Discovery of Possible Gene Relationships through the Application of Self-Organizing Maps to DNA Microarray Databases

DNA microarrays and cell cycle synchronization experiments have made possible the study of the mechanisms of cell cycle regulation of Saccharomyces cerevisiae by simultaneously monitoring the expression levels of thousands of genes at specific time points. On the other hand, pattern recognition techniques can contribute to the analysis of such massive measurements, providing a model of gene expression level evolution through the cell cycle process. In this paper, we propose the use of one of such techniques –an unsupervised artificial neural network called a Self-Organizing Map (SOM)–which has been successfully applied to processes involving very noisy signals, classifying and organizing them, and assisting in the discovery of behavior patterns without requiring prior knowledge about the process under analysis. As a test bed for the use of SOMs in finding possible relationships among genes and their possible contribution in some biological processes, we selected 282 S. cerevisiae genes that have been shown through biological experiments to have an activity during the cell cycle. The expression level of these genes was analyzed in five of the most cited time series DNA microarray databases used in the study of the cell cycle of this organism. With the use of SOM, it was possible to find clusters of genes with similar behavior in the five databases along two cell cycles. This result suggested that some of these genes might be biologically related or might have a regulatory relationship, as was corroborated by comparing some of the clusters obtained with SOMs against a previously reported regulatory network that was generated using biological knowledge, such as protein-protein interactions, gene expression levels, metabolism dynamics, promoter binding, and modification, regulation and transport of proteins. The methodology described in this paper could be applied to the study of gene relationships of other biological processes in different organisms.     

Establishment of an in vitro organoid model of dermal papilla of human hair follicle

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three‐dimensional (3D) DP spheroids (DPS) with or without a silk–gelatin (SG) microenvironment, which showed enhanced DP‐specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil‐treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell–cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial–mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.     

Serum starvation induced cell cycle synchronization facilitates human somatic cells reprogramming

Human induced pluripotent stem cells (iPSCs) provide a valuable model for regenerative medicine and human disease research. To date, however, the reprogramming efficiency of human adult cells is still low. Recent studies have revealed that cell cycle is a key parameter driving epigenetic reprogramming to pluripotency. As is well known, retroviruses such as the Moloney murine leukemia virus (MoMLV) require cell division to integrate into the host genome and replicate, whereas the target primary cells for reprogramming are a mixture of several cell types with different cell cycle rhythms. Whether cell cycle synchronization has potential effect on retrovirus induced reprogramming has not been detailed. In this study, utilizing transient serum starvation induced synchronization, we demonstrated that starvation generated a reversible cell cycle arrest and synchronously progressed through G2/M phase after release, substantially improving retroviral infection efficiency. Interestingly, synchronized human dermal fibroblasts (HDF) and adipose stem cells (ASC) exhibited more homogenous epithelial morphology than normal FBS control after infection, and the expression of epithelial markers such as E-cadherin and Epcam were strongly activated. Futhermore, synchronization treatment ultimately improved Nanog positive clones, achieved a 15-20 fold increase. These results suggested that cell cycle synchronization promotes the mesenchymal to epithelial transition (MET) and facilitates retrovirus mediated reprogramming. Our study, utilization of serum starvation rather than additional chemicals, provide a new insight into cell cycle regulation and induced reprogramming of human cells.     

Mesenchymal–epithelial interactions during hair follicle morphogenesis and cycling

Embryonic hair follicle induction and formation are regulated by mesenchymal–epithelial interactions between specialized dermal cells and epidermal stem cells that switch to a hair fate. Similarly, during postnatal hair growth, communication between mesenchymal dermal papilla cells and surrounding epithelial matrix cells coordinates hair shaft production. Adult hair follicle regeneration in the hair cycle again is thought to be controlled by activating signals originating from the mesenchymal compartment and acting on hair follicle stem cells. Although many signaling pathways are implicated in hair follicle formation and growth, the precise nature, timing, and intersection of these inductive and regulatory signals remains elusive. The goal of this review is to summarize our current understanding and to discuss recent new insights into mesenchymal–epithelial interactions during hair follicle morphogenesis and cycling.     

