Continuation and replacement of Vibrio cholerae non-O1 clonal genomic groups isolated from Plecoglossus altivelis fish in freshwaters.

The dissemination and abundances of Vibrio species in aquatic environments are of interest, as some species cause emerging diseases in humans and in aquatic organisms like fish. It is suggested that Vibrio cholerae non-O1 infections of Plecoglossus altivelis ('ayu') were spread to various parts of Japan through the annual transplantation of juvenile fish. To investigate this, we used genome-aided tracing of 17 V. cholerae strains isolated from ayu between the 1970s and 1990s in different Japanese freshwater systems. The strains formed a genomic clade distinct from all known clades, which we designate as the Ayu clade. Two clonal genomic groups identified within the clade, Ayu-1 and Ayu-2, persisted for a few years (between 1977 to 1979 and 1987 to 1990, respectively), and clonal replacement of Ayu-1 by Ayu-2 took place over an 8-year period. Despite the high similarity between Ayu-1 and Ayu-2 (> 99.9% identity and > 97% fraction of genomes shared), differences in their gene repertoires were found, raising the possibility that they are phenotypically distinct. These results highlight the importance of genome-based studies for understanding the long-term dynamics of populations over the timescale of years.

Genotypic diversity of Streptococcus suis and the S. suis-like bacterium Streptococcus ruminantium in ruminants.

Although Streptococcus suis has attracted public attention as a major swine and human pathogen, this bacterium has also been isolated from other animals, including ruminants. However, recent taxonomic studies revealed the existence of other species that were previously identified as S. suis, and some of these isolates were reclassified as the novel species Streptococcus ruminantium. In Japan, biochemically identified S. suis is frequently isolated from diseased ruminants; however, such isolates have not yet been identified accurately, and their aetiological importance in ruminants is unclear. Therefore, to understand the importance of S. suis and S. suis-like bacteria in ruminants, we reclassified S. suis isolates from ruminants according to the updated classification and investigated their genetic diversity. Although both S. suis and S. ruminantium were isolated from healthy and diseased ruminants, most of the isolates from diseased animals were S. ruminantium, implying that S. ruminantium is more likely to be associated with ruminant disease than S. suis. However, the ruminant S. suis and S. ruminantium isolates from diseased animals were classified into diverse genotypes rather than belonging to certain clonal groups. Genome sequence analysis of 20 S. ruminantium isolates provided information about the antibiotic resistance, potential virulence, and serological diversity of this species. We further developed an S. ruminantium-specific PCR assay to aid in the identification of this bacterium. The information obtained and the method established in this study will contribute to the accurate diagnosis of ruminant streptococcal infections.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

Induction of tumor-specific immune response by gene transfer of Hsp70-cell-penetrating peptide fusion protein to tumors in mice.

To induce a tumor-specific immune response by delivering tumor-associated antigens in tumor cells to antigen-presenting cells (APCs), we designed a fusion protein which consists of heat-shock protein 70 (Hsp70) and the C-terminal 34 amino acids of herpes simplex virus VP22 protein (VP22(268-301)), the former having a peptide binding domain and an ability to be recognized by APCs, and the latter able to achieve cell penetration. Hsp70-VP22(268-301), the fusion protein, was efficiently taken up by mouse dendritic cell (DC) line DC2.4. Major histocompatibility complex (MHC) class I-restricted presentation of an epitope peptide of ovalbumin (OVA) was examined in DC2.4, and Hsp70-VP22(268-301) significantly increased the presentation of the peptide compared with Hsp70. Electroporation-assisted injection of naked plasmid vector expressing Hsp70-VP22(268-301) (pHsp70-VP22(268-301)) into subcutaneous tumors of EG7-OVA, a mouse lymphoma-expressing OVA, significantly increased the survival of mice compared with the same treatment with pHSp70, a plasmid expressing Hsp70. Splenocytes from the pHsp70-VP22(268-301)-treated mice exhibited cytolytic activity against both EG7-OVA and the parent EL4, but not against mouse melanoma B16-F10, suggesting that not only OVA-derived antigens but those common to EG7-OVA and EL4 are delivered to APCs. These results provide a new therapeutic method to induce tumor-specific antitumor immunity without identifying nor isolating tumor-associated antigens.

