Luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, reverses cerebrovascular dysfunction and cognitive impairments in 18-mo-old diabetic animals.

Diabetes mellitus (DM) is a leading risk factor for age-related dementia, but the mechanisms involved are not well understood. We previously discovered that hyperglycemia induced impaired myogenic response (MR) and cerebral blood flow (CBF) autoregulation in 18-mo-old DM rats associated with blood-brain barrier (BBB) leakage, impaired neurovascular coupling, and cognitive impairment. In the present study, we examined whether reducing plasma glucose with a sodium-glucose cotransporter-2 inhibitor (SGLT2i) luseogliflozin can ameliorate cerebral vascular and cognitive function in diabetic rats. Plasma glucose and HbA1c levels of 18-mo-old DM rats were reduced, and blood pressure was not altered after treatment with luseogliflozin. SGLT2i treatment restored the impaired MR of middle cerebral arteries (MCAs) and parenchymal arterioles and surface and deep cortical CBF autoregulation in DM rats. Luseogliflozin treatment also rescued neurovascular uncoupling, reduced BBB leakage and cognitive deficits in DM rats. However, SGLT2i did not have direct constrictive effects on vascular smooth muscle cells and MCAs isolated from normal rats, although it decreased reactive oxygen species production in cerebral vessels of DM rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.NEW & NOTEWORTHY This study demonstrates that luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, improved CBF autoregulation in association with reduced vascular oxidative stress and AGEs production in the cerebrovasculature of 18-mo-old DM rats. SGLT2i also prevented BBB leakage, impaired functional hyperemia, neurodegeneration, and cognitive impairment seen in DM rats. Luseogliflozin did not have direct constrictive effects on VSMCs and MCAs isolated from normal rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.

Reliability and validity of the Chinese version of the biological rhythms interview of assessment in neuropsychiatry in patients with major depressive disorder.

BACKGROUND: Although disturbances in biological rhythms are closely related to the onset of major depressive disorder (MDD), they are not commonly assessed in Chinese clinical practice. The Biological Rhythms Interview of Assessment in Neuropsychiatry (BRIAN) has been used to evaluate disturbances in biological rhythms in MDD. We aimed to assess and confirm the reliability and validity of the Chinese version of the BRIAN (C-BRIAN) in patients with MDD. METHODS: A total of 120 patients with MDD and 40 age- and sex-matched controls were recruited consecutively. Reliability was estimated using Cronbach's alpha, the split-half coefficient, and the test-retest coefficient; test-retest reliability was assessed using Spearman's correlation coefficient. A confirmatory factor analysis was used to determine the construct validity of the scale. The Pittsburgh Sleep Quality Index (PSQI) and the Morningness-Eveningness Questionnaire (MEQ) were used to check concurrent validity by evaluating the correlation between the C-BRIAN, PSQI, and MEQ. RESULTS: The overall Cronbach's α value was 0.898, indicating good internal consistency. The Guttman split-half coefficient was 0.792, indicating good split-half reliability. Moreover, the test-retest reliability for both the total and individual item score was excellent. Confirmatory factor analysis revealed that construct validity was acceptable (χ2/df = 2.117, GFI = 0.80, AGFI = 0.87, CFI = 0.848, and RMSEA = 0.097). Furthermore, total BRIAN scores were found to be negatively correlated with MEQ (r = - 0.517, P < 0.001) and positively correlated with PSQI (r = 0.586, P < 0.001). In addition, patients with MDD had higher BRIAN scores than those in controls. CONCLUSIONS: This study revealed that the C-BRIAN scale has great validity and reliability in evaluating the disturbance of biological rhythms in patients with MDD.

Role of γ-adducin in actin cytoskeleton rearrangements in podocyte pathophysiology.

