Surgical repair of endoscopy-induced colonic perforations: a case-matched study of short-term morbidity and mortality.

BACKGROUND: Our aim was to describe the clinical outcomes of surgical interventions performed for the management of colonoscopy-related perforations and to compare these outcomes with those of matched colorectal surgeries performed in elective and emergency settings. METHODS: We included patients with endoscopic colonic perforation who underwent surgical intervention from the 2014-2017 National Surgery Quality Improvement Program participant use data colorectal targeted procedure file. The primary outcome in this study was short term surgical morbidity and mortality. Patients (group 1) were matched with 1:2 ratio to control patients undergoing same surgical interventions for other indications on an elective (group 2) or emergency basis (group 3). Bivariate analysis was conducted to compare categorical variables between the three groups, and multivariate logistic regression was used to evaluate the association between the surgical indication and 30-day postoperative outcomes. RESULTS: A total of 590 patients were included. The average age of the patients was 66.5±13.6 with female gender predominance (381, 64.6%) The majority of patients underwent open colectomy (365, 61.9%) while the rest had suturing (140, 23.7%) and laparoscopic colectomy (85, 14.4%). Overall mortality occurred in 4.1% and no statistically significant difference in mortality was found between the three techniques (P=0.468). Composite morbidity occurred in 163 patients (27.6%). It was significantly lower in laparoscopic colectomy (14.1%) compared to 30.2% and 29.4% in open colectomy and suturing approaches (P=0.014). Patients undergoing colectomy for iatrogenic colonic perforation had less mortality, infection rates and sepsis, as well as bleeding episodes compared to those who had colectomy on an emergent basis. Outcomes were comparable between the former group and patients undergoing elective colectomy for other indications. CONCLUSIONS: Surgical management of colonoscopy related perforations is safe and effective with outcomes that are similar to that of patients undergoing elective colectomy.

Anesthesia Considerations of a Pregnant Woman With COVID-19 Undergoing Cesarean Delivery: A Case Report.

Physiologic changes during pregnancy may increase the risk of coronavirus disease 2019 (COVID-19) infection. Limited data show serious complications of COVID-19 infection and pregnancy. Severe adverse maternal and perinatal outcomes such as preterm delivery, intensive care unit admission, and neonatal and intrauterine death have been reported. Our knowledge of the epidemiology, pathogenesis, disease progression, and clinical course of COVID-19 is continually changing as more information and evidence emerge. The present case adds further insights on COVID-19 and anesthesia considerations for patients undergoing cesarean delivery. In this case report, we describe a successful spinal anesthetic in a pregnant woman with confirmed COVID-19. To prepare for the likelihood of caring for women during labor and cesarean delivery, anesthesia professionals must know how to provide safe, patient-centered care and how to protect every member of the obstetric team from exposure to the virus. In addition, it is paramount that our profession shares our experiences and practices to help guide our multidisciplinary approach in delivering the best care possible to these women.

Founder events, isolation, and inbreeding: Intercontinental genetic structure of the domestic ferret.

Domestication and breeding for human-desired morphological traits can reduce population genetic diversity via founder events and artificial selection, resulting in inbreeding depression and genetic disorders. The ferret (Mustela putorius furo) was domesticated from European polecats (M. putorius), transported to multiple continents, and has been artificially selected for several traits. The ferret is now a common pet, a laboratory model organism, and feral ferrets can impact native biodiversity. We hypothesized global ferret trade resulted in distinct international genetic clusters and that ferrets transported to other continents would have lower genetic diversity than ferrets from Europe because of extreme founder events and no hybridization with wild polecats or genetically diverse ferrets. To assess these hypotheses, we genotyped 765 ferrets at 31 microsatellites from 11 countries among the continents of North America, Europe, and Australia and estimated population structure and genetic diversity. Fifteen M. putorius were genotyped for comparison. Our study indicated ferrets exhibit geographically distinct clusters and highlights the low genetic variation in certain countries. Australian and North American clusters have the lowest genetic diversities and highest inbreeding metrics whereas the United Kingdom (UK) cluster exhibited intermediate genetic diversity. Non-UK European ferrets had high genetic diversity, possibly a result of introgression with wild polecats. Notably, Hungarian ferrets had the highest genetic diversity and Hungary is the only country sampled with two wild polecat species. Our research has broad social, economic, and biomedical importance. Ferret owners and veterinarians should be made aware of potential inbreeding depression. Breeders in North America and Australia would benefit by incorporating genetically diverse ferrets from mainland Europe. Laboratories using ferrets as biomedical organisms should consider diversifying their genetic stock and incorporating genetic information into bioassays. These results also have forensic applications for conserving the genetics of wild polecat species and for identifying and managing sources of feral ferrets causing ecosystem damage.

