Macrophages and brown adipocytes cross-communicate to modulate a thermogenic program following methamphetamine exposure.

Hyperthermia is a potentially lethal side-effect of Methamphetamine (Meth), a stimulant drug. Activation of non-shivering thermogenesis in brown adipose tissue (BAT) is partly responsible for Meth-induced rise in temperature, with contributing sympathetic neurotransmitters, such as norepinephrine (NE), and reactive oxygen species (ROS). However, the mechanisms controlling the development of a molecular thermogenic program in brown adipocytes (BA) following Meth are unknown. We hypothesize that Meth and NE affect BAT cells, BA and macrophages, to modify their physiology and interactions, with consequences to thermogenic genes. We also hypothesize that ROS play a critical role in signaling transcription of thermogenic genes and their regulatory components. Using primary BA and macrophage cultures, we measured Meth and NE interference with physiological and phenotypic measures that are relevant to thermogenesis in BAT. Meth caused both BA and macrophages to decrease mitochondrial maximal capacity and increase ROS. In BA, the thermogenic genes UCP1, PPARγ, PGC1α and GADD45γ were transcriptionally increased by Meth in a ROS-dependent manner. In macrophages, Meth increased oxidative stress response and caused a predominance of M2 subset markers. BA transcriptional changes in response to Meth and NE were significantly controlled by macrophages. The results suggest that BA and macrophages respond to Meth and NE, with effects on mitochondrial functions and transcription of genes involved in thermogenesis. ROS-dependent signals in BA and cellular interactions between BA and macrophages synergize to regulate the BAT environment and control critical pathways leading to Meth-hyperthermia.

Astrocyte-specific overexpressed gene signatures in response to methamphetamine exposure in vitro.

BACKGROUND: Astrocyte activation is one of the earliest findings in the brain of methamphetamine (Meth) abusers. Our goal in this study was to identify the characteristics of the astrocytic acute response to the drug, which may be critical in pathogenic outcomes secondary to the use. METHODS: We developed an integrated analysis of gene expression data to study the acute gene changes caused by the direct exposure to Meth treatment of astrocytes in vitro, and to better understand how astrocytes respond, what are the early molecular markers associated with this response. We examined the literature in search of similar changes in gene signatures that are found in central nervous system disorders. RESULTS: We identified overexpressed gene networks represented by genes of an inflammatory and immune nature and that are implicated in neuroactive ligand-receptor interactions. The overexpressed networks are linked to molecules that were highly upregulated in astrocytes by all doses of methamphetamine tested and that could play a role in the central nervous system. The strongest overexpressed signatures were the upregulation of MAP2K5, GPR65, and CXCL5, and the gene networks individually associated with these molecules. Pathway analysis revealed that these networks are involved both in neuroprotection and in neuropathology. We have validated several targets associated to these genes. CONCLUSIONS: Gene signatures for the astrocytic response to Meth were identified among the upregulated gene pool, using an in vitro system. The identified markers may participate in dysfunctions of the central nervous system but could also provide acute protection to the drug exposure. Further in vivo studies are necessary to establish the role of these gene networks in drug abuse pathogenesis.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

Membrane-induced changes in the holomyoglobin tertiary structure: interplay with function.

The effect of anionic phospholipid membranes on holomyoglobin (holoMb) conformation and deoxygenation was studied. HoloMb structural changes and behavior in the presence of membranes were monitored by a variety of techniques including far UV and near UV circular dichroism, tryptophan (Trp) fluorescence, absorbance in the Soret region, differential scanning calorimetry, (1)H-NMR spectroscopy, size exclusion chromatography, and macroscopic diffusion. Kinetics of deoxygenation was monitored by absorption at 581 nm. The results gave evidence that proximity to a negatively charged membrane surface can cause destabilization of the structure of holomyoglobin, which delivers oxygen (O2) to mitochondria. It was shown that holoMb undergoes the native-to-intermediate-state transition in the presence of anionic phospholipid membranes at neutral pH, and that in this state it is able to interact with the membranes. When in the intermediate state, holoMb loses its rigid tertiary structure but preserves a pronounced secondary one. The presence of anionic phospholipid membranes substantially accelerates the process of deoxygenation. A possible functional role of the more flexible protein structure acquired in immediate proximity to the membrane surface is discussed.

