CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10.

Brain metastasis (br-met) develops in an immunologically unique br-met niche. Central nervous system-native myeloid cells (CNS-myeloids) and bone-marrow-derived myeloid cells (BMDMs) cooperatively regulate brain immunity. The phenotypic heterogeneity and specific roles of these myeloid subsets in shaping the br-met niche to regulate br-met outgrowth have not been fully revealed. Applying multimodal single-cell analyses, we elucidated a heterogeneous but spatially defined CNS-myeloid response during br-met outgrowth. We found Ccr2+ BMDMs minimally influenced br-met while CNS-myeloid promoted br-met outgrowth. Additionally, br-met-associated CNS-myeloid exhibited downregulation of Cx3cr1. Cx3cr1 knockout in CNS-myeloid increased br-met incidence, leading to an enriched interferon response signature and Cxcl10 upregulation. Significantly, neutralization of Cxcl10 reduced br-met, while rCxcl10 increased br-met and recruited VISTAHi PD-L1+ CNS-myeloid to br-met lesions. Inhibiting VISTA- and PD-L1-signaling relieved immune suppression and reduced br-met burden. Our results demonstrate that loss of Cx3cr1 in CNS-myeloid triggers a Cxcl10-mediated vicious cycle, cultivating a br-met-promoting, immune-suppressive niche.

The role of oculoplastic surgeons in minimally invasive cosmetic injectables.

PURPOSE OF REVIEW: In recent years, the field of aesthetic medicine has witnessed a paradigm shift with an increasing demand for minimally invasive cosmetic procedures, including cosmetic injectables. This review aims to delineate the distinctive role played by oculoplastic surgeons in the administration of cosmetic injectables, comparing their expertise to that of nonphysician practitioners. RECENT FINDINGS: Complications arising from cosmetic injections are discussed, including skin discoloration, inflammation, necrosis, vision loss, retinal pathology, and central nervous system adverse effects. Injector expertise, patient factors, type of filler, location of injection, and management strategies are reviewed. Findings highlight diverse practitioner involvement, common adverse effects like skin necrosis and vision loss, with hyaluronic acid fillers being prominent. Areas at the highest risk for ocular complication include the glabella and nose with potential management involving dissolving fillers and reducing pressure. Emphasis is placed on expert injector selection and patient awareness. SUMMARY: The administration of cosmetic injectables requires a profound understanding of facial anatomy, vasculature, and potential complications. In contrast to nonphysician practitioners, oculoplastic and aesthetic surgeons bring a level of anatomical precision and clinical acumen that is essential for navigating the complexities of cosmetic injectables. Emphasis on training and collaboration among practitioners will be essential in advancing the field while prioritizing patient safety and satisfaction.

Evolutionary transition from blood feeding to obligate nonbiting in a mosquito.

The spread of blood-borne pathogens by mosquitoes relies on their taking a blood meal; if there is no bite, there is no disease transmission. Although many species of mosquitoes never take a blood meal, identifying genes that distinguish blood feeding from obligate nonbiting is hampered by the fact that these different lifestyles occur in separate, genetically incompatible species. There is, however, one unique extant species with populations that share a common genetic background but blood feed in one region and are obligate nonbiters in the rest of their range: Wyeomyia smithii Contemporary blood-feeding and obligate nonbiting populations represent end points of divergence between fully interfertile southern and northern populations. This divergence has undoubtedly resulted in genetic changes that are unrelated to blood feeding, and the challenge is to winnow out the unrelated genetic factors to identify those related specifically to the evolutionary transition from blood feeding to obligate nonbiting. Herein, we determine differential gene expression resulting from directional selection on blood feeding within a polymorphic population to isolate genetic differences between blood feeding and obligate nonbiting. We show that the evolution of nonbiting has resulted in a greatly reduced metabolic investment compared with biting populations, a greater reliance on opportunistic metabolic pathways, and greater reliance on visual rather than olfactory sensory input. W. smithii provides a unique starting point to determine if there are universal nonbiting genes in mosquitoes that could be manipulated as a means to control vector-borne disease.

