Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding.

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.

The spatial analysis of extrapulmonary tuberculosis spreading and its interactions with pulmonary tuberculosis in Samarinda, East Kalimantan, Indonesia.

Background: Extrapulmonary Tubercolosis (EPTB) is an infectious disease that affects tissue outside the lungs. EPTB patients cannot be source of infection, therefore the findings in the community indicate that there are still active pulmonary TB patients acting as a source of infection. Understanding distributions of EPTB can be used as indicator to individuate the unmonitored source of TB transmission in the community. Objectives: The aim of this study is to analyze EPTB using spatial modeling based on patients' location. Methods: This study is an observational research with spatial analysis approach using SatScanv.9.4.4 and ArcGis v.10.4. Involving 46 samples of EPTB patients in Anatomy Pathology Laboratory of RSUD Abdul Wahab Sjahranie in 2017 and 7 pulmonary TB patients who were contacts of EPTB patients. The distribution of EPTB patients is mostly located in areas with high population density. Results: The results showed that the distribution pattern of EPTB patients was mostly in areas with high population densities. Space-time permutation model shows there are 3 clusters of EPTB with a 2.91, 0.97, 1.13 km radius centered on -0.504177 S/117.092132 E, -0.476895 S /117.141700 E, -0.517031 S/117.092132 E. Conclusion: The distribution of patients with EPTB and pulmonary TB indicates there is an interaction between EPTB and pulmonary TB in the cluster area. Bernoulli model shows that there is 1 cluster of EPTB and pulmonary TB with relative risk 5.29, radius of 3.19 km, and centered on - 0.458159 S / 117.149945 E.

Luminal B is the Most Common Intrinsic Molecular Subtypes of Invasive Ductal Breast Carcinoma Patients in East Kalimantan, Indonesia.

Objective: Breast carcinoma has no longer been considered as a single and standalone disease. Its subtypes have been known to vary in terms of risk factors, natural histories, and responses to therapies. In particular, intrinsic molecular subtypes based on St. Gallen International Expert Consensus 2013 have classified breast carcinoma into luminal A, luminal B, HER2+, and triple-negative, depending on the expression of ER, PgR, HER2, and Ki-67. Research on intrinsic molecular subtypes of breast carcinoma in Indonesia, however, are rarely conducted, which then triggers the intention to conduct this study. Methods: In this work, a retrospective study was conducted on 92 formalin-fixed paraffin-embedded samples of invasive ductal breast carcinoma patients. These samples were from patients at Abdul Wahab Sjahranie County General Hospital Samarinda, East Kalimantan, Indonesia, in 2016. Next, immunohistochemical staining using anti-ER, PgR, HER2, and Ki-67 antibodies was applied to classify intrinsic molecular subtypes. Then, an association between clinical and immunohistochemical factors with intrinsic molecular subtypes of breast carcinoma were analyzed using Chi-square test. Results: Looking at results of the retrospective study, luminal B was discovered as the most common intrinsic molecular subtypes of breast carcinoma (42.39%) in East Kalimantan, Indonesia. The next ranks of breast carcinoma subtypes in the region included HER2+ (39.13%), triple-negative (10.87%), and luminal A (7.61%). In fact, there was a significant association between age (p = 0.019) with intrinsic molecular subtypes of breast carcinoma. Conclusion: The study found luminal B as the most common intrinsic molecular subtypes of Indonesian breast carcinoma in the region under investigation. In the future, the higher positivity rate of luminal B in breast carcinoma patients compared to prior studies would require further investigations.

Factors Within the Endoneurial Microenvironment Act to Suppress Tumorigenesis of MPNST.