MRP8/14 Is a Molecular Signature Triggered by Dopamine in HIV Latent Myeloid Targets That Increases HIV Transcription and Distinguishes HIV+ Methamphetamine Users with Detectable CSF Viral Load and Brain Pathology.

There is a significant overlap between HIV infection and substance-use disorders. Dopamine (DA) is the most abundantly upregulated neurotransmitter in methamphetamine abuse, with receptors (DRD1-5) that are expressed by neurons as well as by a large diversity of cell types, including innate immune cells that are the targets of HIV infection, making them responsive to the hyperdopaminergic environment that is characteristic of stimulant drugs. Therefore, the presence of high levels of dopamine may affect the pathogenesis of HIV, particularly in the brain. The stimulation of HIV latently infected U1 promonocytes with DA significantly increased viral p24 levels in the supernatant at 24 h, suggesting effects on activation and replication. Using selective agonists to different DRDs, we found that DRD1 played a major role in activating viral transcription, followed by DRD4, which increased p24 with a slower kinetic rate compared to DRD1. Transcriptome and systems biology analyses led to the identification of a cluster of genes responsive to DA, where S100A8 and S100A9 were most significantly correlated with the early increase in p24 levels following DA stimulation. Conversely, DA increased the expression of these genes' transcripts at the protein level, MRP8 and MRP14, respectively, which form a complex also known as calprotectin. Interestingly, MRP8/14 was able to stimulate HIV transcription in latent U1 cells, and this occurred via binding of the complex to the receptor for an advanced glycosylation end-product (RAGE). Using selective agonists, both DRD1 and DRD4 increased MRP8/14 on the surface, in the cytoplasm, as well as secreted in the supernatants. On the other hand, while DRD1/5 did not affect the expression of RAGE, DRD4 stimulation caused its downregulation, offering a mechanism for the delayed effect via DRD4 on the p24 increase. To cross-validate MRP8/14 as a DA signature with a biomarker value, we tested its expression in HIV+ Meth users' postmortem brain specimens and peripheral cells. MRP8/14+ cells were more frequently identified in mesolimbic areas such as the basal ganglia of HIV+ Meth+ cases compared to HIV+ non-Meth users or to controls. Likewise, MRP8/14+ CD11b+ monocytes were more frequent in HIV+ Meth users, particularly in specimens from participants with a detectable viral load in the CSF. Overall, our results suggest that the MRP8 and MRP14 complex may serve as a signature to distinguish subjects using addictive substances in the context of HIV, and that this may play a role in aggravating HIV pathology by promoting viral replication in people with HIV who use Meth.

Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis.

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca(2+) than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.

Methamphetamine signals transcription of IL1ß and TNFα in a reactive oxygen species-dependent manner and interacts with HIV-1 Tat to decrease antioxidant defense mechanisms.