Targeted mutagenesis in a human-parasitic nematode.

Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements.

BACKGROUND: Regulatory adjustments to acute and chronic temperature changes are highly important for aquatic ectotherms because temperature affects their metabolic rate as well as the already low oxygen concentration in water, which can upset their energy balance. This also applies to severe changes in food supply. Thus, we studied on a molecular level (transcriptomics and/or proteomics) the immediate responses to heat stress and starvation and the acclimation to different temperatures in two clonal isolates of the model microcrustacean Daphnia pulex from more or less stressful environments, which showed a higher (clone M) or lower (clone G) tolerance to heat and starvation. RESULTS: The transcriptomic responses of clone G to acute heat stress (from 20 °C to 30 °C) and temperature acclimation (10 °C, 20 °C, and 24 °C) and the proteomic responses of both clones to acute heat, starvation, and heat-and-starvation stress comprised environment-specific and clone-specific elements. Acute stress (in particular heat stress) led to an early upregulation of stress genes and proteins (e.g., molecular chaperones) and a downregulation of metabolic genes and proteins (e.g., hydrolases). The transcriptomic responses to temperature acclimation differed clearly. They also varied depending on the temperature level. Acclimation to higher temperatures comprised an upregulation of metabolic genes and, in case of 24 °C acclimation, a downregulation of genes for translational processes and collagens. The proteomic responses of the clones M and G differed at any type of stress. Clone M showed markedly stronger and less stress-specific proteomic responses than clone G, which included the consistent expression of a specific heat shock protein (HSP60) and vitellogenin (VTG-SOD). CONCLUSIONS: The expression changes under acute stress can be interpreted as a switch from standard products of gene expression to stress-specific products. The expression changes under temperature acclimation probably served for an increase in energy intake (via digestion) and, if necessary, a decrease in energy expenditures (e.g, for translational processes). The stronger and less stress-specific proteomic responses of clone M indicate a lower degree of cell damage and an active preservation of the energy balance, which allowed adequate proteomic responses under stress, including the initiation of resting egg production (VTG-SOD expression) as an emergency reaction.

Panel sequencing of 264 candidate susceptibility genes and segregation analysis in a cohort of non-BRCA1, non-BRCA2 breast cancer families.

PURPOSE: The main aim of this study was to screen epigenetic modifier genes and known breast cancer driver genes for germline mutations in non-BRCA1/2 (BRCAx) breast cancer families in order to identify novel susceptibility genes of moderate-high penetrance. METHODS: We screened 264 candidate susceptibility genes in 656 index cases from non-BRCA1/2 families. Potentially pathogenic candidate mutations were then genotyped in all available family members for the assessment of co-segregation of the variant with disease in the family in order to estimate the breast cancer risks associated with these mutations. For 11 of the candidate susceptibility genes, we screened an additional 800 non-BRCA1/2 breast cancer cases and 787 controls. RESULTS: Only two genes, CHD8 and USH2A showed any evidence of an increased risk of breast cancer (RR = 2.40 (95% CI 1.0-7.32) and 2.48 (95% CI 1.11-6.67), respectively). CONCLUSIONS: We found no convincing evidence that epigenetic modifier and known breast cancer driver genes carry germline mutations that increase breast cancer risk. USH2A is no longer regarded as a breast cancer driver gene and seems an implausible candidate given its association with Usher syndrome. However, somatic mutations in CHD8 have been recently reported, making it an even more promising candidate, but further analysis of CHD8 in very large cohorts of families or case-control studies would be required to determine if it is a moderate-risk breast cancer susceptibility gene.

Evolutionary divergence of core and post-translational circadian clock genes in the pitcher-plant mosquito, Wyeomyia smithii.

