Transcriptional Reprogramming during Effector-to-Memory Transition Renders CD4+ T Cells Permissive for Latent HIV-1 Infection.

The latent reservoir for HIV-1 in resting memory CD4+ T cells is the major barrier to curing HIV-1 infection. Studies of HIV-1 latency have focused on regulation of viral gene expression in cells in which latent infection is established. However, it remains unclear how infection initially becomes latent. Here we described a unique set of properties of CD4+ T cells undergoing effector-to-memory transition including temporary upregulation of CCR5 expression and rapid downregulation of cellular gene transcription. These cells allowed completion of steps in the HIV-1 life cycle through integration but suppressed HIV-1 gene transcription, thus allowing the establishment of latency. CD4+ T cells in this stage were substantially more permissive for HIV-1 latent infection than other CD4+ T cells. Establishment of latent HIV-1 infection in CD4+ T could be inhibited by viral-specific CD8+ T cells, a result with implications for elimination of latent HIV-1 infection by T cell-based vaccines.

Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space.

Chloroplast division involves the coordination of protein complexes from the stroma to the cytosol. The Min system of chloroplasts includes multiple stromal proteins that regulate the positioning of the division site. The outer envelope protein PLASTID DIVISION1 (PDV1) was previously reported to recruit the cytosolic chloroplast division protein ACCUMULATION AND REPLICATION OF CHLOROPLAST5 (ARC5). However, we show here that PDV1 is also important for the stability of the inner envelope chloroplast division protein PARALOG OF ARC6 (PARC6), a component of the Min system. We solved the structure of both the C-terminal domain of PARC6 and its complex with the C terminus of PDV1. The formation of an intramolecular disulfide bond within PARC6 under oxidized conditions prevents its interaction with PDV1. Interestingly, this disulfide bond can be reduced by light in planta, thus promoting PDV1-PARC6 interaction and chloroplast division. Interaction with PDV1 can induce the dimerization of PARC6, which is important for chloroplast division. Magnesium ions, whose concentration in chloroplasts increases upon light exposure, also promote the PARC6 dimerization. This study highlights the multilayer regulation of the PDV1-PARC6 interaction as well as chloroplast division.

A novel amphiphilic motif at the C-terminus of FtsZ1 facilitates chloroplast division.

In bacteria and chloroplasts, the GTPase filamentous temperature-sensitive Z (FtsZ) is essential for division and polymerizes to form rings that mark the division site. Plants contain two FtsZ subfamilies (FtsZ1 and FtsZ2) with different assembly dynamics. FtsZ1 lacks the C-terminal domain of a typical FtsZ protein. Here, we show that the conserved short motif FtsZ1Carboxyl-terminus (Z1C) (consisting of the amino acids RRLFF) with weak membrane-binding activity is present at the C-terminus of FtsZ1 in angiosperms. For a polymer-forming protein such as FtsZ, this activity is strong enough for membrane tethering. Arabidopsis thaliana plants with mutated Z1C motifs contained heterogeneously sized chloroplasts and parallel FtsZ rings or long FtsZ filaments, suggesting that the Z1C motif plays an important role in regulating FtsZ ring dynamics. Our findings uncover a type of amphiphilic beta-strand motif with weak membrane-binding activity and point to the importance of this motif for the dynamic regulation of protein complex formation.

Chemical inhibition of DPP9 sensitizes the CARD8 inflammasome in HIV-1-infected cells.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) induce pyroptosis of HIV-1-infected CD4+ T cells through induction of intracellular HIV-1 protease activity, which activates the CARD8 inflammasome. Because high concentrations of NNRTIs are required for efficient elimination of HIV-1-infected cells, it is important to elucidate ways to sensitize the CARD8 inflammasome to NNRTI-induced activation. We show that this sensitization can be achieved through chemical inhibition of the CARD8 negative regulator DPP9. The DPP9 inhibitor Val-boroPro (VbP) can kill HIV-1-infected cells without the presence of NNRTIs and act synergistically with NNRTIs to promote clearance of HIV-1-infected cells in vitro and in humanized mice. More importantly, VbP is able to enhance clearance of residual HIV-1 in CD4+ T cells isolated from people living with HIV (PLWH). We also show that VbP can partially overcome NNRTI resistance. This offers a promising strategy for enhancing NNRTI efficacy in the elimination of HIV-1 reservoirs in PLWH.

