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Objective: Radical surgery is the mainstay treatment for adenocarcinoma of the esophagogastric junction. The presence of microscopic residual tumor tissue after curative-intent surgery is associated with recurrence. This study compared the outcomes of patients with microscopic residual tumor (Residual+ group) and those without microscopic residual tumor (Residual- group). Material and Methods: We retrospectively reviewed the medical records of 71 patients with adenocarcinomas of the esophagogastric junction who underwent curative-intent surgery between January 2005 and August 2018. We evaluated the clinical and pathological characteristics and compared recurrences, rates and patterns, between groups. Five-year overall survival (OS) and 3-year disease-free survival (DFS) were analyzed by Kaplan-Meier analysis. Results: Nineteen (26.8%) patients had microscopic residual tumors, consisting of 8 (11.3%) with positive resection margins, 10 (14.0%) with malignant cells from peritoneal washing fluid cytology, and 1 (1.4%) with both. The median OS in the Residual- group was significantly better than that in the Residual+ group (31.3 vs 11.9 months, P = 0.003). The Residual- group had better 5-year OS (26.2% vs 11.9%, P = 0.015) and 3-year DFS (24.4% vs 9.8%, P = 0.003) than the Residual+ group. During follow-up period, 48% of the patients in the Residual+ group experienced recurrent disease, with a median follow-up time at 7.7 months. Distant metastasis was the most common site of recurrence. Conclusion: Microscopic residual tumor after resection is associated with poorer survival outcomes and higher recurrence rates. Curative surgery should aim to achieve R0 resection in all patients with resectable adenocarcinomas of the esophagogastric junction.

Background/Aims The coronavirus disease 2019 pandemic has affected the worldwide practice of upper gastrointestinal endoscopy. Here we designed a modified N95 respirator with a channel for endoscope insertion and evaluated its efficacy in upper gastrointestinal endoscopy. Methods Thirty patients scheduled for upper gastrointestinal endoscopy were randomized into the modified N95 (n=15) or control (n=15) group. The mask was placed on the patient after anesthesia administration and particles were counted every minute before (baseline) and during the procedure by a TSI AeroTrak particle counter (9306-04; TSI Inc.) and categorized by size (0.3, 0.5, 1, 3, 5, and 10 µm). Differences in particle counts between time points were recorded. Results During the procedure, the modified N95 group displayed significantly smaller overall particle sizes than the control group (median [interquartile range], 231 [54–385] vs. 579 [213–1,379]×103/m3; p=0.056). However, the intervention group had a significant decrease in 0.3-µm particles (68 [–25–185] vs. 242 [72–588]×103/m3; p=0.045). No adverse events occurred in either group. The device did not cause any inconvenience to the endoscopists or patients. Conclusions This modified N95 respirator reduced the number of particles, especially 0.3-µm particles, generated during upper gastrointestinal endoscopy.

Peritoneal metastasis in gastric cancer has a poor prognosis and is increasing in prevalence. Neoadjuvant chemotherapy is used for advanced tumors; however, surgery is generally not considered for metastatic and unresectable diseases. Recently, conversion surgery, a treatment which aims for an R0 resection following chemotherapy, has become a novel therapeutic option with better survival rates. In addition to surgery, hyperthermic intraperitoneal chemotherapy (HIPEC) leads to significant tumor reduction, but it is limited by its morbidity. Pressurized intraperitoneal aerosol chemotherapy (PIPAC) delivers high concentrations of chemotherapy, but does not remove the tumor, making it useful mostly in palliative settings. Intraperitoneal (IP) therapy, known for its minimally invasive nature and repeatability, shows promise but requires further research. Ultimately, an integrated approach involving systemic chemotherapy, radical gastrectomy, HIPEC, PIPAC and IP chemotherapy can be used to optimize treatment outcomes of gastric cancer patients with peritoneal metastasis.

Abstract Background Surgical resection followed by indicated adjuvant therapy offers potential curative treatment in colonic adenocarcinoma. Beyond the well-established seed and soil theory of colon cancer progression, the 'normal-appearing' tissues near the tumor are not genuinely normal and remain as remnants in patients following surgery. Our objective was to elucidate the alteration of gene expression and pathways across various distances of resection margins in right-sided colonic adenocarcinoma. Methods Twenty-seven fresh samples of primary cancer and 56 matched non-tumor tissues adjacent to the tumor (NAT) were collected from patients with resectable right-sided colon cancer. NAT were systematically obtained at varying distances (1, 5, and 10 cm) on both proximal and distal sides. Comprehensive gene expression analysis was performed using 770-gene PanCancer Progression Panel, delineating distinctive pathways and functional predictions for each region. Results Distinctive gene signatures and pathways exhibited by normal-appearing tissues were discovered at varying distances from cancer. Notably, SFRP2, PTGDS, COL1A1, IL1B, THBS2, PTGIS, COL1A2, NPR1, and BGN were upregulated, while ENPEP, MMP1, and NRCAM were downregulated significantly in 1-cm tissue compared to farther distances. Substantial alterations in the extracellular matrix (ECM) and prostaglandin/thromboxane synthesis were significantly evident at the 1-cm distance. Functional analysis indicated enhanced cell viability and survival, alongside reduced cellular death and apoptosis. Conclusions Different distances exerted a significant impact on gene alteration within the normal-looking mucosa surrounding primary cancer, influenced by various mechanisms. These findings may highlight potential therapeutic targets related to the ECM and prostaglandin/thromboxane pathways for treatment strategies.

Colorectal cancer (CRC) is the third most common malignancy cause of cancer-related mortality worldwide. Epithelial-mesenchymal transition (EMT) promotes cancer metastasis and a tumour-based Glasgow EMT score was associated with adverse clinical features and poor prognosis. In this study, the impact of using the established five tumour-based EMT markers consisting of E-cadherin (E-cad), β-catenin (β-cat), Snail, Zeb-1, and Fascin in combination with the stromal periostin (PN) on the prediction of CRC patients' prognosis were invesigated. Formalin-fixed paraffin-embedded tissues of 202 CRC patients were studies the expressions of E-cad, β-cat, Snail, Zeb-1, Fascin, and PN by immunohistochemistry. Individually, cytoplasmic Fascin (Fc), cytoplasmic Snail (Sc), nuclear Snail (Sn), stromal Snail (Ss), and stromal PN (Ps) were significantly associated with reduced survival. A combination of Ps with Fc, Fs, and Sn was observed in 2 patterns including combined Fc, Fs, and Ps (FcFsPs) and Fc, Sn, and Ps (FcSnPs). These combinations enhanced the prognostic power compared to individual EMT markers and were independent prognostic markers. As the previously established scoring method required five markers and stringent criteria, its clinical use might be limited. Therefore, using these novel combined prognostic markers, either FcFsPs or FcSnPs, may be useful in predicting CRC patient outcomes.

Abstract Background Single-incision laparoscopic colectomy (SILC) was introduced as a novel minimally invasive technique. The benefits of this technique include reducing number of the incision and cosmetic improvement. Unlike the conventional laparoscopic colectomy, majority of previously reported SILC need to be performed using special curved or articulated instruments. The purpose of this study is to demonstrate our initial experience of SILC, which could be performed using the standard laparoscopic instruments. Material and methods Retrospective review of 14 patients who underwent SILC at Siriraj Hospital from May to December 2010, patient's demographic data, perioperative outcomes, early postoperative complications and pathological data were collected and analyzed. Results The mean age of all patients was 60 years. The most common operation with SILC was sigmoidectomy (n = 9), followed by right hemicolectomy (n = 2), left hemicolectomy (n = 1), anterior resection (n = 1), and total colectomy (n = 1). The trocar insertion techniques were multi-fascial incision using regular port (n = 11) and GelPOINT® (n = 3). The mean operative time was 155 minutes (range 90-280) and the mean estimate blood loss was 32.1 mL (range 10-100). All patients were successfully operated without conversion. The mean length of hospital stay was 9 days (range 5-20). There was no mortality. The pathological results revealed colorectal cancer (n = 12), neoplastic polyp (n = 1) and Familial adenomatous polyposis (FAP) (n = 1). The mean number of lymph nodes retrieval was 16.6 (range 3-34). Conclusion SILC can successfully and safely be performed with standard laparoscopic instruments. This technique might be an alternative procedure to conventional laparoscopic colectomy with better cosmetic result.