A Microarray-Based Analysis Reveals that a Short Photoperiod Promotes Hair Growth in the Arbas Cashmere Goat

Many animals exhibit different behaviors in different seasons. The photoperiod can have effects on migration, breeding, fur growth, and other processes. The cyclic growth of the fur and feathers of some species of mammals and birds, respectively, is stimulated by the photoperiod as a result of hormone-dependent regulation of the nervous system. To further examine this phenomenon, we evaluated the Arbas Cashmere goat (Capra hircus), a species that is often used in this type of research. The goats were exposed to an experimentally controlled short photoperiod to study the regulation of cyclic cashmere growth. Exposure to a short photoperiod extended the anagen phase of the Cashmere goat hair follicle to increase cashmere production. Assessments of tissue sections indicated that the short photoperiod significantly induced cashmere growth. This conclusion was supported by a comparison of the differences in gene expression between the short photoperiod and natural conditions using gene chip technology. Using the gene chip data, we identified genes that showed altered expression under the short photoperiod compared to natural conditions, and these genes were found to be involved in the biological processes of hair follicle growth, structural composition of the hair follicle, and the morphogenesis of the surrounding skin appendages. Knowledge about differences in the expression of these genes as well as their functions and periodic regulation patterns increases our understanding of Cashmere goat hair follicle growth. This study also provides preliminary data that may be useful for the development of an artificial method to improve cashmere production by controlling the light cycle, which has practical significance for livestock breeding.     

The serine protease Corin is a novel modifier of the Agouti pathway

The hair follicle is a model system for studying epithelial-mesenchymal interactions during organogenesis. Although analysis of the epithelial contribution to these interactions has progressed rapidly, the lack of tools to manipulate gene expression in the mesenchymal component, the dermal papilla, has hampered progress towards understanding the contribution of these cells. In this work, Corin was identified in a screen to detect genes specifically expressed in the dermal papilla. It is expressed in the dermal papilla of all pelage hair follicle types from the earliest stages of their formation, but is not expressed elsewhere in the skin. Mutation of the Corin gene reveals that it is not required for morphogenesis of the hair follicle. However, analysis of the ;dirty blonde' phenotype of these mice reveals that the transmembrane protease encoded by Corin plays a critical role in specifying coat color and acts downstream of agouti gene expression as a suppressor of the agouti pathway.     

Modelling Hematopoiesis Mediated by Growth Factors With Applications to Periodic Hematological Diseases

Hematopoiesis is a complex biological process that leads to the production and regulation of blood cells. It is based upon differentiation of stem cells under the action of growth factors. A mathematical approach of this process is proposed to understand some blood diseases characterized by very long period oscillations in circulating blood cells. A system of three differential equations with delay, corresponding to the cell cycle duration, is proposed and analyzed. The existence of a Hopf bifurcation at a positive steady-state is obtained through the study of an exponential polynomial characteristic equation with delay-dependent coefficients. Numerical simulations show that long-period oscillations can be obtained in this model, corresponding to a destabilization of the feedback regulation between blood cells and growth factors, for reasonable cell cycle durations. These oscillations can be related to observations on some periodic hematological diseases (such as chronic myelogenous leukemia, for example). ¹Research was partially supported by the INRIA Futurs, ANUBIS Team. ²Research was partially supported by the NSF and the University of Miami.     

Optimal scheduling for cell synchronization by cycle-phase-specific blockers

On the basis of a general kinetic model of the cell cycle, the time schedule of administration of a blocking agent for cell synchronization was optimized. As blockers we considered agents that slow down the rate of transit through the short phase of the cycle. The Pontryagin maximum principle is used. Only stationary populations (the model of the steady state normal tissues) were considered. For such populations the exact form of optimal protocols may be simplified, without any significant loss in effectiveness, to the periodic alternation of suitably chosen intervals of maximum treatment and intervals of rest. The proper lengths of these intervals were obtained from the optimal protocols; their values for various parameters characterizing the cell cycle and the blocker action are presented. With the periodic form of protocols the synchronous movement of cells through any number of cycles may be obtained. The utilization of periodic protocols of synchronization in multiple cancer chemotherapy is discussed.     

