An autopsy-verified case of FTLD-TDP type A with upper motor neuron-predominant motor neuron disease mimicking MM2-thalamic-type sporadic Creutzfeldt-Jakob disease

Here we report an autopsy-verified case of frontotemporal lobar degeneration (FTLD)-transactivation responsive region (TAR) DNA binding protein (TDP) type A with upper motor neuron-predominant motor neuron disease mimicking MM2-thalamic-type sporadic Creutzfeldt-Jakob disease (sCJD). A 69-year-old woman presented with an 11-month history of progressive dementia, irritability, insomnia, and gait disturbance without a family history of dementia or prion disease. Neurological examination revealed severe dementia, frontal signs, and exaggerated bilateral tendon reflexes. Periodic sharp-wave complexes were not observed on the electroencephalogram. Brain diffusion MRI did not reveal abnormal changes. An easy Z score (eZIS) analysis for ^99mTc-ECD-single photon emission computed tomography (^99mTc-ECD-SPECT) revealed a bilateral decrease in thalamic regional cerebral blood flow (rCBF). PRNP gene analysis demonstrated methionine homozygosity at codon 129 without mutation. Cerebrospinal fluid (CSF) analysis showed normal levels of both 14-3-3 and total tau proteins. Conversely, prion protein was slowly amplified in the CSF by a real-time quaking-induced conversion assay. Her symptoms deteriorated to a state of akinetic mutism, and she died of sudden cardiac arrest, one year after symptom onset.

?Despite the SPECT results supporting a clinical diagnosis of MM2-thalamic-type sCJD, a postmortem assessment revealed that this was a case of FTLD-TDP type A, and excluded prion disease. Thus, this case indicates that whereas a bilateral decreasing thalamic rCBF detected by ^99mTc-ECD-SPECT can be useful for diagnosing MM2-thalamic-type sCJD, it is not sufficiently specific. Postmortem diagnosis remains the gold standard for the diagnosis of this condition.     

固定化微绿球藻去除NH4+-N、PO43——P效果的研究

为了探究固定化微绿球藻（Nannochloropsis oculata）去除污水中NH4+-N、PO43--P的效果,采用海藻酸钠固定化包埋技术进行实验。开展了固定化藻球大小、藻细胞包埋密度、藻球投放质量及充气培养条件对NH4+-N、PO43--P去除效果的单因子试验研究。结果表明,固定化藻球大小、藻细胞包埋密度、藻球投放质量和充气培养条件对NH4+-N、PO43--P的去除效果影响显著（P0.05）。藻球直径3.5 mm时生长速率（K）值最大（0.3320.002）,同时NH4+-N、PO43--P去除率效果最佳,分别为（75.083.83）%和（80.803.81）%;藻细胞包埋密度100104 cells/ball时K值最大（0.3300.033）,而NH4+-N、PO43--P去除率则以藻细胞包埋密度300104 cells/ball组为佳,分别达（87.200.43）%和（82.581.72）%,但考虑单位藻细胞去除率,包埋密度以100104 cells/ball为宜;随着藻球用量的增加K值下降,10 g/L组K值最大（0.3010.02）、50 g/L组K值最小（0.1930.01）,投放量30和50 g/L时NH4+-N去除率较高分别为（84.120.78）%和（84.630.45）%,30 g/L组PO43--P去除率最高达（77.131.43）%。综合考虑,藻球投放量选用30 g/L为宜;充气条件培养K值、NH4+-N和PO43--P去除率显著（P0.05）高于不充气,K值分别为（0.3060.006）和（0.1770.010）;NH4+-N去除率分别为（85.930.45）%和（49.320.45）%;PO43--P去除率分别为（66.665.00）%和（46.292.12）%。研究优化了微绿球藻固定化条件:固定化微绿球藻应进行充气培养,藻球规格3.5 mm、藻细胞包埋密度100104 cells/ball、藻球投放量30 g/L。     

Phosphorylation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Truncated Casein Kinase 1δ Triggers Mislocalization and Accumulation of TDP-43

Intracellular aggregates of phosphorylated TDP-43 are a major component of ubiquitin-positive inclusions in the brains of patients with frontotemporal lobar degeneration and ALS and are considered a pathological hallmark. Here, to gain insight into the mechanism of intracellular TDP-43 accumulation, we examined the relationship between phosphorylation and aggregation of TDP-43. We found that expression of a hyperactive form of casein kinase 1 δ (CK1δ1-317, a C-terminally truncated form) promotes mislocalization and cytoplasmic accumulation of phosphorylated TDP-43 (ubiquitin- and p62-positive) in cultured neuroblastoma SH-SY5Y cells. Insoluble phosphorylated TDP-43 prepared from cells co-expressing TDP-43 and CK1δ1-317 functioned as seeds for TDP-43 aggregation in cultured cells, indicating that CK1δ1-317-induced aggregated TDP-43 has prion-like properties. A striking toxicity and alterations of TDP-43 were also observed in yeast expressing TDP-43 and CK1δ1-317. Therefore, abnormal activation of CK1δ causes phosphorylation of TDP-43, leading to the formation of cytoplasmic TDP-43 aggregates, which, in turn, may trigger neurodegeneration.     

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.     

盐酸胍诱导CP43和CP47变性的太赫兹时域光谱技术研究

太赫兹时域光谱技术(THz-TDS)是近年来新兴的一种研究分子构型状态的技术. 把这项技术运用到两种光合膜蛋白CP43和CP47的研究上, 研究结果表明, 运用太赫兹时域光谱技术能有效地把具有相似结构的蛋白质区分开来, 这项技术也是监测蛋白质变性的一种有力工具. 在盐酸胍(GuHCl)作用下, CP47和游离叶绿素a(Chl a)的太赫兹(THz)振幅谱中都出现一个明显的位于1.8 THz处的峰. 我们认为这个峰来自于叶绿素a和盐酸胍的相互作用. 和CP43相比, CP47中的叶绿素a更易于和盐酸胍发生作用.     

Repeated simulated ischemia and protection against gap junctional uncoupling

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The aim of this study was to investigate the effect of early and delayed preconditioning on gap junction communication, connexin abundance, and phosphorylation in cultured neonatal rat cardiac myocytes. Prolonged ischemia followed 5 minutes after preconditioning in the early protocol, whereas 20 hours separated preconditioning and prolonged ischemia in the delayed preconditioning protocol. Gap junctional intercellular communication (GJIC) was assessed by Lucifer yellow dye transfer. An initial reduction in communication in response to sublethal ischemia was observed. This may be one mechanism whereby neighboring cells are protected from damaging substances produced during the first phase of subsequent regional ischemia in early preconditioning protocols. With respect to delayed preconditioning, the transient decrease in GJIC disappeared prior to prolonged ischemia, indicating that other mechanisms are responsible for delayed protection. Both early and delayed preconditioning preserved intercellular coupling after prolonged ischemia and this correlated with presence of less connexin43 dephosphorylation assessed by immunoblot.     

miR-382-3p suppressed IL-1β induced inflammatory response of chondrocytes via the TLR4/MyD88/NF-κB signaling pathway by directly targeting CX43

miR-382-3p has been reported to be upregulated in synovial membrane in knee osteoarthritis (OA). Nevertheless, its role in OA remains largely unknown. The aim of this study was to investigate the specific function and mechanisms of miR-382-3p in the course of OA. In this study, human OA chondrocytes were pretreated with interleukin-1β (IL-1β) at 5 ng/ml for 12 hr to stimulate inflammatory response and matrix metalloproteinases (MMPs) expression in chondrocytes. Meanwhile, miR-382-3p was downregulated in IL-1β-stimulated chondrocytes. In addition, we found that miR-382-3p directly interacts with connexin 43 (CX43) and attenuates the increase of cytochrome c oxidase polypeptide II, inducible nitric oxide synthase, and MMP-1/13 that is induced by IL-1β. Furthermore, our observations indicated that miR-382-3p inhibited the expression of Toll-like receptor 4 (TLR4), Myeloid differentiation primary response 88 (MyD88) and nuclear factor κB (NF-κB) in IL-1β-stimulated chondrocytes, while CX43 overexpression could partly reverse these decreases. In conclusion, miR-382-3p participated in OA may through the TLR4/MyD88/NF-κB signaling pathway by directly targeting CX43.     

D1:Glu244 and D1:Tyr246 of the bicarbonate-binding environment of Photosystem II moderate high light susceptibility and electron transfer through the quinone-Fe-acceptor complex

In cyanobacteria, Glu-244 and Tyr-246 of the Photosystem II (PS II) D1 protein are hydrogen bonded to two water molecules that are part of a hydrogen-bond network between the bicarbonate ligand to a non-heme iron and the cytosol. Ala substitutions were introduced in Synechocystis sp. PCC 6803 to investigate the roles of these residues and the hydrogen-bond network on electron transfer between the primary plastoquinone acceptor, Q_A, and the secondary plastoquinone acceptor, Q_B, of the quinone-Fe-acceptor complex. All mutants assembled PS II; however, an increase in the PS II to PS I ratio was apparent, particularly in the E244A:Y246A double mutant. The mutants also showed impaired oxygen evolution and retarded chlorophyll a fluorescence decays following single turnover actinic flashes, which appeared to be primarily due to reduced Q_B binding in the E244A strain and an enhanced back reaction with the S2 state of the oxygen-evolving complex in the Y246A mutant. Impaired PS II in the Y246A and E244A:Y246A mutants resulted in inactivation of the psbA gene encoding D1. The Y246A and E244A:Y246A mutants also showed high light sensitivity whereas the E244A mutant showed enhanced resilience towards photodamage. Unlike the control strain, all of the mutants were insensitive to the addition of formate or bicarbonate in assays following chlorophyll decay kinetics that reflect electron transfer between Q_A and Q_B, suggesting the bicarbonate binding environment was perturbed. Our data also indicate that waters W582 and W622 (PDB: 4UB6) have essential roles in maintaining the architecture of the acceptor side of PS II.     

Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels. Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work.     

Histological alterations in the livers of Cx43-deficient mice submitted to a cholestasis model

Gap junction intercellular communication capacity and connexin expression are reportedly involved in cell proliferation. To understand the participation of connexins in biliary duct hyperplasia, a cholestasis model was applied to mice with heterologous deletion of Gja 1, the connexin 43 (Cx43) gene. Heterozygous (Cx43+/-) knockout (KO) and wild-type mice (Cx43+/+) (WT) were submitted to bile duct ligation and euthanized at different time points (48 h, 7 days, and 14 days) after surgery. After euthanasia, the macroscopic and microscopic liver alterations were examined. A histomorphometric study of the livers was performed. For this purpose, a grid containing 100 points was applied to each liver section. The volumetric fraction of bile ducts, hepatocytes, arterioles, and terminal hepatic vein were quantified. Cell proliferation was also quantified by western blot PCNA. High mortality was observed in both genotypes. The heterologous deletion of Cx43 did not affect the biliary duct hyperplasia or most of the other parameters analyzed; however, the Cx43-deficient mice showed decrease in hepatic vein angiogenesis in comparison with the wild-type mice 48 h after surgery. In conclusion, our results indicate that the Cx43 gene heterologous deletion does not affect the biliary duct hyperplasia; on the other hand, connexin 43 deficiencies do affect the hepatic vein angiogenesis, although other studies to understand the details of this influence will be necessary.