Insights into a functional model of key deubiquitinases UBP12/13 in plants.

Understanding the complexities of protein ubiquitination is crucial, as it plays a multifaceted role in controlling protein stability, activity, subcellular localization, and interaction, which are central to diverse biological processes. Deubiquitinases (DUBs) serve to reverse ubiquitination, but research progress in plant DUBs is noticeably limited. Among existing studies, UBIQUITIN-SPECIFIC PROTEASE 12 (UBP12) and UBP13 have garnered attention for their extensive role in diverse biological processes in plants. This review systematically summarizes the recent advancements in UBP12/13 studies, emphasizing their function, and their substrate specificity, their relationship with E3 ubiquitin ligases, and the similarities and differences with their mammalian orthologue, USP7. By unraveling the molecular mechanisms of UBP12/13, this review offers in-depth insights into the ubiquitin-proteasome system (UPS) in plants and aims to catalyze further explorations and comprehensive understanding in this field.

The relationship between postpartum negative life events and postpartum depression: a moderated mediation model of neuroticism and psychological flexibility.

BACKGROUND: Postpartum depression (PPD) is a major public health problem worldwide. Previous studies have shown that postpartum negative life events and neuroticism are both important risk factors for PPD. However, few studies have considered the role of protective factors in the influence of postpartum negative life events and neuroticism on PPD. Based on the diathesis-stress model and Acceptance and Commitment Therapy (ACT), a moderated mediating model was established to examine the mediating role of neuroticism between postpartum negative life events and PPD, as well as the moderating role of psychological flexibility in this mediating effect. METHODS: A sample of 776 parturients from three different Grade A hospitals in China were assessed using the Edinburgh Postpartum Depression Scale, the Postpartum Negative Life Events Scale, the Neuroticism Subscale of the Big Five Personality Scale, and the Acceptance and Action Questionnaire- II. RESULTS: PPD, postpartum negative life events, neuroticism, and experiential avoidance were significantly positively correlated with one another. Neuroticism partially mediated the relationship between postpartum negative life events and PPD. In this mediation model, the direct path and the second half of the mediation path were moderated by psychological flexibility. Specifically, the links between postpartum negative life events and PPD, as well as between neuroticism and PPD, were stronger when psychological flexibility was low, but weaker when psychological flexibility was high. CONCLUSIONS: The results show that psychological flexibility plays an important role in buffering the negative effects of postpartum negative life events and neuroticism on PPD. These findings provide implications for the prevention and intervention of PPD using an ACT approach.

[Current situation and related of overweight and obesity in children aged 3-6 years old in Urumqi City in 2021].

OBJECTIVE: To understand the current situation and related factors of overweight and obesity in children aged 3-6 years old in Urumqi City. METHODS: From October to December 2021, a questionnaire survey was conducted on the general information of 1897 children and their fathers or mothers from 10 kindergartens in Urumqi City by stratified cluster sampling, and the height and weight of the children were measured. SPSS 25.0 was used for χ~2 test and Logistic regression analysis. RESULTS: There were 1897 children out of which 961(50.66%) were boys, 936(49.34%) were girls, 334(17.60%) were 3 years old, 592(31.21%)were 4 years old, 667(35.16%) were 5 years old, and 304(16.03%) were 6 years old. The prevalence rate of obesity and overweight in children aged 3-6 years old was 31.21%(592). Single factor analysis showed that child's age, the child's sex, the child's dietary habits, whether the child's father had a family history of obesity, whether the child's mother had a family history of obesity, whether the child's mother suffered from hypertension during pregnancy, whether the child's father smoked, whether the child's mother smoked during pregnancy(including passive smoking), the child's family per capita monthly income, the child's family structure type, and the child's mother's pregnancy age were all statistically significant(P<0.05 or P<0.01). The logistic regression analysis showed that child's age, the child's sex, the child's dietary habits, whether the child's father had a family history of obesity, whether the child's mother had a family history of obesity, whether the child's mother suffered from hypertension during pregnancy, whether the child's father smoked, whether the child's mother smoked during pregnancy(including passive smoking), the child's family structure type, and the child's mother's pregnancy age were overweight and obesity children aged 3-6 years old of related factors in Urumqi(P<0.05 or P<0.01). CONCLUSION: The detection rate of overweight and obesity in children aged 3-6 years old in Urumqi City is high.

Tanshinone I restrains osteosarcoma progression by regulating circ_0000376/miR-432-5p/BCL2 axis.

Circular RNAs (circRNAs) have been identified as important regulators in cancer progression. Nevertheless, little is known about the biological function of circ_0000376 in the progression of osteosarcoma (OS). Cell viability, colony formation ability, apoptosis, and motility were analyzed by Cell Counting Kit-8 assay, colony formation assay, flow cytometry, and transwell assays. Cellular glycolytic metabolism was analyzed using commercial kits. RT-qPCR and Western blot assay were performed to analyze RNA and protein expression in OS tissues and cells. Starbase software was used to establish circRNA-microRNA (miRNA)-messenger RNA linkage, and intermolecular interaction was verified by dual-luciferase reporter assay. Xenograft tumor assay was conducted to analyze the effects of Tanshinone I (Tan I) and circ_0000376 on xenograft tumor growth in vivo. Tan I treatment suppressed the viability, migration, invasion, and glycolysis and triggered the apoptosis of OS cells. Tan I treatment markedly down-regulated circ_0000376 expression in OS cells. The addition of circ_0000376 plasmid largely rescued the malignant behaviors of OS cells upon Tan I exposure. Circ_0000376 interacted with miR-432-5p in OS cells. Circ_0000376 overexpression-mediated protective effects in Tan I-induced OS cells were partly attenuated by the accumulation of miR-432-5p. miR-432-5p bound to the 3' untranslated region (3'UTR) of B-cell leukemia/lymphoma 2 (BCL2) in OS cells. miR-432-5p interference-induced effects in Tan I-treated OS cells were partly overturned by the silence of BCL2. Circ_0000376 can act as miR-432-5p sponge to up-regulate BCL2 expression in OS cells. Circ_0000376 silencing contributed to the anti-tumor effect of Tan I on the growth of xenograft tumors in vivo. Tan I exerted an anti-tumor role in OS progression by targeting circ_0000376/miR-432-5p/BCL2 axis.

Repeated long sessions of transcranial direct current stimulation reduces seizure frequency in patients with refractory focal epilepsy: An open-label extension study.

OBJECTIVE: Although clinical trials have demonstrated that cathodal transcranial direct current stimulation (tDCS) is effective for seizure reduction, its long-term efficacy is unknown. This study aimed to determine the long-term effects of repeated cathodal long tDCS sessions on seizure suppression in patients with refractory epilepsy. METHODS: Patients were recruited to participate in an extended phase of a previous randomized, double-blind, sham-controlled, three-arm, parallel, multicenter study on tDCS. The patients were divided into an active tDCS group (20 min of tDCS per day) and an intensified tDCS group (2 × 20 min of tDCS per day). Each tDCS session lasted 2 weeks and the patients underwent repeated sessions at intervals of 2 to 6 months. The cathode was placed over the epileptogenic focus with the current intensity set as 2 mA. Seizure frequency reduction from baseline was analyzed using the Wilcoxon signed-rank test for two related samples. A generalized estimating equation model was used to estimate group, time, and interaction effects. RESULTS: Among the 19 patients who participated in the extended phase, 11 were in the active tDCS group and underwent 2-16 active tDCS sessions, and eight were in the intensified tDCS group and underwent 3-11 intensified tDCS sessions. Seizure reduction was significant from the first to the seventh follow-up, with a median seizure frequency reduction of 41.7%-83.3% (p < 0.05). Compared to the regular tDCS protocol, each intensified tDCS session substantially decreased seizure frequency by 0.3680 (p < 0.05). One patient experienced an increase of 8.5%-232.8% in the total number of seizures during three treatment sessions and follow-ups. CONCLUSION: Repeated long cathodal tDCS sessions yielded significant and progressive long-term seizure reductions in patients with refractory focal epilepsy.

[Correlation of neck circumference with body mass index in preschool children]. / å­¦é¾„å‰å„¿ç«¥é¢ˆå›´ä¸Žä½“é‡æŒ‡æ•°çš„ç›¸å ³æ€§.

OBJECTIVES: To study the correlation between neck circumference and body mass index and the value of neck circumference in identifying overweight and obesity in preschool children. METHODS: The stratified cluster sampling method was used to recruit 3 719 children under 7 years from 10 kindergartens in Urumqi, China. General data were collected, and physical measurements were performed. A Pearson correlation analysis was used to evaluate the correlation between neck circumference and body mass index. The receiver operating characteristic (ROC) curve was used to assess the accuracy of neck circumference in identifying overweight/obesity. The Kappa consistency test was used to assess the consistency of neck circumference and body mass index in identifying overweight/obesity. RESULTS: There was a positive correlation between neck circumference and body mass index in boys and girls of all ages (r≥0.50, P<0.001). According to body mass index as the criteria for overweight/obesity, the children were divided into an overweight/obesity group and a non-overweight/obesity group, and the analysis showed that the overweight/obesity group had a significantly larger neck circumference than the non-overweight/obesity group (P<0.001). The ROC curve analysis showed that neck circumference had an area under the ROC curve of >0.7 in identifying overweight/obesity for boys and girls. The Kappa consistency test showed that the neck circumference and body mass index had a Kappa value of >0.40 in identifying overweight/obesity in boys and girls of all ages. CONCLUSIONS: Neck circumference is positively correlated to body mass index, and neck circumference can be used to identify overweight/obesity in preschool children.

Synaptic clustering differences due to different GABRB3 mutations cause variable epilepsy syndromes.

GABRB3 is highly expressed early in the developing brain, and its encoded ß3 subunit is critical for GABAA receptor assembly and trafficking as well as stem cell differentiation in embryonic brain. To date, over 400 mutations or variants have been identified in GABRB3. Mutations in GABRB3 have been increasingly recognized as a major cause for severe paediatric epilepsy syndromes such as Lennox-Gastaut syndrome, Dravet syndrome and infantile spasms with intellectual disability as well as relatively mild epilepsy syndromes such as childhood absence epilepsy. There is no plausible molecular pathology for disease phenotypic heterogeneity. Here we used a very high-throughput flow cytometry assay to evaluate the impact of multiple human mutations in GABRB3 on receptor trafficking. In this study we found that surface expression of mutant ß3 subunits is variable. However, it was consistent that surface expression of partnering γ2 subunits was lower when co-expressed with mutant than with wild-type subunits. Because γ2 subunits are critical for synaptic GABAA receptor clustering, this provides an important clue for understanding the pathophysiology of GABRB3 mutations. To validate our findings further, we obtained an in-depth comparison of two novel mutations [GABRB3 (N328D) and GABRB3 (E357K)] associated with epilepsy with different severities of epilepsy phenotype. GABRB3 (N328D) is associated with the relatively severe Lennox-Gastaut syndrome, and GABRB3 (E357K) is associated with the relatively mild juvenile absence epilepsy syndrome. With functional characterizations in both heterologous cells and rodent cortical neurons by patch-clamp recordings, confocal microscopy and immunoblotting, we found that both the GABRB3 (N328D) and GABRB3 (E357K) mutations reduced total subunit expression in neurons but not in HEK293T cells. Both mutant subunits, however, were reduced on the cell surface and in synapses, but the Lennox-Gastaut syndrome mutant ß3 (N328D) subunit was more reduced than the juvenile absence epilepsy mutant ß3 (E357K) subunit. Interestingly, both mutant ß3 subunits impaired postsynaptic clustering of wild-type GABAA receptor γ2 subunits and prevented γ2 subunits from incorporating into GABAA receptors at synapses, although by different cellular mechanisms. Importantly, wild-type γ2 subunits were reduced and less clustered at inhibitory synapses in Gabrb3+/- knockout mice. This suggests that impaired receptor localization to synapses is a common pathophysiological mechanism for GABRB3 mutations, although the extent of impairment may be different among mutant subunits. The study thus identifies the novel mechanism of impaired targeting of receptors containing mutant ß3 subunits and provides critical insights into understanding how GABRB3 mutations produce severe epilepsy syndromes and epilepsy phenotypic heterogeneity.

Altered inhibitory synapses in de novo GABRA5 and GABRA1 mutations associated with early onset epileptic encephalopathies.

We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5ß3γ2 and α1ß3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes.

Primary central nervous system lymphoma with no enhancement initially and no significant progression over a long term: a case report and review of the literature.

Primary central nervous system lymphoma, a rare primary intracranial tumor, is highly malignant and usually associated with poor prognosis. Recent years, owing to the remarkable progress in intervention techniques, survival time reported has been significantly prolonged. Strategies targeting alleviation and remission are primarily depended on the early diagnosis. However, due to the heterogeneity of the clinical symptoms and imaging features, the disease is frequently misdiagnosed especially in the early phase, rendering a delay of optimal treatment. Herein, we reported the case of a 61-year-old man who was initially misdiagnosed as demyelinating encephalopathy. MRI images showed multifocal lesions across the cerebral cortex and deep white matter, which are not strengthened on contrast enhancements. In respect of clinical symptoms, no significant progress was observed over about 11 months. Finally, the diagnosis was revealed by brain biopsy. After reviewing all the images of the patient, we found that the corpus callosum was involved early in the course of the disease. Therefore, for multifocal intracranial lesions involving the corpus callosum, we should always be vigilant about the possibility of primary central nervous system lymphoma. Histopathological examination of brain biopsy is helpful for early definitive diagnosis.

GABA beyond the synapse: defining the subtype-specific pharmacodynamics of non-synaptic GABAA receptors.

KEY POINTS: Physiologically relevant combinations of recombinant GABAA receptor (GABAR) subunits were expressed in HEK293 cells. Using whole-cell voltage clamp and rapid drug application, we measured the GABAR-subtype-specific properties to convey either synaptic or extrasynaptic signalling in a range of physiological contexts. α4ßδ GABARs are optimally tuned to submicromolar tonic GABA and transient surges of micromolar GABA concentrations. α5ß2γ2l GABARs are better suited to higher tonic GABA levels, but also convey robust responses to brief synaptic and perisynaptic GABA fluctuations. α1ß2/3δ GABARs function well at prolonged, micromolar (>2 µm) GABA levels, but not to low tonic (<1 µm GABA) or synaptic/transient GABAergic signalling. These results help illuminate the context- and isoform-specific modes of GABAergic signalling in the brain. ABSTRACT: GABAA receptors (GABARs) mediate a remarkable diversity of signalling modalities in vivo. Yet most published work characterizing responses to GABA has focused on the properties needed to convey fast, phasic synaptic inhibition. We therefore aimed to characterize the most prevalent (α4ßδ, α5ß3γ2L) and least prevalent (α1ß2δ) non-synaptic GABAR currents, using whole-cell voltage clamp recordings of recombinant GABAR expressed in HEK293 cells and drug application protocols to recapitulate the GABA concentration profiles occurring during both fast synaptic and slow extrasynaptic signalling. We found that α4ßδ GABARs were very sensitive to submicromolar GABA, with a rank order potency of α4ß2δ ≥ α4ß1δ ≈ α4ß3δ GABARs. In comparison, the GABA EC50 was up to 20 times higher for α1ß2γ2L GABARs, with α1ß2δ and α5ß3γ2L GABARs having intermediate GABA potency. Both α4ßδ and α5ß3γ2L GABAR currents exhibited slow, but substantial, desensitization as well as prolonged rates of deactivation. These GABAR current properties defined distinct 'dynamic ranges' of responsiveness to changing GABA for α4ß2δ (0.1-1 µm), α5ß3γ2L (0.5-7 µm) and α1ß2γ2L (0.6-9 µm) GABARs. Finally, α1ß2δ GABARs were notable for their relative lack of desensitization and extremely quick deactivation. In summary, our results help delineate the roles that specific GABARs may play in mediating non-synaptic GABA signals. Since ambient GABA levels may be altered during development as well as by drugs and disease states, these findings may help future efforts to understand disrupted inhibition underlying a variety of neurological illnesses, such as epilepsy.

Potential molecular mechanisms of overgrazing-induced dwarfism in sheepgrass (Leymus chinensis) analyzed using proteomic data.

BACKGROUND: This study was designed to reveal potential molecular mechanisms of long-term overgrazing-induced dwarfism in sheepgrass (Leymus chinensis). METHODS: An electrospray ionisation mass spectrometry system was used to generate proteomic data of dwarf sheepgrass from a long-term overgrazed rangeland and normal sheepgrass from a long-term enclosed rangeland. Differentially expressed proteins (DEPs) between dwarf and normal sheepgrass were identified, after which their potential functions and interactions with each other were predicted. The expression of key DEPs was confirmed by high-performance liquid chromatography mass spectrometry (HPLC-MS) using a multiple reaction monitoring method. RESULTS: Compared with normal sheepgrass, a total of 51 upregulated and 53 downregulated proteins were identified in dwarf sheepgrass. The amino acids biosynthesis pathway was differentially enriched between the two conditions presenting DEPs, such as SAT5_ARATH and DAPA_MAIZE. The protein-protein interaction (PPI) network revealed a possible interaction between RPOB2_LEPTE, A0A023H9M8_9STRA, ATPB_DIOEL, RBL_AMOTI and DNAK_GRATL. Four modules were also extracted from the PPI network. The HPLC-MS analysis confirmed the upregulation and downregulation of ATPB_DIOEL and DNAK_GRATL, respectively in dwarf samples compared with in the controls. CONCLUSIONS: The upregulated ATPB_DIOEL and downregulated DNAK_GRATL as well as proteins that interact with them, such as RPOB2_LEPTE, A0A023H9M8_9STRA and RBL_AMOTI, may be associated with the long-term overgrazing-induced dwarfism in sheepgrass.

De novo GABRG2 mutations associated with epileptic encephalopathies.

Epileptic encephalopathies are a devastating group of severe childhood onset epilepsies with medication-resistant seizures and poor developmental outcomes. Many epileptic encephalopathies have a genetic aetiology and are often associated with de novo mutations in genes mediating synaptic transmission, including GABAA receptor subunit genes. Recently, we performed next generation sequencing on patients with a spectrum of epileptic encephalopathy phenotypes, and we identified five novel (A106T, I107T, P282S, R323W and F343L) and one known (R323Q) de novo GABRG2 pathogenic variants (mutations) in eight patients. To gain insight into the molecular basis for how these mutations contribute to epileptic encephalopathies, we compared the effects of the mutations on the properties of recombinant α1ß2γ2L GABAA receptors transiently expressed in HEK293T cells. Using a combination of patch clamp recording, immunoblotting, confocal imaging and structural modelling, we characterized the effects of these GABRG2 mutations on GABAA receptor biogenesis and channel function. Compared with wild-type α1ß2γ2L receptors, GABAA receptors containing a mutant γ2 subunit had reduced cell surface expression with altered subunit stoichiometry or decreased GABA-evoked whole-cell current amplitudes, but with different levels of reduction. While a causal role of these mutations cannot be established directly from these results, the functional analysis together with the genetic information suggests that these GABRG2 variants may be major contributors to the epileptic encephalopathy phenotypes. Our study further expands the GABRG2 phenotypic spectrum and supports growing evidence that defects in GABAergic neurotransmission participate in the pathogenesis of genetic epilepsies including epileptic encephalopathies.

Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αßγ and αßδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS.

The subunit stoichiometry and arrangement of synaptic αßγ GABAA receptors are generally accepted as 2α:2ß:1γ with a ß-α-γ-ß-α counterclockwise configuration, respectively. Whether extrasynaptic αßδ receptors adopt the analogous ß-α-δ-ß-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or ß2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and ß2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of ß2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1ß2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αßγ and αßδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αßγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical ß-α-γ/δ-ß-α receptors, including ß-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and ß-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

Epileptic encephalopathy de novo GABRB mutations impair γ-aminobutyric acid type A receptor function.

