Consumer food waste in the United States represents substantial amounts of wasted nutrients, as well as needless environmental impact from wasted agricultural inputs, energy use, and greenhouse gas emissions. Efforts to reduce food waste at the consumer level are urgently needed to address the most prominent nutrition and environmental sustainability issues we now face. Importantly, individuals report that saving money is a salient motivator for reducing food waste, yet contemporary evidence on the consumer cost of wasted food is lacking. The objectives of this study are to 1) estimate the daily per capita cost of food wasted, inedible, and consumed 2) at home and away from home, and 3) by food group.This study utilizes cross-sectional, nationally-representative data on food intake from the National Health and Nutrition Examination Survey (2001–2016), linked with nationally representative data on food waste from published literature, as well as data on food prices and food price inflation from multiple publicly-available sources. Survey-weighted procedures estimated daily per capita expenditure on food waste for 39,758 adults aged ≥20 y.Total daily per capita food expenditure was $13.27, representing 27% wasted, 14% inedible, and 59% consumed. The greatest daily food waste expenditures were observed for meat and seafood purchased for consumption outside of the home , and fruits and vegetables purchased for consumption in the home .The most cost-effective ways to reduce food waste at the consumer level are to focus waste reduction efforts on meat and seafood purchased for consumption outside of the home and fruits and vegetables purchased for consumption in the home. A number of strategies are available to help consumers reduce their food waste, which can increase their financial flexibility to purchase more healthy foods while simultaneously reducing environmental impact. Diet quality in the United States remains far below recommended levels –3. Less At the same time, the average American wastes about one pound of food every day, including large amounts of healthy foods like fruits and vegetables . This reEfforts to reduce food waste at the consumer level are urgently needed to help address the most prominent nutrition and environmental sustainability issues in the US and globally. Importantly, many Americans report that saving money is the most salient motivator for reducing food waste . Along wBuzby and Hyman  and VenkThere is an urgent need to better understand the daily cost of consumer food waste in contemporary dollars, and to account for the important difference in food prices between foods purchased for at-home consumption and those purchased for consumption away from home. This information is needed to better understand opportunities to target consumer reductions in food waste so that individuals can have greater financial flexibility to purchase healthier foods while also reducing environmental impact. To address these important research gaps, the objectives of this study are to 1) estimate the daily per capita cost of food wasted, inedible, and consumed 2) at home and away from home, and 3) by food group.Individual-level data on food intake and sociodemography were acquired from the National Health and Nutrition Examination Survey (NHANES) 2001–2016 from 39,758 individuals ≥20 y , and a sNHANES provides data on foods as they were reported consumed, which is often in the form of dishes with multiple ingredients. Each NHANES food was disaggregated into its component ingredients using the Food Commodity Intake Database FCID, 2001–2010), which provides data on the weight of nearly 500 ingredients included in each NHANES dish . Each in01–2010, n = 41 categories; Supplemental Table Data on the price of each NHANES food were derived using multiple sources, as depicted in Fig. CNPP Food Prices Database provides only retail prices (FAH prices), so the price of each FAFH was derived using data from the National Household Food Acquisition and Purchase Survey  was assessed by comparing the main results to three sensitivity models: Model 1 used 2008 food prices, Model 2 used FAH prices, and Model 3 represented a composite of Model 1 and Model 2 (2008 food prices and FAH prices). Uncertainty in food intake data was assessed by comparing the main results to two additional sensitivity models. Specifically, these models were needed to investigate whether the use of FCID to disaggregate NHANES foods into their component ingredients resulted in biased estimates of food intake over time, since FCID has not been updated since 2010 and may not be applicable to NHANES surveys after that date. Separate analyses were conducted for two distinct time points, 2001–2010 (Model 4) and 2011–2016 (Model 5). The main analysis was tested against each of the sensitivity models using Wald tests with The analytic sample included 39,758 individuals ≥20 y Table . Over twP = 0.003), inedible (P < 0.001), and consumed (P < 0.001), yet no difference (P = 0.154) was observed between expenditures on wasted food between FAH  and FAFH .Total daily per capita food expenditure was $13.27 (95% CI: $13.01–13.53), representing 27% wasted , 14% inedible , and 59% consumed , followed by fruits and vegetables (30%), grains (10%), sweets (6%), and dairy (5%); beverages, nuts and seeds, eggs, frozen foods, fats and oils, and other foods each represented less than 5% . Separate sensitivity analyses demonstrated no difference (P = 0.071) in food waste expenditure when restricting the food intake data to 2001–2010 (Model 4), and a decrease of $0.30 (P = 0.006) when restricting the food intake data to 2011–2016 (Model 5).Sensitivity analyses demonstrated that using 2008 food prices (Model 1), FAH food prices (Model 2), and a combination of both (Model 3) resulted in a decrease in food waste expenditures by $0.36, $0.64, and $1.04, respectively  and food consumed away from home (FAFH), but greater expenditure was observed for FAFH meat and seafood and FAH fruits and vegetables.Others have estimated that daily per capita food waste at the consumer level represented approximately $1.07–1.10 , 12, whiAccording to recently-updated food expenditure estimates published by the US Department of Agriculture , the aveThe present study suggests that the average American consumer spends over $1300 per year ($3.62 per day × 365 days) on food that ends up being wasted, which is greater than the annual expenditure on vehicle gasoline ($1250); apparel ($1207); household heating and electricity ($1149); property taxes $1046); and household maintenance, repairs, and insurance ($936) for the average single-person household in 2017 . Since i46; and hThe present study demonstrates that meat and seafood, as well as fruits and vegetables, accounted for the greatest value of consumer food waste by food group, cumulatively representing over 60% of the total, which is consistent with previous findings , 12. ImpThis