Methamphetamine (Meth) abuse is a common HIV co-morbidity that is linked to aggravated Central Nervous System (CNS) inflammation, which accentuates HIV- associated neurological disorders, triggered both directly or indirectly by the drug. We used the well-established human innate immune macrophage cell line system (THP1) to demonstrate that Reactive Oxygen Species (ROS) immediately induced by Meth play a role in the increased transcription of inflammatory genes, in interaction with HIV-1 Tat peptide. Meth and Tat, alone and together, affect early events of transcriptional activity, as indicated by changes in RNA polymerase (RNAPol) recruitment patterns throughout the genome, via ROS-dependent and -independent mechanisms. IL1ß (IL1ß) and TNF α (TNFα), two genes with defining roles in the inflammatory response, were both activated in a ROS-dependent manner. We found that this effect occurred via the activation of the activator protein 1 (AP-1) comprising cFOS and cJUN transcription factors and regulated by the SRC kinase. HIV-1 Tat, which was also able to induce the production of ROS, did not further impact the effects of ROS in the context of Meth, but promoted gene activity independently from ROS, via additional transcription factors. For instance, HIV-1 Tat increased NFkB activation and activated gene clusters regulated by Tata box binding peptide, ING4 and IRF2. Importantly, HIV-1 Tat decreased the expression of anti-oxidant genes, where its suppression of the detoxifying machinery may contribute to the aggravation of oxidative stress induced by ROS in the context of Meth. Our results provide evidence of effects of Meth via ROS and interactions with HIV Tat that promote the transcription of inflammatory genes such as IL1ß and TNFα.

Age-associated changes in microglia activation and Sirtuin-1- chromatin binding patterns.

The aging process is associated with changes in mechanisms maintaining physiology, influenced by genetics and lifestyle, and impacting late life quality and longevity. Brain health is critical in healthy aging. Sirtuin 1 (Sirt1), a histone deacetylase with silencing properties, is one of the molecular determinants experimentally linked to health and longevity. We compared brain pathogenesis and Sirt1-chromatin binding dynamics in brain pre-frontal cortex from 2 groups of elder rhesus macaques, divided by age of necropsy: shorter-lived animals (18-20 years old (yo)), equivalent to 60-70 human yo; and longer-lived animals (23-29 yo), corresponding to 80-100 human yo and modeling successful aging. These were compared with young adult brains (4-7 yo). Our findings indicated drastic differences in the microglia marker Iba1, along with factors influencing Sirt1 levels and activity, such as CD38 (an enzyme limiting NAD that controls Sirt1 activity) and mir142 (a microRNA targeting Sirt1 transcription) between the elder groups. Iba1 was lower in shorter-lived animals than in the other groups, while CD38 was higher in both aging groups compared to young. mir142 and Sirt1 levels were inversely correlated in longer-lived brains (>23yo), but not in shorter-lived brains (18-20 yo). We also found that Sirt1 binding showed signs of better efficiency in longer-lived animals compared to shorter-lived ones, in genes associated with nuclear activity and senescence. Overall, differences in neuroinflammation and Sirt1 interactions with chromatin distinguished shorter- and longer-lived animals, suggesting the importance of preserving microglia and Sirt1 functional efficiency for longevity.

Polygenic networks in peripheral leukocytes indicate patterns associated with HIV infection and context-dependent effects of cannabis use.

In spite of suppressive antiretroviral therapies (ART), Human Immunodeficiency Virus (HIV)-infected subjects still experience the consequences of viral persistence and chronic inflammation. In the brain, where most HIV-1 targets are of innate immune origin, neurological and cognitive impairments are detectable and enhanced by highly prevalent substance use disorders. Cannabis is one of the most prevalent substances among HIV+ â€‹subjects, compared to non-infected populations, either prescribed for improving various symptoms or used recreationally, as well as a component of polysubstance use. The mechanisms by which addictive substances and HIV interact are multifactorial and poorly understood. Importantly, the HIV brain target cells, macrophages and microglia, express receptors to neurotransmitters elevated by such drugs, and express receptors to cannabinoids, particularly CB2R. We have tested a panel of 784 transcripts associated with neurological disorders, digitally multiplexed and detectable in peripheral blood cells from a small cohort (n â€‹= â€‹102) of HIV-positive (HIV+) and HIV-negative (HIV-) specimens, stratified based on criteria of lifetime (LT) dependence of cannabis (CAN+) or not (CAN-). Demographic homogeneity and low incidence of co-morbidities helped increase power and allowed the identification of key differences consistent with HIV infection, cannabis exposure, or their interactions. A small percentage of these subjects used cannabis as well as other drugs. The data was analyzed using robust systems and visualization strategies to detect orchestrated patterns in gene networks connected based on molecular interfaces with higher power than in single genes. We found that the effects of cannabis differed drastically between HIV- and HIV+ â€‹groups, particularly in gene networks playing a role in inflammation, neurodegeneration, apoptosis and leukocyte adhesion and transmigration. At the level of individual genes, we identified detrimental effects that were associated with polysubstance use as a covariate, particularly methamphetamine. Transcription factor usage predictions suggest that the effects of cannabis are associated with transcriptional co-regulation at the gene promoters by multiple factors that vary by context. Overall, we have found that the effects of cannabis may be context-dependent, with potential benefits in the context of HIV reflected by improvements in cognition, but in the absence of the polysubstance use component.