Methanol bioeconomy: promotion of rice crop yield in paddy fields with microbial cells prepared from natural gas-derived C1 compound.

Methylotrophs, which can utilize methanol as a sole carbon source, are promising microorganisms to be exploited in a methanol-based bioeconomy, in which a variety of useful compounds are biotechnologically produced from natural gas-derived methanol. Pink-pigmented facultative methylotrophs (PPFMs) are common plant phyllospheric bacteria and are known to enhance seedling growth and total biomass of various plants. However, improvement of crop yield by inoculation of PPFMs at the field level has not been well investigated. We herein describe improvement of crop yield of several rice cultivars by foliar spraying of PPFMs. After selection of PPFM strains and rice cultivars by the in vitro seedling growth test, we further conducted paddy field experiments. The crop yield of the sake-brewing rice Oryza sativa cultivar Hakutsurunishiki was reproducibly improved in a commercial paddy field for over a 5-year period. A one-time foliar spray of PPFM cells (living or killed) or a cell wall polysaccharide fraction, after the heading date, acted in the phyllosphere and effectively improved crop yield. Our results show that the established process with PPFMs is feasible for improvement of food production in the methanol bioeconomy.

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. There have been advances in genome analysis of MAH from human isolates, but studies of isolates from pigs are limited despite its potential source of infection to human. Here, we obtained 30 draft genome sequences of MAH from pigs reared in Japan. The 30 draft genomes were 4,848,678-5,620,788 bp in length, comprising 4652-5388 coding genes and 46-75 (median: 47) tRNAs. All isolates had restriction modification-associated genes and 185-222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

Mutation of alanine-422 in KaiC leads to a low amplitude of rhythm in the reconstituted cyanobacterial circadian clock.

The cyanobacterial circadian oscillator can be reconstituted by mixing the purified clock proteins KaiA, KaiB, and KaiC with ATP in vitro, leading to a 24-h oscillation of KaiC phosphorylation. The cyanobacterial mutant pr1 carrying valine instead of alanine at position 422 of KaiC (KaiC-A422V) lost the ability to shift the phase of the circadian rhythm. In this study, we analyzed KaiC-A422V to investigate the effect of this single-residue substitution on the in vitro reconstitution of KaiC oscillation. KaiC-A422V exhibited low amplitude oscillations of phosphorylation with a smaller amount of Kai complex than wild-type KaiC (KaiC-WT). Although KaiA can stimulate KaiC phosphorylation, the phosphorylation level of KaiC-A422V is much lower than that of KaiC-WT even at higher KaiA concentrations. It has been suggested that monomer shuffling of KaiC is involved in entraining the in vitro rhythm. To examine whether KaiC-A422V has the capacity for monomer shuffling, we used the difference in the amplitude of the phosphorylation rhythms between KaiC-WT and KaiC-A422V as the indicator of monomer shuffling. When KaiC-A422V and KaiC-WT were mixed, the amplitude of the phosphorylation rhythm changed according to the mixing ratio. This suggests that KaiC-A422V has a reduced ability to shuffle monomers in hexameric KaiC. In addition, the A422V mutation resulted in a change of the stability of the KaiC protein.

Emergence of a Multidrug-Resistant Enterobacter hormaechei Clinical Isolate from Egypt Co-Harboring mcr-9 and blaVIM-4.

