Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.

Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue.

Internal sensory neurons innervate body organs and provide information about internal state to the CNS to maintain physiological homeostasis. Despite their conservation across species, the anatomy, circuitry, and development of internal sensory systems are still relatively poorly understood. A largely unstudied population of larval Drosophila sensory neurons, termed tracheal dendrite (td) neurons, innervate internal respiratory organs and may serve as a model for understanding the sensing of internal states. Here, we characterize the peripheral anatomy, central axon projection, and diversity of td sensory neurons. We provide evidence for prominent expression of specific gustatory receptor genes in distinct populations of td neurons, suggesting novel chemosensory functions. We identify two anatomically distinct classes of td neurons. The axons of one class project to the subesophageal zone (SEZ) in the brain, whereas the other terminates in the ventral nerve cord (VNC). We identify expression and a developmental role of the POU-homeodomain transcription factor Pdm3 in regulating the axon extension and terminal targeting of SEZ-projecting td neurons. Remarkably, ectopic Pdm3 expression is alone sufficient to switch VNC-targeting axons to SEZ targets, and to induce the formation of putative synapses in these ectopic target zones. Our data thus define distinct classes of td neurons, and identify a molecular factor that contributes to diversification of axon targeting. These results introduce a tractable model to elucidate molecular and circuit mechanisms underlying sensory processing of internal body status and physiological homeostasis.SIGNIFICANCE STATEMENT How interoceptive sensory circuits develop, including how sensory neurons diversify and target distinct central regions, is still poorly understood, despite the importance of these sensory systems for maintaining physiological homeostasis. Here, we characterize classes of Drosophila internal sensory neurons (td neurons) and uncover diverse axonal projections and expression of chemosensory receptor genes. We categorize td neurons into two classes based on dichotomous axon target regions, and identify the expression and role of the transcription factor Pdm3 in mediating td axon targeting to one of these target regions. Our results provide an entry point into studying internal sensory circuit development and function, and establish Pdm3 as a regulator of interoceptive axon targeting.

Background Neurological deficits in hypoxic-ischemic encephalopathy, even with therapeutic hypothermia, are partially attributed to white matter injury. We theorized that proteasome insufficiency contributes to white matter injury. Methods and Results Neonatal piglets received hypoxia-ischemia ( HI ) or sham procedure with normothermia, hypothermia, or hypothermia+rewarming. Some received a proteasome activator drug (oleuropein) or white matter-targeted, virus-mediated proteasome knockdown. We measured myelin oligodendrocyte glycoprotein, proteasome subunit 20S (P20S), proteasome activity, and carbonylated and ubiquitinated protein levels in white matter and cerebral cortex. HI reduced myelin oligodendrocyte glycoprotein levels regardless of temperature, and myelin oligodendrocyte glycoprotein loss was associated with increased ubiquitinated and carbonylated protein levels. Ubiquitinated and carbonyl-damaged proteins increased in white matter 29 hours after HI during hypothermia to exceed levels at 6 to 20 hours. In cortex, ubiquitinated proteins decreased. Ubiquitinated and carbonylated protein accumulation coincided with lower P20S levels in white matter; P20S levels also decreased in cerebral cortex. However, proteasome activity in white matter lagged behind that in cortex 29 hours after HI during hypothermia. Systemic oleuropein enhanced white matter P20S and protected the myelin, whereas proteasome knockdown exacerbated myelin oligodendrocyte glycoprotein loss and ubiquitinated protein accumulation after HI . At the cellular level, temperature and HI interactively affected macroglial P20S enrichment in subcortical white matter. Rewarming alone increased macroglial P20S immunoreactivity, but this increase was blocked by HI . Conclusions Oxidized and ubiquitinated proteins accumulate with HI -induced white matter injury. Proteasome insufficiency may drive this injury. Hypothermia did not prevent myelin damage, protect the proteasome, or preserve oxidized and ubiquitinated protein clearance after HI .

This study aimed at investigating the anti-obesity and anti-dyslipidemic effects of Stachys sieboldii Miq. root (SS) powder in rats following a high-fat and high-cholesterol (HFC) diet for 6 weeks. Thirty-two Sprague-Dawley rats were fed one of the following diets: a regular diet (RD), HFC, HFC supplemented with 3% SS (HFC + 3SS) or HFC supplemented with 5% SS (HFC + 5SS). Following an HFC diet increased body weight (BW) gain (p < 0.001) and the food efficiency ratio (FER; p < 0.001); however, SS consumption gradually prevented the HFC-induced BW gain (p < 0.001) and increase in FER (p < 0.01). The HFC diet resulted in increased liver size (p < 0.001) and total adipose tissue weight (p < 0.001), whereas the SS supplementation decreased hepatomegaly (p < 0.05) and body fat mass (p < 0.001). SS consumption prevented the increased activities of serum alanine aminotransferase (ALT; p < 0.001), aspartate aminotransferase (AST; p < 0.001), alkaline phosphatase (ALP; p < 0.01 in HFC + 5SS) and lactate dehydrogenase (LDH; p < 0.001 in HFC + 5SS) induced by the HFC diet (p < 0.001). The SS supplementation improved lipid profiles in the circulation by lowering triglyceride (TG; p < 0.01), total cholesterol (TC; p < 0.001) and non-HDL cholesterol (non-HDL-C; p < 0.001) levels, as well as the atherogenic index (p < 0.01) and cardiac risk factor (p < 0.01). The lipid distribution in the liver (p < 0.05) and white adipose tissues (WAT; p < 0.001) of the HFC + SS diet-consuming rats was remarkably lower than that of the HFC diet-consuming rats. The average size of the epididymal adipose tissue (p < 0.001) was significantly lower in the HFC + SS diet-fed rats than in the HFC diet-fed rats. The fecal lipid (>3% SS; p < 0.001) and cholesterol (5% SS; p < 0.001) efflux levels were significantly elevated by the SS supplementation compared to those measured in the RD or HFC diet-fed groups. In addition, the hepatic lipid and cholesterol metabolism-related gene expressions were affected by SS consumption, as the hepatic anabolic gene expression (Acc; p < 0.001, Fas; p < 0.001 and G6pdh; p < 0.01) was significantly attenuated. The HFC + 5SS diet-fed rats exhibited elevated hepatic Cyp7a1 (p < 0.001), Hmgcr (p < 0.001) and Ldlr (p < 0.001) mRNA expression levels compared to the HFC diet-fed rats. These results suggest that SS may possess anti-adipogenic and lipid-lowering effects by enhancing lipid and cholesterol efflux in mammals.

Neonatal brain injury from hypoxia-ischemia (HI) causes major morbidity. Piglet HI is an established method for testing neuroprotective treatments in large, gyrencephalic brain. Though many neurobehavior tests exist for rodents, such tests and their associations with neuropathologic injury remain underdeveloped and underutilized in large, neonatal HI animal models. We examined whether spatial T-maze and inclined beam tests distinguish cognitive and motor differences between HI and sham piglets and correlate with neuropathologic injury. Neonatal piglets were randomized to whole-body HI or sham procedure, and they began T-maze and inclined beam testing 17 days later. HI piglets had more incorrect T-maze turns than did shams. Beam walking time did not differ between groups. Neuropathologic evaluations at 33 days validated the injury with putamen neuron loss after HI to below that of sham procedure. HI decreased the numbers of CA3 pyramidal neurons but not CA1 pyramidal neurons or dentate gyrus granule neurons. Though the number of hippocampal parvalbumin-positive interneurons did not differ between groups, HI reduced the number of CA1 interneuron dendrites. Piglets with more incorrect turns had greater CA3 neuron loss, and piglets that took longer in the maze had fewer CA3 interneurons. The number of putamen neurons was unrelated to T-maze or beam performance. We conclude that neonatal HI causes hippocampal CA3 neuron loss, CA1 interneuron dendritic attrition, and putamen neuron loss at 1-month recovery. The spatial T-maze identifies learning and memory deficits that are related to loss of CA3 pyramidal neurons and fewer parvalbumin-positive interneurons independent of putamen injury.