A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary glands.

Fibroblast growth factor 10 (FGF10) is well established as a mesenchyme-derived growth factor and a critical regulator of fetal organ development in mice and humans. Using a single-cell RNA sequencing (RNA-seq) atlas of salivary gland (SG) and a tamoxifen inducible Fgf10CreERT2:R26-tdTomato mouse, we show that FGF10pos cells are exclusively mesenchymal until postnatal day 5 (P5) but, after P7, there is a switch in expression and only epithelial FGF10pos cells are observed after P15. Further RNA-seq analysis of sorted mesenchymal and epithelial FGF10pos cells shows that the epithelial FGF10pos population express the hallmarks of ancient ionocyte signature Forkhead box i1 and 2 (Foxi1, Foxi2), Achaete-scute homolog 3 (Ascl3), and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10pos cells are specialized SG ionocytes located in ducts and important for the ionic modification of saliva. In addition, they maintain FGF10-dependent gland homeostasis via communication with FGFR2bpos ductal and myoepithelial cells.

Detection of H3K4me3 Identifies NeuroHIV Signatures, Genomic Effects of Methamphetamine and Addiction Pathways in Postmortem HIV+ Brain Specimens that Are Not Amenable to Transcriptome Analysis.

Human postmortem specimens are extremely valuable resources for investigating translational hypotheses. Tissue repositories collect clinically assessed specimens from people with and without HIV, including age, viral load, treatments, substance use patterns and cognitive functions. One challenge is the limited number of specimens suitable for transcriptional studies, mainly due to poor RNA quality resulting from long postmortem intervals. We hypothesized that epigenomic signatures would be more stable than RNA for assessing global changes associated with outcomes of interest. We found that H3K27Ac or RNA Polymerase (Pol) were not consistently detected by Chromatin Immunoprecipitation (ChIP), while the enhancer H3K4me3 histone modification was abundant and stable up to the 72 h postmortem. We tested our ability to use HeK4me3 in human prefrontal cortex from HIV+ individuals meeting criteria for methamphetamine use disorder or not (Meth +/-) which exhibited poor RNA quality and were not suitable for transcriptional profiling. Systems strategies that are typically used in transcriptional metadata were applied to H3K4me3 peaks revealing consistent genomic activity differences in regions where addiction and neuronal synapses pathway genes are represented, including genes of the dopaminergic system, as well as inflammatory pathways. The resulting comparisons mirrored previously observed effects of Meth on suppressing gene expression and provided insights on neurological processes affected by Meth. The results suggested that H3K4me3 detection in chromatin may reflect transcriptional patterns, thus providing opportunities for analysis of larger numbers of specimens from cases with substance use and neurological deficits. In conclusion, the detection of H3K4me3 in isolated chromatin can be an alternative to transcriptome strategies to increase the power of association using specimens with long postmortem intervals and low RNA quality.

The effect of detergents and lipids on the properties of the outer-membrane protein OmcA from Shewanella oneidensis.

