The spatiotemporal blood vessel formation and specification at the osteogenic and angiogenic interface of murine cranial bone defect repair were examined utilizing a high-resolution multiphoton-based imaging platform in conjunction with advanced optical techniques that allow interrogation of the oxygen microenvironment and cellular energy metabolism in living animals. Our study demonstrates the dynamic changes of vessel types, that is, arterial, venous, and capillary vessel networks at the superior and dura periosteum of cranial bone defect, suggesting a differential coupling of the vessel type with osteoblast expansion and bone tissue deposition/remodeling during repair. Employing transgenic reporter mouse models that label distinct types of vessels at the site of repair, we further show that oxygen distributions in capillary vessels at the healing site are heterogeneous as well as time- and location-dependent. The endothelial cells coupling to osteoblasts prefer glycolysis and are less sensitive to microenvironmental oxygen changes than osteoblasts. In comparison, osteoblasts utilize relatively more OxPhos and potentially consume more oxygen at the site of repair. Taken together, our study highlights the dynamics and functional significance of blood vessel types at the site of defect repair, opening up opportunities for further delineating the oxygen and metabolic microenvironment at the interface of bone tissue regeneration.

Dopamine signals mainly through D1 receptors (D1Rs) and D2 receptors (D2Rs); D1R-expressing or D2R-expressing neurons contribute to distinct reward and addictive behaviors. Traditionally, transgenic mice expressing green fluorescent protein (GFP) under D1R or D2R promoters are used for fluorescent verification in electrophysiology studies, whereas Cre mice are employed for behavioral research. However, it is unknown whether the same neuronal populations are targeted in GFP and Cre mice. Additionally, while D1Rs and D2Rs are known to be expressed in different striatal neurons, their expression patterns outside the striatum remain unclear. The present study addressed these two questions by using several transgenic mouse lines expressing fluorescent proteins (GFP or tdTomato) or Cre under the control of D1R or D2R promoters. We found a high degree of overlap between GFP-positive and Cre-positive neurons in the striatum and hippocampus. Additionally, we discovered that D1Rs and D2Rs were highly segregated in the orbitofrontal cortex, prefrontal cortex, dorsal and ventral hippocampus, and amygdala: ~4-34 percent of neurons co-expressed these receptors. Importantly, slice electrophysiological studies demonstrated that D1R-positive and D1R-negative hippocampal neurons were functionally distinct in a mouse line generated by crossing Drd1a-Cre mice with a Cre reporter Ai14 line. Lastly, we discovered that chronic alcohol intake differentially altered D1R-positive and D2R-positive neuron excitability in the ventral CA1. These data suggest that GFP and Cre mice target the same populations of striatal neurons, D1R-expressing or D2R-expressing neurons are highly segregated outside the striatum, and these neurons in the ventral hippocampal may exert distinct roles in alcohol addiction.

Semilunar valve malformations are common human congenital heart defects. Bicuspid aortic valves occur in 2-3% of the population, and pulmonic valve stenosis constitutes 10% of all congenital heart disease in adults (Brickner et al., 2000) [1]. Semilunar valve defects cause valve regurgitation, stenosis, or calcification, leading to endocarditis or congestive heart failure. These complications often require prolonged medical treatment or surgical intervention. Despite the medical importance of valve disease, the regulatory pathways governing semilunar valve development are not entirely clear. In this report we investigated the spatiotemporal role of calcineurin/Nfatc1 signaling in semilunar valve development. We generated conditional knockout mice with calcineurin gene disrupted in various tissues during semilunar valve development. Our studies showed that calcineurin/Nfatc1 pathway signals in the secondary heart field (SHF) but not in the outflow tract myocardium or neural crest cells to regulate semilunar valve morphogenesis. Without SHF calcineurin/Nfatc1 signaling, the conal endocardial cushions-the site of prospective semilunar valve formation--first develop and then regress due to apoptosis, resulting in a striking phenotype with complete absence of the aortic and pulmonic valves, severe valve regurgitation, and perinatal lethality. This role of calcineurin/Nfatc1 signaling in the SHF is different from the requirement of calcineurin/Nfatc1 in the endocardium for semilunar valve formation (Chang et al., 2004) [2], indicating that calcineurin/Nfatc1 signals in multiple tissues to organize semilunar valve development. Also, our studies suggest distinct mechanisms of calcineurin/Nfat signaling for semilunar and atrioventricular valve morphogenesis. Therefore, we demonstrate a novel developmental mechanism in which calcineurin signals through Nfatc1 in the secondary heart field to promote semilunar valve morphogenesis, revealing a new supportive role of the secondary heart field for semilunar valve formation.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs), a key component of the endosomal sorting complex required for transport (ESCRT), has been implicated in many essential biological processes. However, the physiological role of endogenous Hgs in the vascular system has not previously been explored. Here, we have generated brain endothelial cell (EC) specific Hgs knockout mice to uncover the function of Hgs in EC polarity and cerebrovascular stability.

