Decoding the role of aberrant RNA alternative splicing in hepatocellular carcinoma: a comprehensive review.

During eukaryotic gene expression, alternative splicing of messenger RNA precursors is critical in increasing protein diversity and regulatory complexity. Multiple transcript isoforms could be produced by alternative splicing from a single gene; they could eventually be translated into protein isoforms with deleted, added, or altered domains or produce transcripts containing premature termination codons that could be targeted by nonsense-mediated mRNA decay. Alternative splicing can generate proteins with similar, different, or even opposite functions. Increasingly strong evidence indicates that abnormal RNA splicing is a prevalent and crucial occurrence in cellular differentiation, tissue advancement, and the development and progression of cancer. Aberrant alternative splicing could affect cancer cell activities such as growth, apoptosis, invasiveness, drug resistance, angiogenesis, and metabolism. This systematic review provides a comprehensive overview of the impact of abnormal RNA alternative splicing on the development and progression of hepatocellular carcinoma.

E-commerce development and its contribution to agricultural non-point source pollution control: Evidence from 283 cities in China.

Agricultural non-point source (ANPS) pollution is a pressing environmental issue in developing countries that poses a substantial threat to sustainable development. With the rapid growth of e-commerce and its great penetration and transformation in many socioeconomic sectors, e-commerce plus agriculture is widely regarded as the solution to sustainable agricultural development. However, the environmental impacts of e-commerce on agriculture, as well as the underlying mechanisms have yet to be fully explored and verified. Based on China's practices of e-commerce development and its integration with agriculture, a panel dataset of 283 prefecture-level cities from 2009 to 2019 was collected, and a spatial difference in difference (SDID) model combined with a Durbin model was constructed to examine the local and spatial spillover effects of e-commerce development on ANPS pollution. It is found out that e-commerce development has significant positive environmental impacts achieved by stimulating industrial structure upgrading and promoting green technology innovation, while the mechanism of cultivation scaling up tends to aggravate the ANPS pollution. Spatial analysis demonstrates that e-commerce development also helps alleviate the ANPS pollution of neighboring regions with a decayed effect over a distance. Meanwhile, the impact of e-commerce on mitigating ANPS pollution shows regional heterogeneity. Those developed regions present significant positive effects, while those regions dominated by agriculture economy and without sufficient supporting facilities for e-commerce development, show significant negative effects. Therefore, we argue that the positive environmental contribution of e-commerce is not bound to happen but instead is contingent, while policies should be adapted to local conditions and enhanced to encourage the integration of e-commerce and other socioeconomic sectors, in order to develop beyond the immature pioneering stage.

The Sharing of Costs and Benefits of Rural Environmental Pollution Governance in China: A Qualitative Analysis through Guanxi Networks Perspective.

Concern has been expressed in many parts of the world that community relations in rural areas are breaking down, making issues such as rural environmental degradation harder to resolve without external regulation. Guanxi is a specific Chinese idiom for characterizing social networks, as a broad term to represent existing relations among people, which can be loosely translated as ''relationship''. Based on a case study of an underdeveloped mountainous area of Southern China, this paper examined the problem from the perspective of guanxi, and explored the impacts of internal group differentiation catalyzed by pig farming pollution and the subsequent influences on the distribution of costs and benefits of different shareholders. It was found that the guanxi in the village were changed from blood relationship centered to economic interest centered. This disparity exerts a significant influence on the distribution of costs and benefits of pollution control and exacerbates environmental inequalities. This means that pig farmers dominated the narrative of pig farming pollution, while the ordinary villagers chose to suffer without protesting, which hinders the advancement of pollution control, and pig farmers took the benefits of weak pollution control and managed to transfer the external cost to others, while others became direct victims. The paper concludes that the rich become richer and the poor become poorer in both economic and environmental perspectives. It is strongly suggested that guanxi should be integrated into the consideration and decision-making process of rural environmental governance in order to guarantee the efficiency and efficacy of its implementation.

Surveying the Landscape of Quality-of-Care Measures for Mental and Substance Use Disorders.

