Human Bone Marrow Endothelial Progenitor Cell Transplantation into Symptomatic ALS Mice Delays Disease Progression and Increases Motor Neuron Survival by Repairing Blood-Spinal Cord Barrier.

Convincing evidence demonstrated impairment of the blood-spinal cord barrier (BSCB) in Amyotrophic Lateral Sclerosis (ALS), mainly by endothelial cell (EC) alterations. Replacing damaged ECs by cell transplantation is a potential barrier repair strategy. Recently, we showed that intravenous (iv) administration of human bone marrow CD34+ (hBM34+) cells into symptomatic ALS mice benefits BSCB restoration and postpones disease progression. However, delayed effect on motor function and some severely damaged capillaries were noted. We hypothesized that hematopoietic cells from a restricted lineage would be more effective. This study aimed to establish the effects of human bone marrow-derived endothelial progenitor cells (hBMEPCs) systemically transplanted into G93A mice at symptomatic disease stage. Results showed that transplanted hBMEPCs significantly improved behavioral disease outcomes, engrafted widely into capillaries of the gray/white matter spinal cord and brain motor cortex/brainstem, substantially restored capillary ultrastructure, significantly decreased EB extravasation into spinal cord parenchyma, meaningfully re-established perivascular astrocyte end-feet, and enhanced spinal cord motor neuron survival. These results provide novel evidence that transplantation of hBMEPCs effectively repairs the BSCB, potentially preventing entry of detrimental peripheral factors, including immune/inflammatory cells, which contribute to motor neuron dysfunction. Transplanting EC progenitor cells may be a promising strategy for barrier repair therapy in this disease.

Diversity in CO2-Concentrating Mechanisms among Chemolithoautotrophs from the Genera Hydrogenovibrio, Thiomicrorhabdus, and Thiomicrospira, Ubiquitous in Sulfidic Habitats Worldwide.

Members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus fix carbon at hydrothermal vents, coastal sediments, hypersaline lakes, and other sulfidic habitats. The genome sequences of these ubiquitous and prolific chemolithoautotrophs suggest a surprising diversity of mechanisms for the uptake and fixation of dissolved inorganic carbon (DIC); these mechanisms are verified here. Carboxysomes are apparent in the transmission electron micrographs of most of these organisms but are lacking in Thiomicrorhabdus sp. strain Milos-T2 and Thiomicrorhabdus arctica, and the inability of Thiomicrorhabdus sp. strain Milos-T2 to grow under low-DIC conditions is consistent with the absence of carboxysome loci in its genome. For the remaining organisms, genes encoding potential DIC transporters from four evolutionarily distinct families (Tcr_0853 and Tcr_0854, Chr, SbtA, and SulP) are located downstream of carboxysome loci. Transporter genes collocated with carboxysome loci, as well as some homologs located elsewhere on the chromosomes, had elevated transcript levels under low-DIC conditions, as assayed by reverse transcription-quantitative PCR (qRT-PCR). DIC uptake was measureable via silicone oil centrifugation when a representative of each of the four types of transporter was expressed in Escherichia coli The expression of these genes in the carbonic anhydrase-deficient E. coli strain EDCM636 enabled it to grow under low-DIC conditions, a result consistent with DIC transport by these proteins. The results from this study expand the range of DIC transporters within the SbtA and SulP transporter families, verify DIC uptake by transporters encoded by Tcr_0853 and Tcr_0854 and their homologs, and introduce DIC as a potential substrate for transporters from the Chr family.IMPORTANCE Autotrophic organisms take up and fix DIC, introducing carbon into the biological portion of the global carbon cycle. The mechanisms for DIC uptake and fixation by autotrophic Bacteria and Archaea are likely to be diverse but have been well characterized only for "Cyanobacteria" Based on genome sequences, members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus have a variety of mechanisms for DIC uptake and fixation. We verified that most of these organisms are capable of growing under low-DIC conditions, when they upregulate carboxysome loci and transporter genes collocated with these loci on their chromosomes. When these genes, which fall into four evolutionarily independent families of transporters, are expressed in E. coli, DIC transport is detected. This expansion in known DIC transporters across four families, from organisms from a variety of environments, provides insight into the ecophysiology of autotrophs, as well as a toolkit for engineering microorganisms for carbon-neutral biochemistries of industrial importance.