Hydrogels are attractive soilless media for plant cultivation with strong water and nutrient retention. However, pristine hydrogels contain mostly ultra-micro pores and lack air-filled porosity for root zone aeration. Herein we report a porous hydrogel composite comprising an agarose network and porous growing mix particle (GMP) fillers. The agarose backbone allowed the composite to sustain a 12-d growth cycle for red cabbage microgreens without the need for watering or crew interaction. Moreover, the GMP induced greater total pore volume and increased the prevalence of pores >30 μm by 8-fold. Further investigation suggested that the nutrients from GMP accounted for a 54 % increase in microgreen yield over pristine hydrogel, while the porous structure introduced by GMP improved the yield by another 44 %. Increased air-filled porosity accelerated the water transport and loss of hydrogel but maintained favorable water potential levels for plant extraction. Finally, the hydrogel composite supported microgreen growth satisfyingly under simulated microgravity despite some morphological changes. Results of this study reveal a novel growth substrate that is lightweight, convenient, and water-efficient, while effectively sustaining plant growth for multiple applications including indoor farming and space farming.

Mural cells of the vessel wall, namely pericytes and vascular smooth muscle cells, are essential for vascular integrity. The developmental sources of these cells and molecular mechanisms controlling their progenitors in the heart are only partially understood. Here we show that endocardial endothelial cells are progenitors of pericytes and vascular smooth muscle cells in the murine embryonic heart. Endocardial cells undergo endothelial-mesenchymal transition and convert into primitive mesenchymal progenitors expressing the platelet-derived growth factor receptors, PDGFRα and PDGFRβ. These progenitors migrate into the myocardium, differentiate and assemble the wall of coronary vessels, which requires canonical Wnt signalling involving Frizzled4, β-catenin and endothelial cell-derived Wnt ligands. Our findings identify a novel and unexpected population of progenitors for coronary mural cells with potential relevance for heart function and disease conditions.

Dental implants are major treatment options for restoring teeth loss. Biological processes at the implant-tissue interface are critical for implant osseointegration. Superior mechanical properties of the implant constitute a major challenge for traditional histological techniques. It is imperative to develop new technique to investigate the implant-tissue interface.

Mutations in the transcription factor TBX20 (T-box 20) are associated with congenital heart disease. Germline ablation of Tbx20 results in abnormal heart development and embryonic lethality by embryonic day 9.5. Because Tbx20 is expressed in multiple cell lineages required for myocardial development, including pharyngeal endoderm, cardiogenic mesoderm, endocardium, and myocardium, the cell type-specific requirement for TBX20 in early myocardial development remains to be explored.

Congenital heart disease (CHD) is the major cause of morbidity/mortality in infancy and childhood. Using a mouse model to uncover the mechanism of CHD is essential to understand its pathogenesis. However, conventional 2D phenotyping methods cannot comprehensively exhibit and accurately distinguish various 3D cardiac malformations for the complicated structure of heart cavity. Here, a new automated tool based on microcomputed tomography (micro-CT) image data sets known as computer-assisted cardiac cavity tracking (CACCT) is presented, which can detect the connections between cardiac cavities and identify complicated cardiac malformations in mouse hearts automatically. With CACCT, researchers, even those without expert training or diagnostic experience of CHD, can identify complicated cardiac malformations in mice conveniently and precisely, including transposition of the great arteries, double-outlet right ventricle and atypical ventricular septal defect, whose accuracy is equivalent to senior fetal cardiologists. CACCT provides an effective approach to accurately identify heterogeneous cardiac malformations, which will facilitate the mechanistic studies into CHD and heart development.

Astrocyte atrophy is the main histopathological hallmark of major depressive disorder (MDD) in humans and in animal models of depression. Here we show that electroacupuncture prevents astrocyte atrophy in the prefrontal cortex and alleviates depressive-like behaviour in mice subjected to chronic unpredictable mild stress (CUMS). Treatment of mice with CUMS induced depressive-like phenotypes as confirmed by sucrose preference test, tail suspension test, and forced swimming test. These behavioural changes were paralleled with morphological atrophy of astrocytes in the prefrontal cortex, revealed by analysis of 3D reconstructions of confocal Z-stack images of mCherry expressing astrocytes. This morphological atrophy was accompanied by a decrease in the expression of cytoskeletal linker Ezrin, associated with formation of astrocytic leaflets, which form astroglial synaptic cradle. Electroacupuncture at the acupoint ST36, as well as treatment with anti-depressant fluoxetine, prevented depressive-like behaviours, astrocytic atrophy, and down-regulation of astrocytic ezrin. In conclusion, our data further strengthen the notion of a primary role of astrocytic atrophy in depression and reveal astrocytes as cellular target for electroacupuncture in treatment of depressive disorders.