BACKGROUND: Internal circadian (circa, about; dies, day) clocks enable organisms to maintain adaptive timing of their daily behavioral activities and physiological functions. Eukaryotic clocks consist of core transcription-translation feedback loops that generate a cycle and post-translational modifiers that maintain that cycle at about 24 h. We use the pitcher-plant mosquito, Wyeomyia smithii (subfamily Culicini, tribe Sabethini), to test whether evolutionary divergence of the circadian clock genes in this species, relative to other insects, has involved primarily genes in the core feedback loops or the post-translational modifiers. Heretofore, there is no reference transcriptome or genome sequence for any mosquito in the tribe Sabethini, which includes over 375 mainly circumtropical species. METHODS: We sequenced, assembled and annotated the transcriptome of W. smithii containing nearly 95 % of conserved single-copy orthologs in animal genomes. We used the translated contigs and singletons to determine the average rates of circadian clock-gene divergence in W. smithii relative to three other mosquito genera, to Drosophila, to the butterfly, Danaus, and to the wasp, Nasonia. RESULTS: Over 1.08 million cDNA sequence reads were obtained consisting of 432.5 million nucleotides. Their assembly produced 25,904 contigs and 54,418 singletons of which 62 % and 28 % are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. DISCUSSION: The W. smithii transcriptome includes all nine circadian transcription-translation feedback-loop genes and all eight post-translational modifier genes we sought to identify (Fig. 1). After aligning translated W. smithii contigs and singletons from this transcriptome with other insects, we determined that there was no significant difference in the average divergence of W. smithii from the six other taxa between the core feedback-loop genes and post-translational modifiers. CONCLUSIONS: The characterized transcriptome is sufficiently complete and of sufficient quality to have uncovered all of the insect circadian clock genes we sought to identify (Fig. 1). Relative divergence does not differ between core feedback-loop genes and post-translational modifiers of those genes in a Sabethine species (W. smithii) that has experienced a continual northward dispersal into temperate regions of progressively longer summer day lengths as compared with six other insect taxa. An associated microarray platform derived from this work will enable the investigation of functional genomics of circadian rhythmicity, photoperiodic time measurement, and diapause along a photic and seasonal geographic gradient.

Differential transcriptomic responses of ancient and modern Daphnia genotypes to phosphorus supply.

Little is known about the role of transcriptomic changes in driving phenotypic evolution in natural populations, particularly in response to anthropogenic environmental change. Previous analyses of Daphnia genotypes separated by centuries of evolution in a lake using methods in resurrection ecology revealed striking genetic and phenotypic shifts that were highly correlated with anthropogenic environmental change, specifically phosphorus (P)-driven nutrient enrichment (i.e. eutrophication). Here, we compared the transcriptomes of two ancient (~700-year-old) and two modern (~10-year-old) genotypes in historic (low P) and contemporary (high P) environmental conditions using microarrays. We found considerable transcriptomic variation between 'ancient' and 'modern' genotypes in both treatments, with stressful (low P) conditions eliciting differential expression (DE) of a larger number of genes. Further, more genes were DE between 'ancient' and 'modern' genotypes than within these groups. Expression patterns of individual genes differed greatly among genotypes, suggesting that different transcriptomic responses can result in similar phenotypes. While this confounded patterns between 'ancient' and 'modern' genotypes at the gene level, patterns were discernible at the functional level: annotation of DE genes revealed particular enrichment of genes involved in metabolic pathways in response to P-treatments. Analyses of gene families suggested significant DE in pathways already known to be important in dealing with P-limitation in Daphnia as well as in other organisms. Such observations on genotypes of a single natural population, separated by hundreds of years of evolution in contrasting environmental conditions before and during anthropogenic environmental changes, highlight the important role of transcriptional mechanisms in the evolutionary responses of populations.

Conserved transcriptional responses to cyanobacterial stressors are mediated by alternate regulation of paralogous genes in Daphnia.

