Unusual Removal of T-DNA in T1 Progenies of Rice after Agrobacterium-mediated CRISPR/Cas9 Editing

In the present study, we attempted to knock out the bar gene selection marker in the fixed Bt- and herbicide-resistant transgenic line BT-T07 (T8 generation) to generate a marker-free Bt-resistant rice line. A binary vector containing a CRISPR/Cas9 system targeting the 108–130 bp region of bar was transformed into rice embryonic calli, and plantlets were regenerated under non-selective conditions. Three T0 plants were observed to have non-target point mutations and deletions in the targeted gene and were putatively heterozygous and chimeras. One T0 plant, 130-4, was confirmed to have a 76-nt deletion, from 140 bp to 225 bp, and it showed the segregation of bar in its T1 progenies, with 16 bar-knockout lines and seven normal bar-expressing lines. However, the CRISPR/Cas9 editing vector sequences were not detected in any of the T1 plants. In addition, unusual removal of pre-existing T-DNA was observed in all bar-knockout T1 plants. Illumina sequencing of a bar-knockout line, 130-4-36, revealed a small fraction of read residues of pre-existing T-DNA from the bar sequence to the right border at the original junction site. We speculate that this rare phenomenon might be directed by the homology between pre-existing T-DNA and CRISPR/Cas9 editing vector sequences during meiotic recombination. We report imprecise modifications and unpredictable outcomes of gene-editing techniques, providing valuable perspectives on gene-editing applications.

Importance of the Duodenal Window and Fredet’s Fascia in Laparoscopic Right Hemicolectomy: Technical Note

Background: Laparoscopic right hemicolectomy requires a precise anatomical dissection to mobilise the right and proximal transverse mesocolon, following the avascular fusion planes of Toldt and Fredet. Fredet’s plane is crucial to the preparation of the origin of vessels. Easy access to Fredet’s and Toldt’s fasciae can be obtained through the “duodenal window”, a flimsy area of the root of the proximal transverse mesocolon, the margins of which are the right border of the superior mesenteric pedicle, the ileocolic pedicle, the right colic pedicle and the marginal artery. Method: We propose that dissection of the duodenal window should be the first step in laparoscopic right hemicolectomy, to obtain easy access to the duodenopancreatic plane and prepare the fascia. Results: This “duodenal window-first” technique has been applied in 45 laparoscopic right hemicolectomies and 14 laparoscopic extended right hemicolectomies, with only two conversions to open surgery. The duodenal window was easily identified in all but 3 cases with significant visceral obesity. No significant intra- or postoperative morbidity was recorded in these cases and the median postoperative length of stay was 4 days. All resections were R0 and an adequate number of retrieved lymph nodes were obtained in almost all cases. Conclusion: The duodenal window-first approach is a feasible and safe technique to standardise the first steps of radical laparoscopic right hemicolectomy, allowing prompt and complete anatomical identification and dissection.

The segment IV approach: a useful method for achieving the critical view of safety during laparoscopic cholecystectomy in patients with anomalous bile duct

Background The critical view of safety (CVS) method can be achieved by avoiding vasculo-biliary injury resulting from misidentification during laparoscopic cholecystectomy (LC). Although achieving the CVS has become popular worldwide, there is no established standardized technique to achieve the CVS in patients with an anomalous bile duct (ABD). We recently reported our original approach for securing the CVS using a new landmark, the diagonal line of the segment IV of the liver (D-line). The D-line is an imaginary line that lies on the right border of the hilar plate. The cystic structure can be securely isolated along the D-line without any misidentification, regardless of the existence of an ABD. We named this approach the segment IV approach in LC. Methods In this study, we adopted the segment IV approach in patients with an ABD. Results From October 2015 to June 2020, 209 patients underwent LC using the segment IV approach. Among them, three (1.4%) were preoperatively diagnosed with an ABD. The branching point of the cystic duct was the posterior sectional duct, anterior sectional duct, or left hepatic duct in each patient. The CVS was achieved in all cases without any complications. Conclusion It is a promising technique, especially even for patients with an ABD during LC.

The segment IV approach: a useful method for achieving the critical view of safety during laparoscopic cholecystectomy in patients with anomalous bile duct

Background The critical view of safety (CVS) method can be achieved by avoiding vasculo-biliary injury resulting from misidentification during laparoscopic cholecystectomy (LC). Although achieving the CVS has become popular worldwide, there is no established standardized technique to achieve the CVS in patients with an anomalous bile duct (ABD). We recently reported our original approach for securing the CVS using a new landmark, the diagonal line of the segment IV of the liver (D-line). The D-line is an imaginary line that lies on the right border of the hilar plate. The cystic structure can be securely isolated along the D-line without any misidentification, regardless of the existence of an ABD. We named this approach the segment IV approach in LC. Methods In this study, we adopted the segment IV approach in patients with an ABD. Results From October 2015 to June 2020, 209 patients underwent LC using the segment IV approach. Among them, three (1.4%) were preoperatively diagnosed with an ABD.The branching point of the cystic duct was the posterior sectional duct, anterior sectional duct, or left hepatic duct in each patient. The CVS was achieved in all cases without any complications. Conclusion It is a promising technique, especially even for patients with an ABD during LC.

The Segment IV Approach: A Useful Method for Achieving the Critical View of Safety During Laparoscopic Cholecystectomy in Patients With Anomalous Bile Duct

Background The critical view of safety (CVS) method can be achieved by avoiding vasculo-biliary injury resulting from misidentification during laparoscopic cholecystectomy (LC). Although achieving the CVS has become popular worldwide, there is no established standardized technique to achieve the CVS in patients with an anomalous bile duct (ABD). We recently reported our original approach for securing the CVS using a new landmark, the diagonal line of the segment IV of the liver (D-line). The D-line is an imaginary line that lies on the right border of the hilar plate. The cystic structure can be securely isolated along the D-line without any misidentification, regardless of the existence of an ABD. We named this approach the segment IV approach in LC. Methods In this study, we adopted the segment IV approach in patients with an ABD. From October 2015 to June 2020, 209 patients underwent LC using the segment IV approach. Among them, three (1.4%) were preoperatively diagnosed with an ABD. Results The branching point of the cystic duct was the posterior sectional duct, anterior sectional duct, or left hepatic duct in each patient. The CVS was achieved in all cases without any complications. Conclusion The segment IV approach is useful for achieving CVS, especially even for patients with an ABD during LC.

Genetic structure, function and evolution of capsule biosynthesis loci in Vibrio parahaemolyticus

Capsule-forming extracellular polysaccharides are crucial to bacterial host colonization, invasion, immune evasion and ultimately pathogenicity. Due to warming ocean waters and human encroachment of coastal ecosystems, Vibrio parahaemolyticus has emerged as a globally important food-borne enteropathogen implicated in acute gastroenteritis, wound infections, and septic shock. Conventionally, the antigenic properties of lipopolysaccharide (LPS, O antigen) and capsular polysaccharide (CPS, K antigen) have provided a basis for serotyping V. parahaemolyticus, while disclosure of genetic elements encoding 13 O-serogroups have allowed molecular serotyping methods to be developed. However, the genetic structure of CPS loci for 71 K-serogroups has remained unidentified, limiting progress in understanding its roles in V. parahaemolyticus pathophysiology. In this study, we identified and characterized the genetic structure and their evolutionary relationship of CPS loci of 40 K-serogroups through whole genome sequencing of 443 V. parahaemolyticus strains. We found a distinct pattern of CPS gene cluster across different K-serogroups, and expanded its new right-border by identifying glpX as a key gene conserved across all serotypes. A total of 217 genes involved in CPS biosynthesis were annotated. Functional contents and genetic structure of the 40 K-serogroups were analyzed. Based on inferences from species trees and gene trees, we proposed an evolution model of the CPS gene clusters of 40 K-serogroups. Horizontal gene transfer by recombination from other Vibrio species, gene duplication and nonsense mutations are likely to play instrumental roles in the evolution of CPS in V. parahaemolyticus. It is the first time, to the best of our knowledge, that a large-scale of CPS gene clusters of different K-serogroups in V. parahaemolyticus have been identified and characterized in evolutionary contexts. This work should help advance understanding on the variation of CPS in V. parahaemolyticus, and provide a framework for developing diagnostically relevant serotyping methods.Author summaryDue to warming ocean waters and human encroachment of coastal ecosystems, Vibrio parahaemolyticus has emerged as a globally important food-borne enteropathogen. However, the genetic structure of CPS loci for 71 K-serogroups V. parahaemolyticus have remained unidentified, limiting progress in understanding its roles in V. parahaemolyticus pathophysiology. In this study, we identified and characterized the genetic structure of CPS loci of 40 K-serogroups through whole genome sequencing of 443 V. parahaemolyticus strains. We expanded and identified its new right-border by identifying glpX as a key gene conserved across all serotypes. We proposed an evolution model of the CPS gene clusters of 40 K-serogroups. We also found horizontal gene transfer by recombination from other Vibrio species, gene duplication and nonsense mutations are likely to play instrumental roles in the evolution of CPS in V. parahaemolyticus. It is the first time, to the best of our knowledge, that a large-scale of CPS loci of different K-serogroups in V. parahaemolyticus have been identified and characterized in evolutionary contexts. This work should help advance understanding on the variation of CPS in V. parahaemolyticus, and provide a framework for developing diagnostically relevant serotyping methods.

Parametric Synthesis of a Robust Controller Based on the Method of Dominant Poles

In the paper a linear control system described by its characteristic polynomial with interval coefficients including parameters of controller linearly is considered. Problem of the research is finding parameters of a controller guaranteeing dynamic characteristics of a system despite interval parametric uncertainty of its object. It is proposed to base a controller synthesis on root quality indices: minimal stability degree and maximal oscillability degree. Desired values of these indices will be provided with the help of dominant poles method. Applying this method consists in placing a pair of complex-conjugate dominant poles; all other poles — unrestricted poles — will be placed by defining a right border of their allocation area on a complex plane. To apply dominant poles method, a feature of stability degree and oscillability degree to be determined by images of certain vertices of a parametric polytope was used. To synthesize a controller, it is proposed to divide its parameters in two groups: dependent ones and unrestricted ones. The first group of controller parameters is to provide desired allocation of dominant poles in one of vertices of parametric polytope (a dominant vertex). Unrestricted parameters of a controller are to provide desired distance between dominant poles and allocation area of unrestricted poles. To find coordinates of a dominant vertex and verifying vertices providing unrestricted poles allocation, an interval extension of basic phase equation of a root locus theory was developed. This resulted in interval phase inequalities, whose solution allows finding coordinates of desired vertices of characteristic polynomials coeffi cients polytope. Knowing a dominant vertex polynomial and dominant poles allows expressing dependent parameters of a controller from unrestricted ones. Obtained expressions allow placing unrestricted poles in a desired area of a complex plane. To do this, a D-partition by unrestricted parameters of a controller is performed in all verifying vertices of parametric polytope of a system. After choosing values of unrestricted parameters from intersection of all stability domains obtain during D-partition, dependent parameters of a controller can be calculated. An example of synthesizing a PID-controller guaranteeing desired values of dynamics characteristics for an interval control system of the fourth order is provided.

Identification of a genetic element required for spore killing in Neurospora

ABSTRACTMeiotic drive elements like Spore killer-2 (Sk-2) in Neurospora are transmitted through sexual reproduction to the next generation in a biased manner. Sk-2 achieves this biased transmission through spore killing. Here, we identify rfk-1 as a gene required for the spore killing mechanism. The rfk-1 gene is associated with a 1,481 bp DNA interval (called AH36) near the right border of the 30 cM Sk-2 element, and its deletion eliminates the ability of Sk-2 to kill spores. The rfk-1 gene also appears to be sufficient for spore killing because its insertion into a non-Sk-2 isolate disrupts sexual reproduction after the initiation of meiosis. Although the complete rfk-1 transcript has yet to be defined, our data indicate that rfk-1 encodes a protein of at least 39 amino acids and that rfk-1 has evolved from a partial duplication of gene ncu07086. We also present evidence that rfk-1’s location near the right border of Sk-2 is critical for the success of spore killing. Increasing the distance of rfk-1 from the right border of Sk-2 causes it to be inactivated by a genome defense process called meiotic silencing by unpaired DNA (MSUD), adding to accumulating evidence that MSUD exists, at least in part, to protect genomes from meiotic drive.

Estimation of gestational age using fetal liver morphometry

Background and Aim: The liver is a large organ in the body. Its various parameters can be utilized in determining the gestational age of the fetus and also in diagnosing pathologies affecting liver growth. The aim is to measure the various liver parameters in non deformed fetuses and to develop regression equations between the liver parameters and the gestational age. Materials and methods: The study was done on 30 aborted fetuses with varying gestational age from 13 to 32 weeks. Fetuses with any gross abnormalities were excluded. The crown rump length was measured for all. Dissection was done and the livers were removed weighed, length of right border, length of the border, width of superior surface, distance of porta hepatis to the right and left border were recorded using manual vernier caliper. Result: The various parameters of the liver showed a strong correlation with the CRL and hence the gestational age. Conclusion: The determination of the gestational age of the fetus using the correlation between the crown rump length and the liver morphometry can be done accurately.