Frequent somatic gene conversion as a mechanism for loss of heterozygosity in tumor suppressor genes.

The major processes in carcinogenesis include the inactivation of tumor-suppressor genes (TSGs). Although Knudson's two-hit model requires two independent inactivating mutations, perhaps more frequently, a TSG inactivation can occur through a loss of heterozygosity (LOH) of an inactivating mutation. Deletion and uniparental disomy (UPD) have been well documented as LOH mechanisms, but the role of gene conversion is poorly understood. Here, we developed a simple algorithm to detect somatic gene conversion from short-read sequencing data. We applied it to 6285 cancer patient samples, from which 4978 somatic mutations that underwent gene conversion to achieve LOH were found. This number accounted for 14.8% of the total LOH mutations. We further showed that LOH by gene conversion was enriched in TSGs compared with non-TSG genes, showing a significant contribution of gene conversion to carcinogenesis.

Subclonal accumulation of immune escape mechanisms in microsatellite instability-high colorectal cancers.

BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 µs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 µs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.

Successful treatment of nonunion with teriparatide after failed ankle arthrodesis for Charcot arthropathy.

We describe a case of successful treatment to nonunion after multiple arthrodesis operations for Charcot arthropathy with teriparatide. We describe the case of a 25-year-old woman with severe Type I diabetes mellitus that resulted in nonunion after multiple arthrodesis operations for Charcot arthropathy. The woman sustained a femoral shaft fracture for which she underwent surgery with intramedullary nail fixation. Immediately after surgery, an empiric course of teriparatide was initiated. Femoral shaft fracture healing was observed after 2 weeks, and the woman was able to walk 12 weeks after the surgery, at which point plain film and computed tomography images revealed complete union of the ankle.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

Duplication with structural modification through extrachromosomal circular and lariat DNA in the human genome.

Duplication plays an important role in creating drastic changes in genome evolution. In addition to well-known tandem duplication, duplication can occur such that a duplicated DNA fragment is inserted at another location in the genome. Here, we report several genomic regions in the human genome that could be best explained by two types of insertion-based duplication mechanisms, where a duplicated DNA fragment was modified structurally and then inserted into the genome. In one process, the DNA fragment is turned into an extrachromosomal circular DNA, cut somewhere in the circle, and reintegrated into another location in the genome. And in the other, the DNA fragment forms a "lariat structure" with a "knot", the strand is swapped at the knot, and is then reintegrated into the genome. Our results suggest that insertion-based duplication may not be a simple process; it may involve a complicated procedures such as structural modification before reintegration. However, the molecular mechanism has yet to be fully understood.

Clinical outcome of extrasynovial steroid injection for trigger finger.

A prospective clinical study was performed to investigate the clinical results of extrasynovial (subcutaneous) steroid injection for trigger finger. One hundred and twenty-nine trigger fingers were investigated in 100 adult patients; 76 were women and 24 were men. Their mean age was 60 years (range: 17 to 88 years). We classified trigger fingers into three different grades according to clinical severity at a medical examination. All patients were injected with betamethasone mixed with lidocaine. Surgical release of the A1 pulley was performed at the patients' request if steroid injection therapy was not effective. Pain and snapping were relieved in 98% and 74% of cases, respectively. Recurrence occurred in about half our patients, but the same clinical benefit was obtained after re-injection. Surgery was performed for seven fingers. No complications of steroid injections were observed. This study suggests that extrasynovial steroid injection is a valuable conservative treatment for trigger finger and it is not necessary to try and inject into the tendon sheath to get a good result and markedly reduce the risk of causing damage to tendons and other structures.

Ulnar focal cortical indentation: a previously unrecognised form of ulnar dysplasia.

Deformity of the forearm due to growth disturbance of the ulna occurs in a number of conditions such as ulnar deficiency, multiple exostoses, and neurofibromatosis. We report a previously unrecognised form, caused by focal cortical indentation. We have treated five children with this condition, three girls and two boys; the mean age at presentation was 5 years (2 to 8). The deformity was first recognised about the age of two years, and progressed gradually. The radiological findings were the same in all cases. The focal cortical indentation was seen at the distal end of the ulna with anteromedial bowing and dysplasia. The radial head was dislocated posterolaterally. In one patient the histological findings at the site of indentation were of a fold of tissue resembling periosteum, which interfered with enchondral ossification. Treatment by ulnar lengthening using an external fixator and osteotomy which corrected both the ulnar deformity and reduced the dislocated radial head in two cases gave the best results.

Case of trigger finger related to an intertendinous connection between the flexor tendons.

We report a rare cause of trigger finger related to an anatomical variation of an intertendinous connection between the flexor digitorum superficialis and flexor digitorum profundus tendons in the palmar region.

Palmar lunate transscaphoid fracture-dislocation caused by palmar flexion injury.

The authors report a rare case of palmar lunate transscaphoid fracture-dislocation resulting from a palmar flexion injury. After performing an open reduction of the lunate, they used a dorsal approach to fix the fractured scaphoid with a Herbert screw. A Kirschner wire fixation was also performed to stabilize the lunotriquetral joint.

Correction of dorsi-flexed intercalated segment instability after restoration of scaphoid height in a cadaver model of scaphoid non-union.

Models of scaphoid non-union with static dorsi-flexed intercalated segment instability were produced in five frozen arms from cadavers or subjects following accidents by repetitive mechanical loading of the wrist joints longitudinally after a bone defect has been made at the mid-portion of the scaphoid. We designed four models of reduction: anatomical reduction; reduction with a shortened scaphoid; anatomical reduction but with the radio-lunate ligament sectioned, and a shortened scaphoid with the radio-lunate ligament sectioned. Results suggested that anatomical reduction with rigid fixation with a Herbert screw was most effective for correction of malalignment with DISI. Preservation of the radio-lunate ligament during the palmar approach to the scaphoid seemed to be important to prevent ligamentous carpal instability.

Chondroma within the flexor tendon sheath of the index finger: case report.

Chondroma of soft tissue is rare. We report a patient in whom a chondroma occurred within the flexor tendon sheath of the index finger. Magnetic resonance imaging showed the extent of the tumor, which wrapped around flexor tendons within the sheath, but did not invade either tendons or sheath. Total excision was done with preservation of the flexors and flexor tendon sheath. After the operation, the index finger had a full range of motion, and movement was painless.

Osteomyelitis and arthritis of the wrist caused by Mycobacterium intracellulare in an immunocompetent patient: a case report and literature review.

Mycobacterium intracellulare causes infection in humans. Involvement of joint and bone, however, is extremely rare. We present the case of an immunocompetent 67-year-old female with chronic swelling of the wrist joint diagnosed as rheumatoid arthritis by her previous physician. Examination revealed an unclosed fistula associated with a puncture, and bone and joint destruction on radiographs. She was diagnosed with osteomyelitis and arthritis due to M. intracellulare on histological and microbiological examinations. She was successfully treated with radical surgical debridement and anti-tuberculous drugs for 1 year and there was no recurrence at 3 years postoperatively.

A case of toe macrodactyly treated by application of a vascularised nail graft.

Macrodactyly is a relatively rare congenital abnormality of the fingers and toes and is difficult to treat. We report a new method for treating toe macrodactyly in which a nail with a vascularised pedicle is raised, the toe is shortened to an appropriate length, and the nail transferred to an aesthetically appropriate proximal site. Although this technique is of a higher level of difficulty than conventional procedures involving pedicled nail flaps, allows aesthetically more favourable nail reconstruction by single-stage operation.

Pressure distribution in the radiocarpal joint measured with a densitometer designed for pressure-sensitive film.

To measure the pressure distribution in the radiocarpal joint, a biomechanical study was done with fresh cadavers and pressure-sensitive film. With a load of 100 newtons, the contact locations were identified for 27 different wrist positions and the peak articular pressures and contact area were measured. Normal wrists were studied first, then modified to show different ulnar variances, and then to be models of malunited Colles' fractures of increasing severity. The contact locations of the scaphoid and the lunate were separate, and the mean peak articular pressures were 2.8 and 2.7 megapascals, respectively, for the normal wrists. In the radiolunate articulation, this mean increased by 27% with 2.5 mm ulnar minus variance and decreased by 22% with 2.5 mm ulnar plus variance. In the models of malunited Colles' fracture, the contact locations shifted from volar to dorsal, and the contact area decreased as the deformity became worse.

Cerebral fat embolism after a nondisplaced tibial fracture: case report.

Fat embolism syndrome has been observed after traumatic or nontraumatic events. In traumatic cases, fat embolism syndrome is known to occur in patients with a fracture of a long bone. The case of a patient with a cerebral fat embolism associated with a nondisplaced fracture of the tibial shaft is reported.

Cineradiographic study of wrist motion after fracture of the distal radius.

Residual dorsal tilt is one of the main deformative factors in the malunion of fractures of the distal radius. We investigated the effect of such tilt on the range of wrist motion and on carpal alignment in a cineradiographic study of wrist motion in 30 patients with extra-articular Colles' fracture. With increased dorsal tilt, the range of wrist motion became more restricted and abnormalities of carpal alignment during wrist motion became apparent. When the dorsal tilt was less than 10 degrees, the effects on the range of motion and on the dynamic carpal alignment were small.