OBJECTIVE: Quality measures that are used to evaluate health care services have a central role in monitoring and incentivizing quality improvement and the provision of evidence-based treatment. This systematic scan aimed to catalog quality-of-care measures for mental and substance use disorders and assess gaps and redundancies to inform efforts to develop and retire measures. METHODS: Quality measure inventories were analyzed from six organizations that evaluate health care quality in the United States. Measures were included if they were defined via symptoms or diagnoses of mental and substance use disorders or specialty treatments or treatment settings for adults. RESULTS: Of 4,420 measures analyzed, 635 (14%) met inclusion criteria, and 376 unique quality-of-care measure constructs were cataloged and characterized. Symptoms or diagnoses of disorders were most commonly used to define measures (46%, N=172). Few measures were available for certain disorders (e.g., anxiety disorders), evidence-based treatments (e.g., psychotherapy), and quality domains (e.g., equity). Only one in four measures was endorsed by the National Quality Forum, which independently and critically evaluates quality measures. Among measures that were actively in use for national quality improvement initiatives (N=319), process measures (57%) were most common, followed by outcome measures (30%), the latter of which focused most often on experience of care. CONCLUSIONS: A vast landscape of mental and substance use disorder quality-of-care measures currently exists, and continued efforts to harmonize duplicative measures and to develop measures for underrepresented evidence-based treatments and quality domains are warranted. The authors recommend reinvesting in a national, centralized system for measure curation, with a stakeholder-centered process for independent measure review and endorsement.

Mastery of type 2 diabetes prevention and treatment knowledge by general practitioners in Shanghai: a cross-sectional study.

BACKGROUND: To study the competency of general practitioners (GPs) in Shanghai, China on prevention and management of type 2 diabetes, also understand factors that may prohibit it. METHODS: A survey questionnaire with 25 questions was designed based on 2013 Chinese Type 2 Diabetes Prevention Guidelines and Chinese Type 2 Diabetes Prevention Guidelines (Grassroots Edition) and conducted among 789 GPs who work at 54 community healthcare centers (CHCs) within 16 districts at Shanghai, China. Excel 2016 and SPSS 24.0 were used for data analysis, and a difference of P < 0.05 was considered to be statistically significant. RESULTS: The GPs did poorly on three aspect of diabetes prevention and treatment: (1) treatment goals in elderly patients, (2) screening methods for high-risk population, and (3) aspirin contraindications. The statistical analysis data showed that GPs who finished standardized training had correct answer on 13.58 ± 3.31 questions out of total 25, with mean accuracy rate of 54.32%. Except the questions for high-risk population screening method and the diagnostic criteria for type 2 diabetes, there was no difference in the accuracy of other questions between GPs with or without standardized training (P < 0.05). However, sex, educational level, and subspecialty experience are affective factors on their competency in type 2 diabetes prevention and treatment knowledge. CONCLUSION: The results indicated that communities should strengthen the training of GPs in diabetes management and bidirectional referral. Frequent continuing education and skills training should be provided among GPs at CHCs to ensure their competency of type 2 diabetes prevention and treatment knowledge after obtaining their GP license disregard of their standardized training. In addition, attention should be paid to GPs who had lower education background or non-clinical subspecialty experience to strengthen their clinical knowledge of type 2 diabetes.

Barriers and facilitators to interdisciplinary communication during consultations: a qualitative study.

OBJECTIVE: Communication failures between clinicians lead to poor patient outcomes. Critically injured patients have multiple injured organ systems and require complex multidisciplinary care from a wide range of healthcare professionals and communication failures are abundantly common. This study sought to determine barriers and facilitators to interdisciplinary communication between the consulting trauma, intensive care unit (ICU) team and specialty consultants for critically injured patients at an urban, safety-net, level 1 trauma centre. DESIGN: An observational qualitative study of barriers and facilitators to interdisciplinary communication. SETTING: We conducted observations of daily rounds in two trauma surgical ICUs and recorded the most frequently consulted teams. PARTICIPANTS: Key informant interviews after presenting clinical vignettes as discussion prompts were conducted with a broad range of clinicians from the ICUs and physicians and nurse practitioners from the consultant teams who were identified during the observations. Interviews were recorded and transcribed verbatim. Data of these 10 interviews were combined with primary transcript data from prior study (25 interviews) and analysed together because of the same setting with same themes. Independent coding of the transcripts, with iterative reconciliation, was performed by two coders. OUTCOMES MEASURES: Facilitators and barriers of interdisciplinary communication were identified. RESULTS: A total of 35 interview transcripts were analysed. Cardiology and interventional radiology were the most frequently consulted teams. Consulting and consultant clinicians reported that perceived accessibility from the team seeking a consultation and the consultant team impacted interdisciplinary communication. Accessibility had a physical dimension as well as a psychological dimension. Accessibility was demonstrated by responsiveness between clinicians of different disciplines and in turn facilitated interdisciplinary communication. Social norms, cognitive biases, hierarchy and relationships were reported as both facilitators and barriers to accessibility, and therefore, interdisciplinary communication. CONCLUSION: Accessibility impacted interdisciplinary communication between the consulting and the consultant team. ARTICLE SUMMARY: Elucidates barriers and facilitators to interdisciplinary communication between consulting and consultant teams.