The protein OmcA from the bacterium Shewanella oneidensis was purified to homogeneity, and characterized using UV-vis spectroelectrochemistry, EPR, and circular dichroism (far- and near-UV regions). EPR spectroscopy showed that many different c-type hemes are present in the protein: high-spin, low-spin, and highly anisotropic low-spin hemes were all detected. The protein was shown to bind tightly to artificial membranes (liposomes) composed of dioleoylphosphatidylglycerol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (60:40 molar ratio), which mimic the natural environment. However, on the basis of the spectroscopic and electrochemical studies, binding to liposomes does not appear to significantly alter either the structure or the properties of OmcA. On the other hand, the electrochemical properties of OmcA are noticeably changed in the presence of the detergents used during the initial purification stages. In particular, the reduction potentials of two out of the ten OmcA hemes appear to shift in the presence of detergent, perhaps because these hemes are solvent-exposed.

Systems Biology Analysis of the Antagonizing Effects of HIV-1 Tat Expression in the Brain over Transcriptional Changes Caused by Methamphetamine Sensitization.

Methamphetamine (Meth) abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein, trans-activator of transcription (Tat), has been described to induce changes in brain gene transcription that can result in impaired reward circuitry, as well as in inflammatory processes. In transgenic mice with doxycycline-induced Tat protein expression in the brain, i.e., a mouse model of neuroHIV, we tested global gene expression patterns induced by Meth sensitization. Meth-induced locomotor sensitization included repeated daily Meth or saline injections for seven days and Meth challenge after a seven-day abstinence period. Brain samples were collected 30 min after the Meth challenge. We investigated global gene expression changes in the caudate putamen, an area with relevance in behavior and HIV pathogenesis, and performed pathway and transcriptional factor usage predictions using systems biology strategies. We found that Tat expression alone had a very limited impact in gene transcription after the Meth challenge. In contrast, Meth-induced sensitization in the absence of Tat induced a global suppression of gene transcription. Interestingly, the interaction between Tat and Meth broadly prevented the Meth-induced global transcriptional suppression, by maintaining regulation pathways, and resulting in gene expression profiles that were more similar to the controls. Pathways associated with mitochondrial health, initiation of transcription and translation, as well as with epigenetic control, were heavily affected by Meth, and by its interaction with Tat in anti-directional ways. A series of systems strategies have predicted several components impacted by these interactions, including mitochondrial pathways, mTOR/RICTOR, AP-1 transcription factor, and eukaryotic initiation factors involved in transcription and translation. In spite of the antagonizing effects of Tat, a few genes identified in relevant gene networks remained downregulated, such as sirtuin 1, and the amyloid precursor protein (APP). In conclusion, Tat expression in the brain had a low acute transcriptional impact but strongly interacted with Meth sensitization, to modify effects in the global transcriptome.

Sex differences and Tat expression affect dopaminergic receptor expression and response to antioxidant treatment in methamphetamine-sensitized HIV Tat transgenic mice.

Methamphetamine (Meth) abuse is a common HIV comorbidity. Males and females differ in their patterns of Meth use, associated behaviors, and responses, but the underlying mechanisms and impact of HIV infection are unclear. Transgenic mice with inducible HIV-1 Tat protein in the brain (iTat) replicate many neurological aspects of HIV infection in humans. We previously showed that Tat induction enhances the Meth sensitization response associated with perturbation of the dopaminergic system, in male iTat mice. Here, we used the iTat mouse model to investigate sex differences in individual and interactive effects of Tat and Meth challenge on locomotor sensitization, brain expression of dopamine receptors (DRDs) and regulatory adenosine receptors (ADORAs). Because Meth administration increases the production of reactive oxygen species (ROS), we also determined whether the effects of Meth could be rescued by concomitant treatment with the ROS scavenger N-acetyl cysteine (NAC). After Meth sensitization and a 7-day abstinence period, groups of Tat+ and Tat-male and female mice were challenged with Meth in combination with NAC. We confirmed that Tat expression and Meth challenge suppressed DRD mRNA and protein in males and females' brains, and showed that females were particularly susceptible to the effects of Meth on D1-like and D2-like DRD subtypes and ADORAs. The expression of these markers differed strikingly between males and females, and between females in different phases of the estrous cycle, in a Tat -dependent manner. NAC attenuated Meth-induced locomotor sensitization and preserved DRD expression in all groups except for Tat + females. These data identify complex interactions between sex, Meth use, and HIV infection on addiction responses, with potential implications for the treatment of male and female Meth users in the context of HIV, especially those with cognitive disorders.

