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Receptor‐interacting protein 3 (RIP3) plays an important role in the necroptosis signaling pathway. Our previous studies have shown that the RIP3/mixed lineage kinase domain‐like protein (MLKL)‐mediated necroptosis occurs in retinal ganglion cell line 5 (RGC‐5) following oxygen‐glucose deprivation (OGD). However, upstream regulatory pathways of RIP3 are yet to be uncovered. The purpose of the present study was to investigate the role of p90 ribosomal protein S6 kinase 3 (RSK3) in the phosphorylation of RIP3 in RGC‐5 cell necroptosis following OGD. Our results showed that expression of RSK3, RIP3, and MLKL was upregulated in necroptosis of RGC‐5 after OGD. A computer simulation based on our preliminary results indicated that RSK3 might interact with RIP3, which was subsequently confirmed by co‐immunoprecipitation. Further, we found that the application of a specific RSK inhibitor, LJH685, or rsk3 small interfering RNA (siRNA), downregulated the phosphorylation of RIP3. However, the overexpression of rip3 did not affect the expression of RSK3, thereby indicating that RSK3 could be a possible upstream regulator of RIP3 phosphorylation in OGD‐induced necroptosis of RGC‐5 cells. Moreover, our in vivo results showed that pretreatment with LJH685 before acute high intraocular pressure episodes could reduce the necroptosis of retinal neurons and improve recovery of impaired visual function. Taken together, our findings suggested that RSK3 might work as an upstream regulator of RIP3 phosphorylation during RGC‐5 necroptosis.

Purpose: Evidences suggest that during ischemia/reperfusion events, neuronal loss in ganglion cell layers (GCLs) occurs initially in the peripheral retinae followed by the central. However, which key molecule or factor mediates this selective loss needs elucidation. In the present study, we detected the regional expression of active matrix metalloproteinase 3 (Act-MMP3) in the central and peripheral rat retinae following acute retinal ischemia/reperfusion (RI/R) injury and explored the effects and mechanisms of this regional expression on the selective neuronal loss in GCLs.Methods: QPCR and Western Blotting were used to detect the expression of Act-MMP3 in the central part and peripheral part of the adult rat retinae. Immunofluorescence and double immunofluorescence were used to assess the number of NeuN-positive cells in the GCLs and Iba-1+CD 68-positive cells were determined. Additionally, the Linear-regression analysis was performed to test the correlation between the ODV of Act-MMP3 and the neuronal loss in the GCLs/Iba-1+CD 68 positive cells in retinae.Results: An evident up-regulation of active matrix metalloproteinase 3 (Act-MMP3) in peripheral retinae preceded to that in central region following acute RI/R. We found Act-MMP3 up-regulation to be associated with the selective neuronal loss in GCLs (central: r = 0.7566, p < .0001, r2 = 0.5724; peripheral: r = 0.8241, p < .0001, r2 = 0.6792). Suppressing Act-MMP3 ameliorated the selective neuronal loss in GCLs following acute RI/R. Furthermore, the activation of microglia in the peripheral retinae also preceded to that in the central and was found to be correlated with the regional expression of Act-MMP3 (Central: r = 0.8540, p < .0001, r2 = 0.7294; Peripheral: r = 0.7820, p < .0001, r2 = 0.6116). Suppressing Act-MMP3 ameliorated the microglia regional activation following acute RI/R.Conclusion: The regional expression of Act-MMP3 in the rat retinae may contribute to the selective neuronal loss in GCLs and microglia regional activation in acute RI/R.

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma., The Atr-Check1 pathway is involved in cell cycle and the DNA damage response. Here, the authors show that the Atr-Check1 pathway can inhibit axon regeneration in response to Piezo-mediated mechanosensation, affecting functional recovery.

Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly (ADP-ribose) polymerase 1-mediated parthanatos. Calpains cleave series of key substrates that may lead to cell death or participate in cell death. Regarding the investigation of calpain-mediated programed cell death, it is necessary to identify specific inhibitors that inhibit calpain mediated neuronal death and nervous system diseases.

Background Available data on antibiotic resistance in sub-Saharan Africa is limited despite its increasing threat to global public health. As there is no previous study on antibiotic resistance in patients with clinical features of healthcare-associated infections (HAIs) in Sierra Leone, research is needed to inform public health policies. Our study aimed to assess antibiotic resistance rates from isolates in the urine and sputum samples of patients with clinical features of HAIs. Methodology We conducted a cross-sectional study of adult inpatients aged ≥18 years at Connaught Hospital, an urban tertiary care hospital in Freetown between February and June 2018. Results Over the course of the study, we enrolled 164 patients. Risk factors for HAIs were previous antibiotic use (93.3%), comorbidities (58.5%) and age (≥65 years) (23.9%). Of the 164 samples, 89.6% were urine. Bacterial growth was recorded in 58.8% of cultured specimens; the type of specimen was an independent predictor of bacterial growth (p Escherichia coli and Klebsiella pneumoniae; 29.2% and 19.0% in urine samples and 18.8% and 31.3% in sputum samples, respectively. The overall resistance rates were 58% for all extended-spectrum beta-lactamase (ESBL)-producing organisms, 13.4% for carbapenem-resistant non-lactose fermenting gram-negative bacilli, 8.7% for carbapenem-resistant Acinetobacter baumannii (CRAB) and 1.3% for carbapenem-resistant Enterobacteriaceae (CRE). There were no carbapenem-resistant P. aeruginosa (CRPA) isolates but all Staphylococcus aureus isolates were methicillin-resistant S. aureus. Conclusion We demonstrated a high prevalence rate of ESBL-producing organisms which are a significant burden at the main tertiary hospital in Sierra Leone. Urgent action is needed to strengthen microbiological diagnostic infrastructure, initiate surveillance on antibiotic resistance and develop and implement policy framework on antibiotic stewardship.

In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected.We obtained a blood specimen from the index patient and attempted to isolate and identify a causative pathogen, using genome sequence analysis and electron microscopy. We also initiated a heightened surveillance program in the same hospital to screen for other patients who presented with fever, headache, and a history of tick bites. We used reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cell-culture assays to detect the pathogen and immunofluorescence and neutralization assays to determine the levels of virus-specific antibodies in serum specimens from the patients.We found that the index patient was infected with a previously unknown segmented RNA virus, which we designated Alongshan virus (ALSV) and which belongs to the jingmenvirus group of the family Flaviviridae. ALSV infection was confirmed by RT-PCR assay in 86 patients from Inner Mongolia and Heilongjiang who presented with fever, headache, and a history of tick bites. Serologic assays showed that seroconversion had occurred in all 19 patients for whom specimens were available from the acute phase and the convalescent phase of the illness.A newly discovered segmented virus was found to be associated with a febrile illness in northeastern China. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China.).

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis. The aim of this study was to investigate SFTS virus (SFTSV) infections in ticks in Northeastern China. A total of 6427 ticks, including 5450 Haemaphysalis longicornis, 463 Dermacentor silvarum, 351 Dermacentor nuttalli, and 163 Ixodes persulcatus, were sampled in the Liaoning, Jilin, and Heilongjiang Provinces of Northeastern China. Viral megagenomic analysis of the ticks revealed 25 contigs targeting the M and L segments of the SFTSV genome. H. longicornis collected from Jinxing, Jilin Province, were analyzed by RT-PCR, which showed positive results for SFTSV, with a minimum prevalence of 3.0%. The full-length sequence of the S, M, and L segments of the SFTSV were obtained, and phylogenetic analysis showed that the virus strain found in Jilin formed a monophyletic cluster with the SFTSV strains from Jiangsu, suggesting that SFTSV in the Jilin Province may have spread from the Jiangsu Province. These findings are the first to demonstrate molecular evidence of SFTSV in ticks in the Jilin Province of Northeastern China and indicate the need for measures to prevent and control SFTS.

In our previous study, we reported that peptidyl-prolyl isomerase 1 (Pin1)-modulated regulated necrosis (RN) occurred in cultured retinal neurons after glutamate injury. In the current study, we investigated the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in Pin1-modulated RN in cultured rat retinal neurons, and in an animal in vivo model. We first demonstrated that glutamate might lead to calcium overloading mainly through ionotropic glutamate receptors activation. Furthermore, CaMKII activation induced by overloaded calcium leads to Pin1 activation and subsequent RN. Inactivation of CaMKII by KN-93 (KN, i.e., a specific CaMKII inhibitor) application can decrease the glutamate-induced retinal neuronal RN. Finally, by using an animal in vivo model, we also demonstrated the important role of CaMKII in glutamate-induced RN in rat retina. In addition, flash electroretinogram results provided evidence that the impaired visual function induced by glutamate can recover after CaMKII inhibition. In conclusion, CaMKII is an up-regulator of Pin1 and responsible for the RN induced by glutamate. This study provides further understanding of the regulatory pathway of RN and is a complementary mechanism for Pin1 activation mediated necrosis. This finding will provide a potential target to protect neurons from necrosis in neurodegenerative diseases, such as glaucoma, diabetic retinopathy, and even central nervous system diseases.