Despite a significant increase in genomic data, our knowledge of gene functions and their transcriptional responses to environmental stimuli remains limited. Here, we use the model keystone species Daphnia pulex to study environmental responses of genes in the context of their gene family history to better understand the relationship between genome structure and gene function in response to environmental stimuli. Daphnia were exposed to five different treatments, each consisting of a diet supplemented with one of five cyanobacterial species, and a control treatment consisting of a diet of only green algae. Differential gene expression profiles of Daphnia exposed to each of these five cyanobacterial species showed that genes with known functions are more likely to be shared by different expression profiles, whereas genes specific to the lineage of Daphnia are more likely to be unique to a given expression profile. Furthermore, while only a small number of nonlineage-specific genes were conserved across treatment type, there was a high degree of overlap in expression profiles at the functional level. The conservation of functional responses across the different cyanobacterial treatments can be attributed to the treatment-specific expression of different paralogous genes within the same gene family. Comparison with available gene expression data in the literature suggests differences in nutritional composition in diets with cyanobacterial species compared to diets of green algae as a primary driver for cyanobacterial effects on Daphnia. We conclude that conserved functional responses in Daphnia across different cyanobacterial treatments are mediated through alternate regulation of paralogous gene families.

Functional genomics of acclimation and adaptation in response to thermal stress in Daphnia.

BACKGROUND: Gene expression regulation is one of the fundamental mechanisms of phenotypic plasticity and is expected to respond to selection in conditions favoring phenotypic response. The observation that many organisms increase their stress tolerance after acclimation to moderate levels of stress is an example of plasticity which has been long hypothesized to be based on adaptive changes in gene expression. We report genome-wide patterns of gene expression in two heat-tolerant and two heat-sensitive parthenogenetic clones of the zooplankton crustacean Daphnia pulex exposed for three generations to either optimal (18°C) or substressful (28°C) temperature. RESULTS: A large number of genes responded to temperature and many demonstrated a significant genotype-by-environment (GxE) interaction. Among genes with a significant GxE there were approximately equally frequent instances of canalization, i.e. stronger plasticity in heat-sensitive than in heat-tolerant clones, and of enhancement of plasticity along the evolutionary vector toward heat tolerance. The strongest response observed is the across-the-board down-regulation of a variety of genes occurring in heat-tolerant, but not in heat-sensitive clones. This response is particularly obvious among genes involved in core metabolic pathways and those responsible for transcription, translation and DNA repair. CONCLUSIONS: The observed down-regulation of metabolism, consistent with previous findings in yeast and Drosophila, may reflect a general compensatory stress response. The associated down-regulation of DNA repair pathways potentially creates a trade-off between short-term benefits of survival at high temperature and long-term costs of accelerated mutation accumulation.

Potential for sexual conflict assessed via testosterone-mediated transcriptional changes in liver and muscle of a songbird.

Males and females can be highly dimorphic in metabolism and physiology despite sharing nearly identical genomes, and both sexes respond phenotypically to elevated testosterone, a steroid hormone that alters gene expression. Only recently has it become possible to learn how a hormone such as testosterone affects global gene expression in non-model systems, and whether it affects the same genes in males and females. To investigate the transcriptional mechanisms by which testosterone exerts its metabolic and physiological effects on the periphery, we compared gene expression by sex and in response to experimentally elevated testosterone in a well-studied bird species, the dark-eyed junco (Junco hyemalis). We identified 291 genes in the liver and 658 in the pectoralis muscle that were differentially expressed between males and females. In addition, we identified 1727 genes that were differentially expressed between testosterone-treated and control individuals in at least one tissue and sex. Testosterone treatment altered the expression of only 128 genes in both males and females in the same tissue, and 847 genes were affected significantly differently by testosterone treatment in the two sexes. These substantial differences in transcriptional response to testosterone suggest that males and females may employ different pathways when responding to elevated testosterone, despite the fact that many phenotypic effects of experimentally elevated testosterone are similar in both sexes. In contrast, of the 121 genes that were affected by testosterone treatment in both sexes, 78% were regulated in the same direction (e.g. either higher or lower in testosterone-treated than control individuals) in both males and females. Thus, it appears that testosterone acts through both unique and shared transcriptional pathways in males and females, suggesting multiple mechanisms by which sexual conflict can be mediated.

A synbiotic of Anaerostipes caccae and lactulose prevents and treats food allergy in mice.

Depletion of beneficial microbes by modern lifestyle factors correlates with the rising prevalence of food allergies. Re-introduction of allergy-protective bacteria may be an effective treatment strategy. We characterized the fecal microbiota of healthy and food-allergic infants and found that the anaerobe Anaerostipes caccae (A. caccae) was representative of the protective capacity of the healthy microbiota. We isolated a strain of A. caccae from the feces of a healthy infant and identified lactulose as a prebiotic to optimize butyrate production by A. caccae in vitro. Administration of a synbiotic composed of our isolated A. caccae strain and lactulose increased luminal butyrate in gnotobiotic mice colonized with feces from an allergic infant and in antibiotic-treated specific pathogen-free (SPF) mice, and prevented or treated an anaphylactic response to allergen challenge. The synbiotic's efficacy in two models and microbial contexts suggests that it may be a promising approach for the treatment of food allergy.

Normal and cancer stem cells of the human female reproductive system.

The female reproductive system (FRS) has a great capacity for regeneration. The existence of somatic stem cells (SSC) that are likely to reside in distinct tissue compartments of the FRS is anticipated. Normal SSC are capable of regenerating themselves, produce a progeny of cells that differentiate and maintain tissue architecture and functional characteristics, and respond to homeostatic controls. Among those SSC of the FRS that have been identified are: a) undifferentiated cells capable of differentiating into thecal cells and synthesizing hormones upon transplantation, b) ovarian surface epithelium stem cells, mitotically responsive to ovulation, c) uterine endometrial and myometrial cells, as clonogenic epithelial and stromal cells, and d) epithelial and mesenchymal cells with self-renewal capacity and multipotential from cervical tissues. Importantly, these cells are believed to significantly contribute to the development of different pathologies and tumors of the FRS.It is now widely accepted that cancer stem cells (CSC) are at the origin of many tumors. They are capable of regenerating themselves, produce a progeny that will differentiate aberrantly and do not respond adequately to homeostatic controls. Several cell surface antigens such as CD44, CD117, CD133 and MYD88 have been used to isolate ovarian cancer stem cells. Clonogenic epithelial and stromal endometrial and myometrial cells have been found in normal and cancer tissues, as side population, label-retaining cells, and CD146/PDGF-R beta-positive cells with stem-like features. In summary, here we describe a number of studies supporting the existence of somatic stem cells in the normal tissues and cancer stem cells in tumors of the human female reproductive system.

Characterization of Infectious Keratitis in Opioid Users in a County Hospital Setting.

Purpose: To determine risk factors and clinical course of corneal ulcers in the setting of opioid use. Methods: A retrospective cohort study was performed of patients presenting with bacterial or fungal keratitis at a county hospital from 2010-2021. Subjects were separated into three groups: opioid drug users (heroin, methadone, fentanyl), non-opioid drug users, and non-drug users. 24 opioid users, 77 non-opioid drug users, and 38 non-drug users were included in the study. Chi-square and t-tests were used to compare hospitalization for corneal ulcer treatment; length of hospitalization; loss to follow-up; final best corrected visual acuity (BCVA); medication noncompliance; time to ulcer resolution; and visual disability (defined either by the legal limit for driving in California or the federal limit for blindness). Results: Opioid users had higher rates of unemployment (p=0.002), homelessness (p=0.018), and psychiatric conditions (p=0.024) compared with non-opioid and non-drug users. They had more severe presentations, with worse initial BCVA of the affected eye (p=0.003), larger ulcer size (p=0.023), and higher rates of individuals below the legal vision thresholds for driving (p=0.009) and blindness (p=0.033) at initial presentation. Opioid use was associated with increased rate of hospitalization (p<0.001), higher fortified antibiotic use (p=0.009), worse final BCVA of the affected eye (p=0.020), and increased rates of BCVA worse than the legal vision thresholds for driving (p=0.043) and blindness (p<0.001) on final presentation. Conclusions: Infectious keratitis associated with opioid use is associated with more severe presentations and poorer outcomes, including higher rates of visual disability.