Abstract: This study describes the first full genomic sequence of an mcr-9 and blaVIM-4-carrying multidrug-resistant Enterobacter hormaechei clinical isolate from Egypt. The strain was isolated in April 2015 from the sputum of a patient in Cairo, Egypt. The mcr-9 and blaVIM-4 genes were identified by PCR screening and DNA sequencing; the isolate was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole genomic sequencing. mcr-9 and blaVIM-4 were carried by an IncHI2 plasmid, pAMS-38a (281,121 bp in size); the plasmid also carried genes conferring resistance against sulfonamides (sul1), quinolones (qnrA1), trimethoprim (dfrA1), ß-lactams (blaTEM-1B), aminoglycosides (aac (6')-II, aadA23, aadA2b, and ant(2'')-Ia). The strain was susceptible to colistin (MIC, <0.25 µg/mL); this could be due to the absence of the qseC/qseB regulatory system located downstream of mcr-9 in Enterobacterales, which is involved in the induction of colistin-resistance. The genetic context of mcr-9 and blaVIM-4 was identified as IS1-mcr-9-IS903-pcoS-∆pcoE-rcnA and intI1-blaVIM-4-aac (6')-II-dfrA1-∆aadA23-smr-ISPa21-qacE∆1, respectively. This is the first report of an mcr-9 and blaVIM-4 /IncHI2-carrying multidrug-resistant E. hormaechei clinical isolate from Africa and the Middle East. Plasmids of the IncHI2 group and the two insertion sequences (IS1, and IS903) might be the main vehicles for dissemination of mcr-9. Further screening for mcr-9 is essential for identifying its incidence and to prevent its dissemination.

Efficacy and Safety of the Radiotherapy for Liver Cancer: Assessment of Local Controllability and its Role in Multidisciplinary Therapy.

This study investigated the efficacy and safety of radiotherapy as part of multidisciplinary therapy for advanced hepatocellular carcinoma (HCC). Clinical data of 49 HCC patients treated with radiotherapy were assessed retrospectively. The efficacy of radiotherapy was assessed by progression-free survival, disease control rate, and overall survival. Safety was assessed by symptoms and hematological assay, and changes in hepatic reserve function were determined by Child-Pugh score and albumin-bilirubin (ALBI) score. Forty patients underwent curative radiotherapy, and nine patients with portal vein tumor thrombus (PVTT) underwent palliative radiotherapy as part of multidisciplinary therapy. Local disease control for curative therapy was 80.0% and stereotactic body radiotherapy was 86.7% which was greater than that of conventional radiotherapy (60.0%). Patients with PVTT had a median observation period of 651 days and 75% three-year survival when treated with multitherapy, including radiotherapy for palliative intent, transcatheter arterial chemoembolization, and administration of molecular targeted agents. No adverse events higher than grade 3 and no changes in the Child-Pugh score and ALBI score were seen. Radiotherapy is safe and effective for HCC treatment and can be a part of multidisciplinary therapy.

Draft genome sequences of Mycolicibacterium peregrinum isolated from a pig with lymphadenitis and from soil on the same Japanese pig farm.

OBJECTIVES: Mycolicibacterium peregrinum, a rapidly growing mycobacterial species, can opportunistically infect humans and other animals. Although M. peregrinum infections in animals have been reported, the infection sources are unknown, as is information on its virulence and drug resistant genes, which limits our current understanding of this bacterium. To address this knowledge gap, we obtained draft genome sequences for two M. peregrinum isolates; one from a case of pig lymphadenitis and one from the pig farm's soil. DATA DESCRIPTION: We report here the draft genome sequences of M. peregrinum isolates 131_1 and 138 (6,451,733-bp and 6,479,047-bp). They were isolated from a pig with mesenteric lymph node lymphadenitis and from soil on the Japanese farm where the pig was reared. A sequence alignment identity score of 100% was obtained by in silico DNA-DNA hybridization of the two isolates, while 98.28% (isolate 131_1) and 98.27% (isolate 138) scores were recorded for hybridization with a human isolate. Both isolates carry arr-1, AAC(2')-Ib, RbpA, mtrA and tap drug-resistance genes. Isolates 131_1 and 138 carry 234 and 236 putative virulence genes, respectively. Therefore, environment M. peregrinum is potentially drug resistant and can cause swine lymphadenitis. Our data provides valuable new information for future studies on nontuberculous mycobacteria.