Background: Deciphering avenues to adequately control malignancies in the peripheral nerve will reduce the need for current, largely-ineffective, standards of care which includes the use of invasive, nerve-damaging, resection surgery. By avoiding the need for en bloc resection surgery, the likelihood of retained function or efficient nerve regeneration following the control of tumor growth is greater, which has several implications for long-term health and well-being of cancer survivors. Nerve tumors can arise as malignant peripheral nerve sheath tumors (MPNST) that result in a highly-aggressive form of soft tissue sarcoma. Although the precise cause of MPNST remains unknown, studies suggest that dysregulation of Schwann cells, mediated by the microenvironment, plays a key role in tumor progression. This study aimed to further characterize the role of local microenvironment on tumor progression, with an emphasis on identifying factors within tumor suppressive environments that have potential for therapeutic application. Methods: We created GFP-tagged adult induced tumorigenic Schwann cell lines (iSCs) and transplanted them into various in vivo microenvironments. We used immunohistochemistry to document the response of iSCs and performed proteomics analysis to identify local factors that might modulate divergent iSC behaviors. Results: Following transplant into the skin, spinal cord or epineurial compartment of the nerve, iSCs formed tumors closely resembling MPNST. In contrast, transplantation into the endoneurial compartment of the nerve significantly suppressed iSC proliferation. Proteomics analysis revealed a battery of factors enriched within the endoneurial compartment, of which one growth factor of interest, ciliary neurotrophic factor (CNTF) was capable of preventing iSCs proliferation in vitro. Conclusions: This dataset describes a novel approach for identifying biologically relevant therapeutic targets, such as CNTF, and highlights the complex relationship that tumor cells have with their local microenvironment. This study has significant implications for the development of future therapeutic strategies to fight MPNSTs, and, consequently, improve peripheral nerve regeneration and nerve function.

Macrophages Promote Wound-Induced Hair Follicle Regeneration in a CX3CR1- and TGF-ß1-Dependent Manner.

Hair follicle stem cells are regulated by intrafollicular and extrafollicular niche signals. Appropriate hair follicle regeneration relies on the coordinated release and integration of these signals. How immune cells, particularly cutaneous macrophages, influence the hair follicle stem cell niche and regeneration is not well understood. We took advantage of wound-induced hair growth (WIHG) to explore the relationship between wound macrophages and hair follicle regeneration. First, we showed that WIHG is dependent on CD11b+F4/80+ macrophages at 7-11 days after injury. Next, using CX3CR1gfp/+:CCR2rfp/+ mice to capture the dynamic spectrum of macrophage phenotypes during wound healing, we showed that wound macrophages transition from a CX3CR1lo/med to a CX3CR1hi phenotype at the onset of WIHG. Finally, WIHG is abolished in mice deficient for CX3CR1, delayed with pharmacological inhibition of transforming growth factor-ß receptor type 1, and rescued with exogenous transforming growth factor-ß1. Overall, we propose a model in which transforming growth factor-ß1 and CX3CR1 are critical for recruiting and maintaining the CCR2+CX3CR1hiLy6CloTNFα+ macrophages critical for stimulating WIHG.

Enhanced Expansion and Sustained Inductive Function of Skin-Derived Precursor Cells in Computer-Controlled Stirred Suspension Bioreactors.

Endogenous dermal stem cells (DSCs) reside in the adult hair follicle mesenchyme and can be isolated and grown in vitro as self-renewing colonies called skin-derived precursors (SKPs). Following transplantation into skin, SKPs can generate new dermis and reconstitute the dermal papilla and connective tissue sheath, suggesting they could have important therapeutic value for the treatment of skin disease (alopecia) or injury. Controlled cell culture processes must be developed to efficiently and safely generate sufficient stem cell numbers for clinical use. Compared with static culture, stirred-suspension bioreactors generated fivefold greater expansion of viable SKPs. SKPs from each condition were able to repopulate the dermal stem cell niche within established hair follicles. Both conditions were also capable of inducing de novo hair follicle formation and exhibited bipotency, reconstituting the dermal papilla and connective tissue sheath, although the efficiency was significantly reduced in bioreactor-expanded SKPs compared with static conditions. We conclude that automated bioreactor processing could be used to efficiently generate large numbers of autologous DSCs while maintaining their inherent regenerative function. Stem Cells Translational Medicine 2017;6:434-443.

Improved method to track and precisely count Schwann cells post-transplantation in a peripheral nerve injury model.

BACKGROUND: To optimize survival evaluation of Schwann cells (SCs) in vivo, we tested fluorescent labeling of the nucleus as an improved method of tracking and counting the transplanted SCs at sciatic nerve injury sites in rodents. We also investigated if co-administering cells with the glial growth factor Neuregulin-1 ß (NRG1ß) improves in vivo survival. NEW METHOD: We transduced SCs using a Lentiviral vector with a nuclear localization signal (NLS) fused with mCherry and transplanted them in the sciatic nerve of rat post-crush injury (bilateral) either in the presence or absence of NRG1ß in the injectate media. For comparison, in a separate group of similar injury, GFP-labeled cells were transplanted. After 10 days, nerves were harvested and sections (14µm) were counterstained with Hoechst and imaged. Cells showing co-localization with Hoechst and GFP or mCherry were exhaustively counted and data analyzed. RESULTS: Percentage cells counted in with- and without-NRG condition in both the groups were 0.83±0.13% and 0.06±0.04% (Group 1) & 2.83*±1.95% and 0.23*±0.29% (Group 2). COMPARISON TO EXISTING METHOD: We are introducing fluorescent labeling of the nucleus as a reliable and efficient technique to perform survival assessments in Schwann cell based treatment studies in animal model. This method can overcome the challenges and limitations of the existing method that could result in underestimation of the therapeutic outcome. CONCLUSIONS: Nucleus-restricted fluorescent labeling technique offer improved method of tracking as well as accurately counting transplanted SCs in vivo while NRG1ß in the injectate media can improve survival.

