Genome-Wide CRISPR/Cas9 Screening Unveils a Novel Target ATF7IP-SETDB1 Complex for Enhancing Difficult-to-Express Protein Production.

With the emerging novel biotherapeutics that are typically difficult-to-express (DTE), improvement is required for high-yield production. To identify novel targets that can enhance DTE protein production, we performed genome-wide fluorescence-activated cell sorting (FACS)-based clustered regularly interspaced short palindromic repeats (CRISPR) knockout screening in bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cells. The screen identified the two highest-scoring genes, Atf7ip and Setdb1, which are the binding partners for H3K9me3-mediated transcriptional repression. The ATF7IP-SETDB1 complex knockout in bsAb-producing CHO cells suppressed cell growth but enhanced productivity by up to 2.7-fold. Decreased H3K9me3 levels and an increased transcriptional expression level of the transgene were also observed. Furthermore, perturbation of the ATF7IP-SETDB1 complex in monoclonal antibody (mAb)-producing CHO cells led to substantial improvements in mAb production, increasing the productivity by up to 3.9-fold without affecting the product quality. Taken together, the genome-wide FACS-based CRISPR screen identified promising targets associated with histone methylation, whose perturbation enhanced the productivity by unlocking the transgene expression.

Is the anatomical resection necessary for single hepatocellular carcinoma smaller than 3 cm?: single-center experience of liver resection for a small HCC.

BACKGROUNDS/AIMS: The superiority of anatomical resection (AR) for a small HCC remains controversial. In this study, we investigated the clinical outcomes after AR and non-anatomical liver resection (NAR) for single HCC smaller than 3 cm and the risk factors for HCC recurrence. METHODS: A total of 116 consecutive patients who underwent liver resection for single HCC (<3 cm) between Jan 2006 and Dec 2015 were included in this study. The medical records of these patients were reviewed and analyzed retrospectively. RESULTS: There was no significant difference in tumor recurrence and survival between AR and NAR group. Multivariate analysis showed that hepatitis B (p=0.035, HR=8.72), presence of satellite nodule (p=0.029, HR=3.97) and microvascular invasion (MVI) (p=0.039, HR=2.79) were independent risk factors for early recurrence within 1 year. The overall recurrence was independently related to the presence of satellite nodule (p=0.001, HR=4.98) and background liver cirrhosis (p=0.032, HR=1.96). In patients with MVI, HCC recurrence was significantly more frequent in width of safety margin <1 cm group than ≥1 cm group (p=0.049). CONCLUSIONS: The outcomes of NAR are comparable with those of AR in single HCC smaller than 3 cm. The presence of satellite nodule, MVI and hepatitis B are the independent risk factors for early recurrence, however overall recurrence is correlated with background liver cirrhosis and the presence of satellite nodule rather than pathobiologic factors in single HCC smaller than 3 cm. Hepatic resection with sufficient margin (≥1 cm) is recommended for decreasing risk of recurrence in patients with suspected MVI.

Crystal structure of Pseudomonas aeruginosa N-acetyltransferase PA4534.

The GCN5-related N-acetyltransferase (GNAT) superfamily includes a large and diverse group of enzymes that catalyzes the transfer of an acetyl group from acetyl coenzyme A (Ac-CoA) to the amine group of a substrate. Substrates include protein N-terminus, lysine of histone tails, and other small molecules such as aminoglycoside, serotonin, and glucose-6-phosphate. GNAT superfamily of proteins is involved in many physiologically important reactions in eukaryotes and prokaryotes. However, functions of many GNATs remain unknown and PA4534 is one of those uncharacterized GNAT proteins from Pseudomonas aeruginosa. To investigate functions of the PA4534, we determined the apo and Ac-CoA bound PA4534 structures. Our structures showed that PA4534 shared common characteristic structures with other GNAT family N-acetyltransferases and contained a potential substrate binding tunnel close to the bound Ac-CoA.

Angiogenesis induced by autologous whole bone marrow stem cells transplantation.

BACKGROUND AND OBJECTIVES: It has been presumed that unknown cells and growth factors in bone marrow might promote angiogenesis, so angiogenesis effect could be enhanced by autologous whole bone marrow (WBM) stem cell transplantation. We compared capillary ratio induced by autologous WBM and bone marrow-mononuclear cells (BM-MNCs) to evaluate the anigiogenic effect of auotologous WBM. In addition, the combined effect of WBM transplantation and granulocyte colony-stimulating factor (G-CSF) injection was examined in an ischemic canine model. METHODS AND RESULTS: After creating ischemic limb model, autologous WBM and isolated BM-MNCs were transplanted into the ischemic muscle. In other experiments, autologous WBM with recombinant human G-CSF (rhG-CSF) and autologous WBM without rhG-CSF were transplanted into the ischemic muscle. In this study, normal saline was injected into the contralateral sites in each ischemic model as a control group. After 8 weeks of transplantation, angiography and muscle harvest were performed, and then the anigiographic findings and capillary density, as assessed by immunohistochemical staining, were investigated and analyzed. In comparison with the control group, BM-MNCs and WBM transplantation groups showed higher ratios of the capillary density (1.5±0.01 times, p<0.001 and 1.6±0.15 times, p=0.005, respectively). Between the BM-MNCs and WBM transplantation groups, the capillary ratio was 1.2 folds higher in the WBM group than that in the BM-MNCs group, but there was no significantly different (p=0.116). The angiogensis ratios of both the WBM without G-CSF group and the WBM with G-CSF groups were higher (1.6±0.15 times, p=0.004 and 1.8 ±0.01 times, p=0.005, respectively) than that of the control groups. In comparison with the WBM without G-CSF group, the WBM with G-CSF transplantation group revealed a 1.1 folds higher angiogenesis ratio, but there was no statistically significant difference (p=0.095). CONCLUSIONS: Autologous WBM transplantation is a simpler method and it is not inferior for inducing therapeutic angiogenesis as compared with isolated BM-MNCs transplantation. In addition to autologous WBM transplantation, intravenous G-CSF injection enhances the angiogenic effect of autologous WBM in an ischemic limb.

Angiogenesis facilitated by autologous whole bone marrow stem cell transplantation for Buerger's disease.

We hypothesized that angiogenesis can be triggered by autologous whole bone marrow stem cell transplantation. Twenty-seven patients (34 lower limbs) with Buerger's disease, who were not candidates for surgical revascularization or radiologic intervention, were enrolled in this study. Six sites of the tibia bone were fenestrated using a 2.5-mm-diameter screw under fluoroscopic guidance. Clinical status and outcome were determined using the "Recommended Standards for Reports." To mobilize endothelial progenitor cells (EPCs) from bone marrow, recombinant human granulocyte colony-stimulating factor (r-HuG-CSF) was injected subcutaneously as a dose of 75 microg, preoperatively. During the follow-up period (19.1 +/- 3.5 months), one limb showed a markedly improved outcome (+3), and 26 limbs showed a moderately improved outcome (+2). Thirteen limbs among 17 limbs with nonhealing ulcers healed. Postoperative angiograms were obtained for 22 limbs. Two limbs showed marked (+3), five limbs moderate (+2), and nine limbs slight (+1) collateral development. However, six limbs showed no collateral development (0). Peripheral blood and bone marrow samples were analyzed for CD34 and CD133 molecules to enumerate potential EPCs, and EPC numbers were found to be increased in peripheral blood and in bone marrow after r-HuG-CSF injection. In conclusion, the transplantation of autologous whole BMCs by fenestration of the tibia bone represents a simple, safe, and effective means of inducing therapeutic angiogenesis in patients with Buerger's disease.

Surgical experience of carotid pseudoaneurysm.

An extracranial carotid artery pseudoaneurysm is a rare condition that is caused by various types of arteritis, trauma and infectious causes. Generally, a pseudoaneurysm may be difficult to treat surgically when dissecting the paraaneurysmal fibrotic dense inflammatory tissues. The surgical management of a peudoaneurysm of the carotid artery involves a risk of nerve and arterial injury. This paper reports the repair of a carotid artery pseudoaneurysm after the proximal and distal control of the internal carotid artery using a Pruitt-Inahara shunt (P-I shunt) and the distal control of the external carotid artery using a small sized occlusion balloon catheter.