The posterior dorsomedial striatum (pDMS) is mainly composed of medium spiny neurons (MSNs) expressing either dopamine D1 receptors (D1Rs) or D2Rs. Activation of these two MSN types produces opposing effects on addictive behaviors. However, it remains unclear whether pDMS D1-MSNs or D2-MSNs receive afferent inputs from different brain regions or whether the extrastriatal afferents express distinct dopamine receptors. To assess whether these afferents also contained D1Rs or D2Rs, we generated double transgenic mice, in which D1R-expressing and D2R-expressing neurons were fluorescently labeled. We used rabies virus-mediated retrograde tracing in these mice to perform whole-brain mapping of direct inputs to D1-MSNs or D2-MSNs in the pDMS. We found that D1-MSNs preferentially received inputs from the secondary motor, secondary visual, and cingulate cortices, whereas D2-MSNs received inputs from the primary motor and primary sensory cortices, and the thalamus. We also discovered that the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala (CeA) contained abundant D2R-expressing, but few D1R-expressing, neurons in a triple transgenic mouse model. Remarkably, although limited D1R or D2R expression was observed in extrastriatal neurons that projected to D1-MSNs or D2-MSNs, we found that cortical structures preferentially contained D1R-expressing neurons that projected to D1-MSNs or D2-MSNs, while the thalamus, substantia nigra pars compacta (SNc), and BNST had more D2R-expressing cells that projected to D2-MSNs. Taken together, these findings provide a foundation for future understanding of the pDMS circuit and its role in action selection and reward-based behaviors.

Addiction is proposed to arise from alterations in synaptic strength via mechanisms of long-term potentiation (LTP) and depression (LTD). However, the causality between these synaptic processes and addictive behaviors is difficult to demonstrate. Here we report that LTP and LTD induction altered operant alcohol self-administration, a motivated drug-seeking behavior. We first induced LTP by pairing presynaptic glutamatergic stimulation with optogenetic postsynaptic depolarization in the dorsomedial striatum, a brain region known to control goal-directed behavior. Blockade of this LTP by NMDA-receptor inhibition unmasked an endocannabinoid-dependent LTD. In vivo application of the LTP-inducing protocol caused a long-lasting increase in alcohol-seeking behavior, while the LTD protocol decreased this behavior. We further identified that optogenetic LTP and LTD induction at cortical inputs onto striatal dopamine D1 receptor-expressing neurons controlled these behavioral changes. Our results demonstrate a causal link between synaptic plasticity and alcohol-seeking behavior and suggest that modulation of this plasticity may inspire a therapeutic strategy for addiction.

Antibacterial hand sanitizers could play a prominent role in slowing down the spread and infection of hand bacterial pathogens; luteolin (LUT) is potentially useful as an antibacterial component. Therefore, this study elucidated the antibacterial mechanism of LUT against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and developed an antibacterial hand sanitizer. The results showed that LUT had excellent antibacterial activity against both E. coli (minimum inhibitory concentration (MIC) = 312.5 μg/mL, minimal bactericidal concentration (MBC) = 625 μg/mL), and S. aureus (MIC = 312.5 μg/mL, MBC = 625 μg/mL). Furthermore, LUT induced cell dysfunction in E. coli and S. aureus, changed membrane permeability, and promoted the leakage of cellular contents. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) analysis showed that LUT treatment affected cell structure and disrupted cell membrane integrity. The Fourier transform infrared analysis (FTIR) also confirmed that the LUT acted on the cell membranes of both E. coli and S. aureus. Overall, the application of LUT in hand sanitizer had better inhibition effects. Therefore, this study could provide insight into expanding the application of LUT in the hand sanitizer markets.

The ipsilateral renal parenchymal volume (RPV) experiences a sharp decrease shortly after partial nephrectomy (PN), mainly due to surgical remove or devascularization of kidney tissue. However, the subsequent change of RPV and its association with glomerular filtration rate (GFR) fast decline remains unknown. Our objective was to investigate the change of ipsilateral RPV and renal function status from new baseline (1-12 months after PN) to latest follow-up (≥1 year) after PN, and to explore factors associated with ipsilateral RPV decrease rate and correlation between RPV decrease and GFR fast decline.

