Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries

Background Antibody fragments can be expressed in Escherichia coli, where they are commonly directed to the periplasm via Sec pathway to enable disulphide bridge formations and correct folding. In order to transport antibody fragments to the periplasmic space via Sec pathway, they are equipped with N-terminal signal sequence. Periplasmic expression has many benefits but it's also subjected to many hurdles like inefficient translocation across the inner membrane and insufficient capacity of the translocation system. One solution to overcome these hurdles is a modulation of codon usage of signal sequence which has proved to be an efficient way of tuning the translocation process. Modulation of codon usage of signal sequences has been successfully employed also in improving the expression levels of antibody fragments, but unfortunately the effect of codon usage on the expression has not been thoroughly analyzed. Results In the present study we established three synonymous PelB signal sequence libraries by modulating codon usage of light chain and heavy chain PelB signal sequences of a Fab fragment. Each region (n-region, hydrophobic region and c-region) of the PelB signal sequence in the both chains of the Fab fragment in a bicistronic expression vector was mutated separately. We then screened for clones with improved expression profile. The best source for improved clones was the n-region library but in general, improved clones were obtained from all of the three libraries. After screening, we analyzed the effects of codon usage and mRNA secondary structures of chosen clones on the expression levels of the Fab fragment. When it comes to codon usage based factors, it was discovered that especially codon usage of fifth leucine position of the light chain PelB affects the expression levels of Fab fragment. In addition, we observed that mRNA secondary structures in the translation initiation regions of the light and heavy chain have an effect on expression levels as well. Conclusions In conclusion, the established synonymous signal sequence libraries are good sources for discovering Fab fragments with improved expression profile and obtaining new codon usage related information

Improvements in Hearing and in Quality of Life after Sequential Bilateral Cochlear Implantation in a Consecutive Sample of Adult Patients with Severe-to-Profound Hearing Loss

Bilateral cochlear implantation is increasing worldwide. In adults, bilateral cochlear implants (BICI) are often performed sequentially with a time delay between the first (CI1) and the second (CI2) implant. The benefits of BICI have been reported for well over a decade. This study aimed at investigating these benefits for a consecutive sample of adult patients. Improvements in speech-in-noise recognition after CI2 were followed up longitudinally for 12 months with the internationally comparable Finnish matrix sentence test. The test scores were statistically significantly better for BICI than for either CI alone in all assessments during the 12-month period. At the end of the follow-up period, the bilateral benefit for co-located speech and noise was 1.4 dB over CI1 and 1.7 dB over CI2, and when the noise was moved from the front to 90 degrees on the side, spatial release from masking amounted to an improvement of 2.5 dB in signal-to-noise ratio. To assess subjective improvements in hearing and in quality of life, two questionnaires were used. Both questionnaires revealed statistically significant improvements due to CI2 and BICI. The association between speech recognition in noise and background factors (duration of hearing loss/deafness, time between implants) or subjective improvements was markedly smaller than what has been previously reported on sequential BICI in adults. Despite the relatively heterogeneous sample, BICI improved hearing and quality of life.Peer reviewe

Transcriptomics and Functional Genomics of ROS-Induced Cell Death Regulation by RADICAL-INDUCED CELL DEATH1

Peer reviewe

Humanization, Radiolabeling and Biodistribution Studies of an IgG(1)-Type Antibody Targeting Uncomplexed PSA for Theranostic Applications

Metastatic castration-resistant prostate cancer is today incurable. Conventional imaging methods have limited detection, affecting their ability to give an accurate outcome prognosis, and current therapies for metastatic prostate cancer are insufficient. This inevitably leads to patients relapsing with castration-resistant prostate cancer. Targeting prostate-specific antigens whose expression is closely linked to the activity in the androgen receptor pathway, and thus the pathogenesis of prostate cancer, is a possible way to increase specificity and reduce off-target effects. We have humanized and evaluated radioimmunoconjugates of a previously murine antibody, m5A10, targeting PSA intended for theranostics of hormone-refractory prostate cancer. The humanized antibody h5A10 was expressed in mammalian HEK293 cells transfected with the nucleotide sequences for the heavy and light chains of the antibody. Cell culture medium was filtered and purified by Protein G chromatography, and the buffer was changed to PBS pH 7.4 by dialysis. Murine and humanized 5A10 were conjugated with p-SCN-Bn-CHX-A"-DTPA. Surface plasmon resonance was used to characterize the binding to PSA of the immunoconjugates. Immunoconjugates were labeled with either indium-111 or lutetium-177. Biodistribution studies of murine and humanized 5A10 were performed in mice with LNCaP xenografts. 5A10 was successfully humanized, and in vivo targeting showed specific binding in xenografts. The results thus give an excellent platform for further theranostic development of humanized 5A10 for clinical applications

Improvements in Hearing and in Quality of Life after Sequential Bilateral Cochlear Implantation in a Consecutive Sample of Adult Patients with Severe-to-Profound Hearing Loss

Bilateral cochlear implantation is increasing worldwide. In adults, bilateral cochlear implants (BICI) are often performed sequentially with a time delay between the first (CI1) and the second (CI2) implant. The benefits of BICI have been reported for well over a decade. This study aimed at investigating these benefits for a consecutive sample of adult patients. Improvements in speech-in-noise recognition after CI2 were followed up longitudinally for 12 months with the internationally comparable Finnish matrix sentence test. The test scores were statistically significantly better for BICI than for either CI alone in all assessments during the 12-month period. At the end of the follow-up period, the bilateral benefit for co-located speech and noise was 1.4 dB over CI1 and 1.7 dB over CI2, and when the noise was moved from the front to 90 degrees on the side, spatial release from masking amounted to an improvement of 2.5 dB in signal-to-noise ratio. To assess subjective improvements in hearing and in quality of life, two questionnaires were used. Both questionnaires revealed statistically significant improvements due to CI2 and BICI. The association between speech recognition in noise and background factors (duration of hearing loss/deafness, time between implants) or subjective improvements was markedly smaller than what has been previously reported on sequential BICI in adults. Despite the relatively heterogeneous sample, BICI improved hearing and quality of life

Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert

BACKGROUND: Plants growing in their natural habitat represent a valuable resource for elucidating mechanisms of acclimation to environmental constraints. Populus euphratica is a salt-tolerant tree species growing in saline semi-arid areas. To identify genes involved in abiotic stress responses under natural conditions we constructed several normalized and subtracted cDNA libraries from control, stress-exposed and desert-grown P. euphratica trees. In addition, we identified several metabolites in desert-grown P. euphratica trees. RESULTS: About 14,000 expressed sequence tag (EST) sequences were obtained with a good representation of genes putatively involved in resistance and tolerance to salt and other abiotic stresses. A P. euphratica DNA microarray with a uni-gene set of ESTs representing approximately 6,340 different genes was constructed. The microarray was used to study gene expression in adult P. euphratica trees growing in the desert canyon of Ein Avdat in Israel. In parallel, 22 selected metabolites were profiled in the same trees. CONCLUSION: Of the obtained ESTs, 98% were found in the sequenced P. trichocarpa genome and 74% in other Populus EST collections. This implies that the P. euphratica genome does not contain different genes per se, but that regulation of gene expression might be different and that P. euphratica expresses a different set of genes that contribute to adaptation to saline growth conditions. Also, all of the five measured amino acids show increased levels in trees growing in the more saline soil

Criterios de Aplicación de las Medidas Sustitutivas a la Pena de Prisión Acorde al Cumplimiento de los Fines de la Pena.

Los Fines de la Pena y Principios Aplicables en la Legislación Salvadoreña – Medida sustitutiva a la Pena de Prisión – Criterios utilizados para aplicar el reemplazo o la suspensión de la pena – Atribuciones de las Instituciones encargadas de la Ejecución de las Medidas Sustitutivas de la Pena de Prisión

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material

Health-Related Quality of Life in Metastatic Colorectal Cancer Patients Treated with Curative Resection and/or Local Ablative Therapy or Systemic Therapy in the Finnish RAXO-Study

Simple Summary Metastatic colorectal cancer is the second most common cause of cancer death. Long-term survival and cure can be achieved after intensive treatments, including metastasectomy, i.e., the removal of all metastases. We wanted to clarify whether a patient health-related quality of life (HRQoL) was reduced by treatments that aimed to maximise metastasectomy rates, and whether HRQoL of treated patients is comparable to the general population. In a cross-sectional study of 444 patients (1751 questionnaires) in the RAXO-study population, we show that HRQoL of intensively treated patients, sometimes with multiple and multisite metastasectomies-usually combined with systemic therapy-remains at a high level during and after curative treatment and when compared with the general population. Good HRQoL was also seen during non-curative treatment from first- to later-lines, with an impaired HRQoL only at end-of-life. Thus, we should aim at maximising metastasectomies since they give long-term survival and sometimes cure with a high HRQoL. Metastasectomy and/or local ablative therapy in metastatic colorectal cancer (mCRC) patients often provide long-term survival. Health-related quality of life (HRQoL) data in curatively treated mCRC are limited. In the RAXO-study that evaluated repeated resectability, a multi-cross-sectional HRQoL substudy with 15D, EQ-5D-3L, QLQ-C30, and QLQ-CR29 questionnaires was conducted. Mean values of patients in different treatment groups were compared with age- and gender-standardized general Finnish populations. The questionnaire completion rate was 444/477 patients (93%, 1751 questionnaires). Mean HRQoL was 0.89-0.91 with the 15D, 0.85-0.87 with the EQ-5D, 68-80 with the EQ-5D-VAS, and 68-79 for global health status during curative treatment phases, with improvements in the remission phase (disease-free >18 months). In the remission phase, mean EQ-5D and 15D scores were similar to the general population. HRQoL remained stable during first- to later-line treatments, when the aim was no longer cure, and declined notably when tumour-controlling therapy was no longer meaningful. The symptom burden affecting mCRC survivors' well-being included insomnia, impotence, urinary frequency, and fatigue. Symptom burden was lower after treatment and slightly higher, though stable, through all phases of systemic therapy. HRQoL was high in curative treatment phases, further emphasizing the strategy of metastasectomy in mCRC when clinically meaningful.</p

Resectability, conversion, metastasectomy and outcome according to RAS and BRAF status for metastatic colorectal cancer in the prospective RAXO study

Background Outcomes after metastasectomy for metastatic colorectal cancer (mCRC) vary with RAS and BRAF mutational status, but their effects on resectability and conversion rates have not been extensively studied. Methods This substudy of the prospective RAXO trial included 906 patients recruited between 2011 and 2018. We evaluated repeated centralised resectability assessment, conversion/resection rates and overall survival (OS), according to RAS and BRAF status. Results Patients included 289 with RAS and BRAF wild-type (RAS and BRAFwt), 529 with RAS mutated (RASmt) and 88 with BRAF mutated (BRAFmt) mCRC. Metastatic prevalence varied between the RAS and BRAFwt/RASmt/BRAFmt groups, for liver (78%/74%/61%), lung (24%/35%/28%) and peritoneal (15%/15%/32%) metastases, respectively. Upfront resectability (32%/29%/15%), conversion (16%/13%/7%) and resection/local ablative therapy (LAT) rates (45%/37%/17%) varied for RASa and BRAFwt/RASmt/BRAFmt, respectively. Median OS for patients treated with resection/LAT (n = 342) was 83/69/30 months, with 5-year OS-rates of 67%/60%/24%, while systemic therapy-only patients (n = 564) had OS of 29/21/15 months with 5-year OS-rates of 11%/6%/2% in RAS and BRAFwt/RASmt/BRAFmt, respectively. Resection/LAT was associated with improved OS in all subgroups. Conclusions There were significant differences in resectability, conversion and resection/LAT rates according to RAS and BRAF status. OS was also significantly longer for RAS and BRAFwt versus either mutant. Patients only receiving systemic therapy had poorer long-term survival, with variation according to molecular status.</p