Disease-specific motifs can be identified in circulating nucleic acids from live elk and cattle infected with transmissible spongiform encephalopathies.

To gain insight into the disease progression of transmissible spongiform encephalopathies (TSE), we searched for disease-specific patterns in circulating nucleic acids (CNA) in elk and cattle. In a 25-month time-course experiment, CNAs were isolated from blood samples of 24 elk (Cervus elaphus) orally challenged with chronic wasting disease (CWD) infectious material. In a separate experiment, blood-sample CNAs from 29 experimental cattle (Bos taurus) 40 months post-inoculation with clinical bovine spongiform encephalopathy (BSE) were analyzed according to the same protocol. Next-generation sequencing provided broad elucidation of sample CNAs: we detected infection-specific sequences as early as 11 months in elk (i.e. at least 3 months before the appearance of the first clinical signs) and we established CNA patterns related to BSE in cattle at least 4 months prior to clinical signs. In elk, a progression of CNA sequence patterns was found to precede and correlate with macro-observable disease progression, including delayed CWD progression in elk with PrP genotype LM. Some of the patterns identified contain transcription-factor-binding sites linked to endogenous retroviral integration. These patterns suggest that retroviruses may be connected to the manifestation of TSEs. Our results may become useful for the early diagnosis of TSE in live elk and cattle.

Gastrointestinal complications of diabetes.

Gastrointestinal complications of diabetes include gastroparesis, intestinal enteropathy (which can cause diarrhea, constipation, and fecal incontinence), and nonalcoholic fatty liver disease. Patients with gastroparesis may present with early satiety, nausea, vomiting, bloating, postprandial fullness, or upper abdominal pain. The diagnosis of diabetic gastroparesis is made when other causes are excluded and postprandial gastric stasis is confirmed by gastric emptying scintigraphy. Whenever possible, patients should discontinue medications that exacerbate gastric dysmotility; control blood glucose levels; increase the liquid content of their diet; eat smaller meals more often; discontinue the use of tobacco products; and reduce the intake of insoluble dietary fiber, foods high in fat, and alcohol. Prokinetic agents (e.g., metoclopramide, erythromycin) may be helpful in controlling symptoms of gastroparesis. Treatment of diabetes-related constipation and diarrhea is aimed at supportive measures and symptom control. Nonalcoholic fatty liver disease is common in persons who are obese and who have diabetes. In persons with diabetes who have elevated hepatic transaminase levels, it is important to search for other causes of liver disease, including hepatitis and hemochromatosis. Gradual weight loss, control of blood glucose levels, and use of medications (e.g., pioglitazone, metformin) may normalize hepatic transaminase levels, but the clinical benefit of aggressively treating nonalcoholic fatty liver disease is unknown. Controlling blood glucose levels is important for managing most gastrointestinal complications.

Celecoxib pre-treatment in human colorectal adenocarcinoma patients is associated with gene expression alterations suggestive of diminished cellular proliferation.

Cancer cells treated with the cyclooxygenase-2 inhibitor celecoxib show growth inhibition and induced apoptosis. This study was conducted to determine if the same processes are relevant to celecoxib's effects on human colorectal adenocarcinomas treated in vivo. A cohort of 23 patients with primary colorectal adenocarcinomas was randomised to receive a 7-d course of celecoxib (400mg b.i.d.) or no drug prior to surgical resection. Gene expression profiling was performed on resected adenocarcinomas from the cohort of patients. Using fold change (>1.5) and p-value (<0.05) cut-offs, 190 genes were differentially expressed between adenocarcinomas from patients receiving celecoxib and those that did not. The celecoxib pre-treated samples showed decreased expression levels in multiple genes involved in cellular lipid and glutathione metabolism; changes associated with diminished cellular proliferation. Celecoxib pre-treatment for 7 d in vivo is associated with alterations in colorectal adenocarcinoma gene expression which are suggestive of diminished cellular proliferation.

A mouse model for developing treatment for secondary liver tumors.

Metastatic colorectal cancer is the second most common cause of cancer mortality. The liver is a common site of metastasis and only a minority of patients with liver metastases are candidates for potentially curative surgical resection. The treatment of patients with unresectable liver tumors is a major clinical problem and survival remains low. Many animal models of hepatic metastasis do not result in disease which resembles the advanced cancer setting. The purpose of this study was to establish a murine model for use in the evaluation of therapy for secondary liver cancer. Human colon cancer cells were injected directly into the portal vein of nude mice. Magnetic resonance imaging (MRI), performed weekly, was used to follow the time-course and characteristics of tumor growth. As expected, tumor size increased proportionately with time, following inoculation, and the MRI images correlated well with gross pathology findings at necropsy with respect to both tumor size and location. The tumors retained important morphology and biological characteristics of human colon cancer. Mice bearing liver metastasis were treated with irinotecan or drug vehicle. MRI evaluation pre/post-therapy gave an objective measure of therapeutic response. Irinotecan therapy was able to double the survival (median 76-93 days) compared to vehicle alone (median 43-46 days). This murine model is reproducible, rapid, inexpensive and has an excellent success rate for the development of liver metastasis (100%). When used in conjunction with small animal MRI, this model allows the efficient evaluation of the therapeutics of liver metastasis without the use of repeated laparotomy or splenectomy and without requiring large numbers of animals undergoing terminal experiments.

Identification of a lens-specific cis-acting element within the basal promoter of the human lens intrinsic membrane protein MP19 gene (LIM2).

Using overlapping and mutant oligonucleotides as probes, gel mobility assays and competition experiments identified a sequence from -47 to -32 bp upstream of the LIM2 CAP site, which a lens protein complex bound with high affinity which appeared to bind only to the "sense" strand of the double-stranded DNA molecule. This sequence consisted of a string of four guanine residues followed by seven other nucleotides (AACCTAA) and followed by another four guanines, i.e. GGGGAACCTAAGGGG, called the Hsu element. Promoter-CAT constructs containing this sequence or mutations of the sequence indicated that the Hsu element is located within the basal promoter, and is essential for expression of the LIM2 gene. The trans factors binding to the Hsu element are present throughout development, and appear to be lens-specific. Since the LIM2 gene promoter does not contain a classic TATA box, the Hsu element may serve as the site for binding the RNA polymerase complex.

Does lens intrinsic membrane protein MP19 contain a membrane-targeting signal?

PURPOSE: Lens intrinsic membrane protein MP19 is the second most abundant major protein of the lens fiber cell membrane and appears to be specific to the lens. Different mutations of this protein are known to cause cataract in both humans and mice. To date, the function of MP19 in the lens is not known, nor is the mechanism by which the protein migrates to the cell membrane. The goal of this study was to determine whether or not MP19 distributes to the cell membrane directed by a peptide signal within the sequence of the molecule. METHODS: Using PCR, MP19 cDNA was truncated to yield separate fragments coding for the first 25, 36, and 64 amino acids of the MP19 polypeptide chain. These PCR fragments were further cloned into mammalian expression vector pcDNA4/TO, a tetracycline-regulated vector that, upon induction with tetracycline, allows expression of cDNA inserts within the vector. These vectors expressed each of the MP19 truncated fragments fused to EGFP. Each of the prepared plasmids was transfected into T-REx-293 cells using FuGene 6. Cloned cell lines from each of these transfections were obtained and used in the studies. The fluorescent expressed protein was viewed using confocal microscopy. Proteins from the different cell lines were isolated by different membrane extraction methods and western blot analysis was carried out to further determine the localization of expressed MP19 and MP19 truncated fragments. RESULTS: Cell lines expressing intact MP19/EGFP (with EGFP fused to the COOH-terminal end of MP19, MP19G) fusion protein were observed to traffic MP19 to the cell membrane, where it appeared to sequester in rather large pools. All of the MP19 truncations (with EGFP fused to the COOH-terminal end of each truncation; MP19-25G, MP19-36G, and MP19-64G) appeared to also traffic EGFP to the cell membrane. MP19-25G and MP19-36G did not distribute uniformly on the membrane, but appeared to localize into smaller, punctate "spots" of fluorescent material. MP19-64G distributed on the membrane similarly to MP19-25G and MP19-36G, however, the punctate areas of fluorescent material were considerably larger and similar to that demonstrated by intact MP19G. Western blot analysis of isolated total membranes, intrinsic membranes, and lipid rafts showed that MP19G and MP19-64G were associated with the intrinsic membrane fraction while MP19-25G and MP19-36G were at least 75% associated with the intrinsic membrane fraction. All of the preparations appeared to be at least 50% associated with membrane lipid rafts. However, when EGFP/MP19-25 and EGFP/MP19-36 (with EGFP fused to the NH2-terminal end of the truncated peptide, GMP19-25 or GMP19-36) were expressed, the fusion protein was observed to remain completely soluble in the cytoplasm, identical to expressed EGFP alone. Western blots of these two fusion proteins also indicated that the product did not associate with the cell membrane. In contrast, when EGFP/MP19 (with EGFP fused to the NH2-terminal end of intact MP19, GMP19) was expressed, the fusion protein did integrate into the cell membrane, identical to MP19G. Western blot analysis revealed that GMP19 also associated with lipid rafts, identical to intact MP19G. CONCLUSIONS: It appears that the first 25 amino acids of the MP19 molecule are sufficient to target the protein to the cell membrane, and apparently integrate into the membrane. With the addition of more amino acids, the polypeptide distributes in the membrane similarly to that of the intact MP19 molecule. It appears that the first 25 amino acids of the MP19 molecule is, indeed, a membrane signal and integration sequence. Also, at least part of these 25 amino acids must integrate into the cell membrane, but not extend through the cell membrane.