The aim of the present study was to describe the application of 3D printed templates for intraoperative navigation and simulation of periacetabular osteotomies (PAOs) in a cadaveric model.Five cadaveric specimens (10 sides) underwent thin-slice computed tomographic scans of the ala of ilium downwards to the proximal end of femoral shaft. Bernese PAO was performed. Using Mimics v10.1 software (Materialise, Leuven, Belgium), 3D computed tomographic reconstructions were created and the 4 standard PAO bone cuts-ischial, pubic, anterior, and posterior aspects of the ilium-as well as rotation of the dislocated acetabular bone blocks were simulated for each specimen. Using these data, custom 3D printed bone-drilling templates of the pelvis were manufactured, to guide surgical placement of the PAO bone cuts. An angle fix wedge was designed and printed, to help accurately achieve the predetermined rotation angle of the acetabular bone block. Each specimen underwent a conventional PAO. Preoperative, postsimulation, and postoperative lateral center-edge angles, acetabular indices, extrusion indices, and femoral head coverage were measured and compared; P and t values were calculated for above-mentioned measurements while comparing preoperative and postoperative data, and also in postsimulation and postoperative data comparison.All 10 PAO osteotomies were successfully completed using the 3D printed bone-drilling template and angle fix wedge. No osteotomy entered the hip joint and a single posterior column fracture was observed. Comparison of preoperative and postoperative measurements of the 10 sides showed statistically significant changes, whereas no statistically significant differences between postsimulation and postoperative values were noted, demonstrating the accuracy and utility of the 3D printed templates.The application of patient-specific 3D printed bone-drilling and rotation templates in PAO is feasible and may facilitate improved clinical outcomes, through the use of precise presurgical planning and reduced surgical complications with the precisely guided bone drilling.

Recent studies have pointed out that arthroscopy, the commonly-used surgical procedure for meniscal tears, may lead to an elevated risk of knee osteoarthritis (KOA). The biomechanical factors of KOA can be clarified by the biomechanical analysis after arthroscopic partial meniscectomy (APM). This study aimed to elucidate the cartilage stress and meniscus displacement of the tibiofemoral joint under flexion and rotation loads after APM.

Clinical studies show that chronic pain is accompanied by memory deficits and reduction in hippocampal volume. Experimental studies show that spared nerve injury (SNI) of the sciatic nerve induces long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, but impairs LTP in the hippocampus. The opposite changes may contribute to neuropathic pain and memory deficits, respectively. However, the cellular and molecular mechanisms underlying the functional synaptic changes are unclear. Here, we show that the dendrite lengths and spine densities are reduced significantly in hippocampal CA1 pyramidal neurons, but increased in spinal neurokinin-1-positive neurons in mice after SNI, indicating that the excitatory synaptic connectivity is reduced in hippocampus but enhanced in spinal dorsal horn in this neuropathic pain model. Mechanistically, tumor necrosis factor-alpha (TNF-α) is upregulated in bilateral hippocampus and in ipsilateral spinal dorsal horn, whereas brain-derived neurotrophic factor (BDNF) is decreased in the hippocampus but increased in the ipsilateral spinal dorsal horn after SNI. Importantly, the SNI-induced opposite changes in synaptic connectivity and BDNF expression are prevented by genetic deletion of TNF receptor 1 in vivo and are mimicked by TNF-α in cultured slices. Furthermore, SNI activated microglia in both spinal dorsal horn and hippocampus; pharmacological inhibition or genetic ablation of microglia prevented the region-dependent synaptic changes, neuropathic pain, and memory deficits induced by SNI. The data suggest that neuropathic pain involves different structural synaptic alterations in spinal and hippocampal neurons that are mediated by overproduction of TNF-α and microglial activation and may underlie chronic pain and memory deficits.

Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation.

BAP31 (B-cell receptor-associated protein 31) is an important regulator of intracellular signal transduction and highly expressed in several cancer tissues or testicular tissues. Our previous study had revealed that elevated BAP31 plays a crucial role in the progress and metastasis of cervical cancer. Even so, the precise mechanism of abnormal BAP31 elevation in cervical cancer has not been fully elucidated. We revealed that the expression of BAP31 was mainly regulated by microRNA-362 (miR-362), which was markedly downregulated in cervical cancer tissues and negatively correlated with clinical tumor staging. Overexpression of miR-362 inhibited cervical cancer cell proliferation and increased the proportion of apoptotic cells. Furthermore, miR-362 reduced the tumor sizes and prolonged mice survival time in xenograft nude mice model. Finally, we demonstrated that the BAP31/SPTBN1 complex regulated tumor progression through the Smad 2/3 pathway under the control of miR-362. Collectively, our findings demonstrated that miR-362 could work as an anti-oncomiR that inhibits proliferation and promotes apoptosis in cervical cancer cells via BAP31 and TGFβ/Smad pathway. Overexpression of miR-362 might be a potential therapeutic strategy for cervical cancer.

Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells.