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Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. We developed µMagnify, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. We formulated an enzyme cocktail specifically designed for robust cell wall digestion and expansion of microbial cells without distortion while efficiently retaining biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. Additionally, we developed an associated virtual reality tool to facilitate the visualization and navigation of complex three-dimensional images generated by this method in an immersive environment allowing collaborative exploration among researchers around the world. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables development of new diagnosis strategies against infectious diseases.

With the Texas Central’s high-speed rail fast approaching in Texas, legislators have been presented with an opportunity to reform Texas’ eminent domain laws. The controversial urban-versus-rural project has brought eminent domain policy to the limelight. The Texas Legislature can capitalize on lessons learned from the State’s bout with the Trans-Pecos Pipeline by protecting condemnees and incentivizing good faith efforts by condemnors. This Article proposes five possible reforms for eminent domain law in Texas. First, the Texas Legislature should protect condemnees by aligning their appraisal disclosure requirements with condemnors, who have no duties to disclose appraisals. Second, legislative changes would allow attorney’s fees to be awarded to a condemnee when a condemnor’s offer is significantly lower than the actual value of the property. Third, legislative changes would inform condemnees of exactly which pieces of land that condemnors have the power to take when condemnors make their offer. Fourth, this Article proposes sensible protections for Texas homesteads. Last, this Article explores legislative and judicial blocks that can be used by opponents of the rail.

Background/Aims: Prenatal microcephaly is posited to arise from aberrant mitosis of neural progenitors, which disrupts both neuronal production and survival. Although microcephaly has both a genetic and environmental etiology, the mechanisms by which dysregulation of mitosis causes microcephaly are poorly understood. We previously discovered that prolonged mitosis of mouse neural progenitors, either ex vivo or in vitro, directly alters progeny cell fate, ­resulting in precocious differentiation and apoptosis. This raises questions as to whether prolonged progenitor mitosis affects cell fate and neurogenesis in vivo, and what are the underlying mechanisms? Methods/Results: Towards addressing these knowledge gaps, we developed an in vivo model of mitotic delay. This uses pharmacological inhibition to acutely and reversibly prolong mitosis during cortical development, and fluorescent dyes to label direct progeny. Using this model, we discovered that a causal relationship between mitotic delay of neural progenitors and altered progeny cell fate is evident in vivo. Using transcriptome analyses to investigate the state of delayed cells and their progeny, we uncovered potential molecular mechanisms by which prolonged mitosis induces altered cell fates, including DNA damage and p53 signaling. We then extended our studies to human neural progenitors, demonstrating that lengthened mitosis duration also directly alters neuronal cell fate. Conclusions: This study establishes a valuable new experimental paradigm towards understanding mechanisms whereby lengthened mitosis duration may explain some cases of microcephaly.

Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.

Long-range integration of transcriptional inputs is critical for gene expression, yet the mechanisms remain poorly understood. We investigated the molecular determinants that confer fidelity to expression of the heart identity gene even-skipped (eve). Targeted deletion of regions bound by the repressor Yan defined two novel enhancers that contribute repressive inputs to stabilize tissue-specific output from a third enhancer. Deletion of any individual enhancer reduced Yan occupancy at the other elements, impacting eve expression, cell fate specification, and cardiac function. These long-range interactions may be stabilized by three-dimensional chromatin contacts that we detected between the elements. Our work provides a new paradigm for chromatin-level integration of general repressive inputs with specific patterning information to achieve robust gene expression.

Periodontal manifestations of human immunodeficiency virus (HIV) infection were first described in 1987. Initially, the lesions receiving attention were HIV-associated gingivitis (now known as linear gingival erythema [LGE]) and HIV-associated periodontitis (now known as necrotizing ulcerative periodontitis [NUP]). The true prevalence of LGE was difficult to determine due to variable diagnostic criteria. Recently, LGE has been associated with intraoral Candida infection. The prevalence of NUP is low (or = 5%), and this lesion is associated with pronounced immunosuppression. Current focus on the periodontal manifestations of HIV infection centers on rapid progression of chronic adult periodontitis in HIV+ patients. Attempts to identify the pathogenesis of the increased progression of periodontitis have not proven successful. For example, analysis of subgingival plaque for the presence of bacterial pathogens has failed to detect differences between HIV+ and HIV- patients. Recently our laboratory has identified alterations in the host response in the gingival crevice of HIV+ patients. Comparing HIV+ and HIV- injecting drug users (IDU), levels of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) in gingival crevicular fluid (GCF) were slightly elevated at sites with a probing depth of 1 to 3 mm. At deeper sites (or = 4 mm), total IL-1 beta in GCF was significantly greater in HIV+ individuals. Using the lysosomal acid glycohydrolase beta-glucuronidase (beta G) as a measure of the influx of polymorphonuclear leukocytes (PMN) into the gingival crevice, our data indicated a significant correlation of total beta G in GCF and probing depth in the HIV-IDU (r = 76; P = .02). This result was similar to what we have observed in other studies. In contrast, for HIV+ subjects, total beta G was not associated with probing depth (r = .20; NS). These data suggest that HIV+ patients have altered regulation of PMN recruitment into the gingival crevice. We have begun to investigate the conditions under which subgingival Candida may contribute total periodontal lesions in HIV+ individuals. Candida from subgingival sites has been cultured in HIV+ individuals. Subgingival Candida was distinct from Candida isolated from tongue and buccal mucosal surfaces (as indicated by genomic fingerprinting). We hypothesize the absence of adequate priming of PMN by HIV+ patients. This may be due to a reduced Th1 lymphocyte response. The inability of HIV+ individuals to adequately prime PMN may allow Candida to colonize the subgingival environment. In that milieu, it may act directly or in concert with subgingival bacterial pathogens, or as a cofactor (by inducing production of proinflammatory cytokines) to increase the occurrence of periodontal attachment loss.

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2,868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analyzed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.

Biofilm formation by the opportunistic fungal pathogen Candida albicans is a complex process with significant consequences for human health: it contributes to implanted medical device-associated infections. Recent advances in gene expression profiling and genetic analysis have begun to clarify the mechanisms that govern C. albicans biofilm development and acquisition of unique biofilm phenotypes. Such studies have identified candidate adhesin genes, and have revealed that biofilm drug resistance is multifactorial. Newly defined cell-cell communication pathways also have profound effects on biofilm formation. Future challenges include the elucidation of the structure and function of the extracellular exopolymeric substance that surrounds biofilm cells, and the extension of in vitro biofilm observations to newly developed in vivo biofilm models.

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.