Secondary hypoxia is a known contributor to adverse outcomes in patients with traumatic brain injury (TBI). Based on the evidence that hypoxia and TBI in isolation induce neuroinflammation, we investigated whether TBI combined with hypoxia enhances cerebral cytokine production. We also explored whether increased concentrations of injury biomarkers discriminate between hypoxic (Hx) and normoxic (Nx) patients, correlate to worse outcome, and depend on blood-brain barrier (BBB) dysfunction. Forty-two TBI patients with Glasgow Coma Scale ≤8 were recruited. Cerebrospinal fluid (CSF) and serum were collected over 6 days. Patients were divided into Hx (n=22) and Nx (n=20) groups. Eight cytokines were measured in the CSF; albumin, S100, myelin basic protein (MBP) and neuronal specific enolase (NSE) were quantified in serum. CSF/serum albumin quotient was calculated for BBB function. Glasgow Outcome Scale Extended (GOSE) was assessed at 6 months post-TBI. Production of granulocye macrophage-colony stimulating factor (GM-CSF) was higher, and profiles of GM-CSF, interferon (IFN)-γ and, to a lesser extent, tumor necrosis factor (TNF), were prolonged in the CSF of Hx but not Nx patients at 4-5 days post-TBI. Interleukin (IL)-2, IL-4, IL-6, and IL-10 increased similarly in both Hx and Nx groups. S100, MBP, and NSE were significantly higher in Hx patients with unfavorable outcome. Among these three biomarkers, S100 showed the strongest correlations to GOSE after TBI-Hx. Elevated CSF/serum albumin quotients lasted for 5 days post-TBI and displayed similar profiles in Hx and Nx patients. We demonstrate for the first time that post-TBI hypoxia is associated with prolonged neuroinflammation, amplified extravasation of biomarkers, and poor outcome. S100 and MBP could be implemented to track the occurrence of post-TBI hypoxia, and prompt adequate treatment.

Pregnane steroids have sedative and neuroprotective effects on the brain, due to interactions with the steroid-binding site of the GABA A receptor. In the adult brain, synthesis of the pregnane steroids is increased in response to stress. Therefore, we have used umbilicoplacental embolization to mimic chronic placental insufficiency during late gestation in sheep, to investigate the expression of the steroidogenic enzymes P450scc, 5␣-reductase type I (5␣RI), 5␣-reductase type II (5␣RII), and allopregnanolone (AP) content in the fetal brain. Umbilicoplacental embolization was induced from 114 d gestation (term~147 d) by daily injection of inert microspheres into the umbilical artery and continued for 17-23 d. Fetal arterial oxygen saturation was reduced to~60% of the preembolization value in each fetus, with a significant reduction in blood arterial PO 2 , pH, and plasma glucose concentrations (p Ͻ 0.05) and a significant increase in blood arterial PCO 2 and plasma lactate concentrations (p Ͻ 0.05). At postmortem at 131-137 d gestation, embolized fetuses were growth-restricted (2.10 Ϯ 0.14 kg, n ϭ 5) compared with agematched controls (4.43 Ϯ 0.56 kg, n ϭ 7, p Ͻ 0.05). Umbilicoplacental embolized fetuses showed increased P450scc expression in the primary motor cortex; 5␣RI expression was not changed in any of the regions examined, whereas 5␣RII expression was markedly increased in all brain regions. Brain AP content did not significantly change, whereas plasma concentrations were increased. These findings suggest that the increased expression of P450scc and 5␣RII may be a response that maintains AP concentration in the fetal brain after compromised placental function and/or intrauterine stress. Chronic hypoxemia and placental insufficiency are associated with fetal growth retardation (1). Generally, the brains of these growth-retarded fetuses are either not smaller, or are reduced to a lesser degree compared with the overall size of the fetus, suggesting that the brain is able to adapt to the intrauterine conditions that compromise fetal growth. On the other hand, growth-retarded and premature infants are at a greater risk of perinatal brain damage (2), indicating that some of these adaptive changes leave the brain more vulnerable to cytotoxic damage arising from a range of insults, such as acute hypoxia, asphyxia, or infection-induced inflammation.Pregnane steroids, including AP, modify the excitability of the CNS by interaction with the GABA A receptor at the steroid binding site. In the adult, AP has been shown to have potent anxiolytic (3, 4), anticonvulsant (5, 6), sedative/hypnotic, and anesthetic effects (7) on behavior. A constitutive role for neuroactive steroids in the developing brain was proposed after the observation that progesterone metabolites such as AP appear to maintain the low level of arousal-like behavior that typifies fetal life (8,9). In the rat, stressful stimuli of handling or swim stress have been shown to increase plasma AP content (10, 11), suggesting that modulation of the GABA A rece...