High accuracy model for HBsAg loss based on longitudinal trajectories of serum qHBsAg throughout long-term antiviral therapy.

OBJECTIVE: Hepatitis B surface antigen (HBsAg) loss is the optimal outcome for patients with chronic hepatitis B (CHB) but this rarely occurs with currently approved therapies. We aimed to develop and validate a prognostic model for HBsAg loss on treatment using longitudinal data from a large, prospectively followed, nationwide cohort. DESIGN: CHB patients receiving nucleos(t)ide analogues as antiviral treatment were enrolled from 50 centres in China. Quantitative HBsAg (qHBsAg) testing was prospectively performed biannually per protocol. Longitudinal discriminant analysis algorithm was used to estimate the incidence of HBsAg loss, by integrating clinical data of each patient collected during follow-up. RESULTS: In total, 6792 CHB patients who had initiated antiviral treatment 41.3 (IQR 7.6-107.6) months before enrolment and had median qHBsAg 2.9 (IQR 2.3-3.3) log10IU/mL at entry were analysed. With a median follow-up of 65.6 (IQR 51.5-84.7) months, the 5-year cumulative incidence of HBsAg loss was 2.4%. A prediction model integrating all qHBsAg values of each patient during follow-up, designated GOLDEN model, was developed and validated. The AUCs of GOLDEN model were 0.981 (95% CI 0.974 to 0.987) and 0.979 (95% CI 0.974 to 0.983) in the training and external validation sets, respectively, and were significantly better than those of a single qHBsAg measurement. GOLDEN model identified 8.5%-10.4% of patients with a high probability of HBsAg loss (5-year cumulative incidence: 17.0%-29.1%) and was able to exclude 89.6%-91.5% of patients whose incidence of HBsAg loss is 0. Moreover, the GOLDEN model consistently showed excellent performance among various subgroups. CONCLUSION: The novel GOLDEN model, based on longitudinal qHBsAg data, accurately predicts HBsAg clearance, provides reliable estimates of functional hepatitis B virus (HBV) cure and may have the potential to stratify different subsets of patients for novel anti-HBV therapies.

Sequence variations affect the 5' splice site selection of plant introns.

Introns are noncoding sequences spliced out of pre-mRNAs by the spliceosome to produce mature mRNAs. The 5' ends of introns mostly begin with GU and have a conserved sequence motif of AG/GUAAGU that could base-pair with the core sequence of U1 snRNA of the spliceosome. Intriguingly, ∼ 1% of introns in various eukaryotic species begin with GC. This occurrence could cause misannotation of genes; however, the underlying splicing mechanism is unclear. We analyzed the sequences around the intron 5' splice site (ss) in Arabidopsis (Arabidopsis thaliana) and found sequences at the GC intron ss are much more stringent than those of GT introns. Mutational analysis at various positions of the intron 5' ss revealed that although mutations impair base pairing, different mutations at the same site can have different effects, suggesting that steric hindrance also affects splicing. Moreover, mutations of 5' ss often activate a hidden ss nearby. Our data suggest that the 5' ss is selected via a competition between the major ss and the nearby minor ss. This work not only provides insights into the splicing mechanism of intron 5' ss but also improves the accuracy of gene annotation and the study of the evolution of intron 5' ss.

Quantitative RUBY reporter assay for gene regulation analysis.

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G-box and I-box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost-effective and intuitive strategy for studying the regulatory mechanism of gene expression.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

KEY MESSAGE: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

Novel, high accuracy models for hepatocellular carcinoma prediction based on longitudinal data and cell-free DNA signatures.