This study investigated the effects of partial replacement of dietary fat with krill oil (KO) or coconut oil (CO) on dyslipidemia and lipid metabolism in rats fed with a high-fat diet (HFD). Sprague Dawley rats were divided into three groups as follows: HFD, HFD + KO, and HFD + CO. The rats were fed each diet for 10 weeks and then intraperitoneally injected with phosphate-buffered saline (PBS) or lipopolysaccharide (LPS) (1 mg/kg). The KO- and CO-fed rats exhibited lower levels of serum lipids and aspartate aminotransferases than those of the HFD-fed rats. Rats fed with HFD + KO displayed significantly lower hepatic histological scores and hepatic triglyceride (TG) content than rats fed with HFD. The KO supplementation also downregulated the adipogenic gene expression in the liver. When treated with LPS, the HFD + KO and HFD + CO groups reduced the adipocyte size in the epididymal white adipose tissues (EAT) relative to the HFD group. These results suggest that KO and CO could improve lipid metabolism dysfunction.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes significant morbidity despite treatment with therapeutic hypothermia. Mitochondrial dysfunction may drive the mechanisms underlying neuronal cell death, thereby making mitochondria prime targets for neuroprotection. The mitochondrial permeability transition pore (mPTP) is one such target within mitochondria. In adult animal models, mPTP inhibition is neuroprotective. However, evidence for mPTP inhibition in neonatal models of neurologic disease is less certain. We tested the therapeutic efficacy of the mPTP small molecule inhibitor GNX-4728 and examined the developmental presence of brain mPTP proteins for drug targeting in a neonatal piglet model of hypoxic-ischemic brain injury. Male neonatal piglets were randomized to hypoxia-ischemia (HI) or sham procedure with GNX-4728 (15 mg/kg, IV) or vehicle (saline/cyclodextrin/DMSO, IV). GNX-4728 was administered as a single dose within 5 min after resuscitation from bradycardic arrest. Normal, ischemic, and injured neurons were counted in putamen and somatosensory cortex using hematoxylin and eosin staining. In separate neonatal and juvenile pigs, western blots of putamen mitochondrial-enriched fractions were used to evaluate mitochondrial integrity and the presence of mPTP proteins. We found that a single dose of GNX-4728 did not protect putamen and cortical neurons from cell death after HI. However, loss of mitochondrial matrix integrity occurred within 6h after HI, and while mPTP components are present in the neonatal brain their levels were significantly different compared to that of a mature juvenile brain. Thus, the neonatal brain mPTP may not be a good target for current neurotherapeutic drugs that are developed based on adult mitochondria.

Dry eye syndrome (DES) is a corneal disease often characterized by an irritating, itching feeling in the eyes and light sensitivity. Inflammation and endoplasmic reticulum (ER) stress may play a crucial role in the pathogenesis of DES, although the underlying mechanism remains elusive. Aster koraiensis has been used traditionally as an edible herb in Korea. It has been reported to have wound-healing and inhibitory effects against insulin resistance and inflammation. Here, we examined the inhibitory effects of inflammation and ER stress by A. koraiensis extract (AKE) in animal model and human retinal pigmented epithelial (ARPE-19) cells. Oral administration of AKE mitigated DE symptoms, including reduced corneal epithelial thickness, increased the gap between lacrimal gland tissues in experimental animals and decreased tear production. It also inhibited inflammatory responses in the corneal epithelium and lacrimal gland. Consequently, the activation of NF-κB was attenuated by the suppression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Moreover, AKE treatment ameliorated TNF-α-inducible ocular inflammation and thapsigargin (Tg)-inducible ER stress in animal model and human retinal pigmented epithelial (ARPE-19) cells. These results prove that AKE prevents detrimental functional and histological remodeling on the ocular surface and in the lacrimal gland through inhibition of inflammation and ER stress, suggesting its potential as functional food material for improvement of DES.

Intestinal iron transport requires an iron importer (Dmt1) and an iron exporter (Fpn1). The hormone hepcidin regulates iron absorption by modulating Fpn1 protein levels on the basolateral surface of duodenal enterocytes. In the genetic, iron-loading disorder hereditary hemochromatosis (HH), hepcidin production is low and Fpn1 protein expression is elevated. High Fpn1-mediated iron export depletes intracellular iron, causing a paradoxical increase in Dmt1-mediated iron import. Increased activity of both transporters causes excessive iron absorption, thus initiating body iron loading. Logically then, silencing of intestinal Dmt1 or Fpn1 could be an effective therapeutic intervention in HH. It was previously established that Dmt1 knock down prevented iron-loading in weanling Hamp (encoding hepcidin) KO mice (modeling type 2B HH). Here, we tested the hypothesis that Dmt1 silencing combined with dietary iron restriction (which may be recommended for HH patients) will mitigate iron loading once already established. Accordingly, adult Hamp KO mice were switched to a low-iron (LFe) diet and (non-toxic) folic acid-coupled, ginger nanoparticle-derived lipid vectors (FA-GDLVs) were used to deliver negative-control (NC) or Dmt1 siRNA by oral, intragastric gavage daily for 21 days. The LFe diet reduced body iron burden, and experimental interventions potentiated iron losses. For example, Dmt1 siRNA treatment suppressed duodenal Dmt1 mRNA expression (by ~50%) and reduced serum and liver non-heme iron levels (by ~60% and >85%, respectively). Interestingly, some iron-related parameters were repressed similarly by FA-GDLVs carrying either siRNA, including 59Fe (as FeCl3) absorption (~20% lower), pancreatic non-heme iron (reduced by ~65%), and serum ferritin (decreased 40-50%). Ginger may thus contain bioactive lipids that also influence iron homeostasis. In conclusion, the combinatorial approach of FA-GDLV and Dmt1 siRNA treatment, with dietary iron restriction, mitigated pre-existing iron overload in a murine model of HH.