Risk Factors Predicting Loss to Follow-Up, Medication Noncompliance, and Poor Visual Outcomes Among Patients With Infectious Keratitis at a Public County Hospital.

PURPOSE: Infectious keratitis is a vision-threatening condition requiring close follow-up and disciplined eye drop administration to achieve resolution. Although patients presenting to county hospitals often have more severe presentations, there is a paucity of risk and outcomes data in this setting. This study investigates risk factors predicting loss to follow-up (LTFU), medication noncompliance, and poor outcomes for infectious keratitis in the county hospital setting. METHODS: This was a retrospective case-control study at Zuckerberg San Francisco General Hospital and Trauma Center. Inclusion criteria were patients who had corneal cultures for suspected infectious bacterial or fungal keratitis between 2010 and 2021. Exclusion criteria were patients with viral keratitis only. Multivariable logistic regression was used to analyze the relationship of social and medical risk factors with LTFU, medication noncompliance, worsened visual acuity (VA), and delayed resolution time. RESULTS: Of 174 patients with infectious keratitis in this analysis, 69 (40.0%) had LTFU. Unemployment was associated with increased risk of LTFU (odds ratio 2.58, P = 0.049) and worse final VA ( P = 0.001). Noncompliance trended toward an association with homelessness (odds ratio 3.48, P = 0.095). Increasing age correlated with longer resolution time, with each 1-year increase associated with delayed resolution by 0.549 days ( P = 0.042). CONCLUSIONS: Patients experiencing unemployment, homelessness, or increased age demonstrate higher risk for treatment barriers including loss to follow-up and medication noncompliance, resulting in worse VA and delayed time to resolution. These risk factors should be considered when determining the need for more deliberate follow-up measures in patients with infectious keratitis.

Characterization of Polymicrobial and Antibiotic-Resistant Infectious Keratitis in a County Hospital Setting.

Purpose: Infectious keratitis is a serious cause of visual impairment, particularly in low-income communities. This study examines the associations between social risk factors and polymicrobial keratitis, multidrug resistance, pathogen spectrum, and outcomes at a county hospital. Methods: We performed a retrospective study of Zuckerberg San Francisco General Hospital patients treated for infectious keratitis from 2010-2021. Multivariable regression was performed to analyze the relationships between social, medical, and psychiatric risk factors with polymicrobial growth, multidrug resistance, and clinical outcomes. Results: Of 174 patients with infectious keratitis, 44 (25%) had polymicrobial growth. Six patients (14%) with polymicrobial growth had multidrug-resistant organisms. Homeless patients were more likely to present with polymicrobial infection (OR 3.4, p = 0.023), and polymicrobial infections were associated with multidrug-resistant organisms (p = 0.018). Smoking, drug use, HIV positivity, prior corneal pathology, and contact lens use were not associated with an increased risk of polymicrobial infection. Eleven patients (6.3%) were started on topical antibiotics prior to presentation; of these, none developed polymicrobial infections or multidrug-resistant organisms. Polymicrobial infections increased the likelihood to initiation of fortified antibiotics (OR 2.9, p = 0.011) but did not impact ulcer size, final visual acuity, time to resolution, or likelihood of emergent procedures. Conclusions: Homelessness correlates with an increased risk of polymicrobial keratitis and subsequent multidrug resistance, supporting initiation of broad antibiotic coverage in this population. Prior topical antibiotics did not increase risk of polymicrobial infection. Polymicrobial infection did not significantly worsen clinical outcomes.

Clarifying the Temporal Dynamics of the Circadian Clock and Flowering Gene Network Using Overexpression and Targeted Mutagenesis of Soybean EARLY FLOWERING 3-1 ( GmELF3-1 ).

With progressing climate fluctuations, an understanding of the molecular mechanisms of crop plants that regulate their flowering responses to environments is crucial. To achieve this goal, we aimed at clarifying the gene regulatory networks among the circadian clock and flowering genes in soybean ( Glycine max ). Based on our network inference approach , we hypothesize that GmELF3-1 , one of the Evening Complex (EC) gene homologs in soybean's circadian clock, may have an integrative role in transcriptional regulation of the circadian clock and flowering gene network. In this study, we verify GmELF3-1 ' s regulatory roles in its potential downstream genes by modulating the activity of GmELF3-1 using overexpression and CRISPR-Cas9 in soybean protoplasts. Our results indicate that GmELF3-1 may control the expression of the PRR genes in the circadian clock and the flowering gene GmCOL1a .

Elucidating the Temporal Patterns of Gene Expression in the Inferred Regulatory Interactions of GmCOL1b in Glycine max.

The CONSTANS ( CO ) gene in Arabidopsis thaliana has a central role in photoperiodic regulation of flowering. However, the roles of CO genes in mediating flowering in soybeans ( Glycine max ) remain uncertain. We previously inferred regulatory interactions of a soybean CO homolog, GmCOL1b , using in-house RNA-seq data and the network inference algorithm package CausNet. Here, we identify potential GmCOL1b downstream genes and experimentally verify them by expressing GmCOL1b in soybean protoplast cells. Temporal expression patterns of these genes indicate the regulatory effects of GmCOL1b on the expression of the circadian clock genes GmLCL1 and GmLCL4 and the flowering regulator GmTEM1a .

Identification of maternally-loaded RNA transcripts in unfertilized eggs of Tribolium castaneum.

BACKGROUND: Maternal RNAs play a critical role in early development. Variation in the diversity and levels of maternally derived gene transcripts may be central to the origin of phenotypic novelty -- a longstanding problem in evolution and development. By studying maternal transcriptomes within and between divergent species, a better understanding of the evolutionary forces acting on maternal RNA allocation is possible. RESULTS: We present the first maternal transcriptome of the red flour beetle, Tribolium castaneum. Using a tiled whole-genome microarray, we found that 58.2% of T. castaneum genes are maternally loaded into eggs. Comparison of known Drosophila melanogaster maternal genes to our results showed widespread conservation of maternal expression with T. castaneum. Additionally, we found that many genes previously reported as having sex or tissue specific expression in T. castaneum were also maternally loaded. Identification of such pleiotropy is vital for proper modeling and testing of evolutionary theory using empirical data. The microarray design also allowed the detection of 2315 and 4060 novel transcriptionally active regions greater in length than 100 bp in unfertilized and fertilized T. castaneum eggs, respectively. These transcriptionally active regions represent novel exons of potentially unknown genes for future study. CONCLUSIONS: Our results lay a foundation for utilizing T. castaneum as a model for understanding the role of maternal genes in evolution.

Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance.

BACKGROUND: Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I). METHODS: Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions. RESULTS: CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 10³ dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 105 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR). CONCLUSIONS: We characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, SiHa, Ca Ski and C-4 I) and found that they express characteristic markers of stem cell, EMT and radioresistance. The fact that CICs demonstrated a higher degree of resistance to radiation than differentiated cells suggests that specific detection and targeting of CICs could be highly valuable for the therapy of tumors from the uterine cervix.