Localization of lens intrinsic membrane protein MP19 and mutant protein MP19(To3) using fluorescent expression vectors.

PURPOSE: [corrected] MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific heritable mutation at amino acid 15 in the MP19 protein, termed MP19To3, results in total cataract and microphthalmia in the mouse. The goals of this study were to determine the specific localization of MP19 in the cell membrane and to determine whether the mutant MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19. METHODS: MP19 and MP19To3 cDNAs were cloned into two different sets of expression vectors. The first set was composed of two vectors, pEGFP-N1 and pDsRed2-N1. The first vector expressed green fluorescent protein and the second expressed a red fluorescent protein when transfected into mammalian cells. The two lens membrane protein cDNAs were separately cloned into the vectors so that the cDNA was at the 5'-end of the fluorescent protein coding DNA. These vectors expressed each of the lens proteins fused to the fluorescent protein upon transfection into mammalian cell cultures. The second vector set was a single vector, pcDNA4/TO which must be induced in the transfected cells by tetracycline in order to express the cloned cDNAs. Each of the membrane cDNAs coupled to the fluorescent protein coding region was cut out of the first vector set and cloned into pcDNA4/TO and stable clones were isolated. Each of the prepared plasmids was transfected into human and chick embryo lens epithelial cells and human T-RexTM-293 cells. The fluorescent cells were viewed using confocal and episcopic-fluorescence microscopy. RESULTS: Each of the transfected plasmids expressed fluorescent protein in all three cell lines. MP19 was observed to transport to the cell membrane. When compared to the distribution of another, separate fusion protein consisting of a signal peptide that targets to cell membranes fused to EGFP, MP19 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. In contrast, MP19To3 protein appeared to not distribute to the cell membrane; it instead appeared to collect in a particular subcellular compartment within the cell. CONCLUSIONS: The distribution of MP19 and MP19To3 in the cell appeared to be quite distinct. MP19 was observed to distribute to the cell membrane while MP19To3 did not. The fact that the MP19To3 did not traffic to the membrane, instead appearing to be trapped within a subcellular compartment within the cell sheds further light on the cause of the cataract and microphthalmia observed in the MP19To3 mutation, and further sheds information on the pathway of MP19 transport to the cell membrane.

Temporal expression of three mouse lens fiber cell membrane protein genes during early development.

PURPOSE: Three different lens fiber cell intrinsic membrane proteins, MIP (Major Intrinsic Protein), MP19, and connexin50 (Cx50), have separately been implicated as causative candidates for congenital cataracts. The aim of this study was to determine gene transcript expression of these three proteins during successive stages of mouse embryonic development. METHODS: Total RNA was prepared from mouse embryos taken at days 9-10 (E9-E10) of gestation, heads of day 11-13 (E11-E13) embryos, and lenses of adult mice. Coupled reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine gene transcript expression of MIP, Cx50, and MP19 during embryonic development. The products of RT-PCR were further cloned into the TOPO(TM) TA vector, and further analyzed by double strand nucleotide sequencing. RESULTS: Cx50 gene expression was observed throughout the developmental period observed (E9-E13). MIP transcripts were first observed at mouse embryonic day 11.5 (E11.5) and synthesis continued throughout the developmental period observed. The gene for MP19 (Lim2) begins to express at mouse embryo day 12 (E12) and synthesis continued throughout the developmental period observed. mRNA levels for all three proteins appear to remain steady from these early embryonic stages through adulthood. CONCLUSIONS: The identified early expression of Cx50, MIP, and Lim2 transcripts in mouse embryonic stages suggests that all three proteins play very important, probably quite different, roles in lens fiber cell differentiation. Variation in the temporal expression of these three genes during the course of development suggests a critical gene-coordinated regulation throughout lens fiber cell development. These three genes clearly play important roles in early normal lens development since it is known that mutations in the sequence of each membrane protein results in cataractogenesis.