Pregnane steroids have sedative and neuroprotective effects on the brain as a result of interactions with the steroid-binding site of the GABA A receptor. To determine whether the fetal brain is able to synthesize pregnane steroids de novo from cholesterol, we measured the expression of cytochrome P450 side-chain cleavage (P450scc) and 5␣-reductase type II (5␣RII) enzymes in fetal sheep from 72 to 144 d gestation (term~147 d) and in newborn lambs at 3 and 19-26 d of age. Both P450scc and 5␣RII expression was detectable by 90 d gestation in the major regions of the brain and also in the adrenal glands. Expression increased with advancing gestation and was either maintained at fetal levels or increased further after birth. In contrast, the relatively high content (200-400 pmol/g) of allopregnanolone (5␣-pregnan-3␣-ol-20-one), a major sedative 5␣-pregnane steroid, present throughout the brain from 90 d gestation to term, was reduced significantly (Ͻ50 pmol/g) immediately after birth. These results suggest that although the perinatal brain has the enzymes potentially to synthesize pregnane steroids de novo from cholesterol, either the placenta is a major source of these steroids to the brain or other factors associated with intrauterine life may be responsible for high levels of allopregnanolone production in the fetal brain until birth. Abbreviations AP, allopregnanolone, 5␣-pregnan-3␣-ol-20-one P450scc, P450 side chain cleavage enzyme 5␣RII, 5␣-reductase type II enzyme PMC, primary motor cortex GABA A ,␥-aminobutyric acid/benzodiazepine receptor 5␣-DHP, 5␣-dihydroprogesterone Neuroactive steroids such as allopregnanolone (AP) are potent neuromodulators that modify the excitability of the CNS by interaction with the ␥-aminobutyric acid/benzodiazepine receptor-chloride ionophore (GABA A receptor). In the adult AP is a positive allosteric modulator of the GABA A receptor with potent anxiolytic (1, 2), anticonvulsant (3, 4), sedative/ hypnotic, and anesthetic effects (5) on behavior. Prenatally, neuroactive steroids have been shown to suppress fetal activity in late gestation and seem to have a role in maintaining the low level of arousal-like behavior that typifies fetal life (6, 7). This interaction between AP and the GABA A receptor complex is responsible for the major pharmacologic actions of AP and is distinct from the genomic effects exerted by other steroids such as progesterone (8). In the brain, neuroactive steroids such as AP are synthesized de novo from cholesterol, but a proportion of the pool of steroids may be derived from precursors in blood that enter the brain across the blood-brain barrier (9 -11). Thus, peripheral steroidogenesis may influence neuroactive steroid content in the brain.The cytochrome P450 side-chain cleavage enzyme (P450scc) catalyzes the irreversible conversion of cholesterol to pregnenolone on the inner side of the mitochondrial membrane (12). The primary site of pregnenolone synthesis in the brain seems to be in oligodendrocytes and astrocytes, with significantly less synthesis occ...

