Follow-up of patients who have undergone ileogastrostomy

Patients who have had ileogastrostomy for the treatment of morbid obesity require close, long-term follow-up. From a prospective study of a large number of variables in 12 consecutive patients, and from experience with more than 200 patients who have undergone this procedure since 1982, a protocol has evolved. The authors outline the associated morbid conditions, operative complications and biochemical alterations that are important in the management of these patients.     

Phosphorylation-dependent subfunctionalization of the calcium-dependent protein kinase CPK28

Calcium (Ca²⁺)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca²⁺ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca²⁺ activation at physiological concentrations of Ca²⁺, possibly through stabilization of the Ca²⁺-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca²⁺] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca²⁺-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

Protein kinases represent one of the largest eukaryotic protein superfamilies. While roughly 500 protein kinases have been identified in humans (1), the genomes of Arabidopsis thaliana (hereafter, Arabidopsis) (2) and Oryza sativa (3) encode more than 1,000 and 1,500 protein kinases, respectively, including several families unique to plants. Among these protein kinases are the receptor-like kinases (RLKs), receptor-like cytoplasmic kinases (RLCKs), and calcium-dependent protein kinases (CDPKs or CPKs) that have emerged as key regulators of plant immunity (4–6). Despite encompassing only 2% of most eukaryotic genomes, protein kinases phosphorylate more than 40% of cellular proteins (7, 8), reflecting their diverse roles in coordinating intracellular signaling events. Reversible phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues can serve an array of functions including changes in protein conformation and activation state (9, 10), protein stability and degradation (11, 12), subcellular localization (13–15), and interaction with protein substrates (16–18).Calcium (Ca²⁺) is a ubiquitous secondary messenger that acts cooperatively with protein phosphorylation to propagate intracellular signals. Spatial and temporal changes in intracellular Ca²⁺ levels occur in response to environmental and developmental cues (19–23). In plants, Ca²⁺ transients are decoded by four major groups of calcium sensor proteins, which possess one or more Ca²⁺-binding EF-hand motifs (24, 25): calmodulins (CaM), CaM-like proteins, calcineurin B–like proteins, CDPKs, and Ca²⁺/CaM-dependent protein kinases.At the intersection of phosphorylation cascades and Ca²⁺ signaling are CDPKs, a unique family of Ca²⁺ sensor/kinase-effector proteins. CDPKs have been identified in all land plants and green algae, as well as certain protozoan ciliates and apicomplexan parasites (26, 27). CDPKs have a conserved domain architecture, consisting of a canonical Ser/Thr protein kinase domain and an EF-hand containing Ca²⁺-binding CaM-like domain (CLD), linked together by an autoinhibitory junction (AIJ) and flanked by variable regions on both the amino (N) and carboxyl (C) termini (28, 29). As their name implies, most CDPKs require Ca²⁺ for their activation (30). Upon Ca²⁺ binding to all EF-hands in the CaM-like domain, a dramatic conformational change occurs, freeing the AIJ from the catalytic site of the kinase, rendering the enzyme active (31–33). CDPKs vary in their sensitivity to Ca²⁺ (30), presumably allowing proteins to perceive distinct stimuli through differences in Ca²⁺-binding affinity. For example, Arabidopsis CPK4 displays half maximal kinase activity in the presence of ∼3 μM free Ca²⁺ (30) while CPK5 only requires ∼100 nM (34). Importantly, CDPKs are signaling hubs with documented roles in multiple distinct pathways (4, 24, 35–38) and are therefore likely regulated beyond Ca²⁺ activation.Subfunctionalization is at least partially mediated by protein localization and interaction with pathway-specific binding partners, as is well documented for Arabidopsis CPK3 which functions in response to biotic and abiotic stimuli in distinct cellular compartments (39). Recent attention has been drawn to site-specific phosphorylation as a mechanism to regulate the activity of multifunctional kinases. For example, phosphorylation sites on the RLK BRASSINOSTEROID INSENSITIVE 1–ASSOCIATED KINASE 1 (BAK1) are differentially required for its function as a coreceptor with a subset of leucine-rich repeat –RLKs (40). Phosphoproteomic analyses indicate that CDPKs are differentially phosphorylated following exposure to distinct stimuli (41–48); however, the biochemical mechanisms by which site-specific phosphorylation regulates multifunctional CDPKs is still poorly understood.Arabidopsis CPK28 is a plasma membrane–localized protein kinase with dual roles in plant immune homeostasis (49–51) and phytohormone-mediated reproductive growth (52, 53). In vegetative plants, CPK28 serves as a negative regulator of immune signal amplitude by phosphorylating and activating two PLANT U-BOX–type E3 ubiquitin ligases, PUB25 and PUB26, which target the key immune RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) for proteasomal degradation (50). Owing to elevated levels of BIK1, CPK28 null plants (cpk28-1) have heightened immune responses and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) (51). Upon transition to the reproductive stage, cpk28-1 plants additionally present shorter leaf petioles, enhanced anthocyanin production, and a reduction in stem elongation (52, 53). The molecular basis for developmental phenotypes in the cpk28-1 knockout mutant, beyond hormonal imbalance (52, 53), are comparatively unknown.Our recent work demonstrated that autophosphorylation status dictates Ca²⁺-sensitivity of CPK28 peptide kinase activity in vitro (54). While dephosphorylated CPK28 is stimulated by the addition of 100 μM CaCl₂ compared to untreated protein, hyperphosphorylated CPK28 displayed similar levels of activity at basal Ca²⁺ concentrations (54). These results highlight the interesting possibility that phosphorylation status may control the activation of multifunctional kinases in distinct pathways by allowing CDPKs to respond to stimulus-specific Ca²⁺ signatures.In the present study, we identify a single autophosphorylation site, Ser318, that decouples the activity of CPK28 in immune signaling from its role in reproductive growth. We show that expression of a nonphosphorylatable Ser-to-Ala variant (CPK28^S318A) is unable to complement the immune phenotypes of cpk28-1 mutants but is able to complement defects in stem growth. Further, we uncover a functional role for phosphorylation of Ser318 in priming CPK28 for activation at low free [Ca²⁺]. Together, we demonstrate that site-specific phosphorylation can direct the activity of a multifunctional kinase in distinct pathways and provide evidence for a conserved mechanism in orthologous group IV CDPKs.     