The purpose of the current study was to investigate whether peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) can interact with calpastatin (CAST) and regulate CAST/calpain2, under excessive glutamate conditions, and subsequently regulate necrosis in rat retinal neurons. Glutamate triggered CAST/calpain2-mediated necrosis regulation in primary cultured retinal neurons, as demonstrated by propidium iodide-staining and lactate dehydrogenase assay. Co-IP results and a computer simulation suggested that Pin1 could bind to CAST. Western blot, real-time quantitative polymerase chain reaction, immunofluorescence, and phosphorylation analysis results demonstrated that CAST was regulated by Pin1, as proven by the application of juglone (i.e., a Pin1 specific inhibitor). The retinal ganglion cell 5 cell line, combined with siRNA approach and flow cytometry, was then used to verify the regulatory pathway of Pin1 in CAST/calpain2-modulated neuronal necrosis that was induced by glutamate. Finally, in vivo studies further confirmed the role of Pin1 in CAST/calpain2-modulated necrosis following glutamate excitation, in the rat retinal ganglion cell and inner nuclear layers. In addition, a flash electroretinogram study provided evidence for the recovery of impaired visual function, which was induced by glutamate, with juglone treatment. Our work aims to investigate the involvement of the Pin1-CAST/calpain2 pathway in glutamate-mediated excitotoxicity.

Heat shock protein 90α (HSP90α) maintains cell stabilization and regulates cell death, respectively. Recent studies have shown that HSP90α is involved in receptor interacting protein 3 (RIP3)-mediated necroptosis in HT29 cells. It is known that oxygen and glucose deprivation (OGD) can induce necroptosis, which is regulated by RIP3 in neurons. However, it is still unclear whether HSP90α participates in the process of OGD-induced necroptosis in cultured neurons via the regulation of RIP3. Our study found that necroptosis occurs in primary cultured cortical neurons and PC-12 cells following exposure to OGD insult. Additionally, the expression of RIP3/p-RIP3, MLKL/p-MLKL, and the RIP1/RIP3 complex (necrosome) significantly increased following OGD, as measured through immunofluorescence (IF) staining, Western blotting (WB), and immunoprecipitation (IP) assay. Additionally, data from computer simulations and IP assays showed that HSP90α interacts with RIP3. In addition, HSP90α was overexpressed following OGD in cultured neurons, as measured through WB and IF staining. Inhibition of HSP90α in cultured neurons, using the specific inhibitor, geldanamycin (GA), and siRNA/shRNA of HSP90α, protected cultured neurons from necrosis. Our study showed that the inhibitor of HSP90α, GA, rescued cultured neurons not only by decreasing the expression of total RIP3/MLKL, but also by decreasing the expression of p-RIP3/p-MLKL and the RIP1/RIP3 necrosome. In this study, we reveal that inhibition of HSP90α protects primary cultured cortical neurons and PC-12 cells from OGD-induced necroptosis through the modulation of RIP3 expression.

Long non-coding RNAs (lncRNAs) display multiple functions including regulation of neuronal injury. However, their impact in methamphetamine (METH)-induced neurotoxicity has rarely been reported. Here, using microarray analysis, we investigated the expression profiling of lncRNAs and mRNAs in primary cultured prefrontal cortical neurons after METH treatment. We observed a difference in lncRNA and mRNA expression between the experimental and sham control groups. Using bioinformatics, we analyzed the highest enriched gene ontology (GO) terms of biological process, cellular component, and molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and pathway network analysis. Furthermore, an lncRNA-mRNA co-expression sub-network for aberrantly expressed terms revealed possible interactions of lncRNA NR_110713 and NR_027943 with their related genes. Afterwards, three lncRNAs (NR_110713, NR_027943, GAS5) and two mRNAs (Ddit3, Casp12) were targeted to validate the microarray data by qRT-PCR. This presented an overview of lncRNA and mRNA expression profiling and indicated that lncRNA might participate in METH-induced neuronal apoptosis by regulating the coding genes of neurons.

Tick-borne diseases are considered as emerging infectious diseases in humans and animals in China. In this study, Ixodes persulcatus (n = 1699), Haemaphysalis concinna (n = 412), Haemaphysalis longicornis (n = 390), Dermacentor nuttalli (n = 253), and Dermacentor silvarum (n = 204) ticks were collected by flagging from northeastern China, and detected for infection with Anaplasma, Ehrlichia, Babesia, and Hepatozoon spp. by using nested polymerase chain reaction assays and sequencing analysis. Anaplasma phagocytophilum was detected in all tick species, i.e., I. persulcatus (9.4%), H. longicornis (1.9%), H. concinna (6.5%), D. nuttalli (1.7%), and D. silvarum (2.3%); Anaplasma bovis was detected in H. longicornis (0.3%) and H. concinna (0.2%); Ehrlichia muris was detected in I. persulcatus (2.5%) and H. concinna (0.2%); Candidatus Neoehrlichia mikurensis was only detected in I. persulcatus (0.4%). The Ehrlichia variant (GenBank access number KU921424), closely related to Ehrlichia ewingii, was found in H. longicornis (0.8%) and H. concinna (0.2%). I. persulcatus was infected with Babesia venatorum (1.2%), Babesia microti (0.6%), and Babesia divergens (0.6%). Additionally, four Babesia sequence variants (GenBank access numbers 862303–862306) were detected in I. persulcatus, H. longicornis, and H. concinna, which belonged to the clusters formed by the parasites of dogs, sheep, and cattle (B. gibsoni, B. motasi, and B. crassa). Two Hepatozoon spp. (GenBank access numbers KX016028 and KX016029) associated with hepatozoonosis in Japanese martens were found in the collected ticks (0.1–3.1%). These findings showed the genetic variability of Anaplasma, Ehrlichia, Babesia, and Hepatozoon spp. circulating in ticks in northeastern China, highlighting the necessity for further research of these tick-associated pathogens and their role in human and animal diseases.

Background Tick-borne rickettsioses are considered important emerging zoonoses worldwide, but their etiological agents, rickettsiae, remain poorly characterized in northeastern China, where many human cases have been reported during the past several years. Here, we determined the characteristics of Rickettsia spp. infections in ticks in this area. Methods Ticks were collected by flagging vegetation from Jilin and Heilongjiang provinces of northeastern China followed by morphological identification. The presence of Rickettsia spp. in ticks was detected by PCR targeting the 23S-5S ribosomal RNA intergenic spacer, citrate synthase (gltA) gene, and 190-kDa outer membrane protein gene (ompA). The newly-generated sequences were subjected to phylogenetic analysis using the software MEGA 6.0. Results The overall infection rate of Rickettsia spp. was 6.12 %. Phylogenetic analyses based on the partial gltA and ompA genes demonstrated that rickettsiae detected in the ticks belong to four species, including “Candidatus Rickettsia tarasevichiae”, Rickettsia heilongjiangensis, Rickettsia raoultii, and a potential new species isolate. The associated tick species were also identified, i.e. Dermacentor nuttalli and Dermacentor silvarum for R. raoultii, Haemaphysalis concinna and Haemaphysalis longicornis for R. heilongjiangensis, and Ixodes persulcatus for “Ca. R. tarasevichiae”. All Rickettsia spp. showed significantly high infection rates in ticks from Heilongjiang when compared to Jilin Province. Conclusion Rickettsia heilongjiangensis, R. raoultii and “Ca. R. tarasevichiae” are widely present in the associated ticks in northeastern China, but more prevalent in Heilongjiang Province. The data of this study increase the information on the distribution of Rickettsia spp. in northeastern China, which have important public health implications in consideration of their recent association with human diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1764-2) contains supplementary material, which is available to authorized users.

Summary Background Worldwide, 30% of the world's population have antibodies to the intracellular protozoan parasite Toxoplasma gondii and about 36·7 million people are infected with HIV, but little is known about the prevalence of co-infection with T gondii and HIV. We aimed to characterise the epidemiology and burden of T gondii co-infection in people with HIV infection. Methods In this systematic review and meta-analysis, we searched PubMed, Embase, Google Scholar, ScienceDirect, Chinese Web of Knowledge, Wanfang, and Chongqing VIP databases for studies reporting T gondii infection in HIV-infected people from inception to Feb 29, 2016. Studies were included if they investigated people with HIV infection and presented data that allowed us to establish the prevalence of T gondii infection. We excluded reviews, repeated studies, or animal studies, as well as studies that provided the final results without raw data, had a sample size less than 30 people, had unclear diagnostic methods of T gondii infection, or that included populations with increased risk of T gondii infection. We extracted the numbers of patients with HIV infection and T gondii co-infection from the identified studies. We estimated pooled prevalence of T gondii infection in HIV-infected people by a random-effects model, and evaluated its overall infection burden worldwide. Findings Our search identified 7843 records and after removal of duplicates and initial screening, we reviewed 312 studies in full. Of these articles, 238 were excluded, leaving 74 studies that included 25 989 HIV-infected people from 34 countries. Of these people, 7326 had T gondii co-infection and we estimated the pooled worldwide prevalence of T gondii infection to be 35·8% (95% CI 30·8–40·7). 2353 of 8837 of people in Asia and the Pacific had co-infection with T gondii and HIV (prevalence 25·1%, 95% CI 19·0–31·2), and prevalence was low in this region compared with that in sub-Saharan Africa (44·9%, 32·3–57·5, 2129/5686; odds ratio [OR] 0·61), Latin America and the Caribbean (49·1%, 27·9–70·4, 510/980; OR 0·33), and North Africa and the Middle East (60·7%, 24·1–97·3, 245/439; OR 0·29). 1561 of 3780 people in low-income countries had co-infection (54·7%, 95% CI 35·8–73·5), which was higher than in middle-income countries (34·2%, 27·4–40·9, 3632/11 540; OR 1·53) and high-income countries (26·3%, 20·4–32·2, 2133/10 669; OR 2·82). Worldwide, we calculated that there were roughly 13 138 600 (95% CI 11 303 600–14 936 900) cases of T gondii co-infection in HIV-infected people, with 87·1% in sub-Saharan Africa (11 449 500 cases, 95% CI 8 236 500–14 662 500). Interpretation Our findings suggest that people with HIV infection have a very high burden of T gondii infection, especially in sub-Saharan Africa, and emphasise the importance of routine surveillance for T gondii infection in all HIV-infected people. Funding National Natural Science Foundation and the Special Fund for Agro-scientific Research in the Public Interest in China.

N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of proteasomes and calpain, is widely used to reduce proteasomes or calpain-mediated cell death in rodents. However, ALLN is toxic to retinal neurons to some extent. At the concentration of 10 μM, ALLN is non-toxic to cortical neurons, but induces cell death of retinal neurons in vitro. The tolerance concentration of ALLN for retinal neurons is unclear, and the precise mechanism of cell death induced by ALLN remains elusive. In this study, we investigated the toxic effect of ALLN on primary retinal neurons. The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed no significant changes of cell viability at 1 μM but decreased cell viability after treatment of ALLN at 2.5, 5, and 7.5 μM. Lactate dehydrogenase (LDH) release was highly elevated and propidium iodide (PI)-positive cells were significantly increased at 2.5, 5, and 7.5 μM after all treatment times. Moreover, the protein levels of caspase-3 were up-regulated at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. The ratio of Bax/Bcl-2 was raised and Annexin V-positive cells were increased at 5 and 7.5 μM after 12 and 24 h of ALLN treatment. However, there were no significant changes in either the ratio of microtubule-associated protein 1 light chain 3 (LC3) II/LC3 I or monodansylcadaverine (MDC) staining. Our data clearly show that at the concentrations equal to and higher than 2.5 μM, ALLN may induce cell death of primary retinal neurons by necrosis and apoptosis, but not autophagy. These suggest that primary retinal neurons are more susceptible to ALLN treatment and provide a possible mechanism for the cell death of ALLN-sensitive cells in ALLN injury.

Toxoplasma gondii is prevalent in humans and animals worldwide. The present study aimed to determine the genetic diversity of T. gondii isolates from pigs in Jilin province, northeastern China. A total of 100 DNA samples were extracted from the hilar lymph nodes of slaughtered pigs, and 9 (9.0%, 95% confidence interval: 3.4-14.6%) were detected positive for T. gondii B1 gene by a nested polymerase chain reaction (PCR). The positive DNA samples were typed at 11 genetic markers, including 10 nuclear loci (SAG1, 5'-SAG2, and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, and PK1) and an apicoplast locus (Apico) using the multilocus PCR-restriction fragment length polymorphism technology. Only three isolates were completely typed at all loci, showing that they all belonged to the clonal type I. One isolate was typed at five loci, including 5' +3'-SAG2, SAG2, SAG3, GRA6, and L358, revealing the possible clonal type I. This is the first report of the genetic characterization of T. gondii isolates in pigs in Jilin province, northeastern China, which has implications for better understanding the population structure of T. gondii infection in China.