Laser-Generated Leaky Acoustic Wave Imaging for Interventional Guidewire Guidance.

Ultrasound (US) is widely used to visualize both tissue and the positions of surgical instruments in real time during surgery. Previously we proposed a new method to exploit US imaging and laser-generated leaky acoustic waves (LAWs) for needle visualization. Although successful, that method only detects the position of a needle tip, with the location of the entire needle deduced from knowing that the needle is straight. The purpose of the current study was to develop a beamforming-based method for the direct visualization of objects. The approach can be applied to objects with arbitrary shapes, such as the guidewires that are commonly used in interventional guidance. With this method, illumination by a short laser pulse generates photoacoustic waves at the top of the guidewire that propagate down its metal surface. These waves then leak into the surrounding tissue, which can be detected by a US array transducer. The time of flight consists of two parts: 1) the propagation time of the guided waves on the guidewire and 2) the propagation time of the US that leaks into the tissue. In principle, an image of the guidewire can be formed based on array beamforming by taking the propagation time on the metal into consideration. Furthermore, we introduced directional filtering and a matched filter to compress the dispersion signal associated with long propagation times. The results showed that guidewires could be detected at depths of at least 70 mm. The maximum detectable angle was 56.3°. LAW imaging with a 1268-mm-long guidewire was also demonstrated. The proposed method has considerable potential in new clinical applications.

Promoting agricultural innovation as a means of improving China's rural environment.

Farmer-led agricultural innovation is increasingly viewed as a potential approach to sustainable agriculture especially promoting rural revitalization as well as mitigating agricultural non-point source pollution. However, little research has yet been paid to evaluating the environmental contribution caused by these emerging agricultural innovations. Using data generated in the Qingpu District of Shanghai, this paper focuses on the new agri-business entities and evaluates the impact of agricultural innovation on changes in their use of chemical fertilizers. The findings indicate that different forms of agricultural innovation have radically different outcomes. Innovation of new production technologies and sales tend to have negative impacts on the environment, while both vertical integration with manufacturing-processing-sales activities, and horizontal integration with service activities, are found to make a positive environmental contribution. The paper argues that the different sources of value added generated by innovation provide different incentives for farmers. Those with a narrow concentration on efficiency and market scale tend to intensify their output-maximized production, while those shifting to processing and service activities rely more on the quality and service centered production, which tends to create less damage to the environment.

Impacts of rTMS on Refractory Depression and Comorbid PTSD Symptoms at a Military Treatment Facility.

INTRODUCTION: Repetitive transcranial magnetic stimulation (rTMS) as a treatment for depression has been studied for over two decades. Repetitive TMS was approved by the Food and Drug Administration in 2008 for the treatment of depression after at least one failed trial of an antidepressant medication of adequate dose and duration. This study evaluated whether rTMS treatments may be associated with measurable improvements in depression and post-traumatic stress disorder (PTSD) symptoms for treated military beneficiaries in Hawaii suffering from depression. It also examined the number of failed medication trials that patients underwent before rTMS treatment. MATERIALS AND METHODS: A retrospective chart review of 77 rTMS patients who received and completed treatment between January 1, 2010 and October 31, 2016 was performed. Under a typical treatment regimen, patients receive rTMS for 6 weeks as well as weekly psychiatric assessments, which included completion of Beck's Depression Inventory (BDI) and PTSD Checklist (PCL). A mixed model repeated measures analysis was done assuming an autoregressive order one covariance structure to evaluate changes over time. Adjusted analyses were done to assess whether changes over time differed by age, prior diagnosis of PTSD, active duty status, and gender. RESULTS: The majority of patients were from the army (74%) and 56% were on active duty. Just over half (53%) were male. Most patients (52%) had completed trials of three or more different antidepressant medications before initiation of treatment with rTMS. The mean number of antidepressant trials was 2.7. BDI and PCL scores were significantly lower at end of treatment on average compared to the pretreatment baseline scores. Mean differences for BDI and PCL were significant with P < 0.001 15, 30, and 45 days after TMS treatment was initiated. Overall, 44% of patients experienced a reduction ≥10 points on BDI, and 38% experienced a reduction ≥10 points on PCL. Additionally, scores fell similarly regardless of whether or not patients had a comorbid diagnosis of PTSD. CONCLUSIONS: Our research suggests that rTMS treatments may produce a reduction in symptoms of both depression and PTSD in patients with refractory depression and comorbid PTSD. It may be a useful alternative to antidepressants in the treatment of depression in the military population, including those with comorbid PTSD. Broader implementation of this treatment modality may prove beneficial for the purposes of military readiness, given current policies and restrictions on service members who are initiated on antidepressant medications.