Origin and Lineage Plasticity of Endogenous Lacrimal Gland Epithelial Stem/Progenitor Cells.

The lacrimal gland (LG) is an exocrine organ responsible for the secretion of aqueous tear film. Regenerative and stem cell therapies that target LG repair are coming to the fore, although our understanding of LG cell lineage hierarchy is still incomplete. We utilize the analysis of label-retaining cells (LRCs) and genetic lineage tracing to define LG cell lineage hierarchy. Our study suggests that embryonic LG contains unique long-lived multipotent stem cells that give rise to all postnatal epithelial cell types. Following birth, lineages become established and the fate of progenitor cell descendants becomes restricted. However, some cell lineages retain plasticity after maturation and can trans-differentiate into other cell types upon injury. The demonstration that the LG contains progenitor cells with different levels of plasticity has profound implications for our understanding of LG gland function in homeostasis and disease and will be helpful for developing stem cell-based therapies in the future.

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain.

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.

FGF Gradient Controls Boundary Position Between Proliferating and Differentiating Cells and Regulates Lacrimal Gland Growth Dynamics.

Fibroblast growth factor (FGF) signaling plays an important role in controlling cell proliferation, survival, and cell movements during branching morphogenesis of many organs. In mammals branching morphogenesis is primarily regulated by members of the FGF7-subfamily (FGF7 and FGF10), which are expressed in the mesenchyme, and signal to the epithelial cells through the "b" isoform of fibroblast growth factor receptor-2 (FGFR2). Our previous work demonstrated that FGF7 and FGF10 form different gradients in the extracellular matrix (ECM) and induce distinct cellular responses and gene expression profiles in the lacrimal and submandibular glands. The last finding was the most surprising since both FGF7 and FGF10 bind signal most strongly through the same fibroblast growth factor receptor-2b isoform (FGFR2b). Here we revisit this question to gain an explanation of how the different FGFs regulate gene expression. For this purpose, we employed our ex vivo epithelial explant migration assay in which isolated epithelial explants are grown near the FGF loaded beads. We demonstrate that the graded distribution of FGF induces activation of ERK1/2 MAP kinases that define the position of the boundary between proliferating "bud" and differentiating "stalk" cells of growing lacrimal gland epithelium. Moreover, we showed that gene expression profiles of the epithelial explants exposed to distinct FGFs strictly depend on the ratio between "bud" and "stalk" area. Our data also suggests that differentiation of "stalk" and "bud" regions within the epithelial explants is necessary for directional and persistent epithelial migration. Gaining a better understanding of FGF functions is important for development of new approaches to enhance tissue regeneration.

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (αSMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

Sirtuin 1-Chromatin-Binding Dynamics Points to a Common Mechanism Regulating Inflammatory Targets in SIV Infection and in the Aging Brain.