Transplantation of human bone marrow stem cells into symptomatic ALS mice enhances structural and functional blood-spinal cord barrier repair.

Accumulating evidence shows alterations in the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in ALS patients and in animal models of disease, mainly by endothelial cell (EC) damage. Repair of the altered barrier in the CNS by replacement of ECs via cell transplantation may be a new therapeutic approach for ALS. Recently, we demonstrated positive effects towards BSCB repair by intravenous administration of unmodified human bone marrow CD34+ (hBM34+) cells at different doses into symptomatic ALS mice. However, particular benefits of these transplanted cells on microvascular integrity in symptomatic ALS mice are still unclear. The aim of the present study was to determine the structural and functional spinal cord capillary integrity in symptomatic ALS mice after intravenous administration of hBM34+ cells. The G93A mice at 13 weeks of age intravenously received one of three different cell doses (5 × 104, 5 × 105, or 1 × 106) and were euthanized at 17 weeks of age (4 weeks post-transplant). Control groups were media-treated and non-carrier mutant SOD1 gene mice. Capillary ultrastructural (electron microscopy), immunohistochemical (laminin and HuNu), and histological (myelin and capillary density) analyses were performed in the cervical and lumbar spinal cords. Capillary permeability in the spinal cords was determined by Evans Blue (EB) injection. Results showed significant restoration of ultrastructural capillary morphology, improvement of basement membrane integrity, enhancement of axonal myelin coherence, and stabilization of capillary density in the spinal cords primarily of ALS mice receiving the high dose of 1 × 106 cells. Moreover, substantial reduction of parenchymal EB levels was determined in these mice, confirming our previous results on capillary permeability. Additionally, transplanted cells were detected in blood smears of sacrificed late symptomatic mice by HuNu marker. Altogether, these results provide novel evidence that unmodified bone marrow hematopoietic stem cell treatment at optimal dose might be beneficial for structural and functional repair of the damaged BSCB in advanced stage of ALS, potentially resulting in delayed disease progression by increased motor neuron survival.

Morphologic and Elemental Analysis of Primary Melanosis of the Dentate Nucleus: Review and Correlation With Neuromelanin.

Primary melanosis of the dentate nucleus is a rarely described entity with neither known cause nor definitive clinicopathologic correlation. We revisit this previously reported phenomenon by presenting one such case with a review of the pathology as well as additional investigations including elemental analysis by energy-dispersive X-ray, immunohistochemistry and electron microscopy. The lesion presented macroscopically as a sharply defined, black pigmentation that was restricted to the dentate nucleus of the cerebellum. Other deep nuclei were uninvolved. Similarly, other areas of the cerebellum, brainstem, and supratentorial regions were macroscopically free of pigment. Microscopically, however, the pigment was noted to be present, albeit in microscopic deposits, within layers of the cerebellar cortex. Additionally, immunohistochemistry and electron microscopy defined an intracellular component within astrocytes. X-ray analysis of the pigment showed it to consist almost entirely of sulfur, an element known to be prominent in neuromelanin. This report also describes an association of the pigment with astrocytes by ultrastructural examination. We discuss the results of our findings in the context of etiopathogenetic considerations, seeking to gain a better understanding of this abnormal pigmentation and its relationship to neuromelanin.

Intravenously Transplanted Human Bone Marrow Endothelial Progenitor Cells Engraft Within Brain Capillaries, Preserve Mitochondrial Morphology, and Display Pinocytotic Activity Toward Blood-Brain Barrier Repair in Ischemic Stroke Rats.

