Long read sequencing and expression studies of AHDC1 deletions in Xia-Gibbs syndrome reveal a novel genetic regulatory mechanism.

Xia-Gibbs syndrome (XGS; MIM# 615829) is a rare mendelian disorder characterized by Development Delay (DD), intellectual disability (ID), and hypotonia. Individuals with XGS typically harbor de novo protein-truncating mutations in the AT-Hook DNA binding motif containing 1 (AHDC1) gene, although some missense mutations can also cause XGS. Large de novo heterozygous deletions that encompass the AHDC1 gene have also been ascribed as diagnostic for the disorder, without substantial evidence to support their pathogenicity. We analyzed 19 individuals with large contiguous deletions involving AHDC1, along with other genes. One individual bore the smallest known contiguous AHDC1 deletion (∼350 Kb), encompassing eight other genes within chr1p36.11 (Feline Gardner-Rasheed, IFI6, FAM76A, STX12, PPP1R8, THEMIS2, RPA2, SMPDL3B) and terminating within the first intron of AHDC1. The breakpoint junctions and phase of the deletion were identified using both short and long read sequencing (Oxford Nanopore). Quantification of RNA expression patterns in whole blood revealed that AHDC1 exhibited a mono-allelic expression pattern with no deficiency in overall AHDC1 expression levels, in contrast to the other deleted genes, which exhibited a 50% reduction in mRNA expression. These results suggest that AHDC1 expression in this individual is compensated by a novel regulatory mechanism and advances understanding of mutational and regulatory mechanisms in neurodevelopmental disorders.

AHDC1 missense mutations in Xia-Gibbs syndrome.

Xia-Gibbs syndrome (XGS; MIM: 615829) is a phenotypically heterogeneous neurodevelopmental disorder (NDD) caused by newly arising mutations in the AT-Hook DNA-Binding Motif-Containing 1 (AHDC1) gene that are predicted to lead to truncated AHDC1 protein synthesis. More than 270 individuals have been diagnosed with XGS worldwide. Despite the absence of an independent assay for AHDC1 protein function to corroborate potential functional consequences of rare variant genetic findings, there are also reports of individuals with XGS-like trait manifestations who have de novo missense AHDC1 mutations and who have been provided a molecular diagnosis of the disorder. To investigate a potential contribution of missense mutations to XGS, we mapped the missense mutations from 10 such individuals to the AHDC1 conserved protein domain structure and detailed the observed phenotypes. Five newly identified individuals were ascertained from a local XGS Registry, and an additional five were taken from external reports or databases, including one publication. Where clinical data were available, individuals with missense mutations all displayed phenotypes consistent with those observed in individuals with AHDC1 truncating mutations, including delayed motor milestones, intellectual disability (ID), hypotonia, and speech delay. A subset of the 10 reported missense mutations cluster in two regions of the AHDC1 protein with known conserved domains, likely representing functional motifs. Variants outside the clustered regions score lower for computational prediction of their likely damaging effects. Overall, de novo missense variants in AHDC1 are likely diagnostic of XGS when in silico analysis of their position relative to conserved regions is considered together with disease trait manifestations.

Genetic testing in ambulatory cardiology clinics reveals high rate of findings with clinical management implications.

PURPOSE: Cardiovascular disease (CVD) is the leading cause of death in adults in the United States, yet the benefits of genetic testing are not universally accepted. METHODS: We developed the "HeartCare" panel of genes associated with CVD, evaluating high-penetrance Mendelian conditions, coronary artery disease (CAD) polygenic risk, LPA gene polymorphisms, and specific pharmacogenetic (PGx) variants. We enrolled 709 individuals from cardiology clinics at Baylor College of Medicine, and samples were analyzed in a CAP/CLIA-certified laboratory. Results were returned to the ordering physician and uploaded to the electronic medical record. RESULTS: Notably, 32% of patients had a genetic finding with clinical management implications, even after excluding PGx results, including 9% who were molecularly diagnosed with a Mendelian condition. Among surveyed physicians, 84% reported medical management changes based on these results, including specialist referrals, cardiac tests, and medication changes. LPA polymorphisms and high polygenic risk of CAD were found in 20% and 9% of patients, respectively, leading to diet, lifestyle, and other changes. Warfarin and simvastatin pharmacogenetic variants were present in roughly half of the cohort. CONCLUSION: Our results support the use of genetic information in routine cardiovascular health management and provide a roadmap for accompanying research.

Neptune: an environment for the delivery of genomic medicine.

PURPOSE: Genomic medicine holds great promise for improving health care, but integrating searchable and actionable genetic data into electronic health records (EHRs) remains a challenge. Here we describe Neptune, a system for managing the interaction between a clinical laboratory and an EHR system during the clinical reporting process. METHODS: We developed Neptune and applied it to two clinical sequencing projects that required report customization, variant reanalysis, and EHR integration. RESULTS: Neptune has been applied for the generation and delivery of over 15,000 clinical genomic reports. This work spans two clinical tests based on targeted gene panels that contain 68 and 153 genes respectively. These projects demanded customizable clinical reports that contained a variety of genetic data types including single-nucleotide variants (SNVs), copy-number variants (CNVs), pharmacogenomics, and polygenic risk scores. Two variant reanalysis activities were also supported, highlighting this important workflow. CONCLUSION: Methods are needed for delivering structured genetic data to EHRs. This need extends beyond developing data formats to providing infrastructure that manages the reporting process itself. Neptune was successfully applied on two high-throughput clinical sequencing projects to build and deliver clinical reports to EHR systems. The software is open source and available at https://gitlab.com/bcm-hgsc/neptune .

An immune-competent, replication-permissive Syrian Hamster glioma model for evaluating Delta-24-RGD oncolytic adenovirus.

BACKGROUND: Oncolytic adenoviruses are promising new treatments against solid tumors, particularly for glioblastoma (GBM), and preclinical models are required to evaluate the mechanisms of efficacy. However, due to the species selectivity of adenovirus, there is currently no single animal model that supports viral replication, tumor oncolysis, and a virus-mediated immune response. To address this gap, we took advantage of the Syrian hamster to develop the first intracranial glioma model that is both adenovirus replication-permissive and immunocompetent. METHODS: We generated hamster glioma stem-like cells (hamGSCs) by transforming hamster neural stem cells with hTERT, simian virus 40 large T antigen, and h-RasV12. Using a guide-screw system, we generated an intracranial tumor model in the hamster. The efficacy of the oncolytic adenovirus Delta-24-RGD was assessed by survival studies, and tumor-infiltrating lymphocytes (TILs) were evaluated by flow cytometry. RESULTS: In vitro, hamGSCs supported viral replication and were susceptible to Delta-24-RGD mediated cell death. In vivo, hamGSCs consistently developed into highly proliferative tumors resembling high-grade glioma. Flow cytometric analysis of hamster gliomas revealed significantly increased T-cell infiltration in Delta-24-RGD infected tumors, indicative of immune activation. Treating tumor-bearing hamsters with Delta-24-RGD led to significantly increased survival compared to hamsters treated with phosphate buffered saline (PBS). CONCLUSIONS: This adenovirus-permissive, immunocompetent hamster glioma model overcomes the limitations of previous model systems and provides a novel platform to study the interactions between tumor cells, the host immune system, and oncolytic adenoviral therapy; understanding of which will be critical to implementing oncolytic adenovirus in the clinic.

Phenotypic and protein localization heterogeneity associated with AHDC1 pathogenic protein-truncating alleles in Xia-Gibbs syndrome.

Xia-Gibbs syndrome (XGS) is a rare Mendelian disease typically caused by de novo stop-gain or frameshift mutations in the AT-hook DNA binding motif containing 1 (AHDC1) gene. Patients usually present in early infancy with hypotonia and developmental delay and later exhibit intellectual disability (ID). The overall presentation is variable, however, and the emerging clinical picture is still evolving. A detailed phenotypic analysis of 34 XGS individuals revealed five core phenotypes (delayed motor milestones, speech delay, low muscle tone, ID, and hypotonia) in more than 80% of individuals and an additional 12 features that occurred more variably. Seizures and scoliosis were more frequently associated with truncations that arise before the midpoint of the protein although the occurrence of most features could not be predicted by the mutation position. Transient expression of wild type and different patient truncated AHDC1 protein forms in human cell lines revealed abnormal patterns of nuclear localization including a diffuse distribution of a short truncated form and nucleolar aggregation in mid-protein truncated forms. Overall, both the occurrence of variable phenotypes and the different distribution of the expressed protein reflect the heterogeneity of this syndrome.

[Electroacupuncture relieved neuropathic pain by suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord in rats with spared nerve injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on changes of expression of L-Arg transporter 2 (CAT-2) mRNA and nitric oxide synthase (iNOS) mRNA and protein and contents of NO and cGMP of L4-L6 segments of spinal cord in rats with spared nerve injury (SNI), so as to reveal its mechanism underlying reducing neuropathic pain. METHODS: A total of 120 male SD rats were randomly divided into sham operation, model, EA and NOS inhibitor (N omega-Nitro-L-arginine methyl ester hydrochloride, L-NAME) groups, with 30 rats in each group. The neuropathic pain model was established by ligating and cutting the tibial nerve and the common peroneal nerve. EA (2 Hz, 1-3 mA) was applied to "Weizhong" (BL40) and "Huantiao" (GB30)on the damaged hindlimb for 30 min, once daily from day 11 to 17 after SNI. Rats of the L-NAME group received i.p. of L-NAME (60 mg·kg-1·d-1) for 7 consecutive days. The mechanical pain threshold (PT) was determined before and 10 and 16 d after SNI, respectively. The expression le-vels of CAT-2 mRNA and iNOS mRNA, and iNOS protein in the L4-L6 segments of the spinal cord were detected by using reverse transcription - polymerase chain reaction (RT-PCR) and Western blot, respectively, and the contents of NO and cGMP of L4-L6 assayed using nitrate/nitrite reductase method and radioimmunoassay, respectively. RESULTS: After modeling, the PT was significantly decreased on day 10 and 16 after SNI in comparison with the sham operation group and their own baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05). Compared with the sham operation group, the expression levels of CAT-2 mRNA and iNOS mRNA and protein, as well as the contents of NO2－/NO3－and cGMP were signi-ficantly up-regulated in the model group (P<0.05, P<0.01). Following EA intervention, the levels of CAT-2 mRNA and iNOS mRNA and iNOS protein, and NO2－/NO3－and cGMP contents were all reversed in both EA and L-NAME groups (P<0.05, P<0.01). The effect of EA was significantly superior to that of L-NAME in raising the PT on day 16 after SNI (P<0.05), but obviously inferior to that of L-NAME in down-regulating the expression of CAT-2 mRNA and iNOS mRNA and protein (P<0.05). No significant differences were found between the EA and L-NAME groups in down-regulating NO2－/NO3－ andcGMP contents (P>0.05). CONCLUSION: EA intervention can effectively relieve neuropathic pain in SNI rats, which may be closely related to its function in suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord.

A New Compound Heterozygous Mutation Of BSCL2 In A Chinese Zhuang Ethnic Family With Congenital Generalized Lipodystrophy.

PURPOSE: This study aims to report the clinical features of an infant with CGL in a Chinese Zhuang ethnic family, whose family members were discovered to carry new pathogenic mutations in the BSCL2. PATIENTS AND METHODS: In this study, we report clinical and molecular investigations of CGL disease in a family of 4 members (parents and two sons). We used whole exome sequencing (WES) in the family to examine the genetic cause of the disease. RESULTS: The proband presented with skin pigmentation, hypertriglyceridemia and diabetes. WES identified a previously unreported compound heterozygous mutation in the BSCL2 (c.545_546insCCG heterozygous mutation and exon 3 heterozygous deletion) in the proband. His mother is a heterozygous carrier of the c.545_546insCCG mutation and his father and brother are carriers of the exon 3 heterozygous deletion. CONCLUSION: Compound heterozygous mutation of the BSCL2 (new c.545_546insCCG heterozygous mutation and new exon 3 heterozygous deletion) was detected in the proband with characteristic clinical manifestations of CGL2.

[Effect of Electroacupuncture Intervention on CaMK II-CREB Pathway in Spinal Cord in Rats with Spared Nerve Injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Weizhong" (BL 40)-"Huantiao" (GB 30) on expression of phosphorylated calcium/calmodulin dependent protein kinase II (p-CaMK II) and cAMP response element binding protein (p-CREB) in the spinal cord in rats with spared nerve injury (SNI), so as to explore its mechanism underlying easing neuropathic pain. METHODS: Sixty SD rats were randomly divided into five groups: control (sham-operation) , model, EA, AP-5 (a NMDA receptor antagonist) and L-NAME (a non-selective nitric oxide synthase, NOS inhibitor) (n = 12 in each group). The neuropathic pain model was established by sectioning the right tibal nerve and common peroneal nerve. EA intervention (2 Hz, 1 mA, increasing 1 mA/10 min) was applied to "Weizhong" (BL 40) and "Huantiao" (GB 30) on the injured side for 30 min, once a day for 7 days. Rats of the AP-5 and L-NAME groups were treated by intragastric administration of AP-5 (0.7 mg · kg(-1) · d(-1)) and L-NAME (60 mg · kg(-1) · d(-1)) respectively from the 11 th day after operation, once daily for 7 days. The mechanical pain thresholds were measured before the SNI procedure (baseline) and at the 10th and 16th day after the procedure. The expression of p-CaMK II protein and p-CREB protein and gene of the spinal cord (L4-L6 segments) was determined by Western blot and fluorescence quantitative-polymerase chain reaction (PCR), separately. RESULTS: In comparison to the control group, the mechanical pain threshold was significantly decreased in the model group (P < 0.01). After EA intervention, the mechanical pain thresholds of the EA, AP-5 and L-NAME groups were obviously increased (P < 0.01, P < 0.05) on day 16 post SNI procedure. The expression levels of p-CaMK II and p-CREB proteins and CREB mRNA in the spinal cord were significantly higher in the model group than in the control group (P < 0.05). Compared with the model group, the expression levels of spinal p-CaMK II and p-CREB proteins and CREB mRNA were obviously down-regulated in the EA group (P < 0.05), but not in the AP-5 group and the L-NAME group (P > 0.055. CONCLUSION: EA intervention of BL 40-GB 30 may alleviate pain in neuropathic pain rats, which may be related to its effects in down-regulating spinal CaMK II-CREB pathway function.

[Effect of electroacupuncture intervention on N-methyl-D-aspartic acid receptor expression in spinal cord in rats with chronic constrictive injury of the sciatic nerve].

UNLABELLED: OBJECTIVE; To observe the changes of N-methyl-D-aspartic acid (NMDA) receptor expression of spinal cord after electroacupuncture (EA) intervention in rats with chronic constrictive injury (CCI) of the sciatic nerve so as to reveal the mechanism underlying improvement of neuropathic pain. METHODS: Sixty male SD rats were randomly divided into sham operation (sham) group, CCI model group and EA group (n = 20). CCI model was established by ligature of the right sciatic nerve with a piece of surgical chromic suture. For rats of the sham group, the right sciatic nerve was just isolated without ligature. The rats of the EA group were given with EA stimulation of "Weizhong" (BL 40) and "Huantiao" (GB 30) on the injured side at a frequency of 2 Hz, electric current of 1-3 mA for 30 min (increasing 1 mA every 10 min). The treatment was conducted once a day from the 11th day to the 20th day after modeling. NMDA receptor 2 B subunit (NR 2 B) protein and mRNA expression levels in the spinal cord were determined by immunohistochemistry, Western blot and reverse transcription (RT)-polymerase chain reaction (PCR), respectively, and spinal NMDA receptor subunit 1 (NR 1) protein and mRNA expression levels were measured by Western blot and RT-PCR, respectively. RESULTS: In comparison with the sham group, NR 1 protein and mRNA expression levels of the model group in the spinal cord were considerably upregulated after CCI (P < 0.01, P < 0.05). In comparison with the model group NR 1 protein and mRNA expression levels of the EA group in the spinal cord were evidently down-regulated (P < 0.05). No significant changes of NR 2 B protein and mRNA expression after CCI and EA stimulation were found after immunohistochemistry, Western blot and RT-PCR measurements (P > 0.05). CONCLUSION: EA intervention is effective in alleviating neuropathic pain in CCI rats, which may be closely related to its effects in lowering functional activity of NR 1 protein and mRNA in the spinal cord.

Myxoma virus sensitizes cancer cells to gemcitabine and is an effective oncolytic virotherapeutic in models of disseminated pancreatic cancer.

Myxoma virus (MYXV) is a novel oncolytic virus that has been shown to replicate in pancreatic cancer cells, but its efficacy in animal models of pancreatic cancer has not been determined. The efficacy of MYXV as monotherapy or in combination with gemcitabine was evaluated in intraperitoneal dissemination (IPD) models of pancreatic cancer. The effects of an intact immune system on the efficacy of MYXV therapy was tested by comparing immunodeficient versus immunocompetent murine models and combination therapy with gemcitabine was also evaluated. In cell culture, MYXV replication was robust in a broad range of pancreatic cancer cells and also showed increased oncolysis in combination with gemcitabine. In animal models, MYXV treatment conferred survival benefits over control or gemcitabine-treated cohorts regardless of the cell line or animal model used. MYXV monotherapy was most effective in an immunocompetent IPD model, and resulted in 60% long-term survivors. In Pan02 engrafted immunocompetent IPD models, sequential treatment in which MYXV was administered first, followed by gemcitabine, was the most effective and resulted in 100% long-term survivors. MYXV is an effective oncolytic virus for pancreatic cancer and can be combined with gemcitabine to enhance survival, particularly in the presence of an intact host immune system.

Enhanced expression of Pctk1, Tcf12 and Ccnd1 in hippocampus of rats: Impact on cognitive function, synaptic plasticity and pathology.

We previously identified a set of 50 genes that were differentially transcribed in the hippocampal CA1 region of aged, learning-impaired rats compared to aged, superior learning animals during a Morris water maze paradigm. In the current study, we expressed three of these genes (Pctk1, Tcf12 and Ccnd1), which had shown increased transcription in aged, learning impaired rats, in the hippocampus of young rats using viral gene transfer and tested for learning and memory deficits at age 7-14months. Pctk1 injected animals displayed a modest deficit in acquiring latency in both the Morris water maze and the reverse Morris maze. In the radial arm water maze paradigm, Pctk1, Tcf12 and Ccnd1 expressing animals all showed significant deficits in spatial working memory compared to controls. Rats injected with Ccnd1 and Tcf12, but not Pctk1, also showed a significant deficit in spatial reference memory in the radial arm water maze. Electrophysiological experiments revealed no difference in LTP in Ccnd1 and Pctk1 animals. However, LTD induced by low frequency stimulation was observed in control and Ccnd1 animals, but not in Pctk1 treated animals. In addition, neither Ccnd1 nor Pctk1 expression produced any detectable neuropathology. In contrast Tcf12 expressing animals displayed significant neurodegeneration in both CA1 and dentate gyrus. Several Tcf12 animals also developed tumors that appeared to be glioblastomas, suggesting that aberrant Tcf12 expression in the hippocampus is tumorigenic. Thus, behavioral experiments suggested that overexpression of Pctk1 and Ccnd1 produce a deficit in learning and memory, but electrophysiological experiments do not point to a simple mechanism. In contrast, the learning and memory deficits in Tcf12 animals are likely due to neuropathology associated with Tcf12 gene expression.

Oncolytic virotherapy for ovarian cancer.

In the past two decades, more than 20 viruses with selective tropism for tumor cells have been developed as oncolytic viruses (OVs) for treatments of a variety of malignancies. Of these viruses, eleven have been tested in human ovarian cancer models in preclinical studies. So far, nine phase I or II clinical trials have been conducted or initiated using four different types of OVs in patients with recurrent ovarian cancers. In this article, we summarize the different OVs that are being assessed as therapeutics for ovarian cancer. We also present an overview of recent advances in identification of key genetic or immune-response pathways involved in tumorigenesis of ovarian cancer, which provides a better understanding of the tumor specificities and oncolytic properties of OVs. In addition, we discuss how next-generation OVs could be genetically modified or integrated into multimodality regimens to improve clinical outcomes based on recent advances in ovarian cancer biology.

Oncolytic virotherapy for pancreatic cancer.

Within the past decade, many oncolytic viruses (OVs) have been studied as potential treatments for pancreatic cancer and some of these are currently under clinical trials. The applicability of certain OVs, such as adenoviruses, herpesviruses and reoviruses, for the treatment of pancreatic cancer has been intensively studied for several years, whereas the applicability of other more recently investigated OVs, such as poxviruses and parvoviruses, is only starting to be determined. At the same time, studies have identified key characteristics of pancreatic cancer biology that provide a better understanding of the important factors or pathways involved in this disease. This review aims to summarise the different replication-competent OVs proposed as therapeutics for pancreatic cancer. It also focuses on the unique biology of these viruses that makes them exciting candidate virotherapies for pancreatic cancer and discusses how they could be genetically manipulated or combined with other drugs to improve their efficacy based on what is currently known about the molecular biology of pancreatic cancer.

α-Synuclein expression in rat substantia nigra suppresses phospholipase D2 toxicity and nigral neurodegeneration.

We present genetic evidence that an in vivo role of α-synuclein (α-syn) is to inhibit phospholipase D2 (PLD2), an enzyme that is believed to participate in vesicle trafficking, membrane signaling, and both endo- and exocytosis. Overexpression of PLD2 in rat substantia nigra pars compacta (SNc) caused severe neurodegeneration of dopamine (DA) neurons, loss of striatal DA, and an associated ipsilateral amphetamine-induced rotational asymmetry. Coexpression of human wild type α-syn suppressed PLD2 neurodegeneration, DA loss, and amphetamine-induced rotational asymmetry. However, an α-syn mutant defective for inhibition of PLD2 in vitro also failed to inhibit PLD toxicity in vivo. Further, reduction of PLD2 activity in SNc, either by siRNA knockdown of PLD2 or overexpression of α-syn, both produced an unusual contralateral amphetamine-induced rotational asymmetry, opposite to that seen with overexpression of PLD2, suggesting that PLD2 and α-syn were both involved in DA release or reuptake. Finally, α-syn coimmunoprecipitated with PLD2 from extracts prepared from striatal tissues. Taken together, our data demonstrate that α-syn is an inhibitor of PLD2 in vivo, and confirm earlier reports that α-syn inhibits PLD2 in vitro. Our data also demonstrate that it is possible to use viral-mediated gene transfer to study gene interactions in vivo.

In vivo RNAi-mediated alpha-synuclein silencing induces nigrostriatal degeneration.

Two small-interfering RNAs (siRNAs) targeting alpha-synuclein (alpha-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of alpha-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate alpha-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of alpha-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat alpha-syn and alpha-syn siRNA partially reversed the neurodegenerative and behavioral effects of alpha-syn siRNA alone. Our data show that alpha-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type alpha-syn expression increase the risk of nigrostriatal pathology.

[Influence of acupuncture on the motor ability in training-induced fatigue mice].

OBJECTIVE: To observe the influence of acupuncture on the motor ability of training-induced fatigue mice so as to explore its mechanism underlying acupuncture-induced improvement of physical training fatigue. METHODS: Sixty Kunming mice were divided into normal control, training model and acupuncture groups, with 20 mice in each. The training-fatigue model was established by forcing the mouse to swim in a water tank for 30 min/d in the 1st week, 60 min/d in the 2nd week, 90 min/d in the 3rd week and 120 min/d in the 4th and 5th weeks. "Guanyuan" (CV 4), and bilateral "Zusanli" (ST 36) and "Shenshu" (BL 23) were punctured with filiform needles, and with the needle twirled for about 30 s and retained for 5 min. Thetreatment was conducted once daily for 2 weeks. The duration of exhaustion training was recorded. Serum lactic acid (LA), lactate dehydrogenase (LDH) and creatine kinase (CK) contents were detected by LA oxidase method, immune-suppressive assay and enzyme-linked immunoassay, respectively. RESULTS: Compared with the training-fatigue (model) group, the duration of the exhausted swimming of the acupuncture group was obviously longer (P < 0.05). In comparison with the control group, serum LA, LDH and CK contents were increased considerably in the model and acupuncture groups (P < 0.05, P < 0.01), while compared with the model group, serum LDH level of acupuncture group was increased significantly (P < 0.05), and serum CK level of acupuncture group decreased obviously (P < 0.05). No significant difference was found between the model and acupuncture groups in serum LA content (P > 0.05). CONCLUSION: Acupuncture can effectively improve the motor ability of the training-fatigue mice, which may be closely relevant to its effects in upregulating LDH activity and reducing serum CK.

The phosphorylation state of Ser-129 in human alpha-synuclein determines neurodegeneration in a rat model of Parkinson disease.

Studies have shown that alpha-synuclein (alpha-syn) deposited in Lewy bodies in brain tissue from patients with Parkinson disease (PD) is extensively phosphorylated at Ser-129. We used recombinant Adeno-associated virus (rAAV) to overexpress human wild-type (wt) alpha-syn and two human alpha-syn mutants with site-directed replacement of Ser-129 to alanine (S129A) or to aspartate (S129D) in the nigrostriatal tract of the rat to investigate the effect of Ser-129 phosphorylation state on dopaminergic neuron pathology. Rats were injected with rAAV2/5 vectors in the substantia nigra pars compacta (SNc) on one side of the brain; the other side remained as a nontransduced control. The level of human wt or mutant alpha-syn expressed on the injected side was about four times the endogenous rat alpha-syn. There was a significant reduction of dopaminergic neurons in the SNc and dopamine (DA) and tyrosine hydroxylase (TH) levels in the striatum of all S129A-treated rats as early as 4 wk postinjection. Nigral DA pathology occurred more slowly in the wt-injected animals, but by 26 wk the wt alpha-syn group lost nigral TH neurons equivalent to the mutated S129A group at 8 wk. In stark contrast, we did not observe any pathological changes in S129D-treated animals. Therefore, the nonphosphorylated form of S129 exacerbates alpha-syn-induced nigral pathology, whereas Ser-129 phosphorylation eliminates alpha-syn-induced nigrostriatal degeneration. This suggests possible new therapeutic targets for Parkinson Disease.

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis.

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.

Phosphorylation of specific serine residues in the PKR activation domain of PACT is essential for its ability to mediate apoptosis.

Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser246, was required for stress-induced phosphorylation of the other, Ser287. Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.