Microglia and macrophages are the main non-neuronal subsets of myeloid origin in the brain, and are critical regulators in neurodegenerative disorders, where inflammation is a key factor. Since HIV infection results in neurological perturbations that are similar to those in aging, we examined microglial and infiltrating myeloid subsets in the search for changes that might resemble the ones in aging. For that, we used the SIV infection in rhesus macaques to model neuroAIDS. We found that Sirt-1, a molecule that impacts survival and health in many models, was decreased in cell preparations containing a majority of microglia and myeloid cells from the brain of infected macaques. The role of Sirt-1 in neuroAIDS is unknown. We hypothesized that Sirt-1 silencing functions are affected by SIV. Mapping of Sirt-1 binding patterns to chromatin revealed that the number of Sirt-1-bound genes was 29.6% increased in myeloid cells from infected animals with mild or no detectable neuropathology, but 51% was decreased in severe neuropathology, compared to controls. Importantly, Sirt-1-bound genes in controls largely participate in neuroinflammation. Promoters of type I IFN pathway genes IRF7, IRF1, IFIT1, and AIF1, showed Sirt-1 binding in controls, which was consistently lost after infection, together with higher transcription. Loss of Sirt-1 binding was also found in brains from old uninfected animals, suggesting a common regulation. The role of Sirt-1 in regulating these inflammatory markers was confirmed in two different in vitro models, where Sirt-1 blockage modulated IRF7, IRF1 and AIF1 levels both in human macrophage cell lines and in human blood-derived monocytes from various normal donors, stimulated with a TLR9 agonist. Our data suggests that Sirt-1-inflammatory gene silencing is disturbed by SIV infection, resembling aging in brains. These findings may impact our knowledge on the contribution of myeloid subsets to the neurological consequences of HIV infection, aggravated and overlapping with the aging process.

Modeling the Function of TATA Box Binding Protein in Transcriptional Changes Induced by HIV-1 Tat in Innate Immune Cells and the Effect of Methamphetamine Exposure.

Innate immune cells are targets of HIV-1 infection in the Central Nervous System (CNS), generating neurological deficits. Infected individuals with substance use disorders as co-morbidities, are more likely to have aggravated neurological disorders, higher CNS viral load and inflammation. Methamphetamine (Meth) is an addictive stimulant drug, commonly among HIV+ individuals. The molecular basis of HIV direct effects and its interactions with Meth in host response, at the gene promoter level, are not well understood. The main HIV-1 peptide acting on transcription is the transactivator of transcription (Tat), which promotes replication by recruiting a Tata-box binding protein (TBP) to the virus long-terminal repeat (LTR). We tested the hypothesis that Tat can stimulate host gene expression through its ability to increase TBP, and thus promoting its binding to promoters that bear Tata-box binding motifs. Genes with Tata-box domains are mainly inducible, early response, and involved in inflammation, regulation and metabolism, relevant in HIV pathogenesis. We also tested whether Tat and Meth interact to trigger the expression of Tata-box bearing genes. The THP1 macrophage cell line is a well characterized innate immune cell system for studying signal transduction in inflammation. These cells are responsive to Tat, as well as to Meth, by recruiting RNA Polymerase (RNA Pol) to inflammatory gene promoters, within 15 min of stimulation (1). THP-1 cells, including their genetically engineered derivatives, represent valuable tools for investigating monocyte structure and function in both health and disease, as a consistent system (2). When differentiated, they mimic several aspects of the response of macrophages, and innate immune cells that are the main HIV-1 targets within the Central Nervous System (CNS). THP1 cells have been used to characterize the impact of Meth and resulting neurotransmitters on HIV entry (1), mimicking the CNS micro-environment. Integrative consensus sequence analysis in genes with enriched RNA Pol, revealed that TBP was a major transcription factor in Tat stimulation, while the co-incubation with Meth shifted usage to a distinct and diversified pattern. For validating these findings, we engineered a THP1 clone to be deficient in the expression of all major TBP splice variants, and tested its response to Tat stimulation, in the presence or absence of Meth. Transcriptional patterns in TBP-sufficient and deficient clones confirmed TBP as a dominant transcription factor in Tat stimulation, capable of inducing genes with no constitutive expression. However, in the presence of Meth, TBP was no longer necessary to activate the same genes, suggesting promoter plasticity. These findings demonstrate TBP as mechanism of host-response activation by HIV-1 Tat, and suggest that promoter plasticity is a challenge imposed by co-morbid factors such as stimulant drug addiction. This may be one mechanism responsible for limited efficacy of therapeutic approaches in HIV+ Meth abusers.