Influence of the Tissue Collection Procedure on the Adipogenic Differentiation of Human Stem Cells: Ischemic versus Well-Vascularized Adipose Tissue.

Clinical and basic science applications using adipose-derived stem cells (ADSCs) are gaining popularity. The current adipose tissue harvesting procedures introduce nonphysiological conditions, which may affect the overall performance of the isolated ADSCs. In this study, we elucidate the differences between ADSCs isolated from adipose tissues harvested within the first 5 min of the initial surgical incision (well-vascularized, nonpremedicated condition) versus those isolated from adipose tissues subjected to medications and deprived of blood supply during elective free flap procedures (ischemic condition). ADSCs isolated from well-vascularized and ischemic tissues positively immunostained for several standard stem cell markers. Interestingly, the percent change in the CD36 expression for ADSCs isolated from ischemic versus well-vascularized tissue was significantly lower in males than females (p < 0.05). Upon differentiation and maturation to adipocytes, spheroids formed using ADSCs isolated from ischemic adipose tissue had lower triglyceride content compared to those formed using ADSCs isolated from the well-vascularized tissue (p < 0.05). These results indicate that ADSCs isolated from ischemic tissue either fail to uptake fatty acids or fail to efficiently convert those fatty acids into triglycerides. Therefore, more robust ADSCs suitable to establish in vitro adipose tissue models can be obtained by harvesting well-vascularized and nonpremedicated adipose tissues.

Molecular profiling of the hippocampus of children with autism spectrum disorder.

Several lines of evidence point to a key role of the hippocampus in Autism Spectrum Disorders (ASD). Altered hippocampal volume and deficits in memory for person and emotion related stimuli have been reported, along with enhanced ability for declarative memories. Mouse models have demonstrated a critical role of the hippocampus in social memory dysfunction, associated with ASD, together with decreased synaptic plasticity. Chondroitin sulfate proteoglycans (CSPGs), a family of extracellular matrix molecules, represent a potential key link between neurodevelopment, synaptic plasticity, and immune system signaling. There is a lack of information regarding the molecular pathology of the hippocampus in ASD. We conducted RNAseq profiling on postmortem human brain samples containing the hippocampus from male children with ASD (n = 7) and normal male children (3-14 yrs old), (n = 6) from the NIH NeuroBioBank. Gene expression profiling analysis implicated molecular pathways involved in extracellular matrix organization, neurodevelopment, synaptic regulation, and immune system signaling. qRT-PCR and Western blotting were used to confirm several of the top markers identified. The CSPG protein BCAN was examined with multiplex immunofluorescence to analyze cell-type specific expression of BCAN and astrocyte morphology. We observed decreased expression of synaptic proteins PSD95 (p < 0.02) and SYN1 (p < 0.02), increased expression of the extracellular matrix (ECM) protease MMP9 (p < 0.03), and decreased expression of MEF2C (p < 0.03). We also observed increased BCAN expression with astrocytes in children with ASD, together with altered astrocyte morphology. Our results point to alterations in immune system signaling, glia cell differentiation, and synaptic signaling in the hippocampus of children with ASD, together with alterations in extracellular matrix molecules. Furthermore, our results demonstrate altered expression of genes implicated in genetic studies of ASD including SYN1 and MEF2C.

Extracellular matrix abnormalities in the hippocampus of subjects with substance use disorder.

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.

Extracellular Matrix Abnormalities in the Hippocampus of Subjects with Substance Use Disorder.

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.

Regulation of dendritic spines in the amygdala following sleep deprivation.

The amygdala is a hub of emotional circuits involved in the regulation of cognitive and emotional behaviors and its critically involved in emotional reactivity, stress regulation, and fear memory. Growing evidence suggests that the amygdala plays a key role in the consolidation of emotional memories during sleep. Neuroimaging studies demonstrated that the amygdala is selectively and highly activated during rapid eye movement sleep (REM) and sleep deprivation induces emotional instability and dysregulation of the emotional learning process. Regulation of dendritic spines during sleep represents a morphological correlate of memory consolidation. Several studies indicate that dendritic spines are remodeled during sleep, with evidence for broad synaptic downscaling and selective synaptic upscaling in several cortical areas and the hippocampus. Currently, there is a lack of information regarding the regulation of dendritic spines in the amygdala during sleep. In the present work, we investigated the effect of 5 h of sleep deprivation on dendritic spines in the mouse amygdala. Our data demonstrate that sleep deprivation results in differential dendritic spine changes depending on both the amygdala subregions and the morphological subtypes of dendritic spines. We observed decreased density of mushroom spines in the basolateral amygdala of sleep deprived mice, together with increased neck length and decreased surface area and volume. In contrast, we observed greater densities of stubby spines in sleep deprived mice in the central amygdala, indicating that downscaling selectively occurs in this spine type. Greater neck diameters for thin spines in the lateral and basolateral nuclei of sleep deprived mice, and decreases in surface area and volume for mushroom spines in the basolateral amygdala compared to increases in the cental amygdala provide further support for spine type-selective synaptic downscaling in these areas during sleep. Our findings suggest that sleep promotes synaptic upscaling of mushroom spines in the basolateral amygdala, and downscaling of selective spine types in the lateral and central amygdala. In addition, we observed decreased density of phosphorylated cofilin immunoreactive and growth hormone immunoreactive cells in the amygdala of sleep deprived mice, providing further support for upscaling of dendritic spines during sleep. Overall, our findings point to region-and spine type-specific changes in dendritic spines during sleep in the amygdala, which may contribute to consolidation of emotional memories during sleep.

