Evaluation of ACS-NSQIP and CR-POSSUM risk calculators for the prediction of mortality after colorectal surgery: A retrospective cohort study.

Background: Several risk calculating tools have been introduced into clinical practice to provide patients and clinicians with objective, individualised estimates of procedure-related unfavourable outcomes. The currently available risk calculators (RCs) have been developed by well-endowed health systems in Europe and the USA. Applicability of these RCs in low-middle income country (LMIC) settings with wide disparities in patient population, surgical practice and healthcare infrastructure has not been adequately examined. Patients and Methods: Through this single tertiary care, LMIC-centre, retrospective cohort study, we investigated the accuracy of the two most widely validated RCs - American College of Surgeons-National Surgical Quality Improvement Program (ACS-NSQIP) RC and ColoRectal Physiological and Operative Severity Score for the Enumeration of Mortality and Morbidity (CR-POSSUM) - for the prediction of mortality in patients undergoing elective and emergency colorectal surgery (CRS) from March 2013 to March 2020. Online RCs were used to predict mortality and other outcomes. Accuracy was assessed by Brier score and C statistic. Results: Of 105 patients, 69 (65.71%) underwent elective and 36 (34.28%) underwent emergency CRS. The 30-day overall mortality was 12 - elective 1 (1.4%) and emergency 11 (30.5%). ACS-NSQIP RC performed better for the prediction of overall (C statistic 0.939, Brier score 0.065) and emergency (C statistic 0.840, Brier score 0.152) mortality. However, for elective CRS mortality, Brier scores were similar for both models (0.014), whereas C statistic (0.934 vs. 0.890) value was better for ACS-NSQIP. Conclusions: Both ACS-NSQIP and CR-POSSUM were accurate for the prediction of CRS mortality. However, compared to CR-POSSUM, ACS-NSQIP performed better. The overall performance of both models is indicative of their wider applicability in LMIC centres also.

Diverse Clinical Isolates of Mycobacterium tuberculosis Develop Macrophage-Induced Rifampin Tolerance.

The Mycobacterium tuberculosis lineage 4 strains CDC1551 and H37Rv develop tolerance to multiple antibiotics upon macrophage residence. To determine whether macrophage-induced tolerance is a general feature of clinical M. tuberculosis isolates, we assessed macrophage-induced drug tolerance in strains from lineages 1-3, representing the other predominant M. tuberculosis strains responsible for tuberculosis globally. All 3 lineages developed isoniazid tolerance. While lineage 1, 3, and 4 strains developed rifampin tolerance, lineage 2 Beijing strains did not. Their failure to develop tolerance may be explained by their harboring of a loss-of-function mutation in the Rv1258c efflux pump that is linked to macrophage-induced rifampicin tolerance.

Outcomes of Emergency Laparotomy (EL) Care Protocol at Tertiary Care Center from Low-Middle-Income Country (LMIC).

BACKGROUND: Emergency laparotomy mortality ranges between 10 and 20% in best of Western healthcare systems and is currently a major focus for quality improvement programs. In contrast, emergency surgery scenario in LMIC is largely undefined, often neglected and complex (large burden of diseases but only limited capacity for adequate treatment). We evaluated the efficacy of 'EL care protocol' aimed at cost-effective optimal utilization of best available local expertise and infrastructure. METHODS: One hundred and two consecutive adult patients (≥16 years) who underwent EL from December 2012-December 2015 at a private tertiary hospital were retrospectively analyzed. The patients who underwent emergency laparoscopic procedures were excluded from the analysis. The EL care protocol included. (1) Admission to surgical intensive care unit for pre- and postoperative optimization. (2) Preferred radiologic investigation: abdominal computed tomography (CT) scan. (3) Surgery and critical care by senior surgical gastroenterologists and internists/anesthesiologists, respectively. Outcome measures were procedure-related complications (Clavien-Dindo classification), readmissions and costs. RESULTS: Of the 102 patients, there were 62 males and 40 females with median age of 60 (range 16-93) years. There were no complications in 22 (21.6%) patients, while Clavien-Dindo complications grade I or II occurred in 48 (47%) patients. Grade V Clavien-Dindo complications and the 30-day mortality were similar of 19 (18.6%). The readmission rate was 8 (7.8%). The expected mortality for the study group by P-POSSUM score was 31.2 (30.6%). The ratio (O/E) of observed to expected mortality was 0.61. The all inclusive median cost of treatment was INR 379,255 ($5590). CONCLUSIONS: LMIC centers should develop their own center-specific EL care protocols to improve outcomes of EL.

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media.

The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression.

PURPOSE: The aim of the present study was to determine whether supplementation of resveratrol, a stilbenoid antioxidant with therapeutic significance, influences goat (Capra hircus) oocyte maturation and subsequent embryonic development and expression of apoptosis and early embryonic development-related genes. METHODS: Five different concentrations of resveratrol (0.1, 0.25, 0.5, 2.0 and 5.0 µM) were used in in vitro maturation (IVM) medium. Cell tracker blue and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent stains were used to assay intracellular glutathione and reactive oxygen species levels in mature oocytes. Parthenogenetic activation and hand-made cloning were performed to check the developmental potential following resveratrol treatment. We used quantitative real-time PCR to analyze embryonic gene expression. RESULT: Compared to control, no significant improvement was observed in nuclear maturation in resveratrol-treated groups and at 5.0 µM concentration maturation rate decreased significantly (P < 0.05). But resveratrol treatment at the concentrations of 0.25, 0.5 µM significantly reduced intracellular ROS, and increased GSH concentrations. Oocytes treated with 0.25, 0.5 µM resveratrol when subsequently used for PA and HMC, higher extent of blastocyst yields were observed. Expression analysis of proapoptotic (Bax) gene in mature oocytes, cumulus cells, and HMC-derived blastocysts revealed lesser transcript abundances in various resveratrol-treated groups., however no change in the same was observed for antiapoptotic gene (Bcl2). Differential expression of genes associated with developmental competence and nuclear reprogramming was also observed in HMC-derived blastocysts. CONCLUSION: Our results show that resveratrol treatment at optimum concentrations (0.25 and 0.5 µM) during IVM produced beneficial microenvironment within oocytes by increasing the intracellular GSH, decreasing ROS level and this in turn, stimulated embryonic development and regulated gene expression.

Solid Pseudopapillary Tumor of Pancreas with Sinistral Portal Hypertension: A Rare Presentation.

An elderly male presented with complaint of dull aching pain in left upper abdomen of 2 months' duration. He was well built, and on abdominal examination, a large lump was palpable in left lumbar region. His biochemical, hematological parameters and tumor markers including CA 19.9 were within prescribed normal limits. Contrast enhanced computed tomography (CT) of the abdomen revealed large, well-defined, mixed solid, and cystic lesion arising from distal body-tail of the pancreas extending up to splenic hilum. Chronic thrombosis involving retro pancreatic splenic vein with multiple perisplenic, peripancreatic, and perigastric collaterals were noted. Based on characteristic CT abdomen findings, a diagnosis of solid pseudopapillary tumor (SPT) of the pancreas with sinistral portal hypertension (SPH) was made. The patient was planned for open distal pancreatectomy with splenectomy. At surgery, splenic flexure of colon was densely adhered to the tumor, and hence en bloc resection of colon was also performed. Postoperative period was uneventful, and he was discharged from the hospital on postoperative day 6. Histopathology reported solid pseudopapillary tumor of the pancreas, 22 cm in the largest dimension. SPT of the pancreas rarely present with SPH. At 22-cm size, this may be one of the largest SPTs reported in the English literature to date.

Current Oncologic Standards for Surgery of Small Bowel Cancers.

PURPOSE: The last 2 decades has witnessed efforts towards standardization of surgery for small bowel cancers. The proposed recent guidelines/recommendations pertaining to choice of procedure and extent of lymphadenectomy are based on analysis of data from high volume centres of excellence. We evaluated whether these recently proposed oncologic recommendations can be replicated in the setting of single centre/team. METHODS: This was a retrospective analysis of consecutive adult patients (age ≥ 18 years) who underwent surgery for tumours of small intestine (duodenum, jejunum, and ileum) by the same team of surgeons from 01/01/2010 to 12/31/2019. The procedure performed and lymph nodes harvested during pathologic examination were compared with recent recommendations. RESULTS: Of the 32 patients (20 males and 12 females), mean age was 52.4 (range 31-77) years. Twenty-nine (90.6%) patients underwent surgery for small bowel cancers. Duodenum was the most common site, while NET was the commonest cancer. Whipple's procedure was performed for tumours of II part of duodenum, while for the rest, segmental resection was performed. The median number of lymph nodes examined for duodenal adenocarcinoma and NET was 14 and 9, respectively. For jejunal/ileal adenocarcinoma, median lymph node number examined was 11. CONCLUSION: Our study shows that these recently proposed standards for surgery of small bowel cancers are achievable if basic principles of oncologic surgery are followed.

Derivation of oocyte-like cells from putative embryonic stem cells and parthenogenetically activated into blastocysts in goat.

Germ cells are responsible for the propagation of live animals from generation to generation, but to surprise, a steep increase in infertile problems among livestock poses great threat for economic development of human race. An alternative and robust approach is essential to combat these ailments. Here, we demonstrate that goat putative embryonic stem cells (ESCs) were successfully in vitro differentiated into primordial germ cells and oocyte-like cells using bone morphogenetic protein-4 (BMP-4) and trans-retinoic acid (RA). Oocyte-like cells having distinct zonapellucida recruited adjacent somatic cells in differentiating culture to form cumulus-oocyte complexes (COCs). The putative COCs were found to express the zonapellucida specific (ZP1 and ZP2) and oocyte-specific markers. Primordial germ cell-specific markers VASA, DAZL, STELLA, and PUM1 were detected at protein and mRNA level. In addition to that, the surface architecture of these putative COCs was thoroughly visualized by the scanning electron microscope. The putative COCs were further parthenogenetically activated to develop into healthy morula, blastocysts and hatched blastocyst stage like embryos. Our findings may contribute to the fundamental understanding of mammalian germ cell biology and may provide clinical insights regarding infertility ailments.

Isolated loop pancreatic remnant drainage following pancreaticoduodenal resection.

BACKGROUND/AIMS: Using technology-intensive postoperative critical care, interventional radiology and consequent better management of pancreaticojejunal anastomosis (PJA) leaks, the perioperative mortality of pancreaticoduodenal resection (PDR) at high volume Western centers ranges from 1-5%. Facilities for such sophisticated care are not available in most hospitals in the developing world. We hypothesized that by using an isolated Roux loop for the PJA to minimize the consequences of a leak, it might be feasible to perform PDR with comparable results. METHODOLOGY: From August 1996 to December 2002, 125 consecutive patients (98 males and 27 females with a mean age of 54 years) with peri-ampullary or pancreatic head carcinomas underwent PDR with the PJA made to an isolated Roux loop of jejunum. A prospectively maintained database was analyzed for perioperative mortality, morbidity, hospital stay and costs. RESULTS: The perioperative mortality was 7 (5.6%) and morbidity 52 (42%). Pancreatic fistulae developed in 15 (12%) patients and biliary or intestinal fistulae developed in 1(0.8%) patient each. Five (4%) patients underwent relaparotomy. The median hospital stay was 13 days (6-46 days). CONCLUSIONS: Using an isolated Roux loop for PJA, centers with limited resources can perform PDR to achieve perioperative outcomes comparable to those reported from more sophisticated centers.

Issues in management of pancreatic pseudocysts.

Pancreatic pseudocysts (PPs) comprise more than 80% of the cystic lesions of the pancreas and cause complications in 7-25% of patients with pancreatitis or pancreatic trauma. The first step in the management of PPs is to exclude a cystic tumor. A history of pancreatitis, no septation, solid components or mural calcification on CT scan and high amylase content at aspiration favor a diagnosis of PP. Endoscopic ultrasound (EUS)-guided FNAC is a valuable diagnostic aid. Intervention is indicated for PPs which are symptomatic, in a phase of growth, complicated (infected, hemorrhage, biliary or bowel obstruction) or in those occurring together with chronic pancreatitis and when malignancy cannot be unequivocally excluded. The current options include percutaneous catheter drainage, endoscopy and surgery. The choice depends on the mode of presentation, the cystic morphology and available technical expertise. Percutaneous catheter drainage is recommended as a temporizing measure in poor surgical candidates with immature, complicated or infected PPs. The limitations include secondary infection and pancreatic fistula in 10-20% of patients which increase complications following eventual definitive surgery. Endoscopic therapy for PPs including cystic-enteric drainage (and transpapillary drainage), is an option for PPs which bulge into the enteric lumen which have a wall thickness of less than 1 cm and the absence of major vascular structures on EUS in the proposed tract or those which communicate with the pancreatic duct above a stricture. Surgical internal drainage remains the gold standard and is the procedure of choice for cysts which are symptomatic or complicated or those having a mature wall,. Being more versatile, a cystojejunostomy is preferred for giant pseudocysts (>15 cm) which are predominantly inframesocolic or are in an unusual location. In PPs with coexisting chronic pancreatitis and a dilated pancreatic duct, duct drainage procedures (such as longitudinal pancreaticojejunostomy) should be preferred to a cyst drainage procedure.

Sinistral portal hypertension. A case report.

Sinistral portal hypertension is a clinical syndrome of gastric variceal hemorrhage in the setting of splenic vein thrombosis due to a primary pancreatic pathology. The distinguishing features from other forms of portal hypertension are preserved liver function and a patent extrahepatic portal vein. The important causes include acute and chronic pancreatitis, pancreatic pseudocysts and pancreatic carcinomas. Benign pancreatic neoplasms only rarely cause sinistral portal hypertension. Splenic vein thrombosis complicates 7-20% of patients having pancreatitis or a pancreatic pseudocyst; however, bleeding occurs in only approximately 5% of patients. The diagnosis of sinistral portal hypertension is achieved by a combination of gastroscopy, liver function tests, ultrasound examination (with Doppler) and/or contrast-enhanced CT scan of the abdomen. A mere demonstration of sinistral portal hypertension does not warrant intervention. An expectant management is justifiable in asymptomatic patients with pancreatitis. However, concomitant splenectomy may be considered in patients undergoing operative treatment of symptomatic chronic pancreatitis if sinistral portal hypertension and gastroesophageal varices are present. In patients presenting with gastric variceal hemorrhage, splenectomy (with treatment for the primary pancreatic pathology, e.g. distal pancreatectomy) is curative with excellent long term results.

Distinguishing between periampullary carcinoids and carcinomas--is this possible preoperatively?

It is difficult to distinguish between carcinoid tumors of the pancreatic head and periampullary region and carcinomas preoperatively. Between 1996 and 2002, 125 consecutive pancreaticoduodenectomies done by us for periampullary tumors (14 carcinoids, 111 carcinomas) were analyzed. Patients with carcinoid tumors had significantly younger mean age (48 vs. 54 years), longer history (32 vs. 8 weeks), lower serum total bilirubin levels (1.4 vs. 6.3 mg/dL) and on CT scan, had larger, well-localized tumors (5 cm vs. 2 cm). Their postoperative course was better with no mortality or major morbidity, whereas after resection for carcinoma 7 (6.3%) patients died and 30 (27%) had major postoperative complications. Thus, a tumor of this region in a young patient with indolent history, low bilirubin level and with CT scan depicting a large expansile lesion suggests a carcinoid. Such tumors may be safely resected with low postoperative morbidity and mortality and good long-term prognosis.

Squamous cell carcinoma of stomach.

Squamous cell carcinomas in the gastrointestinal tract are known to occur in oro-pharynx, esophagus and anal canal. Gastric squamous cell carcinoma is a rare epithelial tumour. Surgical treatment is the same as that for an adenocarcinoma, though prognosis is probably worse.

Management of Giant Gastric Ulcer Perforation: Report of a Case and Review of the Literature.

Giant gastric ulcer (GGU) is defined as an ulcer more than 3 cm in diameter. Now infrequent in clinical practice, in the pre-H2 receptor antagonist (H2RA) era, the incidence of GGU varied between 12 and 24% of all gastric ulcers. Proton pump inhibitors reportedly achieve better healing rates and symptom relief in comparison to H2RA. The GGU is associated with high incidence of serious complications such as hemorrhage. A perforated GGU though rare (<2%) offers serious challenges in management. We report one such case wherein the role of multidetector CT scan (MDCT) for diagnosis and treatment planning, surgical options for GGU perforations, and factors affecting outcome are discussed.