We recently reported that the enhanced susceptibility to chronic kidney disease (CKD) in the fawn-hooded hypertensive (FHH) rat is caused, at least in part, by a mutation in γ-adducin (ADD3) that attenuates renal vascular function. The present study explored whether Add3 contributes to the modulation of podocyte structure and function using FHH and FHH.Add3 transgenic rats. The expression of ADD3 on the membrane of primary podocytes isolated from FHH was reduced compared with FHH.Add3 transgenic rats. We found that F-actin nets, which are typically localized in the lamellipodia, replaced unbranched stress fibers in conditionally immortalized mouse podocytes transfected with Add3 Dicer-substrate short interfering RNA (DsiRNA) and primary podocytes isolated from FHH rats. There were increased F/G-actin ratios and expression of the Arp2/3 complexes throughout FHH podocytes in association with reduced synaptopodin and RhoA but enhanced Rac1 and CDC42 expression in the renal cortex, glomeruli, and podocytes of FHH rats. The expression of nephrin at the slit diaphragm and the levels of focal adhesion proteins integrin-α3 and integrin-ß1 were decreased in the glomeruli of FHH rats. Cell migration was enhanced and adhesion was reduced in podocytes of FHH rats as well as in immortalized mouse podocytes transfected with Add3 DsiRNA. Mean arterial pressures were similar in FHH and FHH.Add3 transgenic rats at 16 wk of age; however, FHH rats exhibited enhanced proteinuria associated with podocyte foot process effacement. These results demonstrate that reduced ADD3 function in FHH rats alters baseline podocyte pathophysiology by rearrangement of the actin cytoskeleton at the onset of proteinuria in young animals.

Reduced pericyte and tight junction coverage in old diabetic rats are associated with hyperglycemia-induced cerebrovascular pericyte dysfunction.

Diabetes mellitus (DM) is one of the primary pathological factors that contributes to aging-related cognitive impairments, but the underlying mechanisms remain unclear. We recently reported that old DM rats exhibited impaired myogenic responses of the cerebral arteries and arterioles, poor cerebral blood flow autoregulation, enhanced blood-brain barrier (BBB) leakage, and cognitive impairments. These changes were associated with diminished vascular smooth muscle cell contractile capability linked to elevated reactive oxygen species (ROS) and reduced ATP production. In the present study, using a nonobese T2DN DM rat, we isolated parenchymal arterioles (PAs), cultured cerebral microvascular pericytes, and examined whether cerebrovascular pericyte in DM is damaged and whether pericyte dysfunction may play a role in the regulation of cerebral hemodynamics and BBB integrity. We found that ROS and mitochondrial superoxide production were elevated in PAs isolated from old DM rats and in high glucose (HG)-treated α-smooth muscle actin-positive pericytes. HG-treated pericytes displayed decreased contractile capability in association with diminished mitochondrial respiration and ATP production. Additionally, the expression of advanced glycation end products, transforming growth factor-ß, vascular endothelial growth factor, and fibronectin were enhanced, but claudin 5 and integrin ß1 was reduced in the brain of old DM rats and HG-treated pericytes. Further, endothelial tight junction and pericyte coverage on microvessels were reduced in the cortex of old DM rats. These results demonstrate our previous findings that the impaired cerebral hemodynamics and BBB leakage and cognitive impairments in the same old DM model are associated with hyperglycemia-induced cerebrovascular pericyte dysfunction.NEW & NOTEWORTHY This study demonstrates that the loss of contractile capability in pericytes in diabetes is associated with enhanced ROS and reduced ATP production. Enhanced advanced glycation end products (AGEs) in diabetes accompany with reduced pericyte and endothelial tight junction coverage in the cortical capillaries of old diabetic rats. These results suggest our previous findings that the impaired cerebral hemodynamics, BBB leakage, and cognitive impairments in old DM model are associated with hyperglycemia-induced cerebrovascular pericyte dysfunction.

20-HETE-promoted cerebral blood flow autoregulation is associated with enhanced pericyte contractility.