Functionalized Collagen/Elastin-like Polypeptide Hydrogels for Craniofacial Bone Regeneration.

Critical-sized cranial bone defects fail to re-ossify and require the surgical intervention of cranioplasty. To achieve superior bone healing in such cases, a hydrogel consisting of an interpenetrating network of collagen and elastin-like polypeptide to encapsulate bone morphogenetic protein-2 (BMP-2), doxycycline, and 45S5 Bioglass is developed. This hydrogel has an appropriate elastic modulus of 39 ± 2.2 kPa to allow proper handling during implantation. The hydrogel promotes human adipose-derived stem attachment, proliferation, and differentiation toward the osteogenic lineage, including the deposition of hydroxyapatite particles embedded within a collagenous fibrillar structure after 21 days of in vitro culture. After eight weeks of implantation of the acellular hydrogel in a critical-sized rat cranial defect model, only a small quantity of various pro-inflammatory (< 20 pg mg-1 ) and anti-inflammatory (< 10 pg mg-1 ) factors in the adjacent cranial tissue is noticed, indicating the overall biocompatibility of the hydrogel. Scanning electron microscopy evidenced the presence of new fibrous extracellular matrix and mineral aggregates at the defect site, with calcium/phosphorus ratio of 0.5 and 2.0 by eight and twelve weeks, respectively. Microcomputed tomography (Micro-CT) and histological analyses showed formation of mature mineralized tissue that bridged with the surrounding bone. Taken together, the acellular composite hydrogel shows great promise for superior bone healing after cranioplasty.

Altered expression of somatostatin signaling molecules and clock genes in the hippocampus of subjects with substance use disorder.

Substance use disorders are a debilitating group of psychiatric disorders with a high degree of comorbidity with major depressive disorder. Sleep and circadian rhythm disturbances are commonly reported in people with substance use disorder and major depression and associated with increased risk of relapse. Hippocampal somatostatin signaling is involved in encoding and consolidation of contextual memories which contribute to relapse in substance use disorder. Somatostatin and clock genes also have been implicated in depression, suggesting that these molecules may represent key converging pathways involved in contextual memory processing in substance use and major depression. We used hippocampal tissue from a cohort of subjects with substance use disorder (n = 20), subjects with major depression (n = 20), subjects with comorbid substance use disorder and major depression (n = 24) and psychiatrically normal control subjects (n = 20) to test the hypothesis that expression of genes involved in somatostatin signaling and clock genes is altered in subjects with substance use disorder. We identified decreased expression of somatostatin in subjects with substance use disorder and in subjects with major depression. We also observed increased somatostatin receptor 2 expression in subjects with substance use disorder with alcohol in the blood at death and decreased expression in subjects with major depression. Expression of the clock genes Arntl, Nr1d1, Per2 and Cry2 was increased in subjects with substance use disorder. Arntl and Nr1d1 expression in comparison was decreased in subjects with major depression. We observed decreased expression of Gsk3ß in subjects with substance use disorder. Subjects with comorbid substance use disorder and major depression displayed minimal changes across all outcome measures. Furthermore, we observed a significant increase in history of sleep disturbances in subjects with substance use disorder. Our findings represent the first evidence for altered somatostatin and clock gene expression in the hippocampus of subjects with substance use disorder and subjects with major depression. Altered expression of these molecules may impact memory consolidation and contribute to relapse risk.

Design, Synthesis, and Preliminary Studies of Spiro-isoxazoline-peroxides against Human Cytomegalovirus and Glioblastoma ∥.

The association between glioblastoma (GBM) and human cytomegalovirus (HCMV) infection has been the intensely debated topic over the decades for developing new therapeutic options. In this regard, the peroxides from natural and synthetic sources served as potential antiviral and anticancer agents in the past. Herein, a concise and efficient strategy has been demonstrated to access a novel class of peroxides containing a spiro-isoxazoline to primarily investigate the biological activities. The synthetic compounds were evaluated for in vitro antiviral and antiproliferative activity against HCMV and glioblastoma cell line (GBM6), respectively. While compound 13m showed moderate anti-CMV activity (IC50 = 19 µM), surprisingly, an independent biological assay for compound 13m revealed its antiproliferative activity against the human glioblastoma cell line (GBM6) with an IC50 of 10 µM. Hence, the unification of an isoxazoline and peroxide heterocycles could be a potential direction to initiate the HCMV-GBM drug discovery program.

Dynamin Is Required for Efficient Cytomegalovirus Maturation and Envelopment.

Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate-early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells, as well as in mock-induced parental MEF, at early times postinfection, although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 h postinfection; however, late protein (m55; gB) expression was reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids), indicating successful viral DNA replication, capsid assembly, and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore)-treated MEF showed a phenotype similar to TKO cells upon mouse cytomegalovirus infection, confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation.IMPORTANCE Viruses are known to exploit specific cellular functions at different stages of their life cycle in order to replicate, avoid immune recognition by the host and to establish a successful infection. Cytomegalovirus (CMV)-infected cells are characterized by a prominent cytoplasmic inclusion (virus assembly compartment [vAC]) that is the site of virus maturation and envelopment. While endocytic membranes are known to be the functional components of vAC, knowledge of specific endocytic pathways implicated in CMV maturation and envelopment is lacking. We show here that dynamin, which is an integral part of host endocytic machinery, is largely dispensable for early stages of CMV infection but is required at a late stage of CMV maturation. Studies on dynamin function in CMV infection will help us understand the host-virus interaction pathways amenable to targeting by conventional small molecules, as well as by newer generation nucleotide-based therapeutics (e.g., small interfering RNA, CRISPR/CAS gRNA, etc.).