Genetic relatedness of Mycobacterium avium subsp. hominissuis isolates from bathrooms of healthy volunteers, rivers, and soils in Japan with human clinical isolates from different geographical areas.

Japan reportedly has high incidence rate of nontuberculous mycobacterial lung disease (14.7 cases per 100,000 person in 2014). In Japan, the most common etiology is Mycobacterium avium subsp. hominissuis (MAH). MAH is a typical inhabitant of the environment, especially bathrooms, which are considered as a potential source of infection. To corroborate this hypothesis, we determined the detection rate of MAH in bathrooms of healthy volunteers by an ordinary culture method and we analyzed the genetic relatedness of these isolates with those from patients and other sources. We collected swabs of bathtub inlets, showerheads, bathroom drains, and shower water from 180 residences throughout Japan. The overall MAH detection rate was 16.1%, but the rate varied among regions: it was high in Kanto (9/34, 26.5%) and Kinki (9/33, 27.3%), but low in Kyushu (0/11, 0%), Tohoku (1/23, 4.3%), and Hokkaido (2/23, 8.7%). MAH was detected primarily in bathtub inlet samples (25 out of 170 residences). Variable numbers of tandem repeats (VNTR) analysis was used to examine the genetic relatedness of 57 MAH isolates from bathrooms of the healthy volunteers with human clinical isolates. A minimum spanning tree generated on the basis of the VNTR data indicated that isolates from the bathrooms of the healthy volunteers had a high degree of genetic relatedness with those from Japanese patients, bathrooms of patients, and river water, but not with those from Russian patients and Japanese pigs. These results showed that bathtub inlets in Japan provide an environmental niche for MAH and suggest that bathrooms are one of the important infection sources of MAH in Japan. Understanding country-specific lifestyle habits, such as bathing in Japan, as well as the genetic diversity of MAH, will help in elucidating the sources of this pathogen.

Genetic diversity of environmental Vibrio cholerae O1 strains isolated in Northern Vietnam.

Cholera epidemics have been recorded periodically in Vietnam during the seventh cholera pandemic. Since cholera is a water-borne disease, systematic monitoring of environmental waters for Vibrio cholerae presence is important for predicting and preventing cholera epidemics. We conducted monitoring, isolation, and genetic characterization of V. cholerae strains in Nam Dinh province of Northern Vietnam from Jul 2013 to Feb 2015. In this study, four V. cholerae O1 strains were detected and isolated from 110 analyzed water samples (3.6%); however, none of them carried the cholera toxin gene, ctxA, in their genomes. Whole genome sequencing and phylogenetic analysis revealed that the four O1 isolates were separated into two independent clusters, and one of them diverged from a common ancestor with pandemic strains. The analysis of pathogenicity islands (CTX prophage, VPI-I, VPI-II, VSP-I, and VSP-II) indicated that one strain (VNND_2014Jun_6SS) harbored an unknown prophage-like sequence with high homology to vibriophage KSF-1 phi and VCY phi, identified from Bangladesh and the USA, respectively, while the other three strains carried tcpA gene with a distinct sequence demonstrating a separate clonal lineage. These results suggest that the aquatic environment can harbor highly divergent V. cholera strains and serve as a reservoir for multiple V. cholerae virulence-associated genes which may be exchanged via mobile genetic elements. Therefore, continuous monitoring and genetic characterization of V. cholerae strains in the environment should contribute to the early detection of the sources of infection and prevention of cholera outbreaks as well as to understanding the natural ecology and evolution of V. cholerae.

Comparative Genome Analyses of Streptococcus suis Isolates from Endocarditis Demonstrate Persistence of Dual Phenotypic Clones.

Many bacterial species coexist in the same niche as heterogeneous clones with different phenotypes; however, understanding of infectious diseases by polyphenotypic bacteria is still limited. In the present study, encapsulation in isolates of the porcine pathogen Streptococcus suis from persistent endocarditis lesions was examined. Coexistence of both encapsulated and unencapsulated S. suis isolates was found in 26 out of 59 endocarditis samples. The isolates were serotype 2, and belonged to two different sequence types (STs), ST1 and ST28. The genomes of each of the 26 pairs of encapsulated and unencapsulated isolates from the 26 samples were sequenced. The data showed that each pair of isolates had one or more unique nonsynonymous mutations in the cps gene, and the encapsulated and unencapsulated isolates from the same samples were closest to each other. Pairwise comparisons of the sequences of cps genes in 7 pairs of encapsulated and unencapsulated isolates identified insertion/deletions (indels) ranging from one to 104 bp in different cps genes of unencapsulated isolates. Capsule expression was restored in a subset of unencapsulated isolates by complementation in trans with cps expression vectors. Examination of gene content common to isolates indicated that mutation frequency was higher in ST28 pairs than in ST1 pairs. Genes within mobile genetic elements were mutation hot spots among ST28 isolates. Taken all together, our results demonstrate the coexistence of dual phenotype (encapsulated and unencapsulated) bacterial clones and suggest that the dual phenotypes arose independently in each farm by means of spontaneous mutations in cps genes.

Enhanced generation of cytotoxic T lymphocytes by increased cytosolic delivery of MHC class I epitope fused to mouse heat shock protein 70 via polyhistidine conjugation.

Heat shock protein 70 (Hsp70)-associated antigens in a soluble form have been shown to elicit strong antigen-specific cytotoxic T lymphocyte (CTL) responses following immunization without any adjuvants. In order to improve the potential of Hsp70, we genetically designed a novel Hsp70-based antigen delivery system, in which the model MHC class I epitope of ovalbumin (OVA) (SIINFEKL; OVA257-264) was fused to mouse Hsp70. To facilitate the cytosolic delivery of the peptide following Hsp receptor-mediated endocytosis, polyhistidine of 25 or 50 residues was further fused to the fusion protein. Each fusion protein was then expressed in E. coli and purified. When added to DC2.4 cells, a mouse dendritic cell line, the fusion protein containing polyhistidine of 25 residues was efficiently taken up by the cells and efficiently distributed to the cytosol. The fusion protein also exhibited a significantly improved efficacy of MHC class I-restricted presentation of antigen. Vaccination of mice with the polyhistidine fusion protein generated strong antigen-specific CTL responses and antitumor activity. These findings suggest that polyhistidine fusion is a useful strategy to increase the potential of Hsp-based vaccination.

Xenografting of bovine secondary follicles into ovariectomized female severe combined immunodeficient mice.

Xenografting of ovarian tissue into immunodeficient mice has been used as a model to study the dynamics of follicular development and provides an alternative method for the production of mature oocytes. In a previous experiment, we demonstrated that xenografted bovine secondary follicles developed to the antral stage in severe combined immunodeficient (SCID) mice. In the present study, we examined the development of bovine secondary follicles (140-190 microm in diameter) grafted into ovariectomized mice in comparison with intact female mice as a control. At 4 weeks after grafting, several antral follicles ranging from 350 to 550 microm (457.6 +/- 50.8 microm) in diameter were found in the control mice, while a single large (larger than 2.5 mm) antral follicle and other small follicles were observed in every ovariectomized mouse. At 6 weeks after grafting, the mean diameter of morphologically normal follicles had further increased in the control group (591.8 +/- 132.0 microm). In ovariectomized mice, however, the mean diameter of follicles decreased (4 weeks: 864.2 +/- 988.2 microm; 6 weeks: 496.5 +/- 137.6 microm), since the single large antral follicle observed at 4 weeks had degenerated by 6 weeks. In control mice, more than 70% of follicles were morphologically normal and formed an antrum, and most of the follicles contained morphologically normal oocytes which grew to 122.5 +/- 2.2 microm. In ovariectomized mice, morphologically normal oocytes also grew larger than before grafting, but their survival rate was significantly lower than that in control mice. These results suggest that ovariectomy of host mice alters the developmental pattern of xenografted bovine secondary follicles to accelerate a single follicle to develop in the graft.