Spontaneous Epithelial-Mesenchymal Transition and Resistance to HER-2-Targeted Therapies in HER-2-Positive Luminal Breast Cancer

Resistance to trastuzumab, a rationally designed HER-2-targeting antibody, remains a major hurdle in the management of HER-2-positive breast cancer. Preclinical studies suggest the mechanisms of trastuzumab resistance are numerous. Unfortunately, the majority of these studies are based around HER-2-positive (HER-2+) luminal cell lines. The role of epithelial to mesenchymal transition (EMT), a genetic program that confers a basal phenotype, may represent a novel mechanism of escape for HER-2+ luminal cells from trastuzumab treatment. Here we investigated this possibility using a model of clonal selection in HER-2+ luminal breast cancer cells. Following a random isolation and expansion of “colony clusters” from SKBR-3 cell lines, several colony clusters underwent a spontaneous EMT in-vitro. In addition to expression of conventional EMT markers, all mesenchymal colony clusters displayed a predominant CD44+/CD24- phenotype with decreased HER-2 expression and elevated levels of a β1-integrin isoform with a high degree of N-glycosylation. Treatment with a β1-integrin function-blocking antibody, AIIB2, preferentially decreased the N-glycosylated form of β1-integrin, impaired mammosphere formation and restored epithelial phenotype in mesenchymal colony clusters. Using this model we provide the first clear evidence that resistance to trastuzumab (and lapatinib) can occur spontaneously as HER-2+ cells shift from a luminal to a basal/mesenchymal phenotype following EMT. While the major determinant of trastuzumab resistance in mesenchymal colony clusters is likely the down regulation of the HER-2 protein, our evidence suggests that multiple factors may contribute, including expression of N-glycosylated β1-integrin.     

Periodicity and synchronization in blood-stage malaria infection

Malaria fever is highly periodic and is associated with the parasite replication cycles in red blood cells. The existence of periodicity in malaria infection demonstrates that parasite replication in different red blood cells is synchronized. In this article, rigorous mathematical analysis of an age-structured human malaria model of infected red blood cells (Rouzine and McKenzie, Proc Natl Acad Sci USA 100:3473–3478, 2003) is provided and the synchronization of Plasmodium falciparum erythrocytic stages is investigated. By using the replication rate as the bifurcation parameter, the existence of Hopf bifurcation in the age-structured malaria infection model is obtained. Numerical simulations indicate that synchronization with regular periodic oscillations (of period 48 h) occurs when the replication rate increases. Therefore, Kwiatkowski and Nowak’s observation (Proc Natl Acad Sci USA 88:5111–5113, 1991) that synchronization could be generated at modest replication rates is confirmed.     

Bifurcations in a mathematical model for circadian oscillations of clock genes

Circadian oscillations with a period of about 24h are observed in nearly all living organisms as conspicuous biological rhythms. In this paper, we investigate various kinds of bifurcation phenomena produced in a circadian oscillator model of Drosophila. In Drosophila, it is known that circadian oscillations in the levels of two proteins, PER and TIM, result from the negative feedback exerted by a PER-TIM complex on the expression of the per and tim genes that code for the two proteins. For studying circadian oscillations of proteins in Drosophila, a mathematical model has been proposed. The model cannot only account for regular circadian oscillations in environmental conditions such as constant darkness, but also give rise to more complex oscillatory phenomena including chaos and birhythmicity. By calculating bifurcations using Kawakami's method, we obtain detailed bifurcation diagrams related to stable and unstable invariant sets, and identify parameter regions in which the model generates complex oscillations as well as regular circadian oscillations. Moreover, we study bifurcations observed in the model incorporating the effect on a light-dark (LD) cycle and show that the waveform of the periodic variation in the light-induced parameter has a marked influence on the global bifurcation structure or the type of dynamic behavior resulting from the forcing term of the circadian oscillator by the LD cycles.