BACKGROUND & AIMS: Current hepatocellular carcinoma (HCC) risk scores do not reflect changes in HCC risk resulting from liver disease progression/regression over time. We aimed to develop and validate two novel prediction models using multivariate longitudinal data, with or without cell-free DNA (cfDNA) signatures. METHODS: A total of 13,728 patients from two nationwide multicenter prospective observational cohorts, the majority of whom had chronic hepatitis B, were enrolled. aMAP score, as one of the most promising HCC prediction models, was evaluated for each patient. Low-pass whole-genome sequencing was used to derive multi-modal cfDNA fragmentomics features. A longitudinal discriminant analysis algorithm was used to model longitudinal profiles of patient biomarkers and estimate the risk of HCC development. RESULTS: We developed and externally validated two novel HCC prediction models with a greater accuracy, termed aMAP-2 and aMAP-2 Plus scores. The aMAP-2 score, calculated with longitudinal data on the aMAP score and alpha-fetoprotein values during an up to 8-year follow-up, performed superbly in the training and external validation cohorts (AUC 0.83-0.84). The aMAP-2 score showed further improvement and accurately divided aMAP-defined high-risk patients into two groups with 5-year cumulative HCC incidences of 23.4% and 4.1%, respectively (p = 0.0065). The aMAP-2 Plus score, which incorporates cfDNA signatures (nucleosome, fragment and motif scores), optimized the prediction of HCC development, especially for patients with cirrhosis (AUC 0.85-0.89). Importantly, the stepwise approach (aMAP -> aMAP-2 -> aMAP-2 Plus) stratified patients with cirrhosis into two groups, comprising 90% and 10% of the cohort, with an annual HCC incidence of 0.8% and 12.5%, respectively (p <0.0001). CONCLUSIONS: aMAP-2 and aMAP-2 Plus scores are highly accurate in predicting HCC. The stepwise application of aMAP scores provides an improved enrichment strategy, identifying patients at a high risk of HCC, which could effectively guide individualized HCC surveillance. IMPACT AND IMPLICATIONS: In this multicenter nationwide cohort study, we developed and externally validated two novel hepatocellular carcinoma (HCC) risk prediction models (called aMAP-2 and aMAP-2 Plus scores), using longitudinal discriminant analysis algorithm and longitudinal data (i.e., aMAP and alpha-fetoprotein) with or without the addition of cell-free DNA signatures, based on 13,728 patients from 61 centers across mainland China. Our findings demonstrated that the performance of aMAP-2 and aMAP-2 Plus scores was markedly better than the original aMAP score, and any other existing HCC risk scores across all subsets, especially for patients with cirrhosis. More importantly, the stepwise application of aMAP scores (aMAP -> aMAP-2 -> aMAP-2 Plus) provides an improved enrichment strategy, identifying patients at high risk of HCC, which could effectively guide individualized HCC surveillance.

Mutation of CsARC6 affects fruit color and increases fruit nutrition in cucumber.

KEY MESSAGE: A mutation of CsARC6 not only causes white fruit color in cucumber, but also affects plant growth and fruit quality. Fruit color of cucumber is a very important agronomic trait, but most of the genes affecting cucumber white fruit color are still unknow, and no further studies were reported on the effect of cucumber fruit quality caused by white fruit color genes. Here, we obtained a white fruit mutant em41 in cucumber by EMS mutagenesis. The mutant gene was mapped to a 548 kb region of chromosome 2. Through mutation site analysis, it was found to be a null allele of CsARC6 (CsaV3_2G029290). The Csarc6 mutant has a typical phenotype of arc6 mutant that mesophyll cells contained only one or two giant chloroplasts. ARC6 protein was not detected in em41, and the level of FtsZ1 and FtsZ2 was also reduced. In addition, FtsZ2 could not form FtsZ ring-like structures in em41. Although these are typical arc6 mutant phenotypes, some special phenotypes occur in Csarc6 mutant, such as dwarfness with shortened internodes, enlarged fruit epidermal cells, decreased carotenoid contents, smaller fruits, and increased fruit nutrient contents. This study discovered a new gene, CsARC6, which not only controls the white fruit color, but also affects plant growth and fruit quality in cucumber.

Regulating electron transportation by tungsten oxide nanocapacitors for enhanced radiation therapy.

In the process of radiation therapy (RT), the cytotoxic effects of excited electrons generated from water radiolysis tend to be underestimated due to multiple biochemical factors, particularly the recombination between electrons and hydroxyl radicals (·OH). To take better advantage of radiolytic electrons, we constructed WO3 nanocapacitors that reversibly charge and discharge electrons to regulate electron transportation and utilization. During radiolysis, WO3 nanocapacitors could contain the generated electrons that block electron-·OH recombination and contribute to the yield of ·OH at a high level. These contained electrons could be discharged from WO3 nanocapacitors after radiolysis, resulting in the consumption of cytosolic NAD+ and impairment of NAD+-dependent DNA repair. Overall, this strategy of nanocapacitor-based radiosensitization improves the radiotherapeutic effects by increasing the utilization of radiolytic electrons and ·OH, warranting further validation in multiple tumour models and preclinical experiments.

Drought Stress Tolerance in Vegetables: The Functional Role of Structural Features, Key Gene Pathways, and Exogenous Hormones.