Bovine oocytes in secondary follicles grow and acquire meiotic competence in severe combined immunodeficient mice.

Cortical tissues containing only primordial and primary follicles, or secondary follicles 140-190 microm in diameter, were collected from bovine ovaries and xenografted under the kidney capsules of female severe combined immunodeficient (SCID) mice. Histological examination revealed that all grafts were well vascularised and contained surviving follicles at 4 or 6 weeks after grafting. Primordial and primary follicles survived but did not develop beyond the one-layer stage. Secondary follicles, on the other hand, had formed antra at 4 weeks after grafting. The mean diameter of secondary follicles, which was 165.2 +/- 17.0 microm (n = 42) before grafting, had developed to 442.9 +/- 77.9 microm (n = 37) and 592.9 +/- 116.0 microm (n = 45) in diameter at 4 and 6 weeks after grafting, respectively. The mean diameter of oocytes, which was 55.1 +/- 4.9 microm (n = 42) before grafting, also increased significantly (4 weeks: 105.6 +/- 6.3 microm; 6 weeks: 122.2 +/- 2.6 microm; p < 0.05). Oocytes were recovered from follicles that had developed to more than 400 microm in diameter after 6 weeks, and were subjected to subsequent mature culture. Of these oocytes, 34% (11/32) resumed meiosis and 6% (2/32) matured to the second metaphase. Follicular fluid in bovine antral follicles developed in SCID mice had the 69 kDa protein, which was detected by anti-mouse albumin antibody but not by anti-bovine albumin antibody in immunoblotting analysis. These results demonstrated that bovine secondary follicles develop to the antral stage in SCID mice, and that the oocytes in the follicles acquire the meiotic competence.

Laparoscopic pancreatic surgery: its indications and techniques: from the viewpoint of limiting the indications.

Indications in the field of pancreatic surgery should be limited considering the technical difficulties and the characteristics of pancreatic diseases. Benign or low-grade malignant tumors, including pseudocysts, islet tumors, and cystic tumors, are indications for distal pancreatectomy. Islet tumors such as insulinomas are good candidates for this procedure when they are located near splenic vessels or the main pancreatic duct and enucleation is considered inappropriate. Techniques of laparoscopic distal pancreatectomy with/without splenectomy and laparoscopy-assisted distal pancreatectomy indicated in low-grade malignant tumors such as mucinous cystadenoma are described. Insulinoma is one of the best candidates for enucleation because many of the cases are solitary and benign. The technique of enucleation is also described.

Techniques for difficult cases of laparoscopic cholecystectomy.

Our basic techniques for the management of difficult cases of laparoscopic cholecystectomy (LC) are presented in this article. If access to Calot's triangle cannot be gained safely, dissection should be started at the fundus or body of the gallbladder (GB), rather than the neck (fundus-first method). In cases with a short and wide cystic duct, a transfixing suture should be applied for ligation instead of clipping. EndoGIA is useful for ligating and transecting this case to avoid a subsequent stricture caused by normal method of ligation. Intraoperative cholangiography should be performed near the neck of the GB in cases in which orientation is lost during dissection. More dissection should be performed in the direction of the junction of the bile ducts after orientation is regained. In cases with GB filled with stones accompanied by severe fibrosis, part of the GB is incised to remove the stones and expose the lumen of the GB. Confluence stones can be removed by placing an incision on the GB side of the junction of the duct. The incised part is closed with suture. A cystic tube (C-tube) is placed in the common bile duct through the cystic duct for decompression. In more difficult cases in which dissection cannot be started safely at any location, the body and the fundus of the GB are excised, and a drain is placed at the neck of the GB. Dissection can be carried out from the main surgeon's or the assistant's side depending on the situation, and cooperation between the two surgeons is mandatory to achieve safe LC in difficult cases. When performing the LC, one must have a low threshold for converting to open surgery if injuries cannot be managed safely.