Chronic arm morbidity after curative breast cancer treatment: prevalence and impact on quality of life.

PURPOSE: To determine the prevalence of and contributing factors for chronic arm morbidity including lymphedema in breast cancer patients after treatment and to assess the impact of arm morbidity on quality of life (QOL). PATIENTS AND METHODS: A four-question screening questionnaire was developed and mailed to a random sample of 744 breast cancer patients treated curatively in two cancer centers from 1993 to 1997. Patients were without recurrence and at least 2 years from diagnosis. Respondents were classified as with or without arm-related symptoms on the basis of the survey. Stratified random samples from each group were then invited for a detailed assessment of their symptoms and signs, including the presence of lymphedema. Their QOL was assessed by the European Organization for Research and Treatment of Cancer QOL Questionnaire C-30 and by a detailed arm problem questionnaire that assessed various aspects of daily arm functioning. RESULTS: Approximately half of all screened patients were symptomatic and 12.5% of all assessed patients had lymphedema. Axillary dissection (AD) and axillary radiotherapy (RT) after dissection were statistically significantly related to the occurrence of arm symptoms (odds ratio for AD = 3.3, P <.001; odds ratio for RT = 3.1, P <.001). Symptomatic patients and patients with lymphedema both had impaired QOL compared with asymptomatic patients. CONCLUSION: Treatment for breast cancer is associated with considerable arm morbidity, which has a negative impact on QOL. Arm morbidity should be carefully monitored in future studies involving local treatment modalities for breast cancer.     

Routine shave margins are not necessary in early stage breast cancer treated with Breast Conserving Surgery

Introduction

Breast Conserving Surgery (BCS) is considered standard of care for women with early stage breast cancer. Between 20 and 50% of women treated with BCS will require re-operation for positive or close margins and it has been suggested that routine cavity shave margins may reduce the frequency of positive margins.

