Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues

Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.     

Possible involvement of receptors in the entry of Kunjin virus into Vero cells

Summary The results obtained from electron microscopy, adsorbed and internalised virus assays and immunofluorescence studies supported that the most likely mode of entry of Kunjin virus into Vero cells was by receptor-mediated endocytosis. This was deduced indirectly from the time sequence of events that occurred. Electron microscopy revealed that endocytosis of the virus through coated vesicles had occurred. The adsorbed and internalised virus assay and immunofluorescence studies showed that there were two factors being recycled during endocytosis: the receptor for the virus and clathrin, the protein found on coated pits and vesicles. The study showed that clathrin was recycled first, followed by the receptor.     

Anorectal continence following manual and mechanical anastomosis suture. Results of a controlled study of rectal surgery

In a controlled clinical trial-manual vs. stapler anastomosis in rectal surgery-it was found that both suture techniques per se made no difference in the function of anal continence. The anal pressures at rest and sphincter contraction remained unchanged. A linear reduction of functional reservoir of the "neorectum" could be shown, which depended on the level and healing of the anastomosis. An anastomosis level at 6 cm from anocutaneous line is important for functional reasons. Anastomoses above this level do not cause any consequences for anal continence. Anastomoses below this level result in a reduced functional reservoir for at least 6 months. Within this period a decrease in anal continence is possible, especially in cases of disturbed healing of the anastomosis.     

Use of past care markers in risk-adjustment: accounting for systematic differences across providers

The European Journal of Health Economics - Risk-adjustment models are used to predict the cost of care for patients based on their observable characteristics, and to derive efficient and equitable...     

The Sydney AFF Score: A Simple Tool to Distinguish Females Presenting With Atypical Femur Fractures Versus Typical Femur Fractures

Atypical femur fractures (AFF) are a rare but serious complication of long-term bisphosphonate use. Although clearly defined by ASBMR criteria, a proportion of patients with AFFs may go unrecognized and the use of qualitative fracture criteria may lead to uncertainty in AFF diagnosis, with significant therapeutic implications. A score that rapidly and accurately identifies AFFs among subtrochanteric femur fractures using quantitative, measurable parameters is needed. In a retrospective cohort of 110 female patients presenting with AFFs or typical femur fractures (TFFs), multiple logistic regression and decision tree analysis were used to develop the Sydney AFF score. This score, based on demographic and femoral geometry variables, uses three dichotomized independent predictors and adds one point for each: (age ≤80 years) + (femoral neck width <37 mm) + (lateral cortical width at lesser trochanter ≥5 mm), (score, 0 to 3). In an independent validation set of 53 female patients at a different centre in Sydney, a score ≥2 demonstrated 73.3% sensitivity and 69.6% specificity for AFF (area under the receiver-operating characteristic curve [AUC] 0.775, SE 0.063) and remained independently associated with AFF after adjustment for bisphosphonate use. The Sydney AFF score provides a quantitative means of flagging female patients with atraumatic femur fractures who have sustained an AFF as opposed to a TFF. This distinction has clear management implications and may augment current ASBMR diagnostic criteria. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Guidelines for gastrostomy tube placement and enteral nutrition in patients with severe,refractory hypoglycemia after gastric bypass