Adult skin-derived precursor Schwann cells exhibit superior myelination and regeneration supportive properties compared to chronically denervated nerve-derived Schwann cells.

Functional outcomes following delayed peripheral nerve repair are poor. Schwann cells (SCs) play key roles in supporting axonal regeneration and remyelination following nerve injury, thus understanding the impact of chronic denervation on SC function is critical toward developing therapies to enhance regeneration. To improve our understanding of SC function following acute versus chronic-denervation, we performed functional assays of SCs from adult rodent sciatic nerve with acute- (Day 5 post) or chronic-denervation (Day 56 post), versus embryonic nerves. We also compared Schwann cells derived from adult skin-derived precursors (aSKP-SCs) as an accessible, autologous alternative to supplement the distal (denervated) nerve. We found that acutely-injured SCs and aSKP-SCs exhibited superior proliferative capacity, promotion of neurite outgrowth and myelination of axons, both in vitro and following transplant into a sciatic nerve crush injury model, while chronically-denervated SCs were severely impaired. Acute injury caused re-activation of transcription factors associated with an immature and pro-myelinating SC state (Oct-6, cJun, Sox2, AP2α, cadherin-19), but was diminished with prolonged denervation in vivo and could not be rescued following expansion in vitro suggesting that this is a permanent deficiency. Interestingly, aSKP-SCs closely resembled acutely injured and embryonic SCs, exhibiting elevated expression of these same transcription factors. In summary, prolonged denervation resulted in SC deficiency in several functional parameters that may contribute to impaired regeneration. In contrast, aSKP-SCs closely resemble the regenerative attributes ascribed to acutely-denervated or embryonic SCs emphasizing their potential as an accessible and autologous source of glia cells to enhance nerve regeneration, particularly following delays to surgical repair.

Hair follicle dermal stem cells regenerate the dermal sheath, repopulate the dermal papilla, and modulate hair type.

The dermal papilla (DP) provide instructive signals required to activate epithelial progenitors and initiate hair follicle regeneration. DP cell numbers fluctuate over the hair cycle, and hair loss is associated with gradual depletion/atrophy of DP cells. How DP cell numbers are maintained in healthy follicles remains unclear. We performed in vivo fate mapping of adult hair follicle dermal sheath (DS) cells to determine their lineage relationship with DP and found that a subset of DS cells are retained following each hair cycle, exhibit self-renewal, and repopulate the DS and the DP with new cells. Ablating these hair follicle dermal stem cells and their progeny retarded hair regrowth and altered hair type specification, suggesting that they function to modulate normal DP function. This work identifies a bipotent stem cell within the adult hair follicle mesenchyme and has important implications toward restoration of hair growth after injury, disease, and aging.

Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis.

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.

LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration.

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1-/-) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-alpha and interleukin-1beta) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1-/- chimeric mice using bone marrow transplantation revealed that in mice with Lsp1-/- endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1-/- neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1-/- animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1-/- mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Complexity of inducible nitric oxide synthase: cellular source determines benefit versus toxicity.