Abstract Traditionally, there are several approaches to manage left-sided colonic carcinoma obstruction, such as tumor resection with primary anastomosis, tumor resection with end-colostomy and loop-colostomy. Recently, colonic stent insertion was introduced as a bridge prior to definite surgery. We demonstrated a hybrid approach for obstructed sigmoid carcinoma using colonic stent, followed by single incision laparoscopic colectomy (SILC). A 58 year-old man presented with complete left-sided colonic obstruction. He underwent emergency colonoscopy with metallic stent placement. One week later, he was performed SILC. He recovered well after the operation without any postoperative complications. The pathological result showed adequacy of oncologic resection. This hybrid approach of colonic stent insertion and SILC can be safely performed.

Abstract For Choledochal cyst type I, complete excision of cyst with Roux-en-Y hepaticojejunostomy anastomosis is the treatment of choice. It has been performed laparoscopically with the advancement of laparoscopic skill. Recently, a telemanipulative robotic surgical system was introduced, providing laparoscopic instruments with wrist-arm technology and 3-dimensional visualization of the operative field. We present a case of robot-assisted total excision of a choledochal cyst type I and biliary reconstruction in a 14-year-old girl. No intraoperative complications or technical problems were encountered. An intraabdominal collection occurred and was successfully treated with continuous percutaneous drainage. At one-year follow-up, she is doing well without evidence of recurrent cholangitis.

In the highly contagious coronavirus disease 2019 pandemic, aerosol-generating procedures (AGPs) are associated with high-risk of transmission. Upper gastrointestinal endoscopy is a procedure with the potential to cause dissemination of bodily fluids. At present, there is no consensus that endoscopy is defined as an AGP. This review discusses the current evidence on this topic with additional management. Prevailing publications on coronavirus related to upper gastrointestinal endoscopy and aerosolization from the PubMed and Scopus databases were searched and reviewed. Comparative quantitative analyses showed a significant elevation of particle numbers, implying that aerosols were generated by upper gastrointestinal endoscopy. The associated source events have also been reported. To reduce the dispersion, certain protective measures have been developed. Endoscopic unit protocols are recommended for the concerned personnel. Therefore, upper gastrointestinal endoscopy should be classified as an AGP. Proper practices should be adopted by healthcare workers and patients.

Abstract β-thalassaemia is one of the most common genetic blood diseases worldwide with over 300 mutations in the HBB gene affecting red blood cell functions. Recently, advances in genome editing technology have provided a powerful tool for precise genetic correction. Generation of patient-derived induced pluripotent stem cells (iPSCs) followed by genetic correction of HBB mutations and differentiation into haematopoietic stem/progenitor cells (HSPCs) offers a potential therapy to cure the disease. However, the biggest challenge is to generate functional HSPCs that are capable of self-renewal and transplantable. In addition, functional analyses of iPSC-derived erythroid cells are hampered by poor erythroid expansion and incomplete erythroid differentiation. Previously, we generated an immortalised erythroid cell line (SiBBE) with unique properties, including unlimited expansion and the ability to differentiate into mature erythrocytes. In this study, we report a highly efficient genetic correction of HbE mutation in the SiBBE cells using the CRISPR/Cas9 system. The HbE-corrected clones restored β-globin production with reduced levels of HbE upon erythroid differentiation. Our approach provides a sustainable supply of corrected erythroid cells and represents a valuable model for validating the therapeutic efficacy of gene editing systems.

Objective: The current standard of care for locally advanced rectal cancer is associated with multimodality therapy. Neoadjuvant chemoradiation significantly decreased the locoregional recurrence rate and improved survival. However, distant metastasis develops rather than local recurrence, which becomes the leading cause of death. This study aimed to evaluate the oncological outcomes of total neoadjuvant therapy (TNT) in locally advanced rectal cancer. Materials and Methods: This retrospective study recruited 18 patients diagnosed with locally advanced rectal adenocarcinoma (cT3-4 or cN1-2), treated with consolidation TNT. The primary endpoint was pathological complete response (pCR). The secondary endpoint included postoperative outcomes, local recurrences, and distant metastases. Results: The pathologic complete response was observed in 27.8% of consolidation therapy cases. Downstaging of the T-category was achieved in 10 (55.6%) patients, and downstaging of the N-category was achieved in 14 (77.8%) patients. Only one patient who achieved pCR developed distant metastasis, whereas all patients with pathological stage III developed distant metastasis. Conclusions: TNT is a promising approach for patients with locally advanced rectal cancer. This strategy improved complete pathologic response rates in TNT, and pCR was found to be associated with fewer local recurrences and greater disease-free survival.