BackgroundPostbariatric hypoglycemia (PBH) affects up to 38% of Roux-en-Y gastric bypass (RYGB) patients. Severe cases are refractory to diet and medications. Surgical treatments including bypass reversal and pancreatectomy are highly morbid and hypoglycemia often recurs. We have developed a highly effective method of treatment by which enteral nutrition administered through a gastrostomy (G) tube placed in the remnant stomach replaces oral diet: if done correctly this reverses hyperinsulinemia and hypoglycemia, yielding substantial health and quality of life benefits for severely affected patients.ObjectivesTo provide clinical guidelines for placement of a G-tube to treat postRYGB hypoglycemia, including candidate selection, preoperative evaluation, surgical considerations, and post-RYGB management.SettingStanford University Hospital and Clinics.MethodsBased on our relatively large experience with placing and managing G-tubes for PBH treatment, an interdisciplinary task force developed guidelines for practitioners.ResultsA team approach (endocrinologist, dietitian, surgeon, psychologist) is recommended. Appropriate candidates have a history of RYGB, severe hypoglycemia refractory to medical-nutrition therapy, and significantly affected quality of life. Preoperative requirements include education and expectation setting, determination of initial enteral feeding program, and establishing service with a home enteral provider. Close postoperative follow-up is needed to ensure success and may require adjustments in formula and mode/rate of delivery to optimize tolerance and meet nutritional goals. G-tube nutrition must fully replace oral nutrition to prevent hypoglycemia.ConclusionsG-tube placement in the remnant stomach represents a relatively well-tolerated and effective treatment for severe, refractory hypoglycemia after RYGB.     

Pathogenesis of severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) is a zoonotic infectious disease caused by a novel coronavirus (CoV). The tissue tropism of SARS-CoV includes not only the lung, but also the gastrointestinal tract, kidney and liver. Angiotensin-converting enzyme 2 (ACE2), the C-type lectin CD209L (also known L-SIGN), and DC-SIGN bind SARS-CoV, but ACE2 appears to be the key functional receptor for the virus. There is a prominent innate immune response to SARS-CoV infection, including acute-phase proteins, chemokines, inflammatory cytokines and C-type lectins such as mannose-binding lectin, which plays a protective role against SARS. By contrast there may be a lack of type 1 interferon response. Moreover, lymphopenia with decreased numbers of CD4+ and CD8+ T cells is common during the acute phase. Convalescent patients have IgG-class neutralizing antibodies that recognize amino acids 441-700 of the spike protein (S protein) as the major epitope.     

Glutathione conjugate mediated toxicities

Glutathione (gamma-glutamyl-L-cysteinylglycine: GSH) is present in high concentrations in most living cells and participates in a variety of vital cellular reactions. In particular, GSH protects cells from potentially toxic electrophiles formed via the metabolism of xenobiotics, and such reactions have long been associated with the process of detoxication (Baumann and Preusse, 1879; Jaffe, 1879). Compounds that form GSH conjugates are processed by gamma-glutamyl transpeptidase (gamma-GT) and dipeptidases to cysteine S-conjugates, which are usually excreted in urine as their corresponding mercapturic acids (S-substituted N-acetyl-L-cysteine conjugates). In addition, GSH peroxidase activity, whether catalyzed by the selenium-dependent GSH peroxidase or by the GSH S-transferases, serves to detoxify hydrogen peroxide and organic hydroperoxides. However, in recent years, evidence indicating that GSH conjugation plays an important role in the formation of toxic metabolites from a variety of chemicals has accumulated. Thus, several classes of compounds are converted, via conjugation with GSH, into either cytotoxic, genotoxic, or mutagenic metabolites. The purposes of the symposium on "Glutathione Conjugate Mediated Toxicities" presented at the 1990 Society of Toxicology Annual Meeting were to discuss recent findings in this rapidly moving field, to present ideas on the mechanisms and modulation of GSH conjugate-dependent toxicities, to present a consensus on the broader significance of this work, and to identify directions for future research. This paper summarizes these presentations. GSH conjugation reactions are involved in the bioactivation of several classes of xenobiotics, and four types of GSH-dependent bioactivation reactions can be identified: (1) directly toxic GSH conjugates may be formed from vicinal dihaloalkanes via formation of electrophilic sulfur mustards; (2) cysteine conjugate beta-lyase-dependent bioactivation is involved in the selective nephrotoxicity of haloalkenes; (3) GSH conjugates of hydroquinones and isothiocyanates may serve as transport and targeting metabolites; and (4) GSH-dependent reactions may be involved in the release of toxic agents from precursor organic thiocyanates and nitrosoguanidines (N-methyl-N'-nitro-N-nitroguanidine).