The effect of cirrhosis on trauma outcomes: A systematic review and meta-analysis.

BACKGROUND: The negative effect of cirrhosis on mortality following traumatic injury has been quantified in multiple observational studies. However, to our knowledge, the information contained in these studies has never been synthesized. The aims of this study were: (1) to determine the magnitude of the effect of liver cirrhosis on mortality, morbidity, and hospital course among trauma patients and (2) to analyze sources of study heterogeneity that may lead to differing estimates in the observed mortality rate among patients with cirrhosis. METHODS: A systematic search of EMBASE and PubMed was conducted. Data were extracted from eligible studies and analyzed using a random-effects model to compare trauma outcomes in cirrhotic and noncirrhotic patients (PROSPERO Registration CRD42018088464). Mortality was the primary outcome. Secondary outcomes included complication rate, length of hospital stay, length of intensive care unit stay, and mechanical ventilation days. RESULTS: Title and abstract review of 15,958 articles led to the identification of 31 relevant articles. Ultimately, 18 observational studies were included in this meta-analysis. The pooled effect sizes for mortality (odds ratio [OR], 4.52; 95% confidence interval [CI], 3.13-6.54) and complication rate (OR, 1.92; 95% CI, 1.30-2.85) were higher in the cirrhotic group than the noncirrhotic group. Trauma patients with cirrhosis also incurred longer hospital stays (mean difference, 3.81 days; 95% CI, 1.22-6.41) and longer ICU stays (mean difference, 2.40 days; 95% CI, 0.65-4.15). There was no difference in days spent on mechanical ventilation. CONCLUSION: Preexisting liver cirrhosis is associated with increased mortality rate, complication rate, and length of hospitalization among trauma patients, even after adjusting for confounding factors and potential sources of between-study heterogeneity. Trauma patients with cirrhosis would benefit from heightened surveillance and injury prevention interventions. LEVEL OF EVIDENCE: Systematic review and meta-analysis, level III.

How care decisions are made among interdisciplinary providers caring for critically injured patients: A qualitative study.

BACKGROUND: Injury is the leading cause of death in people under 45 years of age in the United States; however, how care decisions occur in critical injury is poorly understood. This exploratory study sought to generate hypotheses about how care decisions are made among interdisciplinary providers caring for patients who have been critically injured. METHODS: This was a qualitative study conducted at two intensive care units in a level 1 trauma center in an urban, teaching, safety-net hospital. Semistructured interviews consisted of case scenarios with competing clinical priorities presented to 25 interdisciplinary providers, elucidating how decisions are approached. Responses were recorded, transcribed, and coded. Thematic analysis was conducted to discover central themes. Category formulation and sorting was done for data reduction and thematic structuring of the data. The range and central tendency of these themes are reported. RESULTS: The central theme for how care decisions are made among interdisciplinary providers was through the distribution of shared responsibility. The distribution of shared responsibility depended on interdisciplinary communication to navigate the two subthemes of time and roles. Time had to be navigated carefully, because it was both an opportunity for data acquisition and consensus building but also a pressure to decisively progress care. Roles were distinct but interchangeable and consisted of experts, actualizers, and questioners. CONCLUSION: Care decisions are made in the context of shared responsibility among interdisciplinary providers. Interdisciplinary communication is a means of establishing roles and navigating time to distribute shared responsibility among interdisciplinary providers.

Hili Inhibits HIV Replication in Activated T Cells.

P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4+ T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNAArg(UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements.IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4+ T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements.

The spaces and times of community farming.

This paper uses a multiple case study approach to researching people's everyday lives and experiences of six community farms and gardens in diverse settings in China and England. We argue that collective understandings of community are bound up in everyday action in particular spaces and times. Successful community farms and gardens are those that are able to provide suitable spaces and times for these actions so that their members can enjoy multiple benefit streams. These benefits are largely universal: in very different situations in both England and China, CSA members make strong connections with the land, the farmers and other members, even in cases where they rarely visit the farms and gardens. This suggests that community farming and gardening initiatives possess multi-dimensional transformational potential. Not only do they offer a buffer against industrialised and remote food systems, but they also represent therapeutic landscapes valued by those who have experienced time spent at or in connection with them. Our findings indicate that-regardless of location or cultural context-these benefits are durable, so that people who have been engaged in multiple activities at a community farm or garden continue to enjoy these benefits long after most of their engagement has ceased.

Release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein (snRNP) activates hexamethylene bisacetamide-inducible protein (HEXIM1) transcription.

By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.

Diapause, signal and molecular characteristics of overwintering Chilo suppressalis (Insecta: Lepidoptera: Pyralidae).

Diapause is a complex and dynamic process. Chilo suppressalis, an important rice pest in Asia enters facultative diapause as larvae. Our results demonstrated in Yangzhou, China, diapause was initiated between September 4 and 12, 2010. After diapause termination, C. suppressalis remained in quiescence in the field for as long as three months. The average time between collection of field larvae of C. suppressalis and their pupation decreased as the season progressed from fall to next spring. Unexpectedly, the pupated ratio of female to male in the initiation of diapause was 0.22. The abundance of hsp90, hsp70, hsp60 and CsAQP1 all peaked on January 8 or 15, 2011. Nitric oxide (NO) is a secondary messenger that is positively correlated with the diapause of C. suppressalis. Among several geographically separated populations of C. suppressalis, there are no significant differences in the mRNA levels of hsp70, hsp60 or CsAQP1.

Molecular cloning and characterization of the first caspase in the Striped Stem Borer, Chilo suppressalis.

Apoptosis is executed through the activity of the caspases that are aspartyl-specific proteases. In this study, we isolated the caspase gene (Cscaspase-1) of Chilo suppressalis (one of the leading pests responsible for destruction of rice crops). It possesses the open reading frame (ORF) of 295 amino acids including prodomain, large subunit and small subunits, and two cleavage sites (Asp23 and Asp194) were found to be located among them. In addition to these profiles, Cscaspase-1 contains two active sites (His134 and Cys176). Genomic analysis demonstrated there was no intron in the genome of Cscaspase-1. The Cscaspase-1 transcripts were found in all tissues of the fifth instar larvae, and higher levels were found in the midgut, hindgut and Malpighian tubules. Examination of Cscaspase-1 expression in different developmental stages indicated low constitutive levels in the eggs and early larvae stages, and higher abundances were exhibited in the last larvae and pupae stages. The relative mRNA levels of Cscaspase-1 were induced by heat and cold temperatures. For example, the highest increase of Cscaspase-1 transcription was at -3 °C and 36 °C respectively. In a word, Cscaspase-1 plays a role of effector in the apoptosis of C. suppressalis. It also correlates with development, metamorphosis and thermotolerance of C. suppreassalis.

Mechanism of cysteine-dependent inactivation of aspartate/glutamate/cysteine sulfinic acid α-decarboxylases.

Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to ß-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.

Biochemical evaluation of the decarboxylation and decarboxylation-deamination activities of plant aromatic amino acid decarboxylases.

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.

Role of glutamate decarboxylase-like protein 1 (GADL1) in taurine biosynthesis.

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to ß-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no ß-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although ß-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in ß-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Cysteine sulfinic acid decarboxylase activity of Aedes aegypti aspartate 1-decarboxylase: the structural basis of its substrate selectivity.

Insect aspartate 1-decarboxylase (ADC) catalyzes the decarboxylation of aspartate to ß-alanine. Insect ADC proteins share high sequence identity to mammalian cysteine sulfinic acid decarboxylase (CSADC), but there have been no reports indicating any CSADC activity in insect ADC or any ADC activity in mammalian CSADC. Substrate screening of Aedes aegypti ADC (AeADC), however, demonstrates that other than its activity to aspartate, the mosquito enzyme catalyzes the decarboxylation of cysteine sulfinic acid and cysteic acid as efficiently as those of mammalian CSADC under the same testing conditions. Further analysis of Drosophila melanogaster ADC also demonstrated its CSADC activity, suggesting that all insect ADC likely has CSADC activity. This represents the first identification of CSADC activity of insect ADC. On the other hand, HuCSADC displayed no detectable activity to aspartate. Homology modeling of AeADC and substrate docking suggest that residue Q377, localized at the active site of AeADC, could better interact with aspartate through hydrogen bonding, which may play a role in aspartate selectivity. A leucine residue in mammalian CSADC occupies the same position. A mutation at position 377 from glutamine to leucine in AeADC diminished its decarboxylation activity to aspartate with no major effect on its CSADC activity. Comparison of insect ADC sequences revealed that Q377 is stringently conserved among the available insect ADC sequences. Our data clearly established the CSADC activity of mosquito and Drosophila ADC and revealed the primary role Q377 plays in aspartate selectivity in insect ADC.