AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

Background: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results: We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer

Correlation of virulence genes to clinical manifestations and outcome in patients with Streptococcus dysgalactiae subspecies equisimilis bacteremia

Background/PurposeStreptococcus dysgalactiae subsp. equisimilis (SDSE) is increasingly recognized as a human pathogen responsible for invasive infection and streptococcal toxic shock syndrome (STSS). The pathogen possesses virulence genes that resemble those found in Streptococcus pyogenes (GAS). We analyzed the association between these specific toxic genes, clinical presentations, and outcome in patients with SDSE infections.MethodsPatients (older than 18 years) with community-acquired invasive bacteremia caused by SDSE bacteremia who were undergoing treatment at China Medical University Hospital from June 2007 to December 2010 were included in this study. Multiplex polymerase chain reaction was performed to identify virulence genes of the SDSE isolates. Demographic data, clinical presentations, and outcome in patients with SDSE infections were reviewed and analyzed.ResultsForty patients with 41 episodes of SDSE bacteremia were reviewed. The median age of the patients with SDSE infection was 69.7 years; 55% were female and 78% had underlying diseases. Malignancy (13, 33%) and diabetes mellitus (13, 33%) were the most common comorbidities. The 30-day mortality rate was 12%. Compared with the survivors, the non-survivors had a higher rate of diabetes mellitus (80% vs. 26%), liver cirrhosis (60% vs.11%), shock (60% vs.17%), STSS (60% vs. 8%), and a high Pittsburgh bacteremia score >4 (40% vs. 6%). Most isolates had scpA, ska, saga, and slo genes, whereas speC, speG, speH, speI, speK, smez, and ssa genes were not detected. speA gene was identified only in one patient with STSS (1/6, 17%). All isolates were susceptible to penicillin, cefotaxime, levofloxacin, moxifloxacin, vancomycin, and linezolid.ConclusionIn invasive SDSE infections, most isolates carry putative virulence genes, such as scpA, ska, saga, and slo. Clinical SDSE isolates in Taiwan remain susceptible to penicillin cefotaxime, and levofloxacin

Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model

Whole pelvic helical tomotherapy for locally advanced cervical cancer: technical implementation of IMRT with helical tomothearapy

<p>Abstract</p> <p>Background</p> <p>To review the experience and to evaluate the treatment plan of using helical tomotherapy (HT) for the treatment of cervical cancer.</p> <p>Methods</p> <p>Between November 1st, 2006 and May 31, 2009, 10 cervical cancer patients histologically confirmed were enrolled. All of the patients received definitive concurrent chemoradiation (CCRT) with whole pelvic HT (WPHT) followed by brachytherapy. During WPHT, all patients were treated with cisplatin, 40 mg/m²intravenously weekly. Toxicity of treatment was scored according to the Common Terminology Criteria for Adverse Events v3.0 (CTCAE v3.0).</p> <p>Results</p> <p>The mean survival was 25 months (range, 3 to 27 months). The actuarial overall survival, disease-free survival, locoregional control and distant metastasis-free rates at 2 years were 67%, 77%, 90% and 88%, respectively. The average of uniformity index and conformal index was 1.06 and 1.19, respectively. One grade 3 of acute toxicity for diarrhea, thrombocytopenia and three grade 3 leucopenia were noted during CCRT. Only one grade 3 of subacute toxicity for thrombocytopenia was noted. There were no grade 3 or 4 subacute toxicities of anemia, leucopenia, genitourinary or gastrointestinal effects. Compared with conventional whole pelvic radiation therapy (WPRT), WPHT decreases the mean dose to rectum, bladder and intestines successfully.</p> <p>Conclusion</p> <p>HT provides feasible clinical outcomes in locally advanced cervical cancer patients. Long-term follow-up and enroll more locally advanced cervical carcinoma patients by limiting bone marrow radiation dose with WPHT technique is warranted.</p

Association of combination antiretroviral therapy with risk of neurological diseases in patients with HIV/AIDS in Taiwan: a nested case-control study

Heterogeneous neurocognitive impairment remains an important issue, even in the era of combination antiretroviral therapy (cART), with an incidence ranging from 15% to 65%. Although ART drugs with higher penetration scores to the central nervous system (CNS) show better HIV replication control in the CNS, the association between CNS penetration effectiveness (CPE) scores and neurocognitive impairment remains inconclusive. To explore whether ART exposure is associated with the risk of neurological diseases among patients with HIV/AIDS, this study in Taiwan involved 2,571 patients with neurological diseases and 10,284 matched, randomly selected patients without neurological diseases between 2010 and 2017. A conditional logistic regression model was used in this study. The parameters for ART exposure included ART usage, timing of exposure, cumulative defined daily dose (DDD), adherence, and cumulative CPE score. Incident cases of neurological diseases, including CNS infections, cognitive disorders, vasculopathy, and peripheral neuropathy, were obtained from the National Health Insurance Research Database in Taiwan. Odds ratios (ORs) for the risk of neurological diseases were conducted using a multivariate conditional logistic regression model. Patients with a history of past exposure (OR: 1.68, 95% confidence interval [CI]:1.22–2.32), low cumulative DDDs (&lt; 2,500) (OR: 1.28, 95% CI: 1.15–1.42), low adherence (0 &lt; adherence (ADH) ≤ 0.8) (OR: 1.46, 95% CI: 1.30–1.64), or high cumulative CPE scores (&gt;14) (OR: 1.34, 95% CI: 1.14–1.57) had a high risk of neurological diseases. When stratified by classes of ART drugs, patients with low cumulative DDDs or low adherence had a high risk of neurological diseases, including NRTIs, PIs, NNRTIs, INSTIs, and multi-drug tablets. Subgroup analyses also suggested that patients with low cumulative DDDs or low adherence had a high risk of neurological diseases when they had high cumulative CPE scores. Patients with high cumulative DDDs or medication adherence were protected against neurological diseases only when they had low cumulative CPE scores (≤ 14). Patients may be at risk for neurological diseases when they have low cumulative DDDs, low adherence, or usage with high cumulative CPE scores. Continuous usage and low cumulative CPE scores of ART drugs may benefit neurocognitive health in patients with HIV/AIDS