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

Oxidative stress plays a significant role in secondary damage after severe traumatic brain injury (TBI); and melatonin exhibits both direct and indirect antioxidant effects. Melatonin deficiency is deleterious in TBI animal models, and its administration confers neuroprotection, reducing cerebral oedema, and improving neurobehavioural outcome. This study aimed to measure the endogenous cerebrospinal fluid (CSF) and serum melatonin levels post-TBI in humans and to identify relationships with markers of oxidative stress via 8-isoprostaglandin-F 2a (isoprostane), brain metabolism and neurologic outcome. Cerebrospinal fluid and serum samples of 39 TBI patients were assessed for melatonin, isoprostane, and various metabolites. Cerebrospinal fluid but not serum melatonin levels were markedly elevated (7.28±0.92 versus 1.47±0.35 pg/mL, P < 0.0005). Isoprostane levels also increased in both CSF (127.62 ± 16.85 versus 18.28 ± 4.88 pg/mL, P < 0.0005) and serum (562.46±50.78 versus 126.15±40.08 pg/mL (P < 0.0005). A strong correlation between CSF melatonin and CSF isoprostane on day 1 after injury (r = 0.563, P = 0.002) suggests that melatonin production increases in conjunction with lipid peroxidation in TBI. Relationships between CSF melatonin and pyruvate (r = 0.369, P = 0.049) and glutamate (r = 0.373, P = 0.046) indicate that melatonin production increases with metabolic disarray. In conclusion, endogenous CSF melatonin levels increase after TBI, whereas serum levels do not. This elevation is likely to represent a response to oxidative stress and metabolic disarray, although further studies are required to elucidate these relationships.

Allopregnanolone (AP) is a potent modulator of the GABA A receptor. Brain AP concentrations increase in response to stress, which is thought to provide neuroprotection by reducing excitation in the adult brain. Umbilical cord occlusion (UCO) causes hypoxia and asphyxia in the fetus, which are major risk factors associated with poor neurological outcome for the neonate, and may lead to adverse sequelae such as cerebral palsy. The aims of this study were as follows: (i) to determine the effect of 10 min UCO on AP concentrations in the extracellular fluid of the fetal brain using microdialysis, and (ii) to compare the content of the steroidogenic enzymes P450scc and 5α-reductase type II (5αRII) with brain and CSF neurosteroid concentrations. UCO caused fetal asphyxia, hypertension, bradycardia and respiratory acidosis, which returned to normal levels after 1-2 h. AP concentrations in dialysate samples from probes implanted in grey and white matter of the parietal cortex were significantly increased 1 h after UCO from control levels of 10.4 ± 0.4 and 12.4 ± 0.3 to 26.0 ± 5.1 and 27.6 ± 6.4 nmol l −1 , respectively (P < 0.05), before returning to pre-occlusion levels by 3-4 h after UCO. When fetal brains were collected 1 h after a 10 min UCO, the relative increases of AP and pregnenolone content in the parietal cortex were similar to the increase observed in the extracellular (dialysate) fluid. AP, but not pregnenolone, was increased in CSF at this time. P450scc and 5αRII enzyme expression was significantly increased in the cerebral cortex in the UCO fetuses compared to control fetuses. These results suggest that the fetal brain is capable of transiently increasing neurosteroid production in response to asphyxia. The action of the increased neurosteroid content at GABA A receptors may serve to diminish the increased excitation due to excitotoxic amino acid release, and provide short-term protection to brain cells during such stress.

Melatonin is an endogenous hormone mainly produced by the pineal gland whose dysfunction leads to abnormal sleeping patterns. Changes in melatonin have been reported in acute traumatic brain injury (TBI); however, the impact of environmental conditions typical of the intensive care unit (ICU) has not been assessed. The aim of this study was to compare daily melatonin production in three patient populations treated at the ICU to differentiate the role of TBI versus ICU conditions. Forty-five patients were recruited and divided into severe TBI, trauma without TBI, medical conditions without trauma, and compared to healthy volunteers. Serum melatonin levels were measured at four daily intervals at 0400 h, 1000 h, 1600 h, and 2200 h for 7 days post-ICU admission by commercial enzyme linked immunosorbent assay. The geometric mean concentrations (95% confidence intervals) of melatonin in these groups showed no difference being 8.3 (6.3–11.0), 9.3 (7.0–12.3), and 8.9 (6.6–11.9) pg/mL, respectively, in TBI, trauma, and intensive care cohorts. All of these patient groups demonstrated decreased melatonin concentrations when compared to control patients. This study suggests that TBI as well as ICU conditions, may have a role in the dysfunction of melatonin. Monitoring and possibly substituting melatonin acutely in these settings may assist in ameliorating long-term sleep dysfunction in all of these groups, and possibly contribute to reducing secondary brain injury in severe TBI.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.