BACKGROUND: Inducible nitric oxide synthase (iNOS) has been shown to have both beneficial and detrimental effects in sepsis. We focused on a single organ, the heart, and used 2 distinct cell types that express iNOS-the cardiac myocyte and the infiltrating neutrophil-to study the distinct functional effects of iNOS derived from heterogeneous cellular sources. METHODS AND RESULTS: In the first series of experiments, extravascular neutrophils were exposed to isolated single endotoxemic cardiac myocytes. Adhesion of wild-type neutrophils caused a rapid decrease in myocyte shortening and a concomitant increase in neutrophil-derived intracellular oxidative stress within the myocytes that was not observed with neutrophils from iNOS-deficient animals. We previously demonstrated that neutrophil-derived superoxide was essential for myocyte dysfunction; however, superoxide production was not compromised in the iNOS-deficient neutrophils. Because both superoxide and NO were essential for the neutrophil dysfunction, we probed for but could not detect any peroxynitrite assessed by detection of nitrotyrosine. There was a significant increase in length shortening in response to beta-adrenergic stimulation of wild-type myocytes. Surprisingly, myocyte iNOS activity was essential rather than detrimental for the development of beta-adrenergic receptor-mediated increases in shortening in endotoxemic iNOS-deficient myocytes. CONCLUSIONS: These results demonstrate that iNOS, when expressed in isolated cardiac myocytes, can regulate the response to beta-adrenergic stimulation during sepsis. However, as the neutrophils migrate in proximity to myocytes, iNOS now becomes essential for the ability of neutrophils to damage myocytes. These findings demonstrate that cellular source strongly modulates the beneficial and detrimental effect of iNOS.

Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.

The rapid and selective accumulation of neutrophils into the lungs is thought to underlie the pulmonary failure that leads to sepsis-related death. In this study we investigated whether neutrophil TLR4 is important in LPS-induced pulmonary neutrophil recruitment by creating chimeric mice (transferring bone marrow between TLR4(+/+) and TLR4(-/-) mice). In TLR4(+/+) mice receiving TLR4(-/-) bone marrow, 6 weeks after transplant TLR4 was absent in all circulating leukocytes as well as in resident macrophages (these mice were termed LeukocyteTLR4(-/-)), and these cells were completely nonresponsive to LPS. In TLR4(-/-) mice receiving TLR4(+/+) bone marrow, endothelial cells but not leukocytes were deficient in TLR4 (EndotheliumTLR4(-/-)). Surprisingly, systemic LPS (0.5 mg/kg) induced a dramatic increase in neutrophil sequestration into the lungs of LeukocyteTLR4(-/-) mice over the first 4 hours. Concomitantly, numbers of circulating leukocytes decreased by 90%. By contrast, EndotheliumTLR4(-/-) mice showed very little increase in neutrophil sequestration in the lungs, suggesting that endothelium rather than leukocyte TLR4 was important. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte-endothelial cell interactions in LPS-treated EndotheliumTLR4(-/-) mice than in LPS-treated LeukocyteTLR4(-/-) mice. This is consistent with less sequestration of leukocytes into the lungs of EndotheliumTLR4(-/-) mice. In conclusion, our data challenge the view that LPS directly activates neutrophils to trap in lungs and suggest a far more important role than previously appreciated for the endothelial cells.

An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients.

Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium to arrive at a site of infection. By necessity, in the setting of multiple chemoattractants, the neutrophils must prioritize, favoring end target chemoattractants (e.g., fMLP and C5a) emanating from the site of infection over intermediary endogenous chemoattractants (e.g., IL-8 and LTB4) encountered en route to sites of infection. In this study, we propose a hierarchical model of two signaling pathways mediating the decision-making process of the neutrophils, which allows end target molecules to dominate over intermediary chemoattractants. In an under agarose assay, neutrophils predominantly migrated toward end target chemoattractants via p38 MAPK, whereas intermediary chemoattractant-induced migration was phosphoinositide 3-kinase (PI3K)/Akt dependent. When faced with competing gradients of end target and intermediary chemoattractants, Akt activation was significantly reduced within neutrophils, and the cells migrated preferentially toward end target chemoattractants even at 1/1,000th that of intermediary chemoattractants. End target molecules did not require chemotactic properties, since the p38 MAPK activator, LPS, also inhibited Akt and prevented migration to intermediary chemoattractants. p38 MAPK inhibitors not only reversed this hierarchy, such that neutrophils migrated preferentially toward intermediary chemoattractants, but also allowed neutrophils to be drawn out of a local end target chemoattractant environment and toward intermediary chemoattractants unexpectedly in an exaggerated (two- to fivefold) fashion. This was entirely related to significantly increased magnitude and duration of Akt activation. Finally, end target chemoattractant responses were predominantly Mac-1 dependent, whereas nondominant chemoattractants used primarily LFA-1. These data provide support for a two pathway signaling model wherein the end target chemoattractants activate p38 MAPK, which inhibits intermediary chemoattractant-induced PI3K/Akt pathway, establishing an intracellular signaling hierarchy.