PurposePart of the hidden genetic variation in heterogeneous genetic conditions such as inherited retinal diseases (IRDs) can be explained by copy-number variations (CNVs). Here, we explored the ...genomic landscape of IRD genes listed in RetNet to identify and prioritize those genes susceptible to CNV formation.MethodsRetNet genes underwent an assessment of genomic features and of CNV occurrence in the Database of Genomic Variants and literature. CNVs identified in an IRD cohort were characterized using targeted locus amplification (TLA) on extracted genomic DNA.ResultsExhaustive literature mining revealed 1,345 reported CNVs in 81 different IRD genes. Correlation analysis between rankings of genomic features and CNV occurrence demonstrated the strongest correlation between gene size and CNV occurrence of IRD genes. Moreover, we identified and delineated 30 new CNVs in IRD cases, 13 of which are novel and three of which affect noncoding, putative cis-regulatory regions. Finally, the breakpoints of six complex CNVs were determined using TLA in a hypothesis-neutral manner.ConclusionWe propose a ranking of CNV-prone IRD genes and demonstrate the efficacy of TLA for the characterization of CNVs on extracted DNA. Finally, this IRD-oriented CNV study can serve as a paradigm for other genetically heterogeneous Mendelian diseases with hidden genetic variation.
Our objective was to evaluate EUCAST’s ‘rapid antimicrobial susceptibility testing’ (RAST) directly from positive blood culture that delivers antimicrobial results within 6 h for
Staphylococcus ...aureus
,
Enterococcus
spp.,
Escherichia coli
,
Klebsiella pneumoniae
and
Pseudomonas aeruginosa
, using total lab automation. Zone diameters from RAST were compared with MIC results. Furthermore, its influence on time to report was investigated. RAST was performed to all positive aerobic and anaerobic blood culture bottles by subculturing them, i.e. onto Mueller-Hinton agar and adding six antibiotic discs covering Gram-negative and Gram-positive therapy (cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin). RAST was automatically imaged after 6 h. Zone sizes were measured using a TLA software tool and interpreted according to EUCAST clinical breakpoints. Bacteria were identified using MALDI-TOF MS and MIC results were determined using Vitek2 panels. Categorial agreement between agar diffusion and MIC results was investigated. Additionally, time to RAST and time to Vitek were compared for 100 isolates (20 per species). Between November 2018 and April 2019, 3313 positive mono-bacterial blood culture bottles were collected of which 894 bottles with RAST-validated species were investigated. Among these bottles, 2029 individual antibiotic measurements were compared with MIC results from Vitek2 and 14 very major, 28 major and 12 minor errors were found. A median reduction of 17:30 h in time to report was observed. Introduction of RAST with automatic TLA imaging function could reduce time to report by 17:30 h. Excellent accordance between zone diameter and MIC results, particularly for cefoxitin, vancomycin and meropenem, was observed, but drawbacks due to ATU were seen.
Purpose
Minimally invasive surgery is the gold standard treatment for adrenal masses, but it may be a challenging procedure in the case of pheochromocytoma (PHEO). The aim of the present study is to ...report the results of transperitoneal laparoscopic adrenalectomy (TLA) in cases of PHEO in comparison to other types of adrenal lesions.
Methods
From 1994 to 2021, 629 patients underwent adrenalectomy. Twenty-two and thirty-five patients, respectively, were excluded because they underwent bilateral and open adrenalectomy, leaving 572 patients for inclusion. Of these, 114 patients had PHEO (Group A), and 458 had other types of lesions (Group B). To adjust for potential baseline confounders, a propensity score matching (PSM) analysis was conducted.
Results
After PSM, 114 matched pairs of patients were identified from each group. Statistically significant differences were not observed when comparing the median operative time (85 and 90 min in Groups A and B, respectively,
p
= 0.627), conversion rate 6 (5.3%) in each group,
p
= 1.000, transfusion rate 4 (3.5%) and 3 (2.6%) in Groups A and B, respectively,
p
= 1.000, complication rate 7 (6.1%) and 9 (7.9%) in Groups A and B, respectively,
p
= 0.796), median postoperative hospital stay (3.9 and 3.6 days in Groups A and B, respectively,
p
= 0.110), and mortality rate 1 (0.9%) in each group,
p
= 1.000.
Conclusions
Based on this analysis, the results of TLA for PHEO are equivalent to those of TLA for other types of adrenal lesions, but the fundamental requirements are multidisciplinary patient management and adequate surgeon experience. Further prospective studies are required to draw definitive conclusions.
The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison ...with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant. A complementation study identified the “chloroplast signal recognition particle 54” gene (CpSRP54), as the lesion causing the tla4 phenotype. Disruption of this gene resulted in partial failure to assemble and, therefore, lower levels of light-harvesting Chl-binding proteins in the C. reinhardtii thylakoids. A comparative in silico 3-D structure-modeling analysis revealed that the M-domain of the CpSRP54 of C. reinhardtii possesses a more extended finger loop structure, due to different amino acid composition, as compared to that of the Arabidopsis CpSRP54. The work demonstrated that CpSRP54 deletion in microalgae can serve to generate tla mutants with a markedly smaller photosystem Chl antenna size, improved solar energy conversion efficiency, and photosynthetic productivity in high-density cultures under bright sunlight conditions.
•The tla4 mutant of Chlamydomonas reinhardtii possesses a truncated light-harvesting antenna.•Disruption of the signal recognition particle 54 (CpSRP54) gene caused this phenotype.•3D modeling showed a difference in the Chlamydomonas and Arabidopsis CpSRP54 proteins.
The MRP8-Cre-ires/EGFP transgenic mouse (Mrp8cre
, on C57BL/6J genetic background) is popular in immunological and hematological research for specifically expressing Cre recombinase and an EGFP ...reporter in neutrophils. It is often crossed with other transgenic lines carrying
-flanked genes to achieve restricted gene knockout in neutrophils. However, due to the way in which the line was created, basic knowledge about the MRP8-Cre-ires/EGFP transgene in the host genome, such as its integration site(s) and flanking sequences, remains largely unknown, hampering robust experimental design and data interpretation. Here we used a recently developed technique, targeted locus amplification (TLA) sequencing, to fill these knowledge gaps. We found that the MRP8-Cre-ires/EGFP transgene was integrated into chromosome 5 (5qG2) of the host mouse genome. This integration led to a 44 kb deletion of the host genomic sequence, resulting in complete deletion of
and partial deletion of
. Having determined the flanking sequences of the transgene, we designed a new genotyping protocol that can distinguish homozygous, heterozygous, and wildtype Mrp8cre
mice. To our surprise, crossing heterozygous mice produced no homozygous Mrp8cre
mice, most likely due to prenatal lethality resulting from disrupted
gene expression.
Introduction
The high‐sequence homology of the α‐globin‐gene cluster is responsible for microhomology‐mediated recombination events during meiosis, resulting in a high density of deletion breakpoints ...within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation‐dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long‐range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site.
Methods
Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment‐specific inverse primers, creating a library that is suitable for Illumina sequencing.
Results
Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results.
Conclusions
The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha‐globin cluster. The deletion was described only once in an earlier study as the ‐‐gb, but as it was not registered correctly in the available databases, it was not initially recognized as such.
Purpose
The purpose of this study was to evaluate the feasibility and safety of single-incision laparoscopic surgery for totally extraperitoneal inguinal hernia repair (SILS-TEP) with tumescent local ...anesthesia (TLA) at a day-surgery clinic.
Methods
We analyzed, retrospectively, 2148 patients who underwent SILS-TEP under general anesthesia with TLA between April, 2015 and March, 2020 at Gi surgical clinic, to evaluate their operative outcomes. The TLA agent, consisting of normal saline and lidocaine with epinephrine and ropivacaine, was injected during surgery.
Results
The median operative times for unilateral and bilateral hernia were 50 min and 75 min, respectively. Blood loss was minimal in all patients. Conversion to the Lichtenstein method was required in 4% (91/2148) of patients. The median recovery room stay was 125 min and no analgesics were required in the recovery room by 75% (1613/2148) of the patients. All the patients left the clinic on the day of surgery. Complications developed in 6.5% (139/2148) of the patients, as seromas in 6% (125/2148), wound infections in 0.4% (8/2148), and hematomas in 0.2% (4/2148), respectively. Bowel injury and obstruction each occurred in 0.05% (1/2148) of the patients. There were no hernia recurrences.
Conclusion
SILS-TEP with TLA can be performed safely at a day-surgery clinic.
Purpose
Breast surgery is usually performed under general anesthesia. Tumescent local anesthesia (TLA) offers the possibility to anesthetize large areas with highly diluted local anesthetic.
Methods
...In this paper, the implementation, and experiences with TLA in the field of breast surgery are discussed.
Conclusion
For carefully selected indications, breast surgery in TLA represents an alternative to ITN.
Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) T cell therapy, little information has been made ...available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αβT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo.
Straetemans and colleagues characterized αβT cells engineered to express a defined γδT cell receptor (TEG001) used in a first-in-human clinical study, with TLA in combination with NGS and qPCR. They provide a rapid strategy to evaluate the presence, integrity, and persistence of genetic information along the production chain of any GTMP.
Optimisation of microbiological diagnostics in primarily sterile body fluids is required. Our objective was to apply EUCAST’s RAST on primarily sterile body fluids in blood culture bottles with total ...lab automation (TLA) and to compare results to our reference method Vitek2 in order to report susceptibility results earlier. Positive blood culture bottles (BACTEC™ Aerobic/Anaerobic/PEDS) inoculated with primarily sterile body fluids were semi-automatically subcultured onto Columbia 5% SB agar, chocolate agar, MacConkey agar, Schaedler/KV agar and Mueller-Hinton agar. On latter, cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin were added. After 6 h, subcultures and RAST were imaged and MALDI-TOF MS was performed. Zone sizes were digitally measured and interpreted following RAST breakpoints for blood cultures. MIC values were determined using Vitek2 panels. During a 1-year period, 197
Staphylococcus aureus
, 91
Enterococcus
spp., 38
Escherichia coli
, 11
Klebsiella pneumoniae
and 8
Pseudomonas aeruginosa
were found. Categorical agreement between RAST and MIC was 96.5%. Comparison showed no very major errors, 2/7 (28.6%) and 1/7 (14.3%) of major errors for
P. aeruginosa
and meropenem and ciprofloxacin, 1/9 (11.1%) for
K. pneumoniae
and ciprofloxacin, 4/69 (7.0%) and 3/43 (5.8%) for
Enterococcus
spp. and vancomycin and ampicillin, respectively. Minor errors for
P. aeruginosa
and meropenem (1/8; 12.8%) and for
E. coli
and ciprofloxacin (2/29; 6.5%) were found. 30/550 RAST measurements were within area of technical uncertainty. RAST is applicable and performs well for primarily sterile body fluids in blood culture bottles, partially better than blood-based RAST. Official EUCAST evaluation is needed.
PDF