Stroke is a life-threatening disease with limited therapeutic options. Cell therapy has emerged as an experimental stroke treatment. Blood-brain barrier (BBB) impairment is a key pathological manifestation of ischemic stroke, and barrier repair is an innovative target for neurorestoration in stroke. Here, we evaluated via electron microscopy the ability of transplanted human bone marrow endothelial progenitor cells (hBMEPCs) to repair the BBB in adult Sprague-Dawley rats subjected to transient middle cerebral artery occlusion (tMCAO). ß-galactosidase prelabeled hBMEPCs were intravenously transplanted 48 hours post-tMCAO. Ultrastructural analysis of microvessels in nontransplant stroke rats revealed typical BBB pathology. At 5 days post-transplantation with hBMEPCs, stroke rats displayed widespread vascular repair in bilateral striatum and motor cortex, characterized by robust cell engraftment within capillaries. hBMEPC transplanted stroke rats exhibited near normal morphology of endothelial cells (ECs), pericytes, and astrocytes, without detectable perivascular edema. Near normal morphology of mitochondria was also detected in ECs and perivascular astrocytes from transplanted stroke rats. Equally notable, we observed numerous pinocytic vesicles within engrafted cells. Robust engraftment and intricate functionality of transplanted hBMEPCs likely abrogated stroke-altered vasculature. Preserving mitochondria and augmenting pinocytosis in cell-based therapeutics represent a new neurorestorative mechanism in BBB repair for stroke. Stem Cells 2017;35:1246-1258.

Proteomic and Mutant Analysis of the CO2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena.

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) with CO2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira.

Molecular identification and nanoremediation of microbial contaminants in algal systems using untreated wastewater.

Wastewater-algal biomass is a promising option to biofuel production. However, microbial contaminants constitute a substantial barrier to algal biofuel yield. A series of algal strains, Nannochloris oculata and Chlorella vulgaris samples (n = 30), were purchased from the University of Texas, and were used for both stock flask cultures and flat-panel vertical bioreactors. A number of media were used for isolation and differentiation of potential contaminants according to laboratory standards (CLSI). Conventional PCR amplification was performed followed by 16S rDNA sequencing to identify isolates at the species level. Nanotherapeutics involving a nanomicellar combination of natural chitosan and zinc oxide (CZNPs) were tested against the microbial lytic groups through Minimum Inhibitory Concentration (MIC) tests and Transmission Electronic Microscopy (TEM). Results indicated the presence of Pseudomonas spp., Bacillus pumilus/ safensis, Cellulosimicrobium cellulans, Micrococcus luteus and Staphylococcus epidermidis strains at a substantial level in the wastewater-fed algal reactors. TEM confirmed the effectiveness of CZNPs on the lytic group while the average MICs (mg/mL) detected for the strains, Pseudomonas spp, Micrococcus luteus, and Bacillus pumilus were 0.417, 3.33, and 1.458, respectively. Conclusively, CZNP antimicrobials proved to be effective as inhibitory agents against currently identified lytic microbial group, did not impact algae cells, and shows promise for in situ interventions.

Blood-Spinal Cord Barrier Alterations in Subacute and Chronic Stages of a Rat Model of Focal Cerebral Ischemia.

We previously demonstrated blood-brain barrier impairment in remote contralateral brain areas in rats at 7 and 30 days after transient middle cerebral artery occlusion (tMCAO), indicating ischemic diaschisis. Here, we focused on effects of subacute and chronic focal cerebral ischemia on the blood-spinal cord barrier (BSCB). We observed BSCB damage on both sides of the cervical spinal cord in rats at 7 and 30 days post-tMCAO. Major BSCB ultrastructural changes in spinal cord gray and white matter included vacuolated endothelial cells containing autophagosomes, pericyte degeneration with enlarged mitochondria, astrocyte end-feet degeneration and perivascular edema; damaged motor neurons, swollen axons with unraveled myelin in ascending and descending tracts and astrogliosis were also observed. Evans Blue dye extravasation was maximal at 7 days. There was immunofluorescence evidence of reduction of microvascular expression of tight junction occludin, upregulation of Beclin-1 and LC3B immunoreactivities at 7 days and a reduction of the latter at 30 days post-ischemia. These novel pathological alterations on the cervical spinal cord microvasculature in rats after tMCAO suggest pervasive and long-lasting BSCB damage after focal cerebral ischemia, and that spinal cord ischemic diaschisis should be considered in the pathophysiology and therapeutic approaches in patients with ischemic cerebral infarction.