Excision of breast fibroepithelial lesions: when is it still necessary?—A 10-year review of a regional centre

Breast Cancer Research and Treatment - Fibroepithelial lesions (FEL) range from benign fibroadenoma (FA) to malignant phyllodes tumor (PT), but can be difficult to diagnose on core needle biopsy...     

The Mechanism of Weight Loss after Ileo-gastrostomy for Morbid Obesity

Twelve patients (weight 107-178 kg and age range 19-43 years) were investigated following ileo-gastrostomy for morbid obesity. A number of variables were studied prospectively, pre- and postoperatively, to determine the cause of weight loss--previously attributed to malabsorption or decreased caloric intake. Weight loss of 10.9-36.5 kg, mean 22.9 kg, occurred. Three-day calorie counts demonstrated a postoperative decrease in daily caloric consumption of 320-3870, mean 1975 cal. Analysis of body compartment composition after derivation of lean body mass (from calculation of total body water with tritiated water) showed a mean decrease in adipose tissue of 17.7 kg. Postoperative weight loss, mainly fat, could not all be accounted for by decreased caloric consumption or steatorrhoea (72-h stool fat increased by a mean of 30 g). Pulmonary studies showed no significant change in respiratory quotient, but a large decrease in both postoperative utilization of oxygen and the production of carbon dioxide. This may indicate an alternate, anaerobic, energy cycle utilization. Other statistically significant variables included a large fall in cholesterol, LDH cholesterol and triglycerides, and smaller decrease in HDL cholesterol. Changes in gastro-intestinal (GI) hormones and cell counts in stomach and small intestine were also measured and will be reported later.     

De-Escalating Breast Cancer Surgery: Should We Apply Quality Indicators from Other Jurisdictions in Canada?

Quality Indicators (QIs), including the breast-conserving surgery (BCS) rate, were published by the European and American Breast Cancer Societies and this study assesses these in a Canadian population to look for opportunities to de-escalate surgery. A total of 2311 patients having surgery for unilateral, unifocal breast cancer between 2013 and 2017 were identified and BCS QIs calculated. Reasons for mastectomy had been prospectively collected with synoptic operative reporting. Our BCS rate for invasive cancer < 3 cm was 77.1%, invasive cancer < 2 cm was 84.1%, and DCIS < 2 cm was 84.9%. There was no statistically significant change in BCS rates over a five-year period, but there was a reduction in contralateral prophylactic mastectomies (CPM) from 28% in 2013 to 16% in 2017 (p < 0.001). Trend analysis looking at tumour size and medical need for mastectomy indicated that 80% of patients at our centre would be eligible for BCS with tumour cut off of 2.5 cm. Our institution met American but not European QI standards for BCS rates, potentially indicating a difference in patient demographics compared to Europe. Our results support the understanding that BCS rates are influenced by multiple factors and are challenging to compare across jurisdictions. CPM rates may offer a more actionable opportunity to de-escalate surgery for breast cancer.     

Patient navigation reduces time to care for patients with breast symptoms and abnormal screening mammograms

Introduction

Concern has been raised about delays for patients presenting with breast symptoms in Canada. Our objective was to determine if our Rapid Access Breast Clinic (RABC) improved care for patients presenting with breast symptoms compared to the traditional system (TS).

Liver Biopsies Following Ileogastrostomy

Patients who have had ileogastrostomy for the treatment of morbid obesity require close, long-term follow-up. One concern in patients undergoing any form of intestinal bypass surgery is that of possible liver damage. To assess for possible liver damage in morbidly obese patients undergoing ileogastrostomy, we undertook a prospective study of liver biopsies in 12 consecutive patients. Preoperative and 2-year postoperative biopsies of the liver were planned. There were six liver biopsies available for comparison both pre- and post-operatively. The biopsies showed changes of fatty infiltration both pre- and post-operatively. There were no differences in the degree of fatty infiltration, or of other histological parameters which we measured. There were no cases of cirrhosis of the liver recorded.