Objective: Enhanced Recovery After Surgery (ERAS) is a multidisciplinary approach that aims to optimize perioperative management, promote postoperative recovery, reduce postoperative complications, and improve long-term survival. The current study aimed to evaluate and compare the postoperative physical activity after gastrectomy between patients who underwent upper gastrointestinal surgery according to ERAS and those who underwent surgery based on the conventional care (CC) protocol. Materials and Methods: This prospective and retrospective review enrolled 60 patients (n = 31, ERAS group; n = 29, CC protocol group) diagnosed with adenocarcinoma of the stomach and esophagogastric junction who underwent curative surgical resection. Physical outcomes, including body weight, body mass index, body fat percentage, basal metabolic rate, muscle mass, gait speed, and handgrip strength at the preoperative and immediate postoperative periods and at 1, 3, and 6 months postoperatively, were comparedbetween the ERAS and CC protocol groups. Results: One month after surgery, the ERAS group had a lower percentage of body weight loss than the CC protocol group. There was no significant difference in terms of muscle mass loss between the two groups. The hand grip strength of the ERAS group increased after surgery. Further, at 1 month postoperatively, the gait speed of patients who underwent total gastrectomy in the ERAS group was significantly higher than that of patients in the CC protocol group. Conclusion: ERAS for gastrectomy was associated with a lower percentage of weight loss and a trend toward physical activity enhancement in the early postoperative period.

Abstract Background The SARS-CoV2 virus has been identified in abdominal cavity of the COVID-19 patients. Therefore, the potential viral transmission from any surgical created smoke in these patients is of concern especially in laparoscopic surgery. This study aimed to compare the amount of surgical smoke and surgical field contamination between laparoscopic and open surgery in fresh cadavers. Methods Cholecystectomy in 12 cadavers was performed and they were divided into 4 groups: laparoscopic approach with or without smoke evacuator, and open approach with or without smoke evacuator. The increased particle counts in surgical smoke of each group were analyzed. In the model of appendectomy, surgical field contamination under ultraviolet light and visual contamination scale between laparoscopic and open approach were compared. Results Open cholecystectomy significantly produced a greater amount of overall particle sizes, particle sizes

Abstract Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5′-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients’ erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV.

Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.

Objective: Bariatric surgery is considered the most effective treatment for morbid obesity, and is increasingly performed in Thailand and globally. We aimed to establish the outcomes of bariatric surgery performed at Siriraj Hospital, Bangkok. Materials and Methods: This was a retrospective study of patients who underwent bariatric surgery between January 2012 and June 2016. Results: The records of the first 100 patients who underwent bariatric surgery were reviewed, comprising 58 patients who underwent laparoscopic sleeve gastrectomy (LSG) and 42 patients who underwent laparoscopic Roux-en-Y gastric bypass (LRYGB). The median patient age, preoperative body weight, and BMI were 36 years old, 129 kg, and 46.3 kg/m2. All the procedures were performed by a laparoscopic approach. The median operative times for LSG and LRYGB were 156 [85-435] and 265 [180-435] minutes. The median hospital stay was 3 days [3-14]. The major complication rate was 4%. There was no mortality in the 30-day postoperative period. The mean %excess weight loss (%EWL) of LSG was 56.8 ± 19.8%, 59.9 ± 21.7%, and 55.1 ± 21.3%, at 1, 2, and 3 years after surgery. The mean %EWL of LRYGB was 67 ± 18.3%, 66.2 ± 21.4%, and 63.6 ± 19.9%, at 1, 2, and 3 years after surgery. In the patients with type-II diabetes mellitus, 67% had complete diabetic remission at 1 year. The median FBS dropped from 127 to 99 mg/dL (p < 0.001) and HbA1c from 6.6% to 5.5% (p < 0.001). The remission rates of hypertension and dyslipidemia were 58% and 73%. Conclusion: The bariatric procedures are safe with a low complication rate. The procedures also provide good outcomes in postoperative weight loss and comorbidity resolution.

Colorectal cancer (CRC) is the third most common malignancy cause of cancer-related mortality worldwide. Epithelial-mesenchymal transition (EMT) promotes cancer metastasis and a tumour-based Glasgow EMT score was associated with adverse clinical features and poor prognosis. In this study, the impact of using the established five tumour-based EMT markers consisting of E-cadherin (E-cad), β-catenin (β-cat), Snail, Zeb-1, and Fascin in combination with the stromal periostin (PN) on the prediction of CRC patients' prognosis were invesigated. Formalin-fixed paraffin-embedded tissues of 202 CRC patients were studies the expressions of E-cad, β-cat, Snail, Zeb-1, Fascin, and PN by immunohistochemistry. Individually, cytoplasmic Fascin (Fc), cytoplasmic Snail (Sc), nuclear Snail (Sn), stromal Snail (Ss), and stromal PN (Ps) were significantly associated with reduced survival. A combination of Ps with Fc, Fs, and Sn was observed in 2 patterns including combined Fc, Fs, and Ps (FcFsPs) and Fc, Sn, and Ps (FcSnPs). These combinations enhanced the prognostic power compared to individual EMT markers and were independent prognostic markers. As the previously established scoring method required five markers and stringent criteria, its clinical use might be limited. Therefore, using these novel combined prognostic markers, either FcFsPs or FcSnPs, may be useful in predicting CRC patient outcomes. [ABSTRACT FROM AUTHOR]

Abstract Background Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. Methods Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. Results Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. Conclusion This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

Percutaneous coronary intervention (PCI) is a common procedure for the management of coronary artery obstruction. However, it usually causes vascular wall injury leading to restenosis that limits the long-term success of the PCI endeavor. The ultimate objective of this study was to develop the targeting nanoparticles (NPs) that were destined for the injured subendothelium and attract endothelial progenitor cells (EPCs) to the damaged location for endothelium regeneration. Biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs were conjugated with double targeting moieties, which are glycoprotein Ib alpha chain (GPIbα) and human single-chain antibody variable fragment (HuscFv) specific to the cluster of differentiation 34 (CD34). GPIb is a platelet receptor that interacts with the von Willebrand factor (vWF), highly deposited on the damaged subendothelial surface, while CD34 is a surface marker of EPCs. A candidate anti-CD34 HuscFv was successfully constructed using a phage display biopanning technique. The HuscFv could be purified and showed binding affinity to the CD34-positive cells. The GPIb-conjugated NPs (GPIb-NPs) could target vWF and prevent platelet adherence to vWF in vitro. Furthermore, the HuscFv-conjugated NPs (HuscFv-NPs) could capture CD34-positive cells. The bispecific NPs have high potential to locate at the damaged subendothelial surface and capture EPCs for accelerating the vessel repair.

Background/Aims: The coronavirus disease 2019 pandemic has affected the worldwide practice of upper gastrointestinal endoscopy. Here we designed a modified N95 respirator with a channel for endoscope insertion and evaluated its efficacy in upper gastrointestinal endoscopy. Methods: Thirty patients scheduled for upper gastrointestinal endoscopy were randomized into the modified N95 (n=15) or control (n=15) group. The mask was placed on the patient after anesthesia administration and particles were counted every minute before (baseline) and during the procedure by a TSI AeroTrak particle counter (9306-04; TSI Inc.) and categorized by size (0.3, 0.5, 1, 3, 5, and 10 μm). Differences in particle counts between time points were recorded. Results: During the procedure, the modified N95 group displayed significantly smaller overall particle sizes than the control group (median [interquartile range], 231 [54-385] vs. 579 [213-1,379]×103/m3; p=0.056). However, the intervention group had a significant decrease in 0.3-μm particles (68 [-25 to 185] vs. 242 [72-588] ×103/m3; p=0.045). No adverse events occurred in either group. The device did not cause any inconvenience to the endoscopists or patients. Conclusions: This modified N95 respirator reduced the number of particles, especially 0.3-μm particles, generated during upper gastrointestinal endoscopy. [ABSTRACT FROM AUTHOR]

Abstract Background Pluripotent stem cells are attractive progenitor cells for the generation of erythroid cells in vitro as have expansive proliferative potential. However, although embryonic (ESC) and induced pluripotent (iPSC) stem cells can be induced to undergo erythroid differentiation, the majority of cells fail to enucleate and the molecular basis of this defect is unknown. One protein that has been associated with the initial phase of erythroid cell enucleation is the intermediate filament vimentin, with loss of vimentin potentially required for the process to proceed. Methods In this study, we used our established erythroid culture system along with western blot, PCR and interegation of comparative proteomic data sets to analyse the temporal expression profile of vimentin in erythroid cells differentiated from adult peripheral blood stem cells, iPSC and ESC throughout erythropoiesis. Confocal microscopy was also used to examine the intracellular localisation of vimentin. Results We show that expression of vimentin is turned off early during normal adult erythroid cell differentiation, with vimentin protein lost by the polychromatic erythroblast stage, just prior to enucleation. In contrast, in erythroid cells differentiated from iPSC and ESC, expression of vimentin persists, with high levels of both mRNA and protein even in orthochromatic erythroblasts. In the vimentin-positive iPSC orthochromatic erythroblasts, F-actin was localized around the cell periphery; however, in those rare cells captured undergoing enucleation, vimentin was absent and F-actin was re-localized to the enucleosome as found in normal adult orthrochromatic erythroblasts. Conclusion As both embryonic and adult erythroid cells loose vimentin and enucleate, retention of vimentin by iPSC and ESC erythroid cells indicates an intrinsic defect. By analogy with avian erythrocytes which naturally retain vimentin and remain nucleated, retention in iPSC- and ESC-derived erythroid cells may impede enucleation. Our data also provide the first evidence that dysregulation of processes in these cells occurs from the early stages of differentiation, facilitating targeting of future studies.

Human ZNF648 is a novel poly C-terminal C2H2 zinc finger (ZnF) protein identified amongst the most dysregulated proteins in erythroid cells differentiated from induced pluripotent stem cells. Its nuclear localization and structure indicate it is likely a DNA-binding protein. Using a combination of ZNF648 overexpression in an induced pluripotent stem cells line and primary adult erythroid cells, ZNF648 knockdown in primary adult erythroid cells and megakaryocytes, comparative proteomics and transcriptomics we show that ZNF648 is required for both erythroid and megakaryocyte differentiation. Orthologues of ZNF648 were detected across Mammals, Reptilia, Actinopterygii, in some Aves, Amphibia and Coelacanthiformes suggesting the gene originated in the common ancestor of Osteichthyes (Euteleostomi or bony fish). Conservation of the C-terminal ZnF domain is higher, with some variation in ZnF number but a core of at least six ZnF conserved across all groups, with the N-terminus recognisably similar within but not between major lineages. This suggests the N-terminus of ZNF648 evolves faster than the C-terminus, however this is not due to exon-shuffling as the entire coding region of ZNF648 is within a single exon. As for other such transcription factors, the N-terminus likely carries out regulatory functions, but showed no sequence similarity to any known domains. The greater functional constraint on the ZnF domain suggests ZNF648 binds at least some similar regions of DNA in the different organisms. However, divergence of the N-terminal region may enable differential expression, allowing adaptation of function in the different organisms.

BackgroundNo consensus has yet been reached regarding the best anesthetic technique for inguinal hernia repair. This study aimed to compare postoperative clinical outcomes and inflammatory markers among patients who were anesthetized using local, spinal, or general anesthesia for inguinal hernia repair.MethodsThis randomized controlled trial included patients scheduled to undergo elective unilateral inguinal hernioplasty at Siriraj Hospital during November 2014 to September 2015 study period. Patients were randomly assigned to the local (LA), spinal (SA), or general (GA) anesthesia groups. Primary outcomes were postoperative pain at rest and on mobilization at 8 and 24 hours after surgery.ResultsFifty-four patients were included, with 18 patients randomly assigned to each group. Patient demographic and clinical characteristics were similar among groups. There were no significant differences among groups for postoperative pain at rest or on mobilization at 8 and 24 hours after surgery. No significant differences were observed for interleukin-1β, interleukin-6, and interleukin-10 at any time points in any groups. Patients with local anesthesia was associated with less time spent in anesthesia (p = 0.010) and surgery (p = 0.009), lower intraoperative cost (p = 0.003) and total cost in hospital (p = 0.036); however, patient satisfaction in the local anesthesia group (94/100) was statistically significantly lower than the spinal and general anesthesia groups (100/100) (p = 0.010).ConclusionsNo statistically significant difference was observed among groups for postoperative pain scores, duration of hospital stays, complications, or change in inflammatory markers. However, time spent in anesthesia and surgery, the intraoperative cost and total cost for hernia repair, and patient satisfaction were significantly lower in the local anesthesia group than in the other two groups.

Abstract Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle‐cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR‐mediated genome editing of an immortalised human erythroblast cell line (BEL‐A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Fynull), GPB (S−s−U−). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof‐of‐principle demonstration of combinatorial CRISPR‐mediated blood group gene editing to generate customisable or multi‐compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.

Abstract Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. Results The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.

The generation of a sustainable supply of erythroid progenitors is essential for the reliable production of anin vitroderived red blood cell clinical product. Here the authors immortalize early human erythroblasts to generate the first cell line capable of differentiation into functional adult reticulocytes.

Background: Self-expanding metallic stents (SEMS) have been acknowledged in management of acute colorectal obstruction. The surgical approach after SEMS insertion varies from open approach to laparoscopic-assisted approach. The primary objective of this study was to compare the outcomes of laparoscopic approach and open approach after SEMS insertion. Methods: From January 2007 to December 2010, cross-sectional medical records reviewed a total of 76 patients who underwent colorectal stenting with SEMS. Patients and tumor characteristics, complications, morbidity and mortality were obtained. Results: Forty-three patients underwent SEMS placement as a bridge to surgery. Laparoscopic-assisted surgery (LS) was performed in 24 patients (55.8%), and open surgery (OS) was performed in 19 patients (44.2%). All clinicopathological parameters were matched. The technical success of SEMS was found in 42 patients (97.7%), and the clinical stent success was 100%. LS had a higher chance of primary anastomosis than OS (p=0.012; Odd ratio 2.717; 95%CI: 1.79-4.012). LS had a lower permanent ostomy rate (p=0.031; Odd ratio 0.385; 95%CI: 0.259-0.572) and lower estimated blood loss (p=0.024; Odd ratio 0.23; 95%CI: 0.006-0.086). The post-operative complications, mortality rate, recurrence rate, disease free status, and overall survival rates between the two groups were non-significant. Conclusion: Colonic stent is an effective treatment of acute malignant colonic obstruction. The authors suggest the advantage of laparoscopic approach resection after colonic stenting results in a higher primary anastomosis rate, and lower blood loss than open surgery.

Background: Single-incisionlaparoscopiccolectomy(SILC) isanevolvingtechnique withpotentialadvantages by reducing number of incisions that can reduce port-related complications and improve cosmetic results. The purposeof thisstudy wastocomparetheshort-termoutcomesbetweenSILC,hand-assistedlaparoscopiccolectomy (HALC) andstandard multi-port laparoscopic colectomy (MLC). Methods: Retrospectiveanalysesofatotalof90patientsbetweenMay2010and December2011, whounderwent SILC for coloncancer surgery, wereperformedin30patients. Clinicopathologicalparameters were matched1:1 withpatients whounderwent HALC (n=30)andMLC (n=30).Short-term outcomes werecollectedandanalyzed. Results: Operativetime wassignificantlyshorter inSILC comparedto HALC andMLC (p

Objective: Microscopic residual tumor (R1 resection) may have a significant negative impact on prognosis of patients with adenocarcinoma of stomach and esophagogastric junction. Results of R1 resection on survival and recurrence patterns have not yet been thoroughly investigated in Thailand. The aim of this study was to study the oncological outcomes of R1 resection after curative intended surgery. Methods: We retrospectively analyzed the medical records of patients who underwent curative intended surgery from 2005 to 2013 at Siriraj Hospital. Results: 162 patients underwent curative intended gastrectomy with radical lymphadenectomy. 122 (75.3%) patients received R0 resection (non-residual group), 40 (24.7%) patients had microscopic residual tumor (residual group). Microscopic residual tumor was associated with tumor size (p=0.008), tumor extension (p=0.028), pT-stage (p=0.012), pN-stage (p=0.012), and AJCC/TNM stage (p