MIR494 reduces renal cancer cell survival coinciding with increased lipid droplets and mitochondrial changes.

BACKGROUND: miRNAs can regulate cellular survival in various cancer cell types. Recent evidence implicates the formation of lipid droplets as a hallmark event during apoptotic cell death response. It is presently unknown whether MIR494, located at 14q32 which is deleted in renal cancers, reduces cell survival in renal cancer cells and if this process is accompanied by changes in the number of lipid droplets. METHODS: 769-P renal carcinoma cells were utilized for this study. Control or MIR494 mimic was expressed in these cells following which cell viability (via crystal violet) and apoptotic cell numbers (via Annexin V/PI staining) were assessed. By western blotting, MIR494 cellular responses were validated using MIR494 antagomir and Argonaute 2 siRNA. Transmission electron microscopy (TEM) was performed in MIR494-transfected 769-P cells to identify ultrastructural changes. LipidTOX green neutral lipid staining and cholesterol measurements were conducted to assess accumulation of lipids droplets and total cholesterol levels, respectively, in MIR494 expressing 769-P cells. Indirect immunofluorescence and western analyses were also performed to examine changes in mitochondria organization. Co-transfection of MIR494 mimic with siRNA targeting LC3B and ATG7 was conducted to assess their contribution to formation of lipid droplets in MIR494-expressing cells. RESULTS: MIR494 expression reduces viability of 769-P renal cancer cells; this was accompanied by increased cleaved PARP (an apoptotic marker) and LC3B protein. Further, expression of MIR494 increased LC3B mRNA levels and LC3B promoter activity (2.01-fold; 50% increase). Interestingly, expression of MIR494 markedly increased multilamellar bodies and lipid droplets (by TEM and validated by LipidTOX immunostaining) while reducing total cholesterol levels. Via immunocytochemistry, we observed increased LC3B-associated endogenous punctae upon MIR494 expression. In contrast to ATG7 siRNA, knockdown of LC3B reduced the numbers of lipid droplets in MIR494-expressing cells. Our results also identified that MIR494 expression altered the organization of mitochondria which was accompanied by co-localization with LC3B punctae, decreased PINK1 protein, and altered Drp1 intracellular distribution. CONCLUSION: Collectively, our findings indicate that MIR494 reduces cell survival in 769-P renal cancer cells which is accompanied by increased lipid droplet formation (which occurs in a LC3B-dependent manner) and mitochondrial changes.

Studies on the drug resistance profile of Enterococcus faecium distributed from poultry retailers to hospitals.

The multidrug resistant Enterococcus faecium (MEF) strains originating from farm animals are proliferating at a substantial pace to impact downstream food chains and could reach hospitals. This study was conducted to elucidate the drug susceptibility profile of MEF strains collected from poultry products in Ann Arbor, MI area and clinical settings from Michigan State Lab and Moffitt Cancer Center (MCC) in Florida. Presumptive positive Enterococcus isolates at species level were identified by Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) analysis. The antibiotic susceptibility profile for both poultry and clinical strains was determined by the Thermo Scientific's Sensititre conform to the National Committee for Clinical Laboratory Standards (NCCLS) and validated via quantitative real-time PCR (qPCR) methods. Out of 50 poultry samples (Turkey: n = 30; Chicken: n = 20), 36 samples were positive for Enterococcus species from which 20.83% were identified as E. faecium. All the E. faecium isolates were multidrug resistant and displayed resistance to the last alternative drug, quinupristin/dalfopristin (QD) used to treat vancomycin resistant E. faecium (VRE) in hospitals. Results indicate the presence of MEF strains in food animals and clinical settings that are also resistant to QD.

Development of somites and their derivatives in amphioxus, and implications for the evolution of vertebrate somites.

BACKGROUND: Vertebrate somites are subdivided into lineage compartments, each with distinct cell fates and evolutionary histories. Insights into somite evolution can come from studying amphioxus, the best extant approximation of the chordate ancestor. Amphioxus somites have myotome and non-myotome compartments, but development and fates of the latter are incompletely described. Further, while epithelial to mesenchymal transition (EMT) is important for most vertebrate somitic lineages, amphioxus somites generally have been thought to remain entirely epithelial. Here, we examined amphioxus somites and derivatives, as well as extracellular matrix of the axial support system, in a series of developmental stages by transmission electron microscopy (TEM) and in situ hybridization for collagen expression. RESULTS: The amphioxus somite differentiates medially into myotome, laterally into the external cell layer (a sub-dermal mesothelium), ventrally into a bud that forms mesothelia of the perivisceral coelom, and ventro-medially into the sclerotome. The sclerotome forms initially as a monolayered cell sheet that migrates between the myotome and the notochord and neural tube; subsequently, this cell sheet becomes double layered and encloses the sclerocoel. Other late developments include formation of the fin box mesothelia from lateral somites and the advent of isolated fibroblasts, likely somite derived, along the myosepta. Throughout development, all cells originating from the non-myotome regions of somites strongly express a fibrillar collagen gene, ColA, and thus likely contribute to extracellular matrix of the dermal and axial connective tissue system. CONCLUSIONS: We provide a revised model for the development of amphioxus sclerotome and fin boxes and confirm previous reports of development of the myotome and lateral somite. In addition, while somite derivatives remain almost entirely epithelial, limited de-epithelialization likely converts some somitic cells into fibroblasts of the myosepta and dermis. Ultrastructure and collagen expression suggest that all non-myotome somite derivatives contribute to extracellular matrix of the dermal and axial support systems. Although amphioxus sclerotome lacks vertebrate-like EMT, it resembles that of vertebrates in position, movement to surround midline structures and into myosepta, and contribution to extracellular matrix of the axial support system. Thus, many aspects of the sclerotome developmental program evolved prior to the origin of the vertebrate mineralized skeleton.

Iron alters cell survival in a mitochondria-dependent pathway in ovarian cancer cells.

The role of iron in the development of cancer remains unclear. We previously reported that iron reduces cell survival in a Ras/mitogen-activated protein kinase (MAPK)-dependent manner in ovarian cells; however, the underlying downstream pathway leading to reduced survival was unclear. Although levels of intracellular iron, ferritin/CD71 protein and reactive oxygen species did not correlate with iron-induced cell survival changes, we identified mitochondrial damage (via TEM) and reduced expression of outer mitochondrial membrane proteins (translocase of outer membrane: TOM20 and TOM70) in cell lines sensitive to iron. Interestingly, Ru360 (an inhibitor of the mitochondrial calcium uniporter) reversed mitochondrial changes and restored cell survival in HEY ovarian carcinoma cells treated with iron. Further, cells treated with Ru360 and iron also had reduced autophagic punctae with increased lysosomal numbers, implying cross-talk between these compartments. Mitochondrial changes were dependent on activation of the Ras/MAPK pathway since treatment with a MAPK inhibitor restored expression of TOM20/TOM70 proteins. Although glutathione antioxidant levels were reduced in HEY treated with iron, extracellular glutamate levels were unaltered. Strikingly, oxalomalate (inhibitor of aconitase, involved in glutamate production) reversed iron-induced responses in a similar manner to Ru360. Collectively, our results implicate iron in modulating cell survival in a mitochondria-dependent manner in ovarian cancer cells.

Compromised blood-brain barrier competence in remote brain areas in ischemic stroke rats at the chronic stage.

Stroke is a life-threatening disease leading to long-term disability in stroke survivors. Cerebral functional insufficiency in chronic stroke might be due to pathological changes in brain areas remote from the initial ischemic lesion, i.e., diaschisis. Previously, we showed that the damaged blood-brain barrier (BBB) was involved in subacute diaschisis. The present study investigated BBB competence in chronic diaschisis by using a transient middle cerebral artery occlusion (tMCAO) rat model. Our results demonstrated significant BBB damage mostly in the ipsilateral striatum and motor cortex in rats at 30 days after tMCAO. The BBB alterations were also determined in the contralateral hemisphere via ultrastructural and immunohistochemical analyses. Major BBB pathological changes in contralateral remote striatum and motor cortex areas included 1) vacuolated endothelial cells containing large autophagosomes, 2) degenerated pericytes displaying mitochondria with cristae disruption, 3) degenerated astrocytes and perivascular edema, 4) Evans blue extravasation, and 5) appearance of parenchymal astrogliosis. Discrete analyses of striatal and motor cortex areas revealed significantly higher autophagosome accumulation in capillaries of ventral striatum and astrogliosis in dorsal striatum in both cerebral hemispheres. These widespread microvascular alterations in ipsilateral and contralateral brain hemispheres suggest persistent and/or continued BBB damage in chronic ischemia. The pathological changes in remote brain areas likely indicate chronic ischemic diaschisis, which should be considered in the development of treatment strategies for stroke.

Blood-brain barrier impairment in MPS III patients.

BACKGROUND: Mucopolysaccharidosis type III (MPS III) is an autosomal recessive disorder caused by deficiency of a specific enzyme leading to heparan sulfate (HS) accumulation within cells and to eventual progressive cerebral and systemic organ abnormalities. Different enzyme deficiencies comprise the MPS III subcategories (A, B, C, D). Since neuropathological manifestations are common to all MPS III types, determining blood-brain barrier (BBB) condition may be critical to understand potential additional disease mechanisms. METHODS: We investigated BBB integrity in various brain structures of post-mortem tissues from an eleven year old Caucasian female with MPS III A and from a twenty four year old Caucasian female with MPS III D. Control tissues were obtained post-mortem from three Caucasians without neurological deficits: a twelve year old male, a twenty four year old female, and a twenty seven year old female. BBB capillary ultrastructure (electron microscopy) and capillary functional integrity (IgG leakage, tight junction proteins, and lysosomal accumulation within endothelium) were examined. RESULTS: Compromised BBB integrity was found in both MPS III cases. Major study findings were: (1) capillary endothelial and pericyte cell damage; (2) mucopolysaccharide bodies in a majority of endothelial cells and pericytes rupturing cell membranes; (3) severe extracellular edema; (4) IgG microvascular leakage and reductions of occludin and claudin-5 with variations between MPS III types; (5) extensive lysosomal accumulation in capillary endothelium. CONCLUSIONS: These new findings of BBB structural and functional impairment, although from only two cases, MPS III A and III D, may have implications for disease pathogenesis and should be considered in treatment development for MPS III.

Blood-brain barrier alterations provide evidence of subacute diaschisis in an ischemic stroke rat model.

BACKGROUND: Comprehensive stroke studies reveal diaschisis, a loss of function due to pathological deficits in brain areas remote from initial ischemic lesion. However, blood-brain barrier (BBB) competence in subacute diaschisis is uncertain. The present study investigated subacute diaschisis in a focal ischemic stroke rat model. Specific focuses were BBB integrity and related pathogenic processes in contralateral brain areas. METHODOLOGY/PRINCIPAL FINDINGS: In ipsilateral hemisphere 7 days after transient middle cerebral artery occlusion (tMCAO), significant BBB alterations characterized by large Evans Blue (EB) parenchymal extravasation, autophagosome accumulation, increased reactive astrocytes and activated microglia, demyelinization, and neuronal damage were detected in the striatum, motor and somatosensory cortices. Vascular damage identified by ultrastuctural and immunohistochemical analyses also occurred in the contralateral hemisphere. In contralateral striatum and motor cortex, major ultrastructural BBB changes included: swollen and vacuolated endothelial cells containing numerous autophagosomes, pericyte degeneration, and perivascular edema. Additionally, prominent EB extravasation, increased endothelial autophagosome formation, rampant astrogliosis, activated microglia, widespread neuronal pyknosis and decreased myelin were observed in contralateral striatum, and motor and somatosensory cortices. CONCLUSIONS/SIGNIFICANCE: These results demonstrate focal ischemic stroke-induced pathological disturbances in ipsilateral, as well as in contralateral brain areas, which were shown to be closely associated with BBB breakdown in remote brain microvessels and endothelial autophagosome accumulation. This microvascular damage in subacute phase likely revealed ischemic diaschisis and should be considered in development of treatment strategies for stroke.

Impaired blood-brain/spinal cord barrier in ALS patients.

Vascular pathology, including blood-brain/spinal cord barrier (BBB/BSCB) alterations, has recently been recognized as a key factor possibly aggravating motor neuron damage, identifying a neurovascular disease signature for ALS. However, BBB/BSCB competence in sporadic ALS (SALS) is still undetermined. In this study, BBB/BSCB integrity in postmortem gray and white matter of medulla and spinal cord tissue from SALS patients and controls was investigated. Major findings include (1) endothelial cell damage and pericyte degeneration, (2) severe intra- and extracellular edema, (3) reduced CD31 and CD105 expressions in endothelium, (4) significant accumulation of perivascular collagen IV, and fibrin deposits (5) significantly increased microvascular density in lumbar spinal cord, (6) IgG microvascular leakage, (7) reduced tight junction and adhesion protein expressions. Microvascular barrier abnormalities determined in gray and white matter of the medulla, cervical, and lumbar spinal cord of SALS patients are novel findings. Pervasive barrier damage discovered in ALS may have implications for disease pathogenesis and progression, as well as for uncovering novel therapeutic targets.

Chronic autophagy is a cellular adaptation to tumor acidic pH microenvironments.

Tumor cell survival relies upon adaptation to the acidic conditions of the tumor microenvironment. To investigate potential acidosis survival mechanisms, we examined the effect of low pH (6.7) on human breast carcinoma cells. Acute low pH exposure reduced proliferation rate, induced a G1 cell cycle arrest, and increased cytoplasmic vacuolization. Gene expression analysis revealed elevated levels of ATG5 and BNIP3 in acid-conditioned cells, suggesting cells exposed to low pH may utilize autophagy as a survival mechanism. In support of this hypothesis, we found that acute low pH stimulated autophagy as defined by an increase in LC3-positive punctate vesicles, double-membrane vacuoles, and decreased phosphorylation of AKT and ribosomal protein S6. Notably, cells exposed to low pH for approximately 3 months restored their proliferative capacity while maintaining the cytoplasmic vacuolated phenotype. Although autophagy is typically transient, elevated autophagy markers were maintained chronically in low pH conditioned cells as visualized by increased protein expression of LC3-II and double-membrane vacuoles. Furthermore, these cells exhibited elevated sensitivity to PI3K-class III inhibition by 3-methyladenine. In mouse tumors, LC3 expression was reduced by systemic treatment with sodium bicarbonate, which raises intratumoral pH. Taken together, these results argue that acidic conditions in the tumor microenvironment promote autophagy, and that chronic autophagy occurs as a survival adaptation in this setting.

Transcriptional response of the sulfur chemolithoautotroph Thiomicrospira crunogena to dissolved inorganic carbon limitation.

The hydrothermal vent gammaproteobacterium Thiomicrospira crunogena inhabits an unstable environment and must endure dramatic changes in habitat chemistry. This sulfur chemolithoautotroph responds to changes in dissolved inorganic carbon (DIC) (DIC = CO(2) + HCO(3)(-) + CO(3)(-2)) availability with a carbon-concentrating mechanism (CCM) in which whole-cell affinity for DIC, as well as the intracellular DIC concentration, increases substantially under DIC limitation. To determine whether this CCM is regulated at the level of transcription, we resuspended cells that were cultivated under high-DIC conditions in chemostats in growth medium with low concentrations of DIC and tracked CCM development in the presence and absence of the RNA polymerase inhibitor rifampin. Induction of the CCM, as measured by silicone oil centrifugation, was hindered in the presence of rifampin. Similar results were observed for carboxysome gene transcription and assembly, as assayed by quantitative reverse transcription-PCR (qRT-PCR) and transmission electron microscopy, respectively. Genome-wide transcription patterns for cells grown under DIC limitation and those grown under ammonia limitation were assayed via microarrays and compared. In addition to carboxysome genes, two novel genes (Tcr_1019 and Tcr_1315) present in other organisms, including chemolithoautotrophs, but whose function(s) has not been elucidated in any organism were found to be upregulated under low-DIC conditions. Likewise, under ammonia limitation, in addition to the expected enhancement of ammonia transporter and P(II) gene transcription, the transcription of two novel genes (Tcr_0466 and Tcr_2018) was measurably enhanced. Upregulation of all four genes (Tcr_1019, 4-fold; Tcr_131, ∼7-fold; Tcr_0466, >200-fold; Tcr_2018, 7-fold), which suggests that novel components are part of the response to nutrient limitation by this organism, was verified via qRT-PCR.

Blood-brain barrier impairment in an animal model of MPS III B.

BACKGROUND: Sanfilippo syndrome type B (MPS III B) is caused by a deficiency of α-N-acetylglucosaminidase enzyme, leading to accumulation of heparan sulfate within lysosomes and eventual progressive cerebral and systemic multiple organ abnormalities. However, little is known about the competence of the blood-brain barrier (BBB) in MPS III B. BBB dysfunction in this devastating disorder could contribute to neuropathological disease manifestations. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we investigated structural (electron microscope) and functional (vascular leakage) integrity of the BBB in a mouse model of MPS III B at different stages of disease, focusing on brain structures known to experience neuropathological changes. Major findings of our study were: (1) endothelial cell damage in capillary ultrastructure, compromising the BBB and resulting in vascular leakage, (2) formation of numerous large vacuoles in endothelial cells and perivascular cells (pericytes and perivascular macrophages) in the large majority of vessels, (3) edematous space around microvessels, (4) microaneurysm adjacent to a ruptured endothelium, (6) Evans Blue and albumin microvascular leakage in various brain structures, (7) GM3 ganglioside accumulation in endothelium of the brain microvasculature. CONCLUSIONS/SIGNIFICANCE: These new findings of BBB structural and function impairment in MPS III B mice even at early disease stage may have implications for disease pathogenesis and should be considered in current and future development of treatments for MPS III B.

Initial observations of cell-mediated drug delivery to the deep lung.

Using current methodologies, drug delivery to small airways, terminal bronchioles, and alveoli (deep lung) is inefficient, especially to the lower lungs. Urgent lung pathologies such as acute respiratory distress syndrome (ARDS) and post-lung transplantation complications are difficult to treat, in part due to the methodological limitations in targeting the deep lung with high efficiency drug distribution to the site of pathology. To overcome drug delivery limitations inhibiting the optimization of deep lung therapy, isolated rat Sertoli cells preloaded with chitosan nanoparticles were use to obtain a high-density distribution and concentration (92%) of the nanoparticles in the lungs of mice by way of the peripheral venous vasculature rather than the more commonly used pulmonary route. Additionally, Sertoli cells were preloaded with chitosan nanoparticles coupled with the anti-inflammatory compound curcumin and then injected intravenously into control or experimental mice with deep lung inflammation. By 24 h postinjection, most of the curcumin load (∼90%) delivered in the injected Sertoli cells was present and distributed throughout the lungs, including the perialveloar sac area in the lower lungs. This was based on the high-density, positive quantification of both nanoparticles and curcumin in the lungs. There was a marked positive therapeutic effect achieved 24 h following curcumin treatment delivered by this Sertoli cell nanoparticle protocol (SNAP). Results identify a novel and efficient protocol for targeted delivery of drugs to the deep lung mediated by extratesticular Sertoli cells. Utilization of SNAP delivery may optimize drug therapy for conditions such as ARDS, status asthmaticus, pulmonary hypertension, lung cancer, and complications following lung transplantation where the use of high concentrations of anti-inflammatory drugs is desirable, but often limited by risks of systemic drug toxicity.