Existing cerebrovascular blood pressure autoregulation metrics have not been translated to clinical care for pediatric cardiac arrest, in part because signal noise causes high index time-variability. We tested whether a wavelet method that uses near-infrared spectroscopy (NIRS) or intracranial pressure (ICP) decreases index variability compared to that of commonly used correlation indices. We also compared whether the methods identify the optimal arterial blood pressure (ABPopt) and lower limit of autoregulation (LLA). 68 piglets were randomized to cardiac arrest or sham procedure with continuous monitoring of cerebral blood flow using laser Doppler, NIRS and ICP. The arterial blood pressure (ABP) was gradually reduced until it dropped to below the LLA. Several autoregulation indices were calculated using correlation and wavelet methods, including the pressure reactivity index (PRx and wPRx), cerebral oximetry index (COx and wCOx), and hemoglobin volume index (HVx and wHVx). Wavelet methodology had less index variability with smaller standard deviations. Both wavelet and correlation methods distinguished functional autoregulation (ABP above LLA) from dysfunctional autoregulation (ABP below the LLA). Both wavelet and correlation methods also identified ABPopt with high agreement. Thus, wavelet methodology using NIRS may offer an accurate vasoreactivity monitoring method with reduced signal noise after pediatric cardiac arrest.

Cardiac arrest (CA) is the most common cause of acute neurologic insult in children. Many survivors have significant neurocognitive deficits at 1 year of recovery. Epoxyeicosatrienoic acids (EETs) are multifunctional endogenous lipid signaling molecules that are involved in brain pathobiology and may be therapeutically relevant. However, EETs are rapidly metabolized to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH), limiting their bioavailability. We hypothesized that sEH inhibition would improve outcomes after CA in an infant swine model. Male piglets (3-4 kg, 2 weeks old) underwent hypoxic-asphyxic CA. After resuscitation, they were randomized to intravenous treatment with an sEH inhibitor (TPPU, 1 mg/kg; n = 8) or vehicle (10% poly(ethylene glycol); n = 9) administered at 30 min and 24 h after return of spontaneous circulation. Two sham-operated groups received either TPPU (n = 9) or vehicle (n = 8). Neurons were counted in hematoxylin- and eosin-stained sections from putamen and motor cortex in 4-day survivors.

The effects of hypothermia on neonatal encephalopathy may vary topographically and cytopathologically in the neocortex with manifestations potentially influenced by seizures that alter the severity, distribution, and type of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia (HT) with continuous electroencephalography (cEEG) for seizures. Neonatal male piglets received HI-normothermia (NT), HI-HT, sham-NT, or sham-HT treatments. Randomized unmedicated sham and HI piglets underwent cEEG during recovery. Survival was 2-7 days. Normal and pathological neurons were counted in different neocortical areas, identified by cytoarchitecture and connectomics, using hematoxylin and eosin staining and immunohistochemistry for RNA-binding FOX-1 homolog 3 (Rbfox3/NeuN). Seizure burden was determined. HI-NT piglets had a reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to sham-NT and sham-HT piglets with differing severities in the anterior and posterior motor, somatosensory, and frontal cortices. Neocortical neuropathology was attenuated by HT. HT protection was prominent in layer III of the inferior parietal cortex. Rbfox3 immunoreactivity distinguished cortical neurons as: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had an increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT piglets. Neuronal Rbfox3 depletion was partly rescued by HT. Seizure burdens in HI-NT and HI-HT piglets were similar. We conclude that the neonatal HI piglet neocortex has: (1) suprasylvian vulnerability to HI and seizures; (2) a limited neuronal cytopathological repertoire in functionally different regions that engages protective mechanisms with HT; (3) higher seizure burden, insensitive to HT, that is correlated with more panlaminar ischemic-necrotic neurons in the somatosensory cortex; and (4) pathological RNA splicing protein nuclear depletion that is sensitive to HT. This work demonstrates that HT protection of the neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores neuronal depletion of RNA splicing factor.

End-tidal carbon dioxide (ETCO2) correlates with systemic blood flow and resuscitation rate during cardiopulmonary resuscitation (CPR) and may potentially direct chest compression performance. We compared ETCO2-directed chest compressions with chest compressions optimized to pediatric basic life support guidelines in an infant swine model to determine the effect on rate of return of spontaneous circulation (ROSC).

The specific cytopathology that causes abnormal fractional anisotropy (FA) and mean diffusivity (MD) from diffusion tensor imaging (DTI) after neonatal hypoxia-ischemia (HI) is not completely understood. The panoply of cell types in the brain might contribute differentially to changes in DTI metrics. Because glia are the predominant cell type in brain, we hypothesized that changes in FA and MD would signify perturbations in glial microstructure. Using a 3-Tesla clinical scanner, we conducted in vivo DTI MRI in nine neonatal piglets at 20-96 h after excitotoxic brain injury from striatal quinolinic acid injection or global HI. FA and MD from putamen, caudate, and internal capsule in toto were correlated with astrocyte swelling, neuronal excitotoxicity, and white matter injury. Low FA correlated with more swollen astrocytes immunophenotyped by aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), and glutamate transporter-1 (GLT-1). Low FA was also related to the loss of neurons with perineuronal GLT-1+ astrocyte decorations, large myelin swellings, lower myelin density, and oligodendrocyte cell death identified by 2',3'-cyclic nucleotide 3'-phosphodiesterase, bridging integrator-1, and nuclear morphology. MD correlated with degenerating oligodendrocytes and depletion of normal GFAP+ astrocytes but not with astrocyte or myelin swelling. We conclude that FA is associated with cytotoxic edema in astrocytes and oligodendrocyte processes as well as myelin injury at the cellular level. MD can detect glial cell death and loss, but it may not discern subtle pathology in swollen astrocytes, oligodendrocytes, or myelin. This study provides a cytopathologic basis for interpreting DTI in the neonatal brain after HI.

Zinc (Zn) has been reported to mediate leptin secretion, and thus leptin can be an important candidate molecule linking Zn with bone formation. The present study investigated whether zinc deficiency induces leptin secretion by activating a JAK2/STAT3 signaling pathway and leads to osteoblastic apoptosis. MC3T3-E1 cells were incubated for 24 h in normal osteogenic differentiation medium (OSM) or OSM treated with either 1 μM (Low Zn) or 15 μM (High Zn) of ZnCl2 containing 5 μM TPEN (Zn chelator). Our results demonstrated that low Zn stimulated extracellular leptin secretion and increased mRNA and protein expression of leptin in osteoblastic MC3T3-E1 cells. The OB-Rb (long isoform of leptin receptor) expressions were also elevated in osteoblasts under depletion of Zn. Leptin-signaling proteins, JAK2 and p-JAK2 in the cytosol of low Zn osteoblast conveyed leptin signaling, which ultimately induced higher p-STAT3 expression in the nucleus. Apoptotic effects of JAK2/STAT3 pathway were shown by increased caspase-3 in low Zn osteoblasts as well as apoptotic morphological features observed by TEM. Together, these data suggest that low Zn modulates leptin secretion by activating JAK2/STAT3 signaling pathway and induces apoptosis of osteoblastic MC3T3-E1 cells.

Mucosal damage, barrier breach, inflammation, and iron-deficiency anemia (IDA) typify ulcerative colitis (UC) in humans. The anemia in UC appears to mainly relate to systemic inflammation. The pathogenesis of this 'anemia of inflammation' (AI) involves cytokine-mediated transactivation of hepatic Hamp (encoding the iron-regulatory hormone, hepcidin). In AI, high hepcidin represses iron absorption (and iron release from stores), thus lowering serum iron, and restricting iron for erythropoiesis (causing anemia). In less-severe disease states, inflammation may be limited to the intestine, but whether this perturbs iron homeostasis is uncertain. We hypothesized that localized gut inflammation will increase overall iron demand (to support the immune response and tissue repair), and that hepatic Hamp expression will decrease in response, thus derepressing (i.e., enhancing) iron absorption. Accordingly, we developed a rat model of mild, acute colitis, and studied iron absorption and homeostasis. Rats exposed (orally) to DSS (4%) for 7 days had intestinal (but not systemic) inflammation, and biomarker analyses demonstrated that iron utilization was elevated. Iron absorption was enhanced (by 2-3-fold) in DSS-treated, WT rats of both sexes, but unexpectedly, hepatic Hamp expression was not suppressed. Therefore, to gain a better understanding of regulation of iron absorption during acute colitis, Hamp KO rats were used for further experimentation. The severity of DSS-colitis was similar in Hamp KOs as in WT controls. In the KOs, increased iron requirements associated with the physiological response to colitis were satisfied by mobilizing hepatic storage iron, rather than by increasing absorption of enteral iron (as occurred in WT rats). In conclusion then, in both sexes and genotypes of rats, iron absorption was appropriately modulated to match physiological demand for dietary iron during acute intestinal inflammation, but regulatory mechanisms may not involve hepcidin.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection. It produces an unusual intracellular infection in which a vegetative form, called the reticulate body (RB), replicates and then converts into an elementary body (EB), which is the infectious form. Here we use quantitative three-dimensional electron microscopy (3D EM) to show that C. trachomatis RBs divide by binary fission and undergo a sixfold reduction in size as the population expands. Conversion only occurs after at least six rounds of replication, and correlates with smaller RB size. These results suggest that RBs only convert into EBs below a size threshold, reached by repeatedly dividing before doubling in size. A stochastic mathematical model shows how replication-dependent RB size reduction produces delayed and asynchronous conversion, which are hallmarks of the Chlamydia developmental cycle. Our findings support a model in which RB size controls the timing of RB-to-EB conversion without the need for an external signal.

Dysfunctional cerebrovascular autoregulation may contribute to neurologic injury in neonatal hypoxic-ischemic encephalopathy (HIE). Identifying the optimal mean arterial blood pressure (MAPopt) that best supports autoregulation could help identify hemodynamic goals that support neurologic recovery. In neonates who received therapeutic hypothermia for HIE, we hypothesized that the wavelet hemoglobin volume index (wHVx) would identify MAPopt and that blood pressures closer to MAPopt would be associated with less brain injury on MRI. We also tested a correlation-derived hemoglobin volume index (HVx) and single- and multi-window data processing methodology. Autoregulation was monitored in consecutive 3-h periods using near infrared spectroscopy in an observational study. The neonates had a mean MAP of 54 mmHg (standard deviation: 9) during hypothermia. Greater blood pressure above the MAPopt from single-window wHVx was associated with less injury in the paracentral gyri (p = 0.044; n = 63), basal ganglia (p = 0.015), thalamus (p = 0.013), and brainstem (p = 0.041) after adjustments for sex, vasopressor use, seizures, arterial carbon dioxide level, and a perinatal insult score. Blood pressure exceeding MAPopt from the multi-window, correlation HVx was associated with less injury in the brainstem (p = 0.021) but not in other brain regions. We conclude that applying wavelet methodology to short autoregulation monitoring periods may improve the identification of MAPopt values that are associated with brain injury. Having blood pressure above MAPopt with an upper MAP of ~50-60 mmHg may reduce the risk of brain injury during therapeutic hypothermia. Though a cause-and-effect relationship cannot be inferred, the data support the need for randomized studies of autoregulation and brain injury in neonates with HIE.

Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically relevant neonatal piglet model. Protecting and activating proteasomes could be an adjunct therapy to hypothermia. We investigated whether chymotrypsin-like proteasome activity differs regionally and developmentally in the neonatal brain. We also tested whether neonatal brain proteasomes can be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by targeting a proteasome subunit gene using recombinant adeno-associated virus-9 (AAV). During post-HI hypothermia, we treated piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ using AAV-PA28γ with green fluorescent protein (GFP). Neonatal neocortex and subcortical white matter had greater proteasome activity than did liver and kidney. Neonatal white matter had higher proteasome activity than did juvenile white matter. Lower arterial pH 1 h after HI correlated with greater subsequent cortical proteasome activity. With increasing brain homogenate protein input into the assay, the initial proteasome activity increased only among shams, whereas HI increased total kinetic proteasome activity. OLE increased the initial neocortical proteasome activity after hypothermia. AAV drove GFP expression, and white matter PA28γ levels correlated with proteasome activity and subunit levels. However, AAV proteasome modulation varied. Thus, neonatal neocortical proteasomes can be pharmacologically activated. HI slows the initial proteasome performance, but then augments ongoing catalytic activity. AAV-mediated genetic manipulation in the piglet brain holds promise, though proteasome gene targeting requires further development.