OBJECTIVE: The Epi4K Consortium recently identified 4 de novo mutations in the γ-aminobutyric acid type A (GABAA ) receptor ß3 subunit gene GABRB3 and 1 in the ß1 subunit gene GABRB1 in children with one of the epileptic encephalopathies (EEs) Lennox-Gastaut syndrome (LGS) and infantile spasms (IS). Because the etiology of EEs is often unknown, we determined the impact of GABRB mutations on GABAA receptor function and biogenesis. METHODS: GABAA receptor α1 and γ2L subunits were coexpressed with wild-type and/or mutant ß3 or ß1 subunits in HEK 293T cells. Currents were measured using whole cell and single channel patch clamp techniques. Surface and total expression levels were measured using flow cytometry. Potential structural perturbations in mutant GABAA receptors were explored using structural modeling. RESULTS: LGS-associated GABRB3(D120N, E180G, Y302C) mutations located at ß+ subunit interfaces reduced whole cell currents by decreasing single channel open probability without loss of surface receptors. In contrast, IS-associated GABRB3(N110D) and GABRB1(F246S) mutations at ß- subunit interfaces produced minor changes in whole cell current peak amplitude but altered current deactivation by decreasing or increasing single channel burst duration, respectively. GABRB3(E180G) and GABRB1(F246S) mutations also produced spontaneous channel openings. INTERPRETATION: All 5 de novo GABRB mutations impaired GABAA receptor function by rearranging conserved structural domains, supporting their role in EEs. The primary effect of LGS-associated mutations was reduced GABA-evoked peak current amplitudes, whereas the major impact of IS-associated mutations was on current kinetic properties. Despite lack of association with epilepsy syndromes, our results suggest GABRB1 as a candidate human epilepsy gene. Ann Neurol 2016;79:806-825.

Overgrazing induces alterations in the hepatic proteome of sheep (Ovis aries): an iTRAQ-based quantitative proteomic analysis.

BACKGROUND: The degradation of the steppe of Inner Mongolia, due to overgrazing, has resulted in ecosystem damage as well as extensive reductions in sheep production. The growth performance of sheep is greatly reduced because of overgrazing, which triggers massive economic losses every year. The liver is an essential organ that has very important roles in multiple functions, such as nutrient metabolism, immunity and others, which are closely related to animal growth. However, to our knowledge, no detailed studies have evaluated hepatic metabolism adaption in sheep due to overgrazing. The molecular mechanisms that underlie these effects remain unclear. METHODS: In the present study, our group applied isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to investigate changes in the protein profiles of sheep hepatic tissues when nutrition was reduced due to overgrazing (12.0 sheep/ha), with the goal of characterizing the molecular mechanisms of hepatic metabolism adaption in sheep in an overgrazing condition. RESULTS: The body weight daily gain of sheep was greatly decreased due to overgrazing. Overall, 41 proteins were found to be differentially abundant in the hepatic tissue between a light grazing group and an overgrazing group. Most of the differentially expressed proteins identified are involved in protein metabolism, transcriptional and translational regulation, and immune response. In particular, the altered abundance of kynureninase (KYNU) and HAL (histidine ammonia-lyase) involved in protein metabolic function, integrated with the changes of serum levels of blood urea nitrogen (BUN) and glucose (GLU), suggest that overgrazing triggers a shift in energy resources from carbohydrates to proteins, causing poorer nitrogen utilization efficiency. Altogether, these results suggest that the reductions in animal growth induced by overgrazing are associated with liver proteomic changes, especially the proteins involved in nitrogen compounds metabolism and immunity. CONCLUSIONS: This provides new information that can be used for nutritional supplementation to improve the growth performance of sheep in an overgrazing condition.

RISK-FACTORS, PATHOGENESIS, AND PHARMACEUTICAL APPROACHES FOR TREATMENT OF STEROID-INDUCED BONE INFARCTION OF FEMORAL HEAD.

During first year of steroid usage, osteocyte necrosis and blood vessel blockage may occur, which subsequently may produce steroid-induced bone infarction (SIBI) resulting in painful movement of patient. For treatment of SIBI, pharmaceutical strategy is the basic approach. It involves the use of various pharmacologically active compounds including bisphosphonates, hyperbaric oxygen (HBO), coenzyme Q10, erythropoietin, antihyperlipidemics, anticoagulants, antioxidants, and tissue repair protein. Out of these, there is no pharmaceutical agent that may completely treat this disease because many factors are found to be responsible for SIBI development; therefore, there are multiple biomarkers of this disease. This situation argues for need of new therapeutic agents for SIEB1.

Three epilepsy-associated GABRG2 missense mutations at the γ+/ß- interface disrupt GABAA receptor assembly and trafficking by similar mechanisms but to different extents.

We compared the effects of three missense mutations in the GABAA receptor γ2 subunit on GABAA receptor assembly, trafficking and function in HEK293T cells cotransfected with α1, ß2, and wildtype or mutant γ2 subunits. The mutations R82Q and P83S were identified in families with genetic epilepsy with febrile seizures plus (GEFS+), and N79S was found in a single patient with generalized tonic-clonic seizures (GTCS). Although all three mutations were located in an N-terminal loop that contributes to the γ+/ß- subunit-subunit interface, we found that each mutation impaired GABAA receptor assembly to a different extent. The γ2(R82Q) and γ2(P83S) subunits had reduced α1ß2γ2 receptor surface expression due to impaired assembly into pentamers, endoplasmic reticulum (ER) retention and degradation. In contrast, γ2(N79S) subunits were efficiently assembled into GABAA receptors with only minimally altered receptor trafficking, suggesting that N79S was a rare or susceptibility variant rather than an epilepsy mutation. Increased structural variability at assembly motifs was predicted by R82Q and P83S, but not N79S, substitution, suggesting that R82Q and P83S substitutions were less tolerated. Membrane proteins with missense mutations that impair folding and assembly often can be "rescued" by decreased temperatures. We coexpressed wildtype or mutant γ2 subunits with α1 and ß2 subunits and found increased surface and total levels of both wildtype and mutant γ2 subunits after decreasing the incubation temperature to 30°C for 24h, suggesting that lower temperatures increased GABAA receptor stability. Thus epilepsy-associated mutations N79S, R82Q and P83S disrupted GABAA receptor assembly to different extents, an effect that could be potentially rescued by facilitating protein folding and assembly.

Preclinical pharmacology and toxicology study of Ad-hTERT-E1a-Apoptin, a novel dual cancer-specific oncolytic adenovirus.

Clinical studies have demonstrated that conditionally replicating adenovirus is safe. We constructed an oncolytic adenovirus, Ad-hTERT-E1a-Apoptin, using a cancer-specific promoter (human telomerase reverse transcriptase promoter, hTERTp) and a cancer cell-selective apoptosis-inducing gene (Apoptin). Ad-hTERT-E1a-Apoptin was proven effective both in vitro and in vivo in our previous study. In this study, the preclinical safety profiles of Ad-hTERT-E1a-Apoptin in animal models were investigated. At doses of 5.0×10(8), 2.5×10(9), and 1.25×10(10) viral particles (VP)/kg, Ad-hTERT-E1a-Apoptin had no adverse effects on mouse behavior, muscle cooperation, sedative effect, digestive system, and nervous systems, or on beagle cardiovascular and respiratory systems at 5.0×10(8), 2.5×10(9), and 1.25×10(10) VP/kg doses. In acute toxicity tests in mice, the maximum tolerated dose>5×10(10) VP/kg. There was no inflammation or ulceration at the injection sites within two weeks. In repeat-dose toxicological studies, the no observable adverse effect levels of Ad-hTERT-E1a-Apoptin in rats (1.25×10(10) VP/kg) and beagles (2.5×10(9) VP/kg) were 62.5- and 12.5-fold of the proposed clinical dose, respectively. The anti-virus antibody was produced in animal sera. Bone marrow examination revealed no histopathological changes. Guinea pigs sensitized by three repeated intraperitoneal injections of 1.35×10(10) VP/mL Ad-hTERT-E1a-Apoptin each and challenged by one intravenous injection of 1.67×10(8) VP/kg Ad-hTERT-E1a-Apoptin did not exhibit any sign of systemic anaphylaxis. Our data from different animal models suggest that Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent.

Generation of an attenuated Tiantan vaccinia virus by deletion of the ribonucleotide reductase large subunit.

Attenuation of the virulence of vaccinia Tiantan virus (VTT) underlies the strategy adopted for mass vaccination campaigns. This strategy provides advantages of safety and efficacy over traditional vaccines and is aimed at minimization of adverse health effects. In this study, a mutant form of the virus, MVTT was derived from VTT by deletion of the ribonucleotide reductase large subunit (R1) (TI4L). Compared to wild-type parental (VTT) and revertant (VTT-rev) viruses, virulence of the mutant MVTT was reduced by 100-fold based on body weight reduction and by 3,200-fold based on determination of the intracranial 50% lethal infectious dose. However, the immunogenicity of MVTT was equivalent to that of the parental VTT. We also demonstrated that the TI4L gene is not required for efficient replication. These data support the conclusion that MVTT can be used as a replicating virus vector or as a platform for the development of vaccines against infectious diseases and for cancer therapy.