The deleterious effects of drought stress have led to a significant decline in vegetable production, ultimately affecting food security. After sensing drought stress signals, vegetables prompt multifaceted response measures, eventually leading to changes in internal cell structure and external morphology. Among them, it is important to highlight that the changes, including changes in physiological metabolism, signal transduction, key genes, and hormone regulation, significantly influence drought stress tolerance in vegetables. This article elaborates on vegetable stress tolerance, focusing on structural adaptations, key genes, drought stress signaling transduction pathways, osmotic adjustments, and antioxidants. At the same time, the mechanisms of exogenous hormones such as abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) toward improving the adaptive drought tolerance of vegetables were also reviewed. These insights can enhance the understanding of vegetable drought tolerance, supporting vegetable tolerance enhancement by cultivation technology improvements under changing climatic conditions, which provides theoretical support and technical reference for innovative vegetable stress tolerance breeding and food security.

Evaluation of HIV-1 latency reversal and antibody-dependent viral clearance by quantification of singly spliced HIV-1 vpu/env mRNA.

The latent reservoir of HIV-1 is a major barrier for viral eradication. Potent HIV-1 broadly neutralizing antibodies (bNabs) have been used to prevent and treat HIV-1 infections in animal models and clinical trials. Combination of bNabs and latency-reversing agents (LRAs) is considered a promising approach for HIV-1 eradication. PCR-based assays that can rapidly and specifically measure singly spliced HIV-1 vpu/env mRNA are needed to evaluate the induction of the viral envelope production at the transcription level and bNab-mediated reservoir clearance. Here we reported a PCR-based method to accurately quantify the production of intracellular HIV-1 vpu/env mRNA. With the vpu/env assay, we determined the LRA combinations that could effectively induce vpu/env mRNA production in CD4+ T cells from ART-treated individuals. None of the tested LRAs were effective alone. A comparison between the quantitative viral outgrowth assay (Q-VOA) and the vpu/env assay showed that vpu/env mRNA production was closely associated with the reactivation of replication-competent HIV-1, suggesting that vpu/env mRNA was mainly produced by intact viruses. Finally, antibody-mediated in vitro killing in HIV-1-infected humanized mice demonstrated that the vpu/env assay could be used to measure the reduction of infected cells in tissues and was more accurate than the commonly used gag-based PCR assay which measured unspliced viral genomic RNA. In conclusion, the vpu/env assay allows convenient and accurate assessment of HIV-1 latency reversal and bNab-mediated therapeutic strategies.ImportanceHIV-1 persists in individuals on antiretroviral therapy (ART) due to the long-lived cellular reservoirs that contain dormant viruses. Recent discoveries of HIV-1-specific broadly neutralizing antibodies (bNabs) targeting HIV-1 Env protein rekindled the interest in antibody-mediated elimination of latent HIV-1. Latency-reversing agents (LRAs) together with HIV-1 bNabs is a possible strategy to clear residual viral reservoirs, which makes the evaluation of HIV-1 Env expression upon LRA treatment critical. We developed a PCR-based assay to quantify the production of intracellular HIV-1 vpu/env mRNA. Using patient CD4+ T cells, we found that induction of HIV-1 vpu/env mRNA required a combination of different LRAs. Using in vitro, ex vivo and humanized mouse models, we showed that the vpu/env assay could be used to measure antibody efficacy in clearing HIV-1 infection. These results suggest that the vpu/env assay can accurately evaluate HIV-1 reactivation and bNab-based therapeutic interventions.

Inhibition of Rev-erbα ameliorates muscular dystrophy.

Duchene muscular dystrophy leads to progressive muscle structural and functional decline due to chronic degenerative-regenerative cycles. Enhancing the regenerative capacity of dystrophic muscle provides potential therapeutic options. We previously demonstrated that the circadian clock repressor Rev-erbα inhibited myogenesis and Rev-erbα ablation enhanced muscle regeneration. Here we show that Rev-erbα deficiency in the dystrophin-deficient mdx mice promotes regenerative myogenic response to ameliorate muscle damage. Loss of Rev-erbα in mdx mice improved dystrophic pathology and muscle wasting. Rev-erbα-deficient dystrophic muscle exhibit augmented myogenic response, enhanced neo-myofiber formation and attenuated inflammatory response. In mdx myoblasts devoid of Rev-erbα, myogenic differentiation was augmented together with up-regulation of Wnt signaling and proliferative pathways, suggesting that loss of Rev-erbα inhibition of these processes contributed to the improvement in regenerative myogenesis. Collectively, our findings revealed that the loss of Rev-erbα function protects dystrophic muscle from injury by promoting myogenic repair, and inhibition of its activity may have therapeutic utilities for muscular dystrophy.

Differentiation of Prostate Cancer and Stromal Hyperplasia in the Transition Zone With Monoexponential, Stretched-Exponential Diffusion-Weighted Imaging and Diffusion Kurtosis Imaging in a Reduced Number of b Values: Correlation With Whole-Mount Pathology.

OBJECTIVES: The aims of the study were to explore the feasibility of generating a monoexponential model (MEM), stretched-exponential model (SEM) based diffusion-weighted imaging (DWI), and diffusion kurtosis imaging (DKI) by applying the same set of reduced b values and to compare their effectiveness in distinguishing prostate cancer from stromal hyperplasia (SH) in the transition zone (TZ) area. METHODS: An analysis of 75 patients who underwent preoperative DWI ( b values of 0, 700, 1400, 2000 s/mm 2 ) was performed. All lesions were localized on magnetic resonance images according to whole-mount histopathological correlations. The apparent diffusion coefficient (ADC), water molecular diffusion heterogeneity index (α), distributed diffusion coefficient (DDC), mean diffusivity (MD), and mean kurtosis (MK) values were calculated and compared between the TZ cancer and SH groups. Receiver operating characteristic analysis and areas under the receiver operating characteristic curve (AUCs) were carried out for all parameters. RESULTS: Compared with the SH group, the ADC, DDC, α, and MD values of the TZ cancer group were significantly reduced, while the MK value was significantly increased (all P < 0.05). The AUCs of the ADC, DDC, α, MD, and MK were 0.828, 0.801, 0.813, 0.822, and 0.882, respectively. The AUC of MK was significantly higher than that of the other parameters (all P < 0.05). CONCLUSIONS: When using the reduced b -value set, all parameters from MEM, SEM, based DWI, and DKI can effectively distinguish TZ cancer from SH. Among them, DKI demonstrated potential clinical superiority over the others in TZ cancer diagnosis.

High relaxivity Gd3+-based organic nanoparticles for efficient magnetic resonance angiography.

Contrast-enhanced MR angiography (MRA) is a critical technique for vascular imaging. Nevertheless, the efficacy of MRA is often limited by the low rate of relaxation, short blood-circulation time, and metal ion-released potential long-term toxicity of clinical available Gd-based contrast agents. In this work, we report a facile and efficient strategy to achieve Gd-chelated organic nanoparticles with high relaxivity for T1-weighted MRA imaging. The Gd-chelated PEG-TCPP nanoparticles (GPT NPs) have been engineered composite structured consisting of Gd-chelated TCPP and PEG. The spherical structure of TCPP offers more chemical sites for Gd3+ coordination to improve the relaxivity and avoid leakage of the Gd3+ ions. The synthesized GPT NPs exhibit a high relaxation rate of 35.76 mM- 1 s- 1 at 3.0 T, which is higher than the rates for most reported MR contrast agents. Therefore, GPT NPs can be used for MRA with much stronger vascular signals, longer circulation time, and high-resolution arterial vascular visualization than those using clinical MR contrast agents at the same dose. This work may make the T1 MRI contrast agents for high-resolution angiography possible and offer a new candidate for preclinical and clinical applications of MR vascular imaging and vascular disease diagnosis.

Comparative analysis of HBV basic core promoter/pre-core gene mutations and viral quasispecies diversity in HIV/HBV co-infected and HBV mono-infected patients.

Human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection accelerates the progression of HBV-related liver diseases. HBV basic core promoter (BCP)/pre-core (preC) gene mutations may be one of the most important risk factors. In this study, a total of 230 patients were recruited, and 199 patients whose HBV BCP/preC gene were successfully amplified and sequenced, including 99 HIV/HBV co-infected and 100 HBV mono-infected patients. Next-generation sequencing was used for detection of BCP/preC mutations which were then compared in patients with different HBV genotypes and different HBeAg statuses, and 1% and 20% cutoff values were defined to evaluate the mutations. HBV quasispecies diversity was also compared in HIV/HBV co-infected and HBV mono-infected patients. Among the patients infected with HBV genotype C and HBeAg-negative status, the frequency of A1762T/G1764A double mutations was significantly lower in HIV/HBV co-infected patients than in HBV mono-infected patients (53.3% vs. 100.0%, P = 0.008) regardless of the 1% or 20% cutoff value level. However, A1762T/G1764A double mutations did not differ in the other groups (P >0.05). Viral quasispecies diversity was lower in HIV/HBV co-infected patients than in HBV mono-infected patients (P Keywords: human immunodeficiency virus, hepatitis B virus; mutations; viral quasispecies; next-generation sequencing.

