CISD3/MiNT is required for complex I function, mitochondrial integrity, and skeletal muscle maintenance.

Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.

Low-intensity open-field blast exposure effects on neurovascular unit ultrastructure in mice.

Mild traumatic brain injury (mTBI) induced by low-intensity blast (LIB) is a serious health problem affecting military service members and veterans. Our previous reports using a single open-field LIB mouse model showed the absence of gross microscopic damage or necrosis in the brain, while transmission electron microscopy (TEM) identified ultrastructural abnormalities of myelin sheaths, mitochondria, and synapses. The neurovascular unit (NVU), an anatomical and functional system with multiple components, is vital for the regulation of cerebral blood flow and cellular interactions. In this study, we delineated ultrastructural abnormalities affecting the NVU in mice with LIB exposure quantitatively and qualitatively. Luminal constrictive irregularities were identified at 7 days post-injury (DPI) followed by dilation at 30 DPI along with degeneration of pericytes. Quantitative proteomic analysis identified significantly altered vasomotor-related proteins at 24 h post-injury. Endothelial cell, basement membrane and astrocyte end-foot swellings, as well as vacuole formations, occurred in LIB-exposed mice, indicating cellular edema. Structural abnormalities of tight junctions and astrocyte end-foot detachment from basement membranes were also noted. These ultrastructural findings demonstrate that LIB induces multiple-component NVU damage. Prevention of NVU damage may aid in identifying therapeutic targets to mitigate the effects of primary brain blast injury.

New Insights into RPE-Photoreceptor Complex Ultrastructure using Focused Ion Beam-Scanning Election Microscopy (FIB-SEM).

Photoreceptors in the retina are specialized neuronal cells that perceive light and play a central role in the visual system. Damage to photoreceptors is a clinical feature often associated with various retinal degenerative disorders. The photoreceptor bed comprises a unique extracellular matrix (ECM) scaffold often described as the interphotoreceptor matrix (IPM) in the subretinal space, vital during retinal development and homeostasis. In this study, we used focused ion beam scanning electron microscopy (FIB-SEM) and transmission electron microscopy (TEM) to analyze the ultrastructural architecture of the retinal pigmented epithelium (RPE)-photoreceptor complex in mice. Additionally, we describe methods for retinal preparation in EM imaging. TEM images display ultrastructural retina layers, including Bruch's membrane and the interdigitation zone (IZ). The 3-dimensional reconstruction of the outer retina revealed individual photoreceptors, the connection between their inner and outer segment via the photoreceptor cilia, and photoreceptor interaction with the RPE ciliary processes. Our findings highlight the importance of FIB-SEM in deciphering the ultrastructural details of RPE-photoreceptor interactions in the IPM complex which are essential for the maintenance of retinal architecture.

A combination of a cell penetrating peptide and a protein translation inhibitor kills metastatic breast cancer cells.

Cell Penetrating Peptides (CPPs) are promising anticancer and antimicrobial drugs. We recently reported that a peptide derived from the human mitochondrial/ER membrane-anchored NEET protein, Nutrient Autophagy Factor 1 (NAF-1; NAF-144-67), selectively permeates and kills human metastatic epithelial breast cancer cells (MDA-MB-231), but not control epithelial cells. As cancer cells alter their phenotype during growth and metastasis, we tested whether NAF-144-67 would also be efficient in killing other human epithelial breast cancer cells that may have a different phenotype. Here we report that NAF-144-67 is efficient in killing BT-549, Hs 578T, MDA-MB-436, and MDA-MB-453 breast cancer cells, but that MDA-MB-157 cells are resistant to it. Upon closer examination, we found that MDA-MB-157 cells display a high content of intracellular vesicles and cellular protrusions, compared to MDA-MB-231 cells, that could protect them from NAF-144-67. Inhibiting the formation of intracellular vesicles and dynamics of cellular protrusions of MDA-MB-157 cells, using a protein translation inhibitor (the antibiotic Cycloheximide), rendered these cells highly susceptible to NAF-144-67, suggesting that under certain conditions, the killing effect of CPPs could be augmented when they are applied in combination with an antibiotic or chemotherapy agent. These findings could prove important for the treatment of metastatic cancers with CPPs and/or treatment combinations that include CPPs.

CISD3 is required for Complex I function, mitochondrial integrity, and skeletal muscle maintenance.

Mitochondria play a central role in muscle metabolism and function. In skeletal muscles, a unique family of iron-sulfur proteins, termed CISD proteins, support mitochondrial function. The abundance of these proteins declines with aging leading to muscle degeneration. Although the function of the outer mitochondrial proteins CISD1 and CISD2 has been defined, the role of the inner mitochondrial protein CISD3, is currently unknown. Here we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne Muscular Dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscle mitochondria, and that CISD3 interacts with, and donates its clusters to, Complex I respiratory chain subunit NDUFV2. These findings reveal that CISD3 is important for supporting the biogenesis and function of Complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact muscle degeneration syndromes, aging, and related conditions.

Decorin regulates collagen fibrillogenesis during corneal wound healing in mouse in vivo.

A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn-/-) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn-/- corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn-/- and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn-/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn-/- corneas compared to the injured/normal dcn+/+ and uninjured dcn-/- corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn-/- corneas than injured/normal dcn+/+ and uninjured dcn-/- corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn-/- corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.

Starvation at the larval stage increases the vector competence of Aedes aegypti females for Zika virus.

Aedes aegypti is the primary vector of Zika virus (ZIKV), a flavivirus which typically presents itself as febrile-like symptoms in humans but can also cause neurological and pregnancy complications. The transmission cycle of mosquito-borne arboviruses such as ZIKV requires that various key tissues in the female mosquito get productively infected with the virus before the mosquito can transmit the virus to another vertebrate host. Following ingestion of a viremic blood-meal from a vertebrate, ZIKV initially infects the midgut epithelium before exiting the midgut after blood-meal digestion to disseminate to secondary tissues including the salivary glands. Here we investigated whether smaller Ae. aegypti females resulting from food deprivation as larvae exhibited an altered vector competence for blood-meal acquired ZIKV relative to larger mosquitoes. Midguts from small 'Starve' and large 'Control' Ae. aegypti were dissected to visualize by transmission electron microscopy (TEM) the midgut basal lamina (BL) as physical evidence for the midgut escape barrier showing Starve mosquitoes with a significantly thinner midgut BL than Control mosquitoes at two timepoints. ZIKV replication was inhibited in Starve mosquitoes following intrathoracic injection of virus, however, Starve mosquitoes exhibited a significantly higher midgut escape and population dissemination rate at 9 days post-infection (dpi) via blood-meal, with more virus present in saliva and head tissue than Control by 10 dpi and 14 dpi, respectively. These results indicate that Ae. aegypti developing under stressful conditions potentially exhibit higher midgut infection and dissemination rates for ZIKV as adults, Thus, variation in food intake as larvae is potentially a source for variable vector competence levels of the emerged adults for the virus.

Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning.

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Plasmodesmata-localized proteins and ROS orchestrate light-induced rapid systemic signaling in Arabidopsis.

Systemic signaling and systemic acquired acclimation (SAA) are key to the survival of plants during episodes of abiotic stress. These processes depend on a continuous chain of cell-to-cell signaling events that extends from the initial tissue that senses the stress (the local tissue) to the entire plant (systemic tissues). Reactive oxygen species (ROS) and Ca2+ are key signaling molecules thought to be involved in this cell-to-cell mechanism. Here, we report that the systemic response of Arabidopsis thaliana to a local treatment of high light stress, which resulted in local ROS accumulation, required ROS generated by respiratory burst oxidase homolog D (RBOHD). ROS increased cell-to-cell transport and plasmodesmata (PD) pore size in a manner dependent on PD-localized protein 1 (PDLP1) and PDLP5, and this process was required for the propagation of the systemic ROS signals and SAA. Furthermore, aquaporins and several Ca2+-permeable channels in the glutamate receptor-like (GLR), mechanosensitive small conductance-like (MSL), and cyclic nucleotide-gated (CNGC) families were involved in this systemic signaling process. However, we determined that these channels were required primarily to amplify the systemic signal in each cell along the path of the systemic ROS wave, as well as to establish local and systemic acclimation. Thus, PD and RBOHD-generated ROS orchestrate light stress-induced rapid cell-to-cell spread of systemic signals in Arabidopsis.

Expression of a dominant-negative AtNEET-H89C protein disrupts iron-sulfur metabolism and iron homeostasis in Arabidopsis.

Iron-sulfur (Fe-S) clusters play an essential role in plants as protein cofactors mediating diverse electron transfer reactions. Because they can react with oxygen to form reactive oxygen species (ROS) and inflict cellular damage, the biogenesis of Fe-S clusters is highly regulated. A recently discovered group of 2Fe-2S proteins, termed NEET proteins, was proposed to coordinate Fe-S, Fe and ROS homeostasis in mammalian cells. Here we report that disrupting the function of AtNEET, the sole member of the NEET protein family in Arabidopsis thaliana, triggers leaf-associated Fe-S- and Fe-deficiency responses, elevated Fe content in chloroplasts (1.2-1.5-fold), chlorosis, structural damage to chloroplasts and a high seedling mortality rate. Our findings suggest that disrupting AtNEET function disrupts the transfer of 2Fe-2S clusters from the chloroplastic 2Fe-2S biogenesis pathway to different cytosolic and chloroplastic Fe-S proteins, as well as to the cytosolic Fe-S biogenesis system, and that uncoupling this process triggers leaf-associated Fe-S- and Fe-deficiency responses that result in Fe over-accumulation in chloroplasts and enhanced ROS accumulation. We further show that AtNEET transfers its 2Fe-2S clusters to DRE2, a key protein of the cytosolic Fe-S biogenesis system, and propose that the availability of 2Fe-2S clusters in the chloroplast and cytosol is linked to Fe homeostasis in plants.

Zika Virus Dissemination from the Midgut of Aedes aegypti is Facilitated by Bloodmeal-Mediated Structural Modification of the Midgut Basal Lamina.

The arboviral disease cycle requires that key tissues in the arthropod vector become persistently infected with the virus. The midgut is the first organ in the mosquito that needs to be productively infected with an orally acquired virus. Following midgut infection, the virus then disseminates to secondary tissues including the salivary glands. Once these are productively infected, the mosquito is able to transmit the virus to a vertebrate host. Recently, we described the midgut dissemination pattern for chikungunya virus in Aedes aegypti. Here we assess the dissemination pattern in the same mosquito species for Zika virus (ZIKV), a human pathogenic virus belonging to the Flaviviridae. ZIKV infection of secondary tissues, indicative of dissemination from the midgut, was not observed before 72 h post infectious bloodmeal (pibm). Virion accumulation at the midgut basal lamina (BL) was only sporadic, although at 96-120 h pibm, virions were frequently observed between strands of the BL indicative of their dissemination. Our data suggest that ZIKV dissemination from the mosquito midgut occurs after digestion of the bloodmeal. Using gold-nanoparticles of 5 nm and 50 nm size, we show that meal ingestion leads to severe midgut tissue distention, causing the mesh width of the BL to remain enlarged after complete digestion of the meal. This could explain how ZIKV can exit the midgut via the BL after bloodmeal digestion. Ingestion of a subsequent, non-infectious bloodmeal five days after acquisition of an initial, dengue 4 virus containing bloodmeal resulted in an increased number of virions present in the midgut epithelium adjacent to the BL. Thus, subsequent bloodmeal ingestion by an infected mosquito may primarily stimulate de novo synthesis of virions leading to increased viral titers in the vector.

Angiotensin II suppresses autophagy and disrupts ultrastructural morphology and function of mitochondria in mouse skeletal muscle.

Angiotensin II (ANG II)-induced skeletal muscle wasting is characterized by activation of the ubiquitin-proteasome system. However, the potential involvement of proteolytic system macroautophagy/autophagy in this wasting process remains elusive. Autophagy is precisely regulated to maintain cell survival and homeostasis; thus its dysregulation (i.e., overactivation or persistent suppression) could lead to detrimental outcomes in skeletal muscle. Here we show that infusion of ANG II for 7 days in male FVB mice suppressed autophagy in skeletal muscle. ANG II blunted microtubule-associated protein 1 light chain 3B (LC3B)-I-to-LC3B-II conversion (an autophagosome marker), increased p62/SQSTM1 (an autophagy cargo receptor) protein expression, and decreased the number of autophagic vacuoles. ANG II inhibited UNC-51-like kinase 1 via inhibition of 5'-AMP-activated kinase and activation of mechanistic target of rapamycin complex 1, leading to reduced phosphorylation of beclin-1Ser14 and Autophagy-related protein 14Ser29, suggesting that ANG II impairs autophagosome formation in skeletal muscle. In line with ANG II-mediated suppression of autophagy, ANG II promoted accumulation of abnormal/damaged mitochondria, characterized by swelling and disorganized cristae and matrix dissolution, with associated increase in PTEN-induced kinase 1 protein expression. ANG II also reduced mitochondrial respiration, indicative of mitochondrial dysfunction. Together, these results demonstrate that ANG II reduces autophagic activity and disrupts mitochondrial ultrastructure and function, likely contributing to skeletal muscle wasting. Therefore, strategies that activate autophagy in skeletal muscle have the potential to prevent or blunt ANG II-induced skeletal muscle wasting in chronic diseases. NEW & NOTEWORTHY Our study identified a novel mechanism whereby angiotensin II (ANG II) impairs mitochondrial energy metabolism in skeletal muscle. ANG II suppressed autophagosome formation by inhibiting the UNC-51-like kinase 1(ULK1)-beclin-1 axis, resulting in accumulation of abnormal/damaged and dysfunctional mitochondria and reduced mitochondrial respiratory capacity. Therapeutic strategies that activate the ULK1-beclin-1 axis have the potential to delay or reverse skeletal muscle wasting in chronic diseases characterized by increased systemic ANG II levels.

Empagliflozin Ameliorates Type 2 Diabetes-Induced Ultrastructural Remodeling of the Neurovascular Unit and Neuroglia in the Female db/db Mouse.

Type 2 diabetes is associated with diabetic cognopathy. Anti-hyperglycemic sodium glucose transporter 2 (SGLT2) inhibitors have shown promise in reducing cognitive impairment in mice with type 2 diabetes mellitus. We recently described marked ultrastructural (US) remodeling of the neurovascular unit (NVU) in type 2 diabetic db/db female mice. Herein, we tested whether the SGLT-2 inhibitor, empagliflozin (EMPA), protects the NVU from abnormal remodeling in cortical gray and subcortical white matter. Ten-week-old female wild-type and db/db mice were divided into lean controls (CKC, n = 3), untreated db/db (DBC, n = 3), and EMPA-treated db/db (DBE, n = 3). Empagliflozin was added to mouse chow to deliver 10 mg kg-1 day-1 and fed for ten weeks, initiated at 10 weeks of age. Brains from 20-week-old mice were immediately immersion fixed for transmission electron microscopic study. Compared to CKC, DBC exhibited US abnormalities characterized by mural endothelial cell tight and adherens junction attenuation and/or loss, pericyte attenuation and/or loss, basement membrane thickening, glia astrocyte activation with detachment and retraction from mural cells, microglia cell activation with aberrant mitochondria, and oligodendrocyte⁻myelin splitting, disarray, and axonal collapse. We conclude that these abnormalities in the NVU were prevented in DBE. Empagliflozin may provide neuroprotection in the diabetic brain.

Multi-Focal Neuronal Ultrastructural Abnormalities and Synaptic Alterations in Mice after Low-Intensity Blast Exposure.

Service members during military actions or combat training are exposed frequently to primary blast generated by explosive weaponry. The majority of military-related neurotrauma are classified as mild and designated as "invisible injuries" that are prevalent during current conflicts. While the previous experimental blast injury studies using moderate- to high-intensity exposures focused mainly on gross and microscopic neuropathology, our previous studies have shown that low-intensity blast (LIB) exposures resulted in nanoscale subcellular myelin and mitochondrial damages and subsequent behavioral disorders in the absence of gross or detectable cellular damage. In this study, we used transmission electron microscopy to delineate the LIB effects at the ultrastructural level specifically focusing on the neuron perikaryon, axons, and synapses in the cortex and hippocampus of mice at seven and 30 days post-injury (DPI). We found dysmorphic dark neuronal perikaryon and "cytoplasmic aeration" of dendritic processes, as well as increased microtubular fragmentation of the myelinated axons along with biochemically measured elevated tau/phosphorylated tau/Aß levels. The number of cortical excitatory synapses decreased along with a compensatory increase of the post-synaptic density (PSD) thickness both at seven and 30 DPI, while the amount of hippocampal CA1 synapses increased with the reduced PSD thickness. In addition, we observed a significant increase in protein levels of PSD95 and synaptophysin mainly at seven DPI indicating potential synaptic reorganization. These results demonstrated that a single LIB exposure can lead to ultrastructural brain injury with accompanying multi-focal neuronal organelle alterations. This pre-clinical study provides key insights into disease pathogenesis related to primary blast exposure.

Inhibition of regrowth of planktonic and biofilm bacteria after peracetic acid disinfection.

Peracetic acid (PAA) is a promising alternative to chlorine for disinfection; however, bacterial regrowth after PAA disinfection is poorly understood. This study compared the regrowth of bacteria (Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bacillus sp.) after disinfection with PAA or free chlorine. In the absence of organic matter, PAA and free chlorine prevented the regrowth of planktonic cells of P. aeruginosa PAO1 at C·t (= disinfectant concentration × contact time) doses of (28.5 ±â€¯9.8) mg PAA·min·L-1 and (22.5 ±â€¯10.6) mg Cl2·min·L-1, respectively, suggesting that they had comparable efficiencies in preventing the regrowth of planktonic bacteria. For comparison, the minimum C·t doses of PAA and free chlorine to prevent the regrowth of P. aeruginosa PAO1 biofilm cells in the absence of organic matter were (14,000 ±â€¯1,732) mg PAA·min·L-1 and (6,500 ±â€¯2,291) mg Cl2·min·L-1, respectively. PAA was less effective than free chlorine in killing bacteria within biofilms in the absence of organic matter most likely because PAA reacts with biofilm matrix constituents slower than free chlorine. In the presence of organic matter, although the bactericidal efficiencies of both disinfectants significantly decreased, PAA was less affected due to its slower reaction with organic matter and/or slower self-decomposition. For instance, in a dilute Lysogeny broth-Miller, the minimum concentrations of PAA and free chlorine to prevent the regrowth of planktonic P. aeruginosa PAO1 were 20 mg PAA·L-1 and 300 mg Cl2·L-1, respectively. While both disinfectants are strong oxidants disrupting cell membrane, environmental scanning electron microscopy (ESEM) revealed that PAA made holes in the center of the cells, whereas free chlorine desiccated the cells. Overall, this study shows that PAA is a powerful disinfectant to prevent bacterial regrowth even in the presence of organic matter.

Ultrastructural Analysis of Chikungunya Virus Dissemination from the Midgut of the Yellow Fever Mosquito, Aedes aegypti.

The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24⁻32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.

Young Ossabaw Pigs Fed a Western Diet Exhibit Early Signs of Diabetic Retinopathy.

Purpose: Recent clinical data suggest an increasing prevalence of obesity and type 2 diabetes in adolescents, placing them at high risk of developing diabetic retinopathy during adult working years. The present study was designed to characterize the early retinal and microvascular alterations in young Ossabaw pigs fed a Western diet, described as a model of metabolic syndrome genetically predisposed to type 2 diabetes. Methods: Four-month-old Ossabaw miniature pigs were divided into two groups, lean and diet-induced obesity. Obese pigs were fed a Western diet with high-fat/high-fructose corn syrup/high-choleric content for 10 weeks. Blood and retina were collected for biochemical profiling, trypsin digest, flatmounts, Fluoro-Jade C staining, electron microscopy, quantitative PCR, immunohistochemistry, and Western blots. Results: Young Ossabaw pigs had elevated fasting blood glucose after feeding on a Western diet for 10 weeks. Their retina showed disrupted cellular architecture across neural layers, with numerous large vacuoles seen in cell bodies of the inner nuclear layer. Microvessels in the obese animals exhibited thickened basement membrane, along with pericyte ghosts and acellular capillaries. The pericyte to endothelial ratio decreased significantly. Retina flatmounts from obese pigs displayed reduced capillary density, numerous terminal capillary loops, and string vessels, which stained collagen IV but not isolectin IB4. Quantitative PCR and Western blots showed significantly high levels of basement membrane proteins collagen IV and fibronectin in obese pigs. Conclusions: This is the first study to describe the ultrastructural neuronal and vascular changes in the retina of young Ossabaw pigs fed a Western diet, simulating early signs of diabetic retinopathy pathogenesis.

Ultrastructural brain abnormalities and associated behavioral changes in mice after low-intensity blast exposure.

Explosive blast-induced mild traumatic brain injury (mTBI), a "signature wound" of recent military conflicts, commonly affects service members. While past blast injury studies have provided insights into TBI with moderate- to high-intensity explosions, the impact of primary low-intensity blast (LIB)-mediated pathobiology on neurological deficits requires further investigation. Our prior considerations of blast physics predicted ultrastructural injuries at nanoscale levels. Here, we provide quantitative data using a primary LIB injury murine model exposed to open field detonation of 350 g of high-energy explosive C4. We quantified ultrastructural and behavioral changes up to 30 days post blast injury (DPI). The use of an open-field experimental blast generated a primary blast wave with a peak overpressure of 6.76 PSI (46.6 kPa) at a 3-m distance from the center of the explosion, a positive phase duration of approximate 3.0 milliseconds (ms), a maximal impulse of 8.7 PSI × ms and a sharp rising time of 9 × 10-3 ms, with no apparent impact/acceleration in exposed animals. Neuropathologically, myelinated axonal damage was observed in blast-exposed groups at 7 DPI. Using transmission electron microscopy, we observed and quantified myelin sheath defects and mitochondrial abnormalities at 7 and 30 DPI. Inverse correlations between blast intensities and neurobehavioral outcomes including motor activities, anxiety levels, nesting behavior, spatial learning and memory occurred. These observations uncover unique ultrastructural brain abnormalities and associated behavioral changes due to primary blast injury and provide key insights into its pathogenesis and potential treatment.

Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion.

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24-32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus.