BRD2 promotes antibody class switch recombination by facilitating DNA repair in collaboration with NIPBL.

Efficient repair of DNA double-strand breaks in the Ig heavy chain gene locus is crucial for B-cell antibody class switch recombination (CSR). The regulatory dynamics of the repair pathway direct CSR preferentially through nonhomologous end joining (NHEJ) over alternative end joining (AEJ). Here, we demonstrate that the histone acetyl reader BRD2 suppresses AEJ and aberrant recombination as well as random genomic sequence capture at the CSR junctions. BRD2 deficiency impairs switch (S) region synapse, optimal DNA damage response (DDR), and increases DNA break end resection. Unlike BRD4, a similar bromodomain protein involved in NHEJ and CSR, BRD2 loss does not elevate RPA phosphorylation and R-loop formation in the S region. As BRD2 stabilizes the cohesion loader protein NIPBL in the S regions, the loss of BRD2 or NIPBL shows comparable deregulation of S-S synapsis, DDR, and DNA repair pathway choice during CSR. This finding extends beyond CSR, as NIPBL and BRD4 have been linked to Cornelia de Lange syndrome, a developmental disorder exhibiting defective NHEJ and Ig isotype switching. The interplay between these proteins sheds light on the intricate mechanisms governing DNA repair and immune system functionality.

Assessment of the prevalence and associated factors of lower back pain (LBP) among three different professionals in Bangladesh: Findings from a face-to-face survey.

OBJECTIVES: This study aims to evaluate the prevalence and associated factors of lower back pain (LBP) among farmers, rickshaw pullers and office workers in Bangladesh, while also investigating potential contributors within these occupational groups. DESIGN: This cross-sectional study aimed to determine the prevalence of LBP, associated factors and management procedures among farmers, rickshaw pullers and office workers in Bangladesh through face-to-face interviews. SETTING: The study was conducted in different parts of the Bogura district in Bangladesh. PARTICIPANTS: A total of 396 participants were included in the final analysis, all the participants were men and adult in age. MAIN OUTCOME MEASUREMENTS: Data were collected using a semi-structured questionnaire based on previous research. Bivariate and multivariable logistic regression analyses were performed to identify significant associations. RESULTS: The prevalence of LBP was found to be 42.7% among the participants. Farmers and rickshaw pullers had approximately four-times and three-times higher odds of experiencing LBP compared with office workers. Other significant factors associated with LBP included living in a nuclear family, having a normal body weight, lacking professional training, having a chronic disease, having a family history of LBP and experiencing numbness in the legs or feet. The majority of respondents sought medical attention and took medication for their LBP. CONCLUSION: The study underscores occupational differences in LBP prevalence, emphasising the necessity for tailored interventions and occupational health policies. Identifying specific risk factors and management practices in these professions can aid in developing effective prevention strategies and enhancing healthcare services.

Mycorrhizal inoculation enhanced tillering in field grown wheat, nutritional enrichment and soil properties.

To meet food security, commercial fertilizers are available to boost wheat yield, but there are serious ill effects associated with these fertilizers. Amongst various organic alternatives, inoculating crop fields with mycorrhizal species is the most promising option. Although, mycorrhizae are known to enhance wheat yield, but how the mycorrhizae influence different yield and quality parameters of wheat, is not clear. Therefore, this study was undertaken to investigate the influence of indigenous mycorrhizal species on the growth of wheat, its nutritional status and soil properties, in repeated set of field experiments. In total 11 species of mycorrhizae were isolated from the experimental sites with Claroideoglomus, being the most dominant one. Five different treatments were employed during the present study, keeping plot size for each replicate as 6 × 2 m. Introduction of consortia of mycorrhizae displayed a significant increase in number of tillers/plant (49.5%), dry biomass (17.4%), grain yield (21.2%) and hay weight (16.7%). However, there was non-significant effect of mycorrhizal inoculation on 1,000 grains weight. Moreover, protein contents were increased to 24.2%. Zinc, iron, phosphorus and potassium concentrations were also increased to 24%, 21%, 30.9% and 14.8%, respectively, in wheat grains. Enhancement effects were also noted on soil fertility such as soil organic carbon % age, available phosphorus and potassium were increased up to 64.7%, 35.8% and 23.9%, respectively. Herein, we concluded that mycorrhizal introduction in wheat fields significantly increased tillering in wheat and this increased tillering resulted in overall increase in wheat biomass/yield. Mycorrhizae also enhanced nutritional attributes of wheat grains as well as soil fertility. The use of mycorrhizae will help to reduce our dependance on synthetic fertilizers in sustainable agriculture.

HNRNPU facilitates antibody class-switch recombination through C-NHEJ promotion and R-loop suppression.

B cells generate functionally different classes of antibodies through class-switch recombination (CSR), which requires classical non-homologous end joining (C-NHEJ) to join the DNA breaks at the donor and acceptor switch (S) regions. We show that the RNA-binding protein HNRNPU promotes C-NHEJ-mediated S-S joining through the 53BP1-shieldin DNA-repair complex. Notably, HNRNPU binds to the S region RNA/DNA G-quadruplexes, contributing to regulating R-loop and single-stranded DNA (ssDNA) accumulation. HNRNPU is an intrinsically disordered protein that interacts with both C-NHEJ and R-loop complexes in an RNA-dependent manner. Strikingly, recruitment of HNRNPU and the C-NHEJ factors is highly sensitive to liquid-liquid phase separation inhibitors, suggestive of DNA-repair condensate formation. We propose that HNRNPU facilitates CSR by forming and stabilizing the C-NHEJ ribonucleoprotein complex and preventing excessive R-loop accumulation, which otherwise would cause persistent DNA breaks and aberrant DNA repair, leading to genomic instability.

XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation.

The transcriptional coactivator Med12 regulates gene expression through its kinase module. Here, we show a kinase module-independent function of Med12 in CSR. Med12 is essential for super-enhancer activation by collaborating with p300-Jmjd6/Carm1 coactivator complexes. Med12 loss decreases H3K27 acetylation and eRNA transcription with concomitant impairment of AID-induced DNA breaks, S-S synapse formation, and 3'RR-Eµ interaction. CRISPR-dCas9-mediated enhancer activation reestablishes the epigenomic and transcriptional hallmarks of the super-enhancer and fully restores the Med12 depletion defects. Moreover, 3'RR-derived eRNAs are critical for promoting S region epigenetic regulation, synapse formation, and recruitment of Med12 and AID to the IgH locus. We find that XLID syndrome-associated Med12 mutations are defective in both 3'RR eRNA transcription and CSR, suggesting that B and neuronal cells may have cell-specific super-enhancer dysfunctions. We conclude that Med12 is essential for IgH 3'RR activation/eRNA transcription and plays a central role in AID-induced antibody gene diversification and genomic instability in B cells.

Nucleotide Pool Imbalance and Antibody Gene Diversification.

The availability and adequate balance of deoxyribonucleoside triphosphate (dNTP) is an important determinant of both the fidelity and the processivity of DNA polymerases. Therefore, maintaining an optimal balance of the dNTP pool is critical for genomic stability in replicating and quiescent cells. Since DNA synthesis is required not only in genomic replication but also in DNA damage repair and recombination, the abnormalities in the dNTP pool affect a wide range of chromosomal activities. The generation of antibody diversity relies on antigen-independent V(D)J recombination, as well as antigen-dependent somatic hypermutation and class switch recombination. These processes involve diverse sets of DNA polymerases, which are affected by the dNTP pool imbalances. This review discusses the role of the optimal dNTP pool balance in the diversification of antibody encoding genes.

Missing data in randomised controlled trials of rheumatoid arthritis drug therapy are substantial and handled inappropriately.

OBJECTIVES: To analyse the amount, reporting and handling of missing data, approach to intention-to-treat (ITT) principle application and sensitivity analysis utilisation in randomised clinical trials (RCTs) of rheumatoid arthritis (RA). To assess the trend in such reporting 10 years apart (2006 and 2016). METHODS: Parallel group drug therapy RA RCTs with a clinical primary endpoint. RESULTS: 176 studies enrolling a median of 160 (IQR 62-339) patients were eligible. In terms of actual analysis: 81 (46%) RCTs conducted ITT, 42 (23.9%) conducted modified ITT while 53 (30.1%) conducted non-ITT analysis. Only 58 of 97 (59.8%) RCTs reporting an ITT analysis actually performed it. The median (IQR) numbers of participants completing the trial and included in analysis for primary outcome were 86% (74%-91%) and 100% (97.1%-100%), respectively. 53 (32.7%) and 65 (40.1%) RCTs had >20% and 10%-20% missing primary outcome data, respectively. Missing data handling was unreported by 58 of 171 (33.9%) RCTs. When reported, vast majority used simple imputation methods. No significant trend towards improved reporting was seen between 2006 and 2016. Sensitivity analysis numerically improved from 2006 to 2016 (14.7% vs 21.4%). CONCLUSIONS: There is significant discrepancy in the reported and the actual performed analysis in RA drug therapy RCTs. Nearly one-third of RCTs had >20% missing data. The reporting and methods of missing data handling remain inadequate with high usage of non-preferred simple imputation methods. Sensitivity analysis utilisation was low. No trend towards better missing data reporting and handling was seen.

Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.

Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

Inclusion of Veterinary Services in national emergency management plans.

With human population growth, rapid urbanisation, increasing globalisation, and climate change, the interdependency of human health and animal health is mounting. Therefore, the importance of national emergency management plans (NEMPs) for the mitigation of, and preparedness for, all hazards, including disease epidemics, both zoonotic and zootic, is ever increasing. The authors decided to take a One Health approach by assessing the inclusion of Veterinary Services and animal health in NEMPs, based on geographical region, the date of the NEMP, national income status, and the proportion of the agricultural sector in national gross domestic product (GDP). To carry out the assessment, the authors analysed the publicly available NEMPs of 86 Members of the World Organisation for Animal Health. Of the 86 NEMPs reviewed, only a third expressly mentioned Veterinary Services, almost 60% mentioned zoonotic and/or zootic diseases, and about two-thirds mentioned animals to some extent. The highest correlating factor to the inclusion of animal health in NEMPs was the level of the agricultural sector's contributions to the national GDP. Fisheries and aquaculture were not a major consideration in any of the reviewed NEMPs, especially not in relation to diseases. Based on region, Latin America and the Caribbean exhibited the lowest inclusion rate of animal health in NEMPs. The results demonstrate that the omission of animal health is still a problem. A multi-disciplinary approach that includes veterinary medicine as well as human medicine is vital in the construction and/or revision of NEMPs. Future studies should consider whether or not there is a connection between countries' veterinary capacities and the inclusion of Veterinary Services in their NEMPs and whether or not they have the infrastructure and human resources to put into operation the roles of Veterinary Services as identified in their NEMPs.

La croissance démographique humaine, l'urbanisation accélérée, la mondialisation accrue et le changement climatique sont autant de facteurs qui intensifient l'interdépendance de la santé humaine et de la santé animale. De ce fait, les plans nationaux de gestion des urgences jouent un rôle de plus en plus important pour atténuer les dangers, quels qu'ils soient, et pour se préparer à leur survenue, y compris les dangers liés aux épidémies zoonotiques ou zootiques. Les auteurs ont entrepris d'évaluer le niveau d'intégration des Services vétérinaires et de la santé animale dans les plans nationaux de gestion des urgences dans une perspective Une seule santé, en se basant sur les critères suivants : la région géographique, la date du plan national de gestion des urgences, le niveau de revenu du pays et la part du secteur agricole dans le produit intérieur brut (PIB). Pour les besoins de cette évaluation, les auteurs ont analysé les plans nationaux de gestion des urgences publiés par 86 Membres de l'Organisation mondiale de la santé animale. Parmi ces 86 plans nationaux, un tiers seulement mentionnait expressément les Services vétérinaires, près de 60 % mentionnaient les maladies zoonotiques ou les épizooties et environ deux tiers prenaient en compte les animaux pour une raison ou pour une autre. Le facteur présentant la corrélation la plus élevée avec la prise en compte de la santé animale dans les plans nationaux de gestion des urgences était le niveau de contribution du secteur agricole dans le PIB national. Aucun des plans nationaux de gestion des urgences examinés ne prenait en compte la pêche et l'aquaculture en tant qu'aspect important, en particulier en lien avec des maladies. À l'échelle régionale, c'est en Amérique latine et aux Caraïbes que l'intégration de la santé animale dans les plans nationaux de gestion des urgences était la plus faible. Ces résultats montrent que le problème de l'omission de la santé animale est toujours d'actualité. Il est d'une importance capitale qu'une approche pluridisciplinaire intégrant la médecine vétérinaire et la médecine humaine soit adoptée lors de la conception et/ou de la révision des plans nationaux de gestion des urgences. Il conviendrait que de nouvelles études déterminent à l'avenir s'il existe ou non un lien entre les capacités vétérinaires des pays et la prise en compte des Services vétérinaires dans les plans nationaux de gestion des urgences, et si les pays disposent ou non des infrastructures et des ressources humaines permettant à leurs Services vétérinaires de mener à bien les interventions prévues dans les plans nationaux de gestion des urgences.

El crecimiento demográfico, la rápida urbanización, la creciente mundialización y el cambio climático son otros tantos factores que traen consigo una dependencia recíproca cada vez más acusada entre la salud humana y la sanidad animal. De ahí la creciente importancia que van adquiriendo los planes nacionales de gestión de emergencias destinados a prepararse para todo tipo de peligros, incluidas las enfermedades epidémicas, tanto zoonóticas como epizoóticas, y, llegado el caso, a mitigar sus consecuencias. Los autores, partiendo de las premisas de Una sola salud, decidieron evaluar la integración de los Servicios Veterinarios y la sanidad animal en los planes nacionales de gestión de emergencias, utilizando como criterios de evaluación la región geográfica, la fecha del plan nacional en cuestión, el nivel de renta del país y el porcentaje del producto interno bruto (PIB) que representa el sector agrícola. Para llevar a cabo la evaluación los autores analizaron los planes nacionales de gestión de emergencias que están a disposición pública de 86 Miembros de la Organización Mundial de Sanidad Animal. De esos 86 planes nacionales examinados, solo en un tercio se mencionaban explícitamente los Servicios Veterinarios, en casi un 60% se aludía a enfermedades zoonóticas y/o epizoóticas y en cerca de dos tercios se hablaba en alguna medida de los animales. El factor que mayor correlación presentaba con la integración de la sanidad animal en los planes nacionales de gestión de emergencias era la aportación del sector agrícola al PIB. En ninguno de los planes examinados ocupaban un lugar relevante ni la pesca ni la acuicultura, especialmente en relación con las enfermedades. Por regiones, América Latina y el Caribe presentaba el menor porcentaje de integración de la sanidad animal en los planes nacionales de gestión de emergencias. Los resultados demuestran que la omisión de la sanidad animal sigue suponiendo un problema. A la hora de elaborar o revisar los planes nacionales de gestión de emergencias es crucial hacerlo desde planteamientos multidisciplinares que incluyan tanto la medicina veterinaria como la humana. En estudios ulteriores convendría determinar si existe una correlación entre la capacidad veterinaria de los países y la integración de los Servicios Veterinarios en su plan nacional de gestión de emergencias y si los países disponen de la infraestructura y el personal requeridos para que los Servicios Veterinarios cumplan las funciones que se les asignan en el plan nacional de gestión de emergencias.

SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

RNA-binding motifs of hnRNP K are critical for induction of antibody diversification by activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) to generate antibody memory. Previously, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was shown to be required for AID-dependent DNA breaks. Here, we defined the function of major RNA-binding motifs of hnRNP K, GXXGs and RGGs in the K-homology (KH) and the K-protein-interaction (KI) domains, respectively. Mutation of GXXG, RGG, or both impaired CSR, SHM, and cMyc/IgH translocation equally, showing that these motifs were necessary for AID-dependent DNA breaks. AID-hnRNP K interaction is dependent on RNA; hence, mutation of these RNA-binding motifs abolished the interaction with AID, as expected. Some of the polypyrimidine sequence-carrying prototypical hnRNP K-binding RNAs, which participate in DNA breaks or repair bound to hnRNP K in a GXXG and RGG motif-dependent manner. Mutation of the GXXG and RGG motifs decreased nuclear retention of hnRNP K. Together with the previous finding that nuclear localization of AID is necessary for its function, lower nuclear retention of these mutants may worsen their functional deficiency, which is also caused by their decreased RNA-binding capacity. In summary, hnRNP K contributed to AID-dependent DNA breaks with all of its major RNA-binding motifs.

Native Co-immunoprecipitation Assay to Identify Interacting Partners of Chromatin-associated Proteins in Mammalian Cells.

Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were then diluted, pooled, and used for Co-IP. This protocol can be used to identify protein-interactome of other chromatin-associated proteins in a variety of mammalian cells.

Liquiritin elicitation can increase the content of medicinally important glucosinolates and phenolic compounds in Chinese kale plants.

BACKGROUND: Brassica oleracea var. alboglabra (Chinese kale) is an important vegetable grown in southern China. This study was aimed at searching for environmentally friendly and affordable approaches to increase the production of medicinally relevant glucosinolates and phenolic compounds in Chinese kale plants. For this purpose, the foliar application of liquiritin at 0 (control), 250, 500 and 750 ppm was tested starting from the four-leaf stage and repeated every two weeks until plants were two months old. RESULTS: Foliar application of liquiritin in Chinese kale plants significantly increased glucosinolates and total phenolic content, in a dose-dependent manner. Compared with control plants, 2.3- and 1.9-fold increases in yields of glucosinolates and total phenolic content, respectively, were corroborated in Chinese kale plants treated with 750 ppm of liquiritin. Along with rises in the content of eight different glucosinolates, liquiritin elicitation effectively increased the concentration of glycosilated and acylated flavonoids and hydroxycinnamic acids. The expression of genes involved in glucosinolate and phenolic biosynthesis was significantly higher in liquiritin-treated plants as compared to controls. CONCLUSIONS: Liquiritin elicitation is a feasible and environmentally friendly practice for increasing the production of medicinally important glucosinolates and phenolic compounds in Chinese kale, which may improve this plant's value as a nutraceutical food. This study also contributes to understanding the molecular mechanisms underlying liquiritin elicitation. This is the first report documenting the use of liquiritin for an elicitation purpose in plants. © 2019 Society of Chemical Industry.

Scientific productivity: An exploratory study of metrics and incentives.

Competitive pressure to maximize the current bibliometric measures of productivity is jeopardizing the integrity of the scientific literature. Efforts are underway to address the 'reproducibility crisis' by encouraging the use of more rigorous, confirmatory methods. However, as long as productivity continues to be defined by the number of discoveries scientists publish, the impact factor of the journals they publish in and the number of times their papers are cited, they will be reluctant to accept high quality methods and consistently conduct and publish confirmatory/replication studies. This exploratory study examined a sample of rigorous Phase II-IV clinical trials, including unpublished studies, to determine if more appropriate metrics and incentives can be developed. The results suggest that rigorous procedures will help reduce false positives, but to the extent that higher quality methods are accepted as the standard of practice, the current bibliometric incentives will discourage innovative studies and encourage scientists to shift their research to less informative studies of subjects that are already being more actively investigated. However, the results also suggest that it is possible to develop a more appropriate system of rewards. In contrast to the current bibliometric incentives, evaluations of the quality of the methods and reproducibility of the results, innovation and diversity of thought, and amount of information produced may serve as measures and incentives that maintain the integrity of the scientific literature and maximize scientific progress.

Fast and Label-Free Isolation of Circulating Tumor Cells from Blood: From a Research Microfluidic Platform to an Automated Fluidic Instrument, VTX-1 Liquid Biopsy System.

Tumor tissue biopsies are invasive, costly, and collect a limited cell population not completely reflective of patient cancer cell diversity. Circulating tumor cells (CTCs) can be isolated from a simple blood draw and may be representative of the diverse biology from multiple tumor sites. The VTX-1 Liquid Biopsy System was designed to automate the isolation of clinically relevant CTC populations, making the CTCs available for easy analysis. We present here the transition from a cutting-edge microfluidic innovation in the lab to a commercial, automated system for isolating CTCs directly from whole blood. As the technology evolved into a commercial system, flexible polydimethylsiloxane microfluidic chips were replaced by rigid poly(methyl methacrylate) chips for a 2.2-fold increase in cell recovery. Automating the fluidic processing with the VTX-1 further improved cancer cell recovery by nearly 1.4-fold, with a 2.8-fold decrease in contaminating white blood cells and overall improved reproducibility. Two isolation protocols were optimized that favor either the cancer cell recovery (up to 71.6% recovery) or sample purity (≤100 white blood cells/mL). The VTX-1's performance was further tested with three different spiked breast or lung cancer cell lines, with 69.0% to 79.5% cell recovery. Finally, several cancer research applications are presented using the commercial VTX-1 system.

Depletion of recombination-specific cofactors by the C-terminal mutant of the activation-induced cytidine deaminase causes the dominant negative effect on class switch recombination.

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes. Studies on in vitro mutagenized AID as well as its mutations in human patients with hyper-IgM (HIGM)-syndrome type II revealed that C-terminal AID mutations were defective in CSR whereas their DNA cleavage and SHM activities remained intact. The C-terminal mutants of AID were speculated to exert the dominant negative effect on wild-type (WT) AID whereas its mechanism remains unknown. We generated the JP41 (R190X) mutation in one allele and a null mutation on the other allele in a mouse B cell line (CH12F3-2A) using CRISPR/Cas9 genome-editing tools and studied the effect of JP41 expression on the function of exogenously introduced WT AID fused with estrogen receptor (AIDER) in AIDJP41/∆/AIDER CH12F3-2A cells. We found that JP41 expression strongly suppressed not only CSR but also Igh/c-Myc chromosomal translocations by AIDER. We showed that the dominant negative effect is not evident at the DNA cleavage step but obvious at both deletional and inversional recombination steps. We also confirmed the dominant negative effect of other C-terminal mutants, JP8Bdel (R183X) and P20 (34-aa insertion at residue 182) in AID-deficient spleen B cells. Finally, we showed that the expression of JP41 reduced the binding of AIDER with its cofactors (hnRNP L, SERBP1 and hnRNP U). Together, these data indicate that dominant negative effect of JP41 on CSR is likely due to the depletion of the CSR-specific RNA-binding proteins from WT AID.

Transdermal Scopolamine Withdrawal Syndrome Case Report in the Pediatric Cerebral Palsy Population.

Sialorrhea in children with cerebral palsy (CP) results in aspiration, decreased social integration, and poor quality of life. Management options include transdermal anticholinergics such as the scopolamine patch. A controlled clinical trial has proven botulinum toxin (BTX) injections into the salivary glands are an effective alternative to transdermal anticholinergics with a safer side effect profile. Multiple studies of the injections in diverse populations demonstrate reduction in saliva production with improvement in quality of life and decrease in hospitalization-associated costs. The authors describe a 15-year-old boy with spastic quadriplegic CP who developed emesis, nausea, and lethargy 1 day after the first injection of botulinum toxin A (BTX-A) to his salivary glands for sialorrhea management. The authors ascribed his symptoms to scopolamine withdrawal. Given the lack of exposure in the medical literature, there is minimal awareness of the withdrawal syndrome from transdermal scopolamine in children with or without CP, resulting in delayed diagnosis and potential complications. Treatment of the withdrawal syndrome has been successful with meclizine though safety and efficacy has not been established in children younger than 12 despite frequent clinical and over-the-counter use. Prompt diagnosis of the transdermal scopolamine withdrawal syndrome can result in quicker treatment and a shorter hospital stay.

Tomato Plant Proteins Actively Responding to Fungal Applications and Their Role in Cell Physiology.

The pattern of protein induction in tomato plants has been investigated after the applications of pathogenic and non-pathogenic fungal species. Moreover, particular roles of the most active protein against biological applications were also determined using chromatographic techniques. Alternaria alternata and Penicillium oxalicum were applied as a pathogenic and non-pathogenic fungal species, respectively. Protein profile analysis revealed that a five protein species (i.e., protein 1, 6, 10, 12, and 13) possessed completely coupled interaction with non-pathogenic inducer application (P. oxalicum). However, three protein species (i.e., 10, 12, and 14) recorded a strong positive interaction with both fungal species. Protein 14 exhibited the maximum interaction with fungal applications, and its role in plant metabolism was studied after its identification as protein Q9M1W6. It was determined that protein Q1M1W6 was involved in guaiacyl lignin biosynthesis, and its inhibition increased the coumarin contents in tomato plants. Moreover, it was also observed that the protein Q9M1W6 takes significant part in the biosynthesis of jasmonic acid and Indole acetic acid contents, which are defense and growth factors of tomato plants. The study will help investigators to design fundamental rules of plant proteins affecting cell physiology under the influence of external fungal applications.

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.