Role of lipocalin-2 in surgery-induced cognitive decline in mice: a signal from neuron to microglia.

BACKGROUND: Perioperative neurocognitive disorders (PNDs) are common complications observed among surgical patients. Accumulating evidence suggests that neuroinflammation is one of the major contributors to the development of PNDs, but the underlying mechanisms remain unclear. METHODS: qPCR and ELISA analysis were used for detecting LCN2 and cytokine levels. cx3cr1CreER/-:: R26iDTR/- crossed mouse line was used for microglia depletion; intracranial injection of recombinant LCN2 (rLCN2) and adeno-associated viruses (AAV)-mediated shRNA silencing approaches were used for gain and loss of function, respectively. Combing with in vitro microglia cell culture, we have studied the role of LCN2 in surgery-induced cognitive decline in mice. RESULTS: We revealed that Lcn2 mRNA and protein levels were greatly increased in mouse hippocampal neurons after surgery. This surgery-induced elevation of LCN2 was independent of the presence of microglia. Gain of function by intracranial injection of rLCN2 protein into hippocampus disrupted fear memory in naive mice without surgery. Conversely, silencing LCN2 in hippocampus by AAV-shRNA protected mice from surgery-induced microglia morphological changes, neuroinflammation and cognitive decline. In vitro, application of rLCN2 protein induced the expression of several pro-inflammatory cytokines in both BV-2 and primary microglia culture. CONCLUSIONS: These data suggest LCN2 acts as a signal from neuron to induce proinflammatory microglia, which contributes to surgery-induced neuroinflammation and cognitive decline in mice.

YebC regulates variable surface antigen VlsE expression and is required for host immune evasion in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease pathogen causes persistent infection by evading the host immune response. Differential expression of the surface-exposed lipoprotein VlsE that undergoes antigenic variation is a key immune evasion strategy employed by B. burgdorferi. Most studies focused on the mechanism of VlsE antigen variation, but little is known about VlsE regulation and factor(s) that regulates differential vlsE expression. In this study, we investigated BB0025, a putative YebC family transcriptional regulator (and hence designated BB0025 as YebC of B. burgdorferi herein). We constructed yebC mutant and complemented strain in an infectious strain of B. burgdorferi. The yebC mutant could infect immunocompromised SCID mice but not immunocompetent mice, suggesting that YebC plays an important role in evading host adaptive immunity. RNA-seq analyses identified vlsE as one of the genes whose expression was most affected by YebC. Quantitative RT-PCR and Western blot analyses confirmed that vlsE expression was dependent on YebC. In vitro, YebC and VlsE were co-regulated in response to growth temperature. In mice, both yebC and vlsE were inversely expressed with ospC in response to the host adaptive immune response. Furthermore, EMSA proved that YebC directly binds to the vlsE promoter, suggesting a direct transcriptional control. These data demonstrate that YebC is a new regulator that modulates expression of vlsE and other genes important for spirochetal infection and immune evasion in the mammalian host.

Inhibiting Caspase-12 Mediated Inflammasome Activation protects against Oxygen-Glucose Deprivation Injury in Primary Astrocytes.

Stroke is one of the leading causes of death worldwide. Accumulating evidence suggests that NLRP3 inflammasome activation plays an important role in ischemic stroke injury. However, the existence of the NLRP3 inflammasome in astrocytes remains controversial. In this study, we demonstrated the presence of the NLRP3 inflammasome in primary mouse astrocytes and investigated the role of caspase-12 in NLRP3 inflammasome activation and cell injury in an in vitro astrocyte oxygen-glucose deprivation (OGD) model. Astrocytes exposed to 2, 3, and 4 h of OGD exhibited increased cell injury and apoptosis, and the protein levels of caspase-12, cleaved caspase-3, NLRP3 inflammasome components, and IL-1ß were also significantly elevated. Interestingly, pretreatment with the caspase-12-specific inhibitor Z-ATAD-FMK attenuated cell injury and apoptosis and decreased the levels of NLRP3, caspase-1, IL-1ß, and cleaved caspase-3 in the OGD group. In conclusion, Z-ATAD-FMK protected astrocytes against OGD-induced cell death and inhibited NLPR3-inflammasome activation. Our results indicate that caspase-12 and its potential regulation of NLRP3 inflammasome activation might be a promising target for treatment of ischemic stroke.