We previously reported that deficiency in 20-HETE or CYP4A impaired the myogenic response and autoregulation of cerebral blood flow (CBF) in rats. The present study demonstrated that CYP4A was coexpressed with alpha-smooth muscle actin (α-SMA) in vascular smooth muscle cells (VSMCs) and most pericytes along parenchymal arteries (PAs) isolated from SD rats. Cell contractile capabilities of cerebral VSMCs and pericytes were reduced with a 20-HETE synthesis inhibitor, HET0016, but restored with 20-HETE analog WIT003. Similarly, intact myogenic responses of the middle cerebral artery and PA of SD rats decreased with HET0016 and were rescued by WIT003. The myogenic response of the PA was abolished in SS and was restored in SS.BN5 and SS.Cyp4a1 rats. HET0016 enhanced CBF and impaired its autoregulation in the surface and deep cortex of SD rats. These results demonstrate that 20-HETE has a direct effect on cerebral mural cell contractility that may play an essential role in controlling cerebral vascular function.

Novel Mechanistic Insights and Potential Therapeutic Impact of TRPC6 in Neurovascular Coupling and Ischemic Stroke.

Ischemic stroke is one of the most disabling diseases and a leading cause of death globally. Despite advances in medical care, the global burden of stroke continues to grow, as no effective treatments to limit or reverse ischemic injury to the brain are available. However, recent preclinical findings have revealed the potential role of transient receptor potential cation 6 (TRPC6) channels as endogenous protectors of neuronal tissue. Activating TRPC6 in various cerebral ischemia models has been found to prevent neuronal death, whereas blocking TRPC6 enhances sensitivity to ischemia. Evidence has shown that Ca2+ influx through TRPC6 activates the cAMP (adenosine 3',5'-cyclic monophosphate) response element-binding protein (CREB), an important transcription factor linked to neuronal survival. Additionally, TRPC6 activation may counter excitotoxic damage resulting from glutamate release by attenuating the activity of N-methyl-d-aspartate (NMDA) receptors of neurons by posttranslational means. Unresolved though, are the roles of TRPC6 channels in non-neuronal cells, such as astrocytes and endothelial cells. Moreover, TRPC6 channels may have detrimental effects on the blood-brain barrier, although their exact role in neurovascular coupling requires further investigation. This review discusses evidence-based cell-specific aspects of TRPC6 in the brain to assess the potential targets for ischemic stroke management.

The clinical characteristics and risk factors for necrotizing pneumonia caused by Mycoplasma pneumoniae in children.

BACKGROUND: The incidence of necrotizing pneumonia (NP) caused by Mycoplasma pneumoniae (MP) is increasing. We analyzed the clinical characteristics and the risk factors for NP caused by MP. METHODS: A retrospective observational study was conducted in 37 patients with NP caused by MP (NP group) and 74 patients diagnosed with lobar M. pneumoniae pneumonia with no necrosis (control group) who were admitted to our hospital between January 2013 and December 2017. The clinical manifestations, laboratory data, imaging findings, treatments and outcomes were analyzed. RESULTS: The proportion of females, the incidence of pleural effusion, fever duration, hospitalization days, white blood cell count, neutrophil ratio, D-dimer level and use of other types of antibiotics were higher in the NP group than in the control group (P < 0.05). The control group exhibited a greater use of low molecular weight heparin (LMWH) than the NP group (P < 0.05). According to the multivariate logistic regression analysis, a white blood cell count > 12.3 × 109/L (Odds ratio, OR = 6.412), a neutrophil ratio > 73.9% (OR = 6.081) and D-dimer level > 1367.5 ng/mL (OR = 8.501) were risk factors for pulmonary necrosis caused by MP. Furthermore, the use of LMWH (OR = 0.074) reduced the risk of pulmonary necrosis. CONCLUSIONS: NP is a rare complication of severe Mycoplasma pneumoniae pneumonia (SMPP), and although the clinical course is longer than common MP infection, the necrotic area is absorbed gradually. In patients with SMPP presenting with lobar consolidation, a white blood cell count > 12.3 × 109/L, a neutrophil ratio > 73.9% and D-dimer level > 1367.5 ng/mL are risk factors for pulmonary necrosis, and the use of LMWH reduces the risk of pulmonary necrosis.

Female mice with apolipoprotein E4 domain interaction demonstrated impairments in spatial learning and memory performance and disruption of hippocampal cyto-architecture.

We have previously reported cognitive impairments in both young and old mice, particularly in female mice expressing mouse Arg-61 apoE, with a point mutation to mimic the domain interaction feature of human apoE4, as compared to the wildtype mouse (C57BL/6J) apoE. In this study, we further evaluated water maze performance in the female Arg-61 mice at an additional time point and then investigated related hippocampal cyto-architecture in these young female Arg-61 apoE mice vs. the wildtype mice. The results of behavioral performance consistently support our previous report that the young female Arg-61 apoE showed cognitive impairment versus C57BL/6J at the same age. The cyto-architectural results showed that volume of the granular cell layer (GCL) was significantly larger in both 5- and 10-month old Arg-61 apoE mice versus C57BL/6J mice. While the number of newborn calretinin-positive neurons was greater in the sub-granular zone (SGZ) in 5-month old Arg-61 mice, this number dropped significantly in 10-month old Arg-61 mice to a lower level than in age-matched C57BL/6J mice. In addition, the amyloid ß species was significantly higher in 5-month old Arg-61 mice versus age-matched C57BL/6J mice. In conclusion, impaired cognitive functions in female Arg-61 apoE mice appear correlated with larger GCL volume and higher calretinin-positive cell number and suggest a compensatory cellular response that may be related to amyloid beta perturbations early in life. Therefore this study suggests a novel cyto-architectural mechanism of apoE4-dependent pathologies and increased susceptibility of APOEε4 subjects to Alzheimer's disease.

ERß agonist alters RNA splicing factor expression and has a longer window of antidepressant effectiveness than estradiol after long-term ovariectomy

Background: Estrogen therapy (ET), an effective treatment for perimenopausal depression, often fails to ameliorate symptoms when initiated late after the onset of menopause. Our previous work has suggested that alternative splicing of RNA might mediate these differential effects of ET. Methods: Female Sprague­Dawley rats were treated with estradiol (E2) or vehicle 6 days (early ET) or 180 days (late ET) after ovariectomy (OVX). We investigated the differential expression of RNA splicing factors and tryptophan hydroxylase 2 (TPH2) protein using a customized RT2 Profiler PCR Array, reverse-transcription polymerase chain reaction, immunoprecipitation and behaviour changes in clinically relevant early and late ET. Results: Early ET, but not late ET, prolonged swimming time in the forced swim test and reduced anxiety-like behaviours in the elevated plus maze. It reversed OVX-increased (SFRS7 and SFRS16) or OVX-decreased (ZRSR2 and CTNNB1) mRNA levels of splicing factors and ERß splicing changes in the brains of OVX rats. Early ET, but not late ET, also increased the expression of TPH2 and decreased monoamine oxidase A levels in the dorsal raphe in the brains of OVX rats. In late ET, only diarylpropionitrile (an ERß-specific agonist) achieved similar results ­ not E2 (an ERα and ERß agonist) or propylpyrazoletriol (an ERα-specific agonist). Limitations: Our experimental paradigm mimicked early and late ET in the clinical setting, but the contribution of age and OVX might be difficult to distinguish. Conclusion: These findings suggest that ERß alternative splicing and altered responses in the regulatory system for serotonin may mediate the antidepressant efficacy of ET associated with the timing of therapy initiation. It is likely that ERß-specific ligands would be effective estrogen-based antidepressants late after the onset of menopause.

Human LRRK2 G2019S mutation represses post-synaptic protein PSD95 and causes cognitive impairment in transgenic mice.

BACKGROUND: LRRK2 G2019S mutation is associated with increased kinase activity and is the most common mutation associated with late-onset PD. However, the transgenic mouse model has not recapitulated cardinal PD-related motor phenotypes. Non-motor symptoms of PD including cognitive impairments are very common and may appear earlier than the motor symptoms. The objective of this study was to determine whether human LRRK2 with G2019S mutation causes hippocampus-dependent cognitive deficits in mice. RESULTS: Male (LRRK2-G2019S) LRRK2-Tg mice showed impairments in the early portion of the Two-day radial arm water maze acquisition trial as well as in the reversal learning on the third day. However, their performance was similar to Non-Tg controls in the probe trial. LRRK2-Tg mice also displayed impairments in the novel arm discrimination test but not in the spontaneous alternation test in Y-maze. Interestingly, there was no statistically significant locomotor impairment during any of these cognitive test, nor in the locomotor tests including open field, accelerating rotarod and pole tests. Expression of the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin was lower in hippocampal homogenates of LRRK2-Tg mice. CONCLUSION: Consistent with previous reports in human LRRK2 G2019S carriers, the current data suggests that cognitive dysfunctions are present in LRRK2-Tg mice even in the absence of locomotor impairment. LRRK2 G2019S mutation represses the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin. This study also suggests that mild cognitive impairment may appear earlier than motor dysfunctions in LRRK2-G2019S mutation carriers.

Up-Regulation of PKR Signaling Pathway by Ethanol Displays an Age of Onset-Dependent Relationship.

BACKGROUND: Ethanol (EtOH) neurotoxicity can result in devastating effects on brain and behavior by disrupting homeostatic signaling cascades and inducing cell death. One such mechanism involves double-stranded RNA activated protein kinase (PKR), a primary regulator of protein translation and cell viability in the presence of a virus or other external stimuli. EtOH-mediated up-regulation of interferon-gamma (IFN-γ; the oxidative stress-inducible regulator of PKR), PKR, and its target, p53, are still being fully elucidated. METHODS: Using Western blot analysis, immunofluorescence, and linear regression analyses, changes in the IFN-γ-PKR-p53 pathway following chronic EtOH treatment in the frontal cortex of rodents were examined. The role of PKR on cell viability was also assessed in EtOH-treated cells using PKR overexpression vector and PKR inhibitor (PKRI). RESULTS: In rats chronically fed EtOH, PKR, phosphorylated PKR (p-PKR), IFN-γ, and p53 were significantly increased following chronic EtOH exposure. Linear regression revealed a significant correlation between IFN-γ and p-PKR protein levels, as well as p-PKR expression and age of EtOH exposure. Overexpression of PKR resulted in greater cell death, while use of PKRI enhanced cell viability in EtOH-treated cells. CONCLUSIONS: Chronic EtOH exposure activates the IFN-γ-PKR-p53 pathway in the frontal cortex of rodents. p-PKR expression is greater in brains of rodents exposed to EtOH at earlier ages compared to later life, suggesting a mechanism by which young brains could be more susceptible to EtOH-related brain injury. PKR and p-PKR were also colocalized in neurons and astrocytes of rats. This study provides additional insight into biochemical mechanisms underlying alcohol use disorder related neuropathology and warrants further investigation of PKR as a potential pharmacotherapeutic target to combat EtOH-related neurotoxicity, loss of protein translation and brain injury.

Cognitive deficits and disruption of neurogenesis in a mouse model of apolipoprotein E4 domain interaction.

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to "repair" itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.

Exposure to serotonin adversely affects oligodendrocyte development and myelination in vitro.

Serotonin (5-hydroxytryptamine, 5-HT) has been implicated to play critical roles in early neural development. Recent reports have suggested that perinatal exposure to selective serotonin reuptake inhibitors (SSRIs) resulted in cortical network miswiring, abnormal social behavior, callosal myelin malformation, as well as oligodendrocyte (OL) pathology in rats. To gain further insight into the cellular and molecular mechanisms underlying SSRIs-induced OL and myelin abnormalities, we investigated the effect of 5-HT exposure on OL development, cell death, and myelination in cell culture models. First, we showed that 5-HT receptor 1A and 2A subtypes were expressed in OL lineages, using immunocytochemistry, Western blot, as well as intracellular Ca(2+) measurement. We then assessed the effect of serotonin exposure on the lineage development, expression of myelin proteins, cell death, and myelination, in purified OL and neuron-OL myelination cultures. For pure OL cultures, our results showed that 5-HT exposure led to disturbance of OL development, as indicated by aberrant process outgrowth and reduced myelin proteins expression. At higher doses, such exposure triggered a development-dependent cell death, as immature OLs exhibited increasing susceptibility to 5-HT treatment compared to OL progenitor cells (OPC). We showed further that 5-HT-induced immature OL death was mediated at least partially via 5-HT2A receptor, since cell death could be mimicked by 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride, (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride, but atten-uated by pre-treatment with 5-HT2A receptor antagonist ritanserin. Utilizing a neuron-OL myelination co-culture model, our data showed that 5-HT exposure significantly reduced the number of myelinated internodes. In contrast to cell injury observed in pure OL cultures, 5-HT exposure did not lead to OL death or reduced OL density in neuron-OL co-cultures. However, abnormal patterns of contactin-associated protein (Caspr) clustering were observed at the sites of Node of Ranvier, suggesting that 5-HT exposure may affect other axon-derived factors for myelination. In summary, this is the first study to demonstrate that manipulation of serotonin levels affects OL development and myelination, which may contribute to altered neural connectivity noted in SSRIs-treated animals. The current in vitro study demonstrated that exposure to high level of serotonin (5-HT) led to aberrant oligodendrocyte (OL) development, cell injury, and myelination deficit. We propose that elevated extracellular serotonin levels in the fetal brain, such as upon the use of selective serotonin reuptake inhibitors (SSRIs) during pregnancy, may adversely affect OL development and/or myelination, thus contributing to altered neural connectivity seen in Autism Spectrum Disorders. OPC = oligodendrocyte progenitor cell.

Oral Application of Mother's Own Milk for Reducing Necrotizing Enterocolitis in Preterm Infants: An Updated Meta-Analysis of RCTs.

Background: Necrotizing enterocolitis (NEC) and late-onset sepsis (LOS) are the major contributors to mortality and morbidity in preterm infants. This updated meta-analysis was aimed to assess the effects of mother's milk on the incidence of NEC, LOS, and other clinical outcomes in preterm infants. Methods: PubMed, Embase, and the Cochrane library were searched for papers published up to October 2022. Results: A total of 13 RCTs with 1330 infants were included in the final analysis. Significant difference in NEC (stage 2 or 3) was found between the intervention group and the control group (RR = 0.508, 95% CI: 0.314-0.822, and P=0.008). The incidence of proven LOS (RR = 0.809, 95% CI: 0.610-1.071, and P=0.139) and death (RR = 0.800, 95% CI: 0.571-1.122, and P=0.196) was comparable between the two groups. Statistical differences in the incidence of proven or probable LOS (RR = 0.705, 95% CI: 0.577-0.862, and P=0.001) and length of hospitalization (WMD = -4.868, 95% CI: -6.608 to -3.128, and P < 0.001) between the intervention group and the control group were observed. Conclusions: The results of this updated meta-analysis showed that compared to the placebo, mother's milk provides better effects in reducing the incidences of NEC, proven or probable LOS, and the length of stay, whereas no significant benefit of mother's milk was observed in reducing the incidence of proven LOS and death.

A CRISPR-Cas12a-Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application.

Mycoplasma pneumoniae (MP), which is responsible for a majority of community-acquired pneumonia (CAP) in children, has been largely underestimated. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform termed MP-MCDA-CRISPR assay for MP infection diagnosis and clinical application. The MP-MCDA-CRISPR assay amplified the CARDS gene of MP by MCDA method, followed by trans-cleavage of the reporter molecular upon the formation of CRISPR-Cas12a-gRNA-target DNA complex, which was confirmed by the release of fluorescent signals. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the CARDS gene of MP. The optimal temperature for MCDA pre-amplification is 64°C, and the time for CRISPR-Cas12a-gRNA biosensing process is 5 min. The limit of detection (LoD) of the MP-MCDA-CRISPR assay is 50 fg per reaction without any cross-reaction with other non-MP pathogens. The MP-MCDA-CRISPR assay accurately identified the 50 real time-PCR positive clinical samples and 78 negative ones. Taken together, the MP-MCDA-CRISPR assay designed here is a promising diagnostic tool for point-of care (POC) testing of MP infection.

Inhibition of angiotensin II type 1 receptor agonistic autoantibodies by direct binding does not impact reduced uterine perfusion pressure offspring birthweight and blood pressure at adulthood.

BACKGROUND: Preeclampsia, a new-onset hypertension with end-organ damage in pregnancy, is associated with maternal death and morbidity, low birthweight, and B cells producing agonistic autoantibodies to the angiotensin II type 1 receptor. Angiotensin II type 1 receptor agonistic autoantibodies are produced during pregnancy and after delivery and are in the fetal circulation of women with preeclampsia. Angiotensin II type 1 receptor agonistic autoantibodies are shown to contribute to endothelial dysfunction, renal dysfunction, hypertension, fetal growth restriction, and chronic inflammation in women with preeclampsia. The reduced uterine perfusion pressure rat model of preeclampsia exhibits these features. In addition, we have shown that the administration of a 'n7AAc', which blocks the actions of the angiotensin II type 1 receptor autoantibodies, improves preeclamptic features in the rat with reduced uterine perfusion pressure. However, the effect of a 'n7AAc' on the long-term health of the offspring of rats with reduced uterine perfusion pressure is unknown. OBJECTIVE: This study aimed to test the hypothesis that inhibition of angiotensin II type 1 receptor autoantibodies during pregnancy will improve offspring birthweight and prevent increased cardiovascular risk in offspring in adulthood. STUDY DESIGN: To test our hypothesis, a 'n7AAc' (24 µg/d) or vehicle (saline) was given on gestation day 14 via miniosmotic pumps to sham-operated (sham) and Sprague-Dawley rat dams with reduced uterine perfusion pressure. Dams were allowed to deliver naturally, and pup weights were recorded within 12 hours after birth. Pups were aged to 16 weeks, at which time mean arterial pressure was measured and whole blood was collected to measure immune cells by flow cytometry, cytokines by enzyme-linked immunosorbent assay, and angiotensin II type 1 receptor autoantibodies by bioassay. A 2-way analysis of variance with the Bonferroni multiple comparison posthoc test was used for statistical analysis. RESULTS: There was no significant change in offspring birthweight of 'n7AAc'-treated male (5.63±0.09 g) or female (5.66±0.14 g) offspring from reduced uterine perfusion pressure dams compared with vehicle male (5.51±0.17 g) or female (5.74±0.13 g) offspring from reduced uterine perfusion pressure dams. In addition, 'n7AAc' treatment did not affect the birthweight of sham male (5.83±0.11 g) or female (5.64±0.12) offspring compared with vehicle sham male (5.811±0.15 g) or female (5.40±0.24 g) offspring. At adulthood, mean arterial pressure was unchanged in 'n7AAc' treated-male (133±2 mm Hg) and female (127±3 mm Hg) offspring from reduced uterine perfusion pressure dams compared with vehicle male (142±3 mm Hg) and female (133±5 mm Hg) offspring from reduced uterine perfusion pressure dams, the 'n7AAc'-treated sham male (133±3 mm Hg) and female (135±3 mm Hg) offspring, and vehicle sham male (138±4 mm Hg) and female (130±5 mm Hg) offspring. The circulating angiotensin II type 1 receptor autoantibodies were increased in vehicle male (10±2 ΔBPM) and female (14±2 ΔBPM) offspring from reduced uterine perfusion pressure dams and 'n7AAc'-treated male (11±2 ΔBPM) and female (11±2 ΔBPM) offspring from reduced uterine perfusion pressure dams compared with vehicle sham male (1±1 ΔBPM) and female (-1±1 ΔBPM) offspring and 'n7AAc'-treated sham male (-2±2 ΔBPM) and female (-2±2 ΔBPM) offspring. CONCLUSION: Our findings indicated that perinatal 7-amino acid sequence peptide treatment does not negatively impact offspring survival or weight at birth. Perinatal 'n7AAc' treatment did not prevent increased cardiovascular risk in offspring, but it also did not cause an increased cardiovascular risk in offspring with reduced uterine perfusion pressure compared with controls. Furthermore, perinatal 'n7AAc' treatment did not affect endogenous immunologic programming as observed by no change in circulating angiotensin II type 1 receptor autoantibodies in either sex of adult offspring from reduced uterine perfusion pressure dams.

Intracerebral lipopolysaccharide induces neuroinflammatory change and augmented brain injury in growth-restricted neonatal rats.

INTRODUCTION: Intrauterine growth restriction (IUGR) alters fetal development and is associated with neurodevelopmental abnormalities. We hypothesized that growth restriction from reduced intrauterine perfusion would predispose neonatal rats to subsequent inflammatory brain injury. METHODS: In this study, IUGR was achieved by induced placental insufficiency in pregnant rats at 14 days of gestation. IUGR offspring and sham-operated control pups were subsequently injected with intracerebral lipopolysaccharide (LPS) as a model of periventricular leukomalacia (PVL). RESULTS: LPS similarly elevates proinflammatory cytokines in the brains of both IUGR and control rat pups. However, the chemokines cytokine-induced neutrophil chemoattractant-1 (CINC-1) and macrophage chemoattractant protein-1 (MCP-1), as well as microglia activation, were significantly higher in LPS-treated IUGR rat pups as compared with LPS-treated controls. In addition to the unique brain inflammatory response, IUGR rat pups demonstrated increased brain damage with an increased number of apoptotic cells, larger lateral ventricular size, and more severe impairment of myelination. DISCUSSION: This study provides evidence that placental insufficiency may sensitize the innate immune system in the immature brain and reveals a possible link between brain inflammation and injury.

Dexamethasone and betamethasone protect against lipopolysaccharide-induced brain damage in neonatal rats.

INTRODUCTION: The aim of this study was to test whether dexamethasone (Dex) and betamethasone (Beta), two of the most commonly used corticosteroids, protect against lipopolysaccharide (LPS)-induced white matter damage and neurobehavioral dysfunction. METHODS: LPS or sterile saline was injected into the brain white matter of rat pups at postnatal day 5 (P5), and Dex or Beta was given intraperitoneally to the rat pups 1 h before the LPS microinjection. Brain inflammatory response, brain damage, and myelination were examined at P6, P8, and P14. Neurobehavioral tests were performed from P3 through P22. RESULTS: Our results demonstrate that Dex and Beta markedly diminish the LPS-induced brain inflammatory response, restore myelin basic protein (MBP) expression, and alleviate lateral ventricle dilation. Both corticosteroids demonstrate significant protection against most LPS-induced behavioral deficits, including those in rearing, vibrissa-elicited forelimb-placing, beam walking, learning, and elevated plus-maze test. Of note, only Beta improved the locomotion and stereotype dysfunction. In contrast to their beneficial effects, neither drug prevented LPS-induced delay in body weight gain from P6 through P21. DISCUSSION: Our study suggests that if their adverse effects are minimized, corticosteroids may be the potential candidate drugs to prevent brain damage in premature infants.

Loop-Mediated Isothermal Amplification Coupled With Nanoparticle-Based Biosensor: A Rapid and Sensitive Method to Detect Mycoplasma pneumoniae.

Mycoplasma pneumoniae (MP), the causative agent of MP pneumonia (MPP), has posed a substantial burden to public health owing to a lack of rapid and effective diagnostic methods. Here, we designed a loop-mediated isothermal amplification (LAMP)-based assay, termed LAMP, combined with a nanoparticle-based lateral flow biosensor (LAMP-LFB) for rapid and sensitive diagnosis of MP.-LAMP-LFB included a set of six primers targeting the community-acquired respiratory distress syndrome (CARDS) toxin gene and was performed optimally at 63°C for only 30 min. The resulting LAMP products could be visually indicated by LFB within 2 min, thus the whole process could be accomplished within an hour. MP-LAMP-LFB's sensitivity was 50 fg per reaction, which was in complete accordance with these results obtained from real-time turbidity and visual detection reagent (VDR). MP-LAMP-LFB had no cross-reactivity with other pathogens that had similar clinical presentations. Our assay was further validated using 100 nasopharyngeal swab samples collected from children suspected of MPP, and the result was compared with the real-time PCR method. With a positive rate of 50%, the data indicated that MP-LAMP-LFB is a sensitive test for MP detection in clinical settings. Collectively, the MP-LAMP-LFB assay targeting the CARDS toxin gene was a rapid, highly sensitive, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas.

Dopaminergic neuronal injury in the adult rat brain following neonatal exposure to lipopolysaccharide and the silent neurotoxicity.

Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague-Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1ß and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity.