 genes identified by the two tests do not overlap beyond random expectation. Because the non-concordance could be due to low powers leading to high false negatives, we merge every 20-30 genes into a 'supergene'. At the supergene level, the power of detection is large but the calls still do not overlap. We rule out methodological reasons for the non-concordance. In particular, extensive simulations fail to find scenarios whereby positive selection can only be detected by either MK or PAML, but not both. Since molecular evolution is governed by positive and negative selection concurrently, a fundamental assumption for estimating one of these (say, positive selection) is that the other is constant. However, in a broad survey of primates, birds, Drosophila and Arabidopsis, we found that negative selection rarely stays constant for long in evolution. As a consequence, the variation in negative selection is often misconstrued as a signal of positive selection. In conclusion, MK, PAML and any method that examines genomic sequence evolution has to explicitly address the variation in negative selection before estimating positive selection. In a companion study, we propose a possible path forward in two stages-first, by mapping out the changes in negative selection and then using this map to estimate positive selection. For now, the large literature on positive selection between species has to await reassessment.

Dynamic global analysis of transcription reveals the role of miRNAs in synergistic stabilization of gene expression.

Buffering exogenous perturbation is crucial to maintain transcriptional homeostasis during development. While miRNAs have been speculated to play a role in stability maintenance, previous studies seeking to check this conjecture focused on measurements of transcript levels at steady state or involved individual miRNA targets. We measured whole-genome expression dynamics by introducing a transient perturbation and establishing a perturbation and recovery system in Drosophila larvae. We inhibited all transcription and assayed transcriptomes at several time points during recovery from inhibition. We performed these experiments in the wild type and miRNA-deficient genetic backgrounds. Consistent with theories about miRNAs' function in stabilizing the transcriptome, we find that attenuating miRNA expression leads to weak impairment in degradation of targets but strong destabilization of target genes when transcription is re-activated. We further fitted a model that captures the essential aspects of transcription dynamics in our experiments and found that the miRNA target transcripts uniformly overshoot the original steady state as they recover from a general inhibition of transcription if global miRNA levels are reduced. Collectively, our results provide experimental evidence for the idea that miRNAs act cumulatively to stabilize the transcriptional regulatory network. We therefore found a promising approach to assess the effect of these molecules on transcription dynamics.

Efficacy of an ankle brace with a subtalar locking system in inversion control in dynamic movements.

STUDY DESIGN: Controlled laboratory study. OBJECTIVES: To examine effectiveness of an ankle brace with a subtalar locking system in restricting ankle inversion during passive and dynamic movements. BACKGROUND: Semirigid ankle braces are considered more effective in restricting ankle inversion than other types of brace, but a semirigid brace with a subtalar locking system may be even more effective. METHODS: Nineteen healthy subjects with no history of major lower extremity injuries were included in the study. Participants performed 5 trials of an ankle inversion drop test and a lateral-cutting movement without wearing a brace and while wearing either the Element (with the subtalar locking system), a Functional ankle brace, or an ASO ankle brace. A 2-way repeated-measures analysis of variance (ANOVA) was used to assess brace differences (P?.05). RESULTS: All 3 braces significantly reduced total passive ankle frontal plane range of motion (ROM), with the Element ankle brace being the most effective. For the inversion drop the results showed significant reductions in peak ankle inversion angle and inversion ROM for all 3 braces compared to the no brace condition; and the peak inversion velocity was also reduced for the Element brace and the Functional brace. In the lateral-cutting movement, a small but significant reduction of the peak inversion angle in early foot contact and the peak eversion velocity at push-off were seen when wearing the Element and the Functional ankle braces compared to the no brace condition. Peak vertical ground reaction force was reduced for the Element brace compared to the ASO brace and the no brace conditions. CONCLUSIONS: These results suggest that the tested ankle braces, especially the Element brace, provided effective restriction of ankle inversion during both passive and dynamic movements.

Molecular Evolution in Small Steps under Prevailing Negative Selection: A Nearly Universal Rule of Codon Substitution.

The widely accepted view that evolution proceeds in small steps is based on two premises: 1) negative selection acts strongly against large differences and 2) positive selection favors small-step changes. The two premises are not biologically connected and should be evaluated separately. We now extend a previous approach to studying codon evolution in the entire genome. Codon substitution rate is a function of the physicochemical distance between amino acids (AAs), equated with the step size of evolution. Between nine pairs of closely related species of plants, invertebrates, and vertebrates, the evolutionary rate is strongly and negatively correlated with a set of AA distances (ΔU, scaled to [0, 1]). ΔU, a composite measure of evolutionary rates across diverse taxa, is influenced by almost all of the 48 physicochemical properties used here. The new analyses reveal a crucial trend hidden from previous studies: ΔU is strongly correlated with the evolutionary rate (R2 > 0.8) only when the genes are predominantly under negative selection. Because most genes in most taxa are strongly constrained by negative selection, ΔU has indeed appeared to be a nearly universal measure of codon evolution. In conclusion, molecular evolution at the codon level generally takes small steps due to the prevailing negative selection. Whether positive selection may, or may not, follow the small-step rule is addressed in a companion study.