Insight into the Dual Functions of Bacterial Enhancer-Binding Protein Rrp2 of Borrelia burgdorferi.

UNLABELLED: It is well established that the RpoN-RpoS sigma factor (σ(54)-σ(S)) cascade plays an essential role in differential gene expression during the enzootic cycle of Borrelia burgdorferi, the causative agent of Lyme disease. The RpoN-RpoS pathway is activated by the response regulator/σ(54)-dependent activator (also called bacterial enhancer-binding protein [bEBP]) Rrp2. One unique feature of Rrp2 is that this activator is essential for cell replication, whereas RpoN-RpoS is dispensable for bacterial growth. How Rrp2 controls cell replication, a function that is independent of RpoN-RpoS, remains to be elucidated. In this study, by generating a series of conditional rrp2 mutant strains, we demonstrated that the N-terminal receiver domain of Rrp2 is required for spirochetal growth. Furthermore, a D52A point mutation at the phosphorylation site within the N terminus of Rrp2 abolished cell replication. Mutation of the ATPase motif within the central domain of Rrp2 did not affect spirochetal replication, indicating that phosphorylation-dependent ATPase activity of Rrp2 for σ(54) activation is not required for cell growth. However, deletion of the C-terminal domain or a 16-amino-acid truncation of the helix-turn-helix (HTH) DNA-binding motif within the C-terminal domain of Rrp2 abolished spirochetal replication. It was shown that constitutive expression of rpoS is deleterious to borrelial growth. We showed that the essential nature of Rrp2 is not due to an effect on rpoS These data suggest that phosphorylation-dependent oligomerization and DNA binding of Rrp2 likely function as a repressor, independently of the activation of σ(54), controlling an essential step of cell replication in B. burgdorferi IMPORTANCE: Bacterial enhancer-binding proteins (bEBPs) are a unique group of transcriptional activators specifically required for σ(54)-dependent gene transcription. This work demonstrates that the B. burgdorferi bEBP, Rrp2, has an additional function that is independent of σ(54), that of its essentiality for spirochetal growth, and such a function is dependent on its N-terminal signal domain and C-terminal DNA-binding domain. These findings expand our knowledge on bEBP and provide a foundation to further study the underlying mechanism of this new function of bEBP.

HtrA, a Temperature- and Stationary Phase-Activated Protease Involved in Maturation of a Key Microbial Virulence Determinant, Facilitates Borrelia burgdorferi Infection in Mammalian Hosts.

High-temperature requirement protease A (HtrA) represents a family of serine proteases that play important roles in microbial biology. Unlike the genomes of most organisms, that of Borrelia burgdorferi notably encodes a single HtrA gene product, termed BbHtrA. Previous studies identified a few substrates of BbHtrA; however, their physiological relevance could not be ascertained, as targeted deletion of the gene has not been successful. Here we show that BbhtrA transcripts are induced during spirochete growth either in the stationary phase or at elevated temperature. Successful generation of a BbhtrA deletion mutant and restoration by genetic complementation suggest a nonessential role for this protease in microbial viability; however, its remarkable growth, morphological, and structural defects during cultivation at 37°C confirm a high-temperature requirement for protease activation and function. The BbhtrA-deficient spirochetes were unable to establish infection of mice, as evidenced by assessment of culture, PCR, and serology. We show that transcript abundance as well as proteolytic processing of a borrelial protein required for cell fission and infectivity, BB0323, is impaired in BbhtrA mutants grown at 37°C, which likely contributed to their inability to survive in a mammalian host. Together, these results demonstrate the physiological relevance of a unique temperature-regulated borrelial protease, BbHtrA, which further enlightens our knowledge of intriguing aspects of spirochete biology and infectivity.

Self-Sacrificed Additive in Preparing PbI2 Film Enables the Oriented Growth of Perovskite Crystals for Improved Solar Cell Performance.

Due to the limitation of the diffusion kinetics of organic amine salts on the PbI2 layer in the two-step method, prepared perovskite particles are small in size, have many defects, and are randomly oriented, and the cell efficiency and stability are difficult to guarantee due to PbI2 residues. Here, we added a volatile additive, N,N,N',N'-tetramethylethylenediamine (TMEDA), to the PbI2 precursor solution and formed preaggregated atomic clusters with PbI2 through TMEDA, which reduced the Gibbs free energy of nucleation to obtain a porous PbI2 layer, and finally obtained a perovskite film with large particles, few defects, ideal crystal plane orientation, and no additive residues. The results show that the photoelectric conversion efficiency of the optimized device is increased by 1.68% (from 21.68% to 23.36%), and the unpackaged optimized device still maintains the maximum efficiency of 77% after being placed in the air for 1200 h. This study provides an effective way to fabricate efficient and stable perovskite solar cells by promoting the nucleation-induced crystallization orientation by volatile additives.

Tailoring the Buried Interface by Dipolar Halogen-Substituted Arylamine for Efficient and Stable Perovskite Solar Cells.

Improving the quality of the buried interface is decisive for achieving stable and high-efficiency perovskite solar cells. Herein, we report the interface engineering by using dipolar 2,4-difluoro-3,5-dichloroaniline (DDE) as the adhesive between titanium dioxide (TiO2) and MAPbI3. By manipulation of the anchoring groups of DDE, this molecule not only passivated defects of TiO2 but also optimized the energy level alignment. Furthermore, the perovskite film on the modified TiO2 surface showed improved crystallinity, released residual stress, and reduced trap states. Therefore, these benefits directly contribute to achieving a power conversion efficiency of up to 22.10%. The unencapsulated device retained 90% of initial power conversion efficiencies (PCE) after continuous light illumination for 1000 h and 93% of initial PCE after exposure to air with a relative humidity of 30-40% for over 3000 h. Moreover, the performance of PSCs based on FA0.15MA0.85PbI3 has also increased from 20.48 to 23.51%. Our results demonstrate the effectiveness and universality of dipolar halogen-substituted arylamine (DDE) for enhancing PSC performance.

Generalized modality responses in primary sensory neurons of awake mice during the development of neuropathic pain.

Introduction: Peripheral sensory neurons serve as the initial responders to the external environment. How these neurons react to different sensory stimuli, such as mechanical or thermal forces applied to the skin, remains unclear. Methods: Using in vivo two-photon Ca2+ imaging in the lumbar 4 dorsal root ganglion (DRG) of awake Thy1.2-GCaMP6s mice, we assessed neuronal responses to various mechanical (punctate or dynamic) and thermal forces (heat or cold) sequentially applied to the paw plantar surface. Results: Our data indicate that in normal awake male mice, approximately 14 and 38% of DRG neurons respond to either single or multiple modalities of stimulation. Anesthesia substantially reduces the number of responsive neurons but does not alter the ratio of cells exhibiting single-modal responses versus multi-modal responses. Following peripheral nerve injury, DRG cells exhibit a more than 5.1-fold increase in spontaneous neuronal activity and a 1.5-fold increase in sensory stimulus-evoked activity. As neuropathic pain resulting from nerve injury progresses, the polymodal nature of sensory neurons intensifies. The polymodal population increases from 39.1 to 56.9%, while the modality-specific population decreases from 14.7 to 5.0% within a period of 5 days. Discussion: Our study underscores polymodality as a significant characteristic of primary sensory neurons, which becomes more pronounced during the development of neuropathic pain.

[Study on MSO/GO-based determination method for trace amount of aqueous Hg2+].

OBJECTIVE: To establish a highly sensitive fluorometric nanobiosensor for determination of aqueous mercury ions (Hg(2+)) using optimized mercury-specific oligonucleotide (MSO) probes and graphene oxide (GO). METHODS: The nanobiosensor was assembled by attaching the self-designed MSO(1) (5' end labeled with fluorophore carboxyfluorescein (FAM), denoted as FAM-MSO(1)) and MSO(2) to the surface of GO through strong non-covalent bonding forces. Upon the addition of Hg(2+), the formation of the T-Hg(2+)-T configuration desorbed the FAM-MSO(1) and MSO(2) from the surface of GO, resulting in a restoration of the fluorescence of FAM-MSO(1). Using the specific mispairing of T-Hg(2+)-T and the changes in fluorescent signals in solutions, quantitative analysis of Hg(2+) could be performed. RESULTS: The average thickness of the prepared GO sheets was only 1.4 nm. For the Hg(2+) nanobiosensor, the optimum concentrations of FAM-MSO(1) and MSO(2) were both 1 µmol/L, the optimum volume of 0.5 g/L GO was 5 µL, and the limit of detection was 10 pmol/L; it had low cross-reactivity with 10 other kinds of non-specific metal ions; the fluorescence recovery efficiency was up to 65% in the re-determination of Hg(2+) after addition of Na(2)S(2)O(3). CONCLUSION: The MSO/GO-based nanobiosensor is convenient to operate, highly sensitive, highly specific, highly accurate, and reusable. It can be applied to determine trace amount of Hg(2+) in aqueous solutions.

[Detection of mercury (II) ions (Hg(2+)) in water by a nanobiosensor].

OBJECTIVE: To develop a nanobiosensor for rapid colorimetric detecting Mercury (II) Ions (Hg(2+)) in water by mercury-specific oligonucleotides (MSOs) probe and gold nanoparticles. METHODS: The nanobiosensor was assembled by adsorbing the optimized MSOs on the surface of gold nanoparticles. A direct colorimetric probe of Hg(2+) which relied on the T-T mismatches in DNA duplexes was used to selectively and strongly capture Hg(2+). Hg(2+) induces the aggregation of gold nanoparticles with appropriate amount of salts, resulting the color change (red to blue). RESULTS: The diameter and concentration of the gold nanoparticle preparation were 15 nm and 2.97 nmol/L, respectively. Truncated MSOs (9 bp) showed the similar Hg(2+)-binding activity. The optimum concentration of the NaNO3 solution was 0.5 mol/L. The nanobiosensor could detect Hg(2+)in a range of 10 ∼ 1000 µmol/L within few minutes and the specificity was 100%. CONCLUSION: A new nanobiosensor is developed successfully for rapid colorimetric detecting Hg(2+) in water, avoiding either MSOs labeling or gold nanoparticles modification. This technique is simple, convenient and rapid detecting method with high sensitivity and specificity.

TRPC6 Attenuates Cortical Astrocytic Apoptosis and Inflammation in Cerebral Ischemic/Reperfusion Injury.

Transient receptor potential canonical 6 (TRPC6) channel is an important non-selective cation channel with a variety of physiological roles in the central nervous system. Evidence has shown that TRPC6 is involved in the process of experimental stroke; however, the underlying mechanisms remain unclear. In the present study, the role of astrocytic TRPC6 was investigated in an oxygen-glucose deprivation cell model and middle cerebral artery occlusion (MCAO) mouse model of stroke. HYP9 (a selective TRPC6 agonist) and SKF96365 (SKF; a TRPC antagonist) were used to clarify the exact functions of TRPC6 in astrocytes after ischemic stroke. TRPC6 was significantly downregulated during ischemia/reperfusion (IR) injury in cultured astrocytes and in cortices of MCAO mice. Application of HYP9 in vivo alleviated the brain infarct lesion, astrocytes population, apoptosis, and interleukin-6 (IL-6) and IL-1ß release in mouse cortices after ischemia. HYP9 dose-dependently inhibited the downregulation of TRPC6 and reduced astrocytic apoptosis, cytotoxicity and inflammatory responses in IR insult, whereas SKF aggravated the damage in vitro. In addition, modulation of TRPC6 channel diminished IR-induced Ca2+ entry in astrocytes. Furthermore, decreased Ca2+ entry due to TRPC6 contributed to reducing nuclear factor kappa light chain enhancer of activated B cells (NF-κB) nuclear translocation and phosphorylation. Overexpression of astrocytic TRPC6 also attenuated apoptosis, cytotoxicity, inflammatory responses, and NF-κB phosphorylation in modeled ischemia in astrocytes. The results of the present study indicate that the TRPC6 channel can act as a potential target to reduce both inflammatory responses and apoptosis in astrocytes during IR injury, subsequently attenuating ischemic brain damage. In addition, we provide a novel view of stroke therapy by targeting the astrocytic TRPC6 channel.

Transcriptome Profile in Hippocampus During Acute Inflammatory Response to Surgery: Toward Early Stage of PND.

Perioperative neurocognitive disorders (PND) are common complications observed in surgical patients, but there are no effective treatments and the detailed mechanisms remain largely unknown. In this study, transcriptome analysis was performed to investigate the hippocampal changes after surgery and underlying molecular mechanisms of PND. Tibial fracture surgery was performed in 3-4 months old C57BL/6J mice to mimic human orthopedic surgery. We demonstrated that memory consolidation of the hippocampal-dependent trace-fear conditioning task was significantly impaired. By using ELISA, a significant elevated IL-6 was observed both in circulating system and central nervous system and peaked at 6 h post-surgery, but transiently returned to baseline thereafter. Hippocampus were collected at 6 h post-surgery then processed for RNA-Seq. A total of 268 genes were screened differentially expressed between the Surgery and Control group, including 170 up-regulated genes and 98 down-regulated genes. By functional enrichment analysis of differently expressed genes, several KEGG pathways involved in inflammatory mediator regulation of TRP channels, neuroactive ligand-receptor interaction and cholinergic synapse were overrepresented. Quantitative real-time PCR confirmed 15 dysregulated genes of interest. These results provide a comprehensive insight into global gene expression changes during the acute presence of hippocampal inflammation and a better understanding on early stage of PND.

LtpA, a CdnL-type CarD regulator, is important for the enzootic cycle of the Lyme disease pathogen.

Little is known about how Borrelia burgdorferi, the Lyme disease pathogen, adapts and survives in the tick vector. We previously identified a bacterial CarD N-terminal-like (CdnL) protein, LtpA (BB0355), in B. burgdorferi that is preferably expressed at lower temperatures, which is a surrogate condition mimicking the tick portion of the enzootic cycle of B. burgdorferi. CdnL-family proteins, an emerging class of bacterial RNAP-interacting transcription factors, are essential for the viability of Mycobacterium tuberculosis and Myxococcus xanthus. Previous attempts to inactivate ltpA in B. burgdorferi have not been successful. In this study, we report the construction of a ltpA mutant in the infectious strain of B. burgdorferi, strain B31-5A4NP1. Unlike CdnL in M. tuberculosis and M. xanthus, LtpA is dispensable for the viability of B. burgdorferi. However, the ltpA mutant exhibits a reduced growth rate and a cold-sensitive phenotype. We demonstrate that LtpA positively regulates 16S rRNA expression, which contributes to the growth defects in the ltpA mutant. The ltpA mutant remains capable of infecting mice, albeit with delayed infection. Additionally, the ltpA mutant produces markedly reduced spirochetal loads in ticks and was not able to infect mice via tick infection. Overall, LtpA represents a novel regulator in the CdnL family that has an important role in the enzootic cycle of B. burgdorferi.

Investigation of ospC Expression Variation among Borrelia burgdorferi Strains.

Outer surface protein C (OspC) is the most studied major virulence factor of Borrelia burgdorferi, the causative agent of Lyme disease. The level of OspC varies dramatically among B. burgdorferi strains when cultured in vitro, but little is known about what causes such variation. It has been proposed that the difference in endogenous plasmid contents among strains contribute to variation in OspC phenotype, as B. burgdorferi contains more than 21 endogenous linear (lp) and circular plasmids (cp), and some of which are prone to be lost. In this study, we analyzed several clones isolated from B. burgdorferi strain 297, one of the most commonly used strains for studying ospC expression. By taking advantage of recently published plasmid sequence of strain 297, we developed a multiplex PCR method specifically for rapid plasmid profiling of B. burgdorferi strain 297. We found that some commonly used 297 clones that were thought having a complete plasmid profile, actually lacked some endogenous plasmids. Importantly, the result showed that the difference in plasmid profiles did not contribute to the ospC expression variation among the clones. Furthermore, we found that B. burgdorferi clones expressed different levels of BosR, which in turn led to different levels of RpoS and subsequently, resulted in OspC level variation among B. burgdorferi strains.