Secondary Mutation-Induced Alternative Splicing Suppresses RNA Splicing Defect of the jhs1 Mutant.

jing he sheng1 (jhs1) is a mutant of the DNA2 homolog in Arabidopsis (Arabidopsis thaliana), which was previously identified as being involved in DNA damage repair, cell cycle regulation, and meristem maintenance. A mutation at the 3' intron splice site of the 11th intron causes alternative splicing of this intron at two other sites, which results in frame shifts and premature stop codons. Here, we screened suppressors of jhs1 to further study the function and regulatory networks of JHS1 Three suppressors with wild-type-like phenotypes were obtained. Sequencing analysis results showed that each of the suppressors has a second mutation in jhs1 that causes further alternative splicing of the intron and corrects the shifted reading frame with small insertions. Precursor mRNA sequence analysis and intron splice site evaluation results suggested that intron splicing was disturbed in the suppressors, and this switched the splice site, resulting in small insertions in the coding regions of JHS1. Structural analysis of JHS1 suggested that the insertions are in a disordered loop region of the DNA2 domain and do not seem to have much deleterious effect on the function of the protein. This work not only has implications for the evolution of protein sequences at exon junctions but also provides a strategy to study the mechanism of precursor mRNA splicing.

The clock regulator Bmal1 protects against muscular dystrophy.

The muscle-intrinsic clock machinery is required for the maintenance of muscle growth, remodeling and function. Our previous studies demonstrated that the essential transcription activator of the molecular clock feed-back loop, Brain and Muscle Arnt-Like 1(Bmal1), plays a critical role in myogenic progenitor behavior to promote and regenerative myogenesis. Using genetic approaches targeting Bmal1 in the DMDmdx (mdx) dystrophic mouse model, here we report that the loss of Bmal1 function significantly accelerated dystrophic disease progression. In contrast to the mild dystrophic changes in mdx mice, the genetic loss-of-function of Bmal1 aggravated muscle damage in this dystrophic disease background, as indicated by persistently elevated creatine kinase levels, increased injury area and reduced muscle grip strength. Mechanistic studies revealed that markedly impaired myogenic progenitor proliferation and myogenic response underlie the defective new myofiber formation in the chronic dystrophic milieu. Taken together, our study identified the function of pro-myogenic clock gene Bmal1 in protecting against dystrophic damage, suggesting the potential for augmenting Bmal1 function to ameliorate dystrophic or degenerative muscle diseases.

Andrographolide Induces Apoptosis in Gastric Cancer Cells through Reactivation of p53 and Inhibition of Mdm-2.

Andrographolide is a labdane diterpenoid isolated from Andrographis paniculata. The plant extract and andrographolide has long been used in traditional medicine practices mainly for gastrointestinal diseases and improving liver function. Andrographolide has shown various pharmacological properties including anti-inflammatory, antioxidant and anticancer activity. This study evaluated the effect of andrographolide on proliferation of human gastric carcinoma cells in relevance to p53 and Mdm-2 pathways. Andrographolide inhibited the proliferation of SGC7901 and AGS cells in a dose-dependent manner with estimated IC50 values 38 and 44 µM respectively. Effect of andrographolide on p53 activity was ascertained by using a p53 activator (RITA) which showed synergistic inhibition of cell proliferation. While andrographolide when used in combination with a p53 inhibitor (pifithrin-α) showed potent restriction over its response. Andrographolide caused decrease in mitochondrial membrane potential as an indicator of apoptotic activity. Andrographolide activated the expression of p53 protein and gene and downregulated the levels of Mdm-2 (negative regulator of p53). Andrographolide inhibited the colony formation abilities in SGC7901 in a p53-dependent manner followed by induction of mitochondrial intrinsic apoptosis through activation of caspases-9 and -3, cleavage of PARP, and inhibition of pro-apoptotic Bcl-2. Andrographolide induced p53 mediated apoptosis in gastric carcinoma cells which adds to a novel approach in anticancer therapies.