study is not without limitations. Data on food price inflation were available for only 15 major food categories (from CPI), whereas data on food intake were available for over 8500 individual foods (from NHANES), so linking these data may have contributed to over-generalized estimates of contemporary prices for individual foods. Lack of data availability also prevented analyses of food waste across divergent geographic regions and subpopulations, and more research is needed to estimate variability of food waste across these domains. Estimates of food waste were derived from the LAFA data series, which provides a single estimate of the proportion of each food wasted across years. This limitation prevented a time trend analysis, and required that the same food-specific waste proportions were applied consistently to FAH and FAFH. Furthermore, this limitation made it necessary to apply food-specific waste proportions consistently to all individuals in NHANES, which has precedent in previous studies , 36–38. The strengths of this study should be considered when interpreting the findings. For the first time, nationally-representative datasets on food intake, food waste, food prices, eating location, and food price inflation were linked to provide a robust measure of the daily per capita cost of consumer food wasted, inedible, and consumed in contemporary dollars. Importantly, this study also introduced a novel method of accounting for the important price differences between FAH and FAFH, thereby filling an important research gap . The larThis study presents a fresh look at the daily per capita cost of consumer food waste, inedible portions, and consumed food in the US. We use an innovative approach that links dietary data collected from nearly 40 thousand adults over a 16-year period with nationally-representative data on food waste, food prices, eating location, and food price inflation. The average US adult spends over one-quarter of their food budget on food that ends up being wasted, more than the annual expenditure on vehicle gasoline, apparel, household heating and electricity, property taxes, and household maintenance and insurance. These results also shed new light on cost-effective ways to reduce food waste at the consumer level, by targeting waste reduction efforts on meat and seafood consumed outside of the home and fruits and vegetables consumed in the home. A number of strategies are available to help consumers reduce their food waste, which can increase their financial flexibility to purchase more healthy foods while simultaneously reducing environmental impact.Additional file 1. Methodological uncertainty and assumptions embedded in data source linkages.Additional file 2 : Figure S1. Steps to derive the proportion of food waste from the edible weight of food. Figure adapted, with permission, from Conrad, Zach; Niles, Meredith; Neher, Deb; Roy, Eric; Tichenor, Nicole; Jahns, Lisa. (2018). Relationship between diet quality, food waste, and environmental sustainability. PLoS ONE, 13:e0195405. Text boxes with solid outlines represent data aquired from USDA Loss-adjusted Food Availability data series (LAFA); text boxes with dashed outlines represent derived data.Additional file 3 : Table S1. Foods included in analysis. NHANES, National Health and Nutrition Examination Survey; CPI, Consumer Price Index; FoodAPS, Food Acquisition and Purchase Survey. 1Approximately 80% of FoodAPS codes come from the USDA Food and Nutrient Database for Dietary Studes (FNDDS), and the remaining codes are manually assigned by FoodAPS staff (FoodAPS). 2Leading digits in each 8-digit food code. 3All codes begin with the prefix “CUUR0000S”. 4Leading digits in each 8- or 10-digit food code. 5Includes crackers and other grain-based snacks. 6Includes French toast and other sweet grain-based foods. 7Includes imitation milk, flavored milk and milk drinks, evaporated and condensed milk, and dry and powdered milk. 8Includes dairy-based sauces. 9Includes lamb, goat, and game. 10Includes sandwiches made from all meat and seafood sources, luncheon meats, and burgers. 11Includes butter and margarine. 12Includes water, alcohol, and beverage concentrates.Additional file 4 : Table S2. Retail prices for foods purchased for consumption at home and away from home, 2012–2013 (n = 4305). Data acquired from the USDA National Household Food Acquisition and Purchase Survey, 2012–2013. 1Ratio of food away from home to food at home.Additional file 5 : Table S3. Consumer Price Index and inflation coefficients, 2001–2016. NA, Not applicable. 1Represents 1 + percent change in Consumer Price Index from 2001 to 2016. 2Derived by (CFAFH/CFAH)Ci, where C represents the inflation coefficient for all food away from home (FAFH), all food at home (FAH), and each individual food category (i).Additional file 6 : Table S4. Daily per capita cost of food waste by food type, 2001–2016 . 95% confidence intervals not listed for foods with waste <$0.01. 1Paired Wald test used to estimate the difference between food at home and food away from home. 2Includes crackers and other grain-based snacks. 3Includes French toast and other sweet grain-based foods. 4Includes imitation milk, flavored milk and milk drinks, evaporated and condensed milk, and dry and powdered milk. 5Includes dairy-based sauces. 6Includes lamb, goat, and game. 7Includes sandwiches made from all meat and seafood sources, luncheon meats, and burgers. 8Includes beverage concentrates. 9Includes butter and margarine. 10Includes water, alcohol, and beverage concentrates.Additional file 7 : Table S5. Sensitivity analysis for food price assumptions, 2001–2016 . Food purchased = wasted + inedible + consumed. Model 1: Expenditures estimated using 2008 dollar values for all foods reported consumed. Model 2: Expenditures estimated using food at home prices for all foods reported consumed. Model 3: Model 1 + Model 2. 1Difference between the estimated expenditure and each of the modeled expenditures tested using Wald tests at P < 0.05, with Bonferroni adjustment for multiple comparisons. Superscript letters indicate statistical significance for independent comparisons between estimated expenditure and Model 1 (a), Model 2 (b), and Model 3 (c).Additional file 8 : Table S6. Sensitivity analysis for food intake assumptions, 2001–2016 . Food purchased = wasted + inedible + consumed. Model 4: Expenditures estimated for individuals included in the National Health and Nutrition Examination Survey, 2001–2010 only. Model 5: Expenditures estimated for individuals included in the National Health and Nutrition Examination Survey, 2011–2016 only. 1Difference between the estimated expenditure and each of the modeled expenditures tested using Wald tests at P < 0.05, with Bonferroni adjustment for multiple comparisons. Superscript letters indicate statistical significance for independent comparisons between estimated expenditure and Model 4 (a) and Model 5 (b).


We evaluated oncological changes in patients with squamous cell carcinoma of the anus (SCCA) treated by chemoradiotherapy (CRT) from a large UK institute, to derive estimates of contemporary outcomes.We performed a treatment-cohort analysis in 560 patients with non-metastatic SCCA treated with CRT over 25 years. The primary outcomes were 3-year loco-regional failure (LRF), 5-year overall survival (OS), and 5-year cancer-specific survival (CSS). We developed prediction models; and overlaid estimates on published results from historic trials.Ptrends < 0.001); 5-year OS increased from 60% to 76% (Ptrends = 0.001); and 5-year CCS increased from 62% in to 80% (Ptrends = 0.001). For 2020, the models predicted a 3-year LRF of 14.7% (95% CIs: 0–31.3); 5-year OS of 74.7% (95% CIs: 54.6–94.9); and 5-year CSS of 85.7% (95% CIs: 75.3–96.0). Reported oncological outcomes from historic trials generally underestimated contemporary outcomes.Age distributions, proportions by gender and cT stage remained stable over time. The median follow-up was 61 (IQR: 36–79) months. Comparing the first period (1990–1994) with the last period (2010–2014), 3-year LRF declined from 33 to 16% (Current and predicted rates for 3-year LRF and 5-year survivals are considerably improved compared with those in historic trials. These data are informative for policy-makers seeking information on net survival improvements but generally lag behind contemporary management strategies ; focus mainly on common cancers; and generally fail to capture detailed treatment and stage information, necessary to interpret whether survival improvements reflect introductions of new treatments or stage migrations.Large-scale population-based studies in developed countries, such as EUROCARE,2 For trialists, there is an additional need to forecast expected number of events based on current standard of care. But a new problem is emerging in trials—namely that outcomes from contemporary standard arm management exceed expectations . Thus, trials reach target recruitment but findings appear to lack power.3 This issue is exemplified in recent non-oncology (ARRIVE4) and oncology (COLOFOL5 and ROLAAR6) trials.In contrast, patients and oncologists generally seek to understand prognosis, namely the chance of surviving from a specific cancer, in the context of contemporary treatment options.7 ESMO-ESSO-ESTRO,8 and ACPGBI.9 Approximately three-quarters of patients with SCCA receive CRT as initial treatment.10 Through systematic review,11 we recently reported on 45 studies of patients with SCCA who received either radiotherapy alone (RT) or CRT and noted that 5-year overall survival increased from a mean estimate of 64% in 1980 to 75% in 2010 (p = 0.046). It is conceivable that this temporal improvement might be driven by improvements in loco-regional control, but might also be due to unmeasured factors, such as general improvement in healthcare, centralisation, improved imaging and radiotherapy delivery, and more effective management of toxicity. It might also reflect early tumour stage at presentation or younger mean age at diagnosis.Here, we address the above problem in the setting of an uncommon cancer, namely squamous cell carcinoma cancer of the anus (SCCA), treated with chemo-radiotherapy (CRT). The latter is standard of care in many countries as reflected by guidelines, for example, from NCCN,In this study, we confirmed the observation of significant temporal improvement in survivals and aimed to use these striking temporal changes to derive models to estimate contemporary outcomes.We performed a treatment-cohort analysis, using a prospectively maintained clinical database of patients with SCCA treated at the Christie NHS Foundation Trust, Manchester, United Kingdom, seen between 1 January 1990 and 31 December 2014, and followed to 30 April 2018. The Christie anal cancer multi-disciplinary team (MDT) meeting was centralised for the Greater Manchester and North Cheshire geographical areas (approximate 1.8 million) in 2007. From 2004, pre-treatment HIV testing was performed selectively .12 We recognised a change in pre-treatment staging assessment through the study period and categorised this as follows: 1990 to 2003 assessment was physical examination and CT imaging; 2004 to 2010 assessment added MR imaging;13 and from 2011 to 2014, assessment additionally added Fluoro-Deoxy-Glucose Positron Emission Tomography/Computed Tomography. TNM staging was in accordance with the American Joint Committee on Cancer (AJCC) staging 7th Edition.14Patients were included if they had histologically confirmed squamous cell carcinoma arising from the anal canal or margin treated with CRT with curative intent. For sensitivity analyses, patients treated curatively with RT alone were added. Standard clinical, pathological and treatment-related variables were collected, as previously published.15 radiotherapy regimen was prescribed and described elsewhere.12 After 2001, the treatment protocol followed that used in the ACT II trial16—namely, radiotherapy of 50.4 Gy was delivered over 5.5 weeks with a two phase technique, without a mandatory break. Phase 1 included 30.6 Gy in 17 daily fractions with non-conformal rectangular parallel-opposed fields. Phase 2 required conformal planning and delivered 19.8 Gy in 11 daily fractions over 15 days to the primary tumour with a 3 cm margin and any involved lymph nodes. From 2005, we reported median duration of radiotherapy treatment.From 1990 to 2001, a split ACT I2 on day 1, and continuous infusion of 5-fluorouracil (5-FU) 1000 mg/m2 on days 1–4 and days 29–32 or cisplatin (60 mg/m2 on days 1 and 29) with 5-FU (as above), the latter regimen as part of the ACT II trial16 (2001–2008). The selection to RT or CRT was randomized as part of the ACT I trial15 until 1994. Thereafter, selection for RT was the exception, and based on contra-indications to the use of CRT, typically co-morbidities or increasing age.Chemotherapy regimens were administered concurrently with radiotherapy as either: mitomycin-C (MMC) 12 mg/m13 From 6 to 60 months, patients were assessed clinically on a six-monthly basis and imaging follow-up based on risk of local relapse—in patients deemed at high-risk for local relapse , MR scans were generally performed at 12, 18, 24 and 36 months; in the remainder (low-risk), MR scans were performed at 36 months. Prior to 2004, surveillance was by clinical examination.Since 2004, post-treatment follow-up was typically clinical assessment at 6 weeks after completion of CRT and again at clinical visits paralleling the 3- and 6-month MR scans.17 core outcome measures. Time-to-events were from the date of start of first treatment. LRF was defined as the presence of either residual or recurrent disease within the inguinal/pelvic anatomic sites. OS was defined as the period of time until death from any cause; CSS was defined as the period of time until death from anal cancer.For this analysis, the primary outcomes were 3-year loco-regional failure (LRF); 5-year overall survival (OS); and 5-year cancer-specific survival (CSS). These are CORMACAll statistical analyses were performed using Stata software, Version 14 . The main analysis was based on patients receiving curative CRT; sensitivity analyses included all patients treated with curative intent—namely CRT and RT, over the time period. In order to test for a period effect, we divided the cohort into five groups of five-year intervals, spanning the 25-year study period. Differences in baseline characteristics across the five periods were explored using the Cuzick’s non-parametric test and the Cochran-Armitage test for trends (2 × n tables) as appropriate. For cT stage, we used ordinal regression to account for the multinomial stage proportions and examine whether overall stage distribution and stage-specific proportions changed significantly.To derive predicted contemporary (2020) estimates, we used a two-stage approach. First, we assessed for key confounders in this cohort and evaluated the associations between patient and tumour factors with the three outcomes of 3-year LRF; 5-year OS; and 5-year CSS. We derived Kaplan–Meier (K–M) estimates and then performed univariable and multivariable analyses using Cox models, adjusted for year of treatment. Proportionality assumptions were tested using Schoenfeld residuals.Second, we sought to relate changes in key outcomes with study periods. For this analysis, we estimated the three outcomes using K–M methods, in two-year bands , and related these over time using regression models, weighted for period sample size. Initial exploration revealed that linear models might predict implausible outcomes . Therefore, non-linear splines were used. A range of cut-off points from years 2000 to 2010 were tested as pivots for each scenario. The optimal cut-point was determined based on three criteria: (i) visual inspection of plots; (ii) lowest AIC (Akaike Information Criteria) value per model; and (iii) clinically plausible coefficients. For example, if LRF rates were declining (negative regression coefficient), we rejected models where the regression coefficient ‘right’ of the cut-point was positive. Once the optimum regression spline model was determined, we used it to predict options to extrapolate estimates with 95% confidence intervals (95% CIs) for 2020. We additionally tested for the presence of competing risk of death bias by visually comparing the predictions for 5-year OS vs. 5-year CSS over time.21 of CRT in patients with SCCA, and visually inspected for model fit.Finally, once we established the optimal regression models, we superimposed the equivalent estimates for the three primary outcomes from the six reported trialsBetween 1990 and 2014, there were 1040 referrals to the anal cancer MDT of which 930 (89%) were primary SCCA. Numbers of patients with SCCA doubled from 134 in 1990–94 to 266 in 2010–14. Of the 930 patients, there were 701 patients treated with curative intent, with either RT (N: 141) or CRT (N: 560) . By contrast, nodal positivity increased from 17% in the first study period to 41% in the last study period (P < 0.001). The baseline characteristics for the 701 patients undergoing either RT or CRT with curative intent are detailed in Table Table Ptrends < 0.001) (Table p = 0.003), and higher with increasing cT stage . Anatomic site, tumour differentiation and nodal positivity were not associated with LRF.With a median follow-up of 61 (IQR: 36–79) months, there were 119 LRFs among the 560 patients who underwent CRT. The 3-year LRF rate was 33% in the first study period (1990–1994), declining to 16% in the last study period 2010–2014) (010–2014 Ptrends = 0.001) (Table p < 0.001), node positivity (p = 0.012), and poorer performance status . Gender, anatomic site, tumour differentiation and HIV positivity were not associated with OS.There were 230 deaths among the 560 patients who underwent CRT. The 5-year OS rate was 60% in the first study period (1990–1994), increasing to 76% in the last study period (2010–2014) (Ptrends = 0.001) (Table p < 0.001), node positivity (p = 0.024), and poorer performance status . Gender, anatomic site, tumour differentiation and HIV positivity were not associated with CSS.There were 143 deaths from anal cancer. The 5-year CCS rate was 62% in the first study period (1990–1994), increasing to 80% in the last study period (2010–2014) ,24 and Tomaszewski et al. 25 present institutional series over 25–30 years. However, the treatments within the cohorts from Myerson23 and Kim24 were heterogeneous, and although Tomaszewski et al.25 concentrated on patients treated with CRT, this study did not account for the potential effect of time on presentation and outcome. Whitford et al. 26 explored for a time period effect on outcome but the study was too small (76 cases over 30 years) to take account of varying presentations with time. Recently, Guren et al.27 reported that 5-year net (or relative) survival in 1548 patients from the Cancer Registry of Norway increased from 63 to 73% (1987–2016). However, while the registry reported that 82% were treated with curative intent, detailed treatment details were lacking. Furthermore, relative survival represents a modelled survival estimate taking account underlying period changes, and does not equal observed patient survival estimates, which is required by patients and trialists.A small number of institute-level studies have described the presentation and outcomes of anal cancer over time. Myerson et al. (Washington University),19 demonstrated the use of CRT improved local control compared with RT alone. Further trials,21 reported between 2008 and 2013, established the combination of radiotherapy with 5-fluorouracil and mitomycin-C as the optimal therapy. While the use of CRT is associated with improved loco-regional disease control , it is unclear whether this translates into improvements in overall survival . To the best of our knowledge, our study is the first to illustrate parallel temporal improvement for LRF and survivals.In the 1990s, two randomised trials11 For example, this might explain why nodal positivity was not a predictor of loco-regional relapse. We caution against the interpretation that the increased proportion of nodal positivity reflects a ‘true’ shift to more advanced stage disease, as the proportions of T stages remained constant over the study period.In our analysis, there were striking increases in the proportions of pre-treatment nodal positivity from 17% in 1990–94 to 41% in 2010–14. We believe that most of this is driven by the introduction into clinical practice of modern imaging modalities, a type of the Will-Rogers phenomenon. We have written extensively about this and described the added phenomenon of ‘reduced prognostic discrimination’.28 in the 1990s to only 23% in the ACT II trial from the mid-2000s.29 This is an area of ongoing research in this cohort.Our study has limitations. First, there may be selection bias. Over the study period, improvements might reflect stricter criteria for curative intent. This seems unlikely as the proportions treated by curative intent were broadly 80% throughout. Similarly, improvements might reflect proportionately increased use of CRT (rather than RT). This is true—though our sensitivity analyses demonstrate that the same patterns of oncological outcomes were seen for the combined RT and CRT cohort. Second, there may be unmeasured confounding. For example, we did not routinely capture performance status data before 2005. It is likely that our patients’ general health status improved with time, though it seems less plausible that this alone accounts for the observed 16% absolute improvement in overall survival. Third, there was a lack of treatment-related toxicity data. It is conceivable that grade 3 and 4 toxicities lessened, and their management improved. Again, it seems unlikely that this alone accounted for the magnitude of observed improvements in overall survival. Fourth, we did not capture technical refinements in salvage surgery over time, which might account for some increases in long-term disease-free states. However, as primary locoregional failure rates have reduced substantially, salvage surgery is now less often required. Furthermore, among patients with local relapses, the proportion that proceed to salvage surgery has decreased from more than 70% in historic series23 50 patients;24 284 patients;25 76 patients26)—were smaller. Third, we concentrated our analysis on a homogenous treatment—namely CRT. The details of this treatment varied with time, but the backbone was 50–55 Gy radiotherapy and a 5-FU-mitomycin based concurrent chemotherapy. Fourth, there was appropriate length follow-up. Fifth, our definition of 3-year LRF is equivalent to that currently used in the UK PLATO trial,30 and many of the patient population in the primary analysis of this study are equivalent to those eligible for modern trials, like PLATO.There are several study strengths. First, we used a prospectively maintained database, where for example, key prognostic factors such as pre-treatment stage were consistently recorded. Second, this is the largest temporal clinical institute-level dataset of its type. Other datasets  are likely contributors. Centralisation of anal cancer management is likely to have contributed to improvements through use of defined patient protocols. The culmination of these changes is the probable driver of improved oncological outcomes, although near-impossible to quantify. Human Papilloma Virus (HPV) is the aetiological agent in most SCCA tumours, but is also a marker of radio-sensitivity.The current and predicted rates for 3-year LRF and 5-year survivals are more optimistic than those in the historic trials. It is important that ongoing and future trials are appropriately powered to reflect event rates for current standard of care (the control arm). We illustrate this as follows. Consider a hypothetical trial based on clinical practice 25 years ago. We assume that the LRF rate was 30% and the new intervention aimed to improve LRF by (relative) 25% i.e. to 24%. Assuming an alpha = 0.05 and power = 0.80, a 1:1 head-to-head trial would require 675 in each arm  with 365 events. Now consider a similar trial today. We assume that the LRF rate is 20% and the new intervention aimed to improve LRF by (relative) 25% i.e. to 15%. Assuming an alpha = 0.05 and power = 0.80, a 1:1 head-to-head trial would require 715 in each arm  with 251 events.11 may partly explained the increase in proportions of SCCA patients with node positivity, there might be other factors. Nodal positivity is clinically important as this is used as treatment stratification in clinical practice and in trials. The relevance of this is still not clear.First, while the Will Rogers phenomenon32 RTOG 052933 was a phase 2 evaluation of dose-painted intensity modulated radiation therapy in combination with 5-FU and MMC, which not only showed a reduction of acute morbidity but also improved LRF. If there is a causal relationship between LRF and OS, these new treatment modalities might further improve LRF, reduce treatment-related toxicity, and ultimately, further reduce the death burden from this cancer.Second, we are now in an era of accurate radiotherapy delivery with VMAT and IMRT, which has become standard RT for anal cancer.Supplemental material


EZH2, CUX1, TET2, and BCOR. After initial therapy with hydroxyurea and interferon-α, the patient underwent allogeneic hematopoietic stem cell transplantation, with reduced intensity conditioning and a matched sibling donor. He had no signs of relapsed disease 2 years after the transplant. Based on the patient outcome, we summarize the diagnostic and therapeutic approaches for patients diagnosed with myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome, and review the current literature, emphasizing the role of genetic mutations and allogeneic hematopoietic stem cell transplantation. Larger and more detailed clinical studies are strongly needed to optimize and standardize diagnostic and therapeutic approaches for this disease.The entity myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome is characterized by the coexistence of both myeloproliferative and myelodysplastic features in the bone marrow. Risk assessment and treatment recommendations have not been standardized, and clinicians rely on updated patient studies and reviews to make decisions for treatment approaches. Histopathological features have traditionally been important, although in the last decade, several studies have reported mutational profiles of this rare disease. Here, we present a case, wherein the patient presented with leukocytosis and the diagnostic work-up revealed features of myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome. Mutational profiling revealed mutations in four genes associated with myeloid malignancies, namely,  By definition, these cases show morphologic myeloproliferative features and signs of dysplastic bone marrow changes. The karyotype is often normal or shows abnormalities similar to MDS. Cases of CMML display peripheral blood monocytosis and dysplasia in one or more myeloid lineages. aCML is characterized by dysgranulopoiesis along with peripheral blood leukocytosis, mimicking chronic myeloid leukemia (CML). These patients are by definition BCR–ABL1 negative, and the classical MPN mutations—JAK2, CALR, and MPL—are usually absent.3 The diagnostic work-up of MDS/MPN overlap syndrome is challenging due to heterogeneity and similarity to other MDS and MPN entities, for example, chronic neutrophil leukemia (CNL), accelerated phase of MPN, or prefibrotic primary myelofibrosis (prePMF).4 Thus, the use of myeloid mutation panels should be encouraged for proper classification of prognoses and identify subgroups that would benefit from targeted treatment.5Myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap syndrome is a rare disease, recognized by the 2016 World Health Organization (WHO) classification of myeloid malignancies.6 Here, we present the case of a 62-year-old man, who was diagnosed with MDS/MPN-U, and harbored mutations in four genes associated with myeloid malignancies: EZH2, CUX1, TET2, and BCOR. He was initially treated with induction therapy and then successfully allografted with a matched sibling donor.No standard treatment or protocol exists for the patients with MDS/MPN overlap syndrome. Allogeneic hematopoietic stem cell transplantation  has been suggested as the only curative option for eligible patients with a suitable donor.9/L). During hospitalization, he did not show any discomfort or signs of illness. The only constitutional symptom was light sweating during night over the previous 3 months, which had possibly worsened over the last week. His vital signs were normal. Clinical examination revealed a palpable spleen and three fingerbreadths below the costal margin. Chest radiography was normal. The spleen was 17.3 cm in length by ultrasonographic measurement, and no hepatomegaly was noted. The results of the blood tests at time of hospitalization are demonstrated in The patient was a 62-year-old man who was previously a smoker and had a medical history of hypertension. During a routine health check, a complete blood count was performed, revealing a severe leukocytosis (white blood cell (WBC): 62 × 10The blood smear revealed neutrophils at all stages of differentiation, mostly mature neutrophils without any clear signs of dysplasia . There were 1% blasts and no basophils or monocytes .JAK2, CALR, and MPL were negative. Moreover, fluorescence in situ hybridization (FISH) analysis for the Philadelphia chromosome, t, and real-time polymerase chain reaction (RT-PCR) for BCR–ABL1 fusion transcripts, which was initiated before the results of FISH analysis due to high clinical suspicion of CML, also turned out negative. Furthermore, G-banding demonstrated a normal male karyotype , and FISH analysis for PDGFRA, PDGFRB, PCM1-JAK2, and FGFR1 rearrangements was negative. Bone marrow biopsy confirmed signs of MDS/MPN, with dominant mature granulocytopoiesis and an increased number of individually spread atypical megakaryocytes  93.5%), CUX1 (VAF 83.5%), TET2 (VAF 53.9%), and BCOR (VAF 12.6%) mutations. These gene mutations are commonly observed in MDS/MPN overlap syndrome, but they are not specific for any entity within the syndrome.7 According to the 2016 WHO classification system for myeloid malignancies, we classified the patient as having MDS/MPN-U.1To further confirm the diagnosis, analysis of a myeloid mutation panel  was performed on the bone marrow aspirate. The panel confirmed clonal hematopoiesis in the presence of The patient’s leukocytosis and thrombocytosis (a myeloproliferative feature of the disease) expanded over the next few weeks, and he had light constitutional symptoms. Treatment with hydroxyurea was initiated, and gradually increased to a dose of 1500 mg/day; the treatment was well tolerated. Pegylated interferon-α was added to the treatment and gradually increased to a dose of 100 mg once weekly. Consequently, while the leukocytosis improved, precursors still comprised >10% of nucleated cells. 8 Graft-versus-host disease (GvHD) prophylaxis was as follows: methotrexate at days +1, +3, +6, and +11, and cyclosporin A from day −1 with a target serum concentration of 200–300 µg/L.9 Engraftment occurred with platelets >20 × 109/L on day +15, >50 × 109/L on day +20, and neutrophils >0.2 × 109/L on day +16. The patient developed acute skin and intestinal GvHD  on day +60, which was successfully treated with steroids and addition of alpha-1-proteinase inhibitor. The therapeutic range of cyclosporin A was raised. Due to a rise in cytomegalovirus (CMV) DNA transcript levels without signs of CMV-related disease, pre-emptive intravenous ganciclovir was administered from day +90 until CMV DNA transcripts were no longer detected.10 Evaluation at 3, 6, 9, 12 months, and 2 years confirmed complete remission (CR) with normal blood and bone marrow smears, and donor chimerism >99%.Reduced intensity conditioning (RIC) allo-HSCT with a matched sibling donor was performed 6 months after initial diagnosis, and the disease was stable during this period. The conditioning regimen consisted of fludarabine and treosulfan.BCR–ABL1-negative MDS/MPN mimicking CML prompts close collaboration among clinicians, pathologists, and geneticists.3 Our patient presented with expanded granulopoiesis without dysplasia in peripheral blood and bone marrow, and without monocytosis, eosinophilia, or basophilia. Blasts accounted for 1%–2%, and neutrophil precursors were >10% in peripheral blood. In the bone marrow, dysplasia was noted in the megakaryocytes. The lack of monocytosis excluded CMML diagnosis. Regarding bone marrow morphology, more grouped megakaryocytes and obvious fibrotic changes are expected in primary myelofibrosis, although reticulin staining demonstrated focalized increase grade 1  model successfully risk stratified 135 MDS/MPN-U patients at 3 years follow-up. Palomo et al.5 found that the presence of cytogenetic abnormalities was associated with an inferior overall survival (OS) in MDS/MPN neoplasms, except for aCML. The presence of specific somatic mutations is suggested to negatively impact survival. An example is the presence of a TET2 mutation in aCML,15 or ASXL1 mutation in CMML.16 Palomo et al.5 argued that ASXL1 mutation is not associated with poorer outcome in aCML since the disease already has an aggressive course and harbors a high percentage of ASXL1 mutations. In their study, EZH2 was associated with a poorer OS in patients with aCML.5 They also aimed to characterize a molecular subtype of MDS/MPN-U as “aCML-like,” which actually has poorer OS compared to the “CMML-like” subtype of MDS/MPN-U.5 Mangaonkar et al.11 demonstrated the adverse prognostic impact of CBL and TP53 mutations in MDS/MPN-U patients. Bose et al.12 reported that results positive for ⩾1 gene mutation in their panel associated with worse OS, but none of them predicted an inferior outcome individually. For aCML and MDS/MPN-U diagnoses, age, severe leukocytosis, and the presence of somatic mutations are outlined as adverse factors.15There are no standardized risk stratification scores for MDS/MPN overlap syndromes. The International Prognostic Scoring System–Revised (IPSS-R) developed for MDS combined with cytogenetics is often used in clinical practice and studies, but its prognostic usefulness has been uncertain.TET2, EZH2, CUX1, and BCOR. Interestingly, they belong to four different functional classes  of 24 months in an unselected patient cohort with MDS/MPN-U. The only curative option proposed is allo-HSCT, although a few evidence-based recommendations have been made.19 A retrospective analysis by Onida et al.6 described CR in 87% of patients with aCML reported to the European Society for Blood and Marrow Transplantation (EBMT) registry between 1997 and 2006, following allo-HSCT for aCML, 5 years OS in 51% patients and a relapse FS in 36% patients. Kurosawa et al.14 reported a 3-year OS for 48.3% patients in a cohort of 86 patients with MDS/MPN-U treated with allo-HSCT between 2001 and 2017 in Japan using registry data. The OS was worse in patients >50 years of age and those with disease progression according to the International Working Group (IWG) response criteria for MDS at the time of allo-HSCT. Both relapse and non-relapse mortality rates were approximately 25%. Mutational profiles of patients were not assessed.14The natural history of aCML suggests a median survival of 25 months, and a transformation to acute myeloid leukemia (AML) in 40% within 18 months of diagnosis.24 the adverse prognostic factors related to the mutation profile, and the availability of a suitable donor led to a relatively rapid allo-HSCT. Allo-HSCT was accomplished during the chronic phase of the disease, which is regarded favorable.14 In recent reviews, both Smith et al.13 and Shallis and Zeidan3 emphasized risk stratification and incorporation of genetic data for prognostic models in MDS/MPN overlap syndrome. It must be emphasized that the decision to initiate treatment and to transplant in the setting of a low-risk and stable-phase disease is not straightforward and should be the result of a thorough overall assessment.Despite being asymptomatic and in low-risk categories according to both the IPSS-R and the MDA model, our patient’s age, low hematopoietic cell transplantation–specific comorbidity index (HCT-CI),14 although it has demonstrated efficacy in classical MPNs to reduce WBC with tolerated side effects.25 There were no JAK2 or CSF3R mutations to support the use of ruxolitinib.2 There are ongoing studies regarding selective inhibitors of EZH2 in myeloid malignancies,26 although this approach was not available in this setting. There was no increase in blasts in the marrow or dominant cytopenia, which would have prompted the use of a hypomethylating agent as a bridge to allo-HSCT.2 Due to the age of the patient, an RIC regimen was chosen.8Limited data exist on treatment up-front allo-HSCT. For our patient, the main goal was to avoid proliferative symptoms and progression to the blast phase. The choices of administering interferon-α and hydroxyurea were empirical, and dosages were guided with near normalization of leukocyte counts. Interferon-α is rarely mentioned as a therapeutic option in case series,19 trying to unite possible dysplastic as well as proliferative aspects of the heterogeneous group. The 3-month evaluation after RIC allo-HSCT showed complete remission according to these defined response criteria. However, there is no possibility of monitoring minimal residual disease (MRD) for MDS/MPN patients although TET2, EZH2, and CSF3R are all potential markers.4The MDS/MPN International Working Group (MDS/MPN IWG) published proposed response criteria for MDS/MPN in 2015,This case report highlights the challenging diagnostic work-up required for MDS/MPN overlap syndrome. Close collaboration among clinicians, pathologists, and geneticists is essential. It is challenging since there are no standardized diagnostic or therapeutic options for this disease entity, and that somatic mutations would be incorporated in the risk assessment and considered while choosing a treatment strategy; however, their applicability in clinical settings may not be immediate. Mutational profiling will be a part of the MRD-assessment post-allo-HSCT in the future. Through our report, we show that RIC allo-HSCT is feasible in patients with MDS/MPN-U, and that complete remission is achievable.


Cardiac damage triggered by severe hypocalcemia is well known. However, the role of chronic hypoparathyroidism (HP) and pseudohypoparathyroidism (PHP) in cardiac health is still unclear. We investigated the effect of chronic HP and PHP on cardiac structure and conductive function in patients compiling with treatment.The study included 18 patients with HP and eight with PHP aged 45.4 ± 15.4 and 22.1 ± 6.4 years, respectively with a previously regular follow-up. In addition, 26 age- and sex-matched healthy controls were included. General characteristics and biochemical indices were recorded. Cardiac function and structure were assessed by estimation of myocardial enzymes, B-type natriuretic peptide (BNP), and echocardiography. The 12-lead electrocardiogram and 24-h Holter electrocardiography were performed to evaluate the conductive function.Levels of serum calcium in HP and PHP were 2.05 ± 0.16 mmol/L and 2.25 ± 0.19 mmol/L, respectively. The levels of myocardial enzyme and BNP were within the normal range. Adjusting for age at evaluation and body mass index, all M-mode measurements, left ventricular mass (LVM), LVM index (LVMI) and relative wall thickness (RWT) were comparable between patients and controls. Prolongation of corrected QT (QTc) intervals occurred in 52.6% (10/19) of patients, and 6.7% (1/15) of patients manifested more than 100 episodes of supraventricular and ventricular extrasystoles, as well as supraventricular tachycardia. None of the above arrhythmias was related to a severe clinical event.From this pilot study, patients diagnosed with HP and PHP and well-controlled serum calcium levels manifested normal cardiac morphology and ventricular function, except for prolonged QTc intervals, and a small percentage of mild arrhythmias needing further investigation. Hypoparathyroidism (HP) and pseudohypoparathyroidism (PHP) are rare diseases due to parathyroid hormone (PTH) insufficiency or resistance leading to hypocalcemia, hyperphosphatemia and related symptoms. The conventional treatment for HP and PHP includes calcium supplementation combined with vitamin D analogs. To avoid hypercalciuria-related renal damage and maintain optimal levels of calcium phosphate synthesis, the serum calcium levels were regulated slightly below normal or within the low-normal range in HP patients. Hyperphosphatemia often persists among HP and PHP patients receiving conventional therapy.High serum phosphorus directly triggers vascular injury, increases the mineral deposition in vascular smooth muscle cells in studies in vitro , and incThus, the objective of the current pilot study was to directly investigate the effect of long-standing HP and PHP on cardiac morphology and function, and cardiac conductive system in patients compling with treatment of calcium and vitamin D analogs.n = 1) and those diagnosed with post-surgical hypoparathyroidism due to parathyroidectomy (n = 1) were excluded. The remaining 26 patients and 26 age- and sex-matched healthy controls with normal thyroid function and with no CVD or associated risk factors during the same period were finally included in the analysis. The controls came from the health examination center in PUMCH. The patients were evaluated with a 12-lead electrocardiogram (ECG), cardiac markers, echocardiography and 24-h Holter electrocardiography. The controls were evaluated with echocardiography by the same physician as the patient group. All patients received calcium combined with vitamin D analogs  at the time of investigation independently to enrollment. The study was conducted according to the principles of the Declaration of Helsinki and approved by the Ethics Committee of PUMCH. Written informed consent was obtained from all included subjects or the parents for subjects aged below 18 years. The diagnosis of HP and PHP was established by the presence of hypocalcemia, and hyperphosphatemia with inappropriately low and normal-to-high intact PTH levels, respectively, while the secondary hyperparathyroidism including chronic kidney disease and vitamin D deficiency (serum 25OHD < 20 ng/ml) } + 0.6. The LVM index  was calculated by dividing LVM by the BSA. The relative wall thickness (RWT) was calculated using the formula: (2 × LVPWd) / LVEDD.All the included subjects were evaluated using a non-invasive transthoracic echocardiogram  with an m5s-D probe. M-mode measurements and conventional Doppler echocardiographic examinations were performed according to the American Society of Echocardiography (ASE) guidelines . The parResting standard 12-lead ECG was obtained only in 19 patients. Resting heart rate, PR intervals, QRS duration, and corrected QT (QTc) intervals were determined via ECG analysis.Baseline cardiac rhythm and potential arrhythmias were evaluated using an ambulatory 12-lead electrocardiography in 15 patients over a 24-h period during their normal daily activities . The total 24-h ventricular rates, and the maximal and minimal ventricular rates were measured automatically.Data were analyzed using SPSS software version 22.0 . Categorical variables are expressed as numbers or percentages. Continuous variables are expressed as mean ± standard deviation or median and inter-quartile ranges, as appropriate. The clinical demographics and laboratory results of patients were compared with those of the healthy controls, and between patients diagnosed with HP and PHP using the Student’s t-test or Wilcoxon test. The cardiac parameters were compared using a general linear model adjusted for age at the evaluation, gender and other confounders. Associations between serum calcium, serum phosphorus, PTH and cardiac parameters were evaluated via partial correlation analysis.P = 0.045; age at cardiac evaluation 45.4 ± 15.4 vs. 22.1 ± 6.4 years, p <  0.001, respectively). The disease courses from onset of hypocalcemia were longer in patients diagnosed with HP compared with those with PHP at the evaluation. One patient with HP suffered from diabetes, and four patients with HP had hypertension. No subject had self-reported CAD or related symptoms. The average levels of serum calcium were 2.05 ± 0.16 mmol/L in HP patients and 2.25 ± 0.19 mmol/L in patients with PHP (P = 0.011) during the evaluation. Serum magnesium, serum potassium, fasting blood glucose, and lipid components were within normal limits. The serum phosphorus levels (×ULN) were higher  and the eGFR was lower  in HP than in PHP . Partial correlation analysis showed that serum calcium, serum P and PTH were not correlated with cardiac parameters after adjusting for age at evaluation and gender  with supraventricular extrasystoles. Among the 12/15 patients with supraventricular extrasystoles, one (6.7%) female patient with non-surgical HP  manifested 243 episodes of supraventricular extrasystole in 24 h (the incidence in the remaining 11 subjects ranged from 2 to 57 episodes), as well as supraventricular tachycardia (SVT)  with complaints of palpitations. Among 7/15 patients (46.7%) with ventricular extrasystoles, one (6.7%) female patient with post-surgical HP  presented 4063 episodes of ventricular extrasystoles noticed by 24 h (the remaining six subjects presented with 2–16 episodes) with shortness of breath during activity. No patient showed ventricular tachycardia. No atrioventricular block was found  and PHP undergoing conventional treatment. The study demonstrated that except for mild QTc prolongation as well as a small percentage of mild arrhythmias, there was no significant difference in left ventricular morphology, systolic dysfunction, or severe cardiac events such as syncope and heart failure in patients with HP and PHP compared with age- and sex-matched healthy controls.Previous studies suggested that both hypocalcemia and hyperphosphatemia had adverse effects on cardiovascular system , 5, 17. n = 73, mean age 46.0 ± 13.3 years; 32% with hypertension) with frequent and complex ventricular extrasystoles (63%) for 6.5 ± 1.8 years (from 1 to 9.5 years) was not increased according to US death rates during the same period. In another study, Maurer et al. [In addition, supraventricular and ventricular extrasystoles were detected in 80% and 46.7% of our subjects evaluated by 24-h Holter electrocardiography. Two patients presented with palpitations or shortness of breath accompanied by supraventricular tachycardia or frequent ventricular extrasystoles. Studies found that ventricular extrasystoles, supraventricular extrasystoles, and supraventricular tachycardia occurred in 40–75%, 60.8%, and 2.2% respectively in healthy subjects via 24- to 48 h Holter examination –24. Evenr et al.  found thP <  0.01) in patients with HP [In this case-control study, no significant differences in left ventricular structure and systolic function were observed between HP and PHP patients with low-normal or normal levels of serum calcium and healthy controls, based on echocardiography. Left ventricular hypertrophy was not observed in all patients, although a few female patients may display borderline concentric remodeling and eccentric hypertrophy, as shown in Fig.  with HP . LVEF waCompared with PHP patients, RWT appeared to be larger in HP patients in our study after adjusting for age at evaluation, BMI, and levels of serum calcium and serum phosphorus. However, they were all within the normal range. The levels of serum calcium, serum phosphorus, and PTH were not related to RWT. In addition, due to the small sample size, patients with post-surgical and non-surgical HP were not separated in our study. Therefore, further classification of large samples of patients diagnosed with HP and PHP into subgroups for comparative analyses may facilitate the determination of differences and clinical significance accurately.This study has limitations. The major limitation is the small sample size due to the rarity of non-surgical HP and PHP. The cross-sectional design is another limitation, which prevented the analysis of changes in cardiac structure and function during long-term follow-up and precluded any inferences of causal relationship. Also, we have not included patients with previous heart damage due to hypocalcemia, and therefore, the cardiac characteristics of such patients may have been missed. We only included patients who consented to cardiac examination, which suggested a selection bias. Another possible weakness is the relative youth of the included patients since the effects of HP on the cardiovascular system take longer and may be more dramatic in older patients. Meanwhile, date from the controls including biochemical indicators, 12 lead ECG and 24-h Holter electrocardiography were incomplete, although we speculated that the results of the study were not significantly affected. Finally, the patients were not categorized into subgroups according to different etiologies and the small sample size prevented any comparison of differences between subgroups.The results of this preliminary study indicate no definitive evidence of a negative effect of cardiac structure or systolic function among patients diagnosed with HP and PHP receiving regular calcium and vitamin D analogs, except for a significant proportion of subjects with prolonged QTc and a few subjects of arrhythmia, warranting further investigation. Studies with a larger sample size are warranted to verify or further explore this situation.


B. subtilis, cellular (p)ppGpp level is determined by the bifunctional (p)ppGpp synthetase/hydrolase RelA and two small alarmone synthetases (SASs) YjbM (SasB) and YwaC (SasA). However, it is less clear whether these enzymes are also involved in regulation of alarmones outside of (p)ppGpp. Here we developed an improved LC-MS-based method to detect a broad spectrum of metabolites and alarmones from bacterial cultures with high efficiency. By characterizing the metabolomic signatures of SasA expressing B. subtilis, we identified strong accumulation of the (p)ppGpp analog pGpp, as well as accumulation of ppApp and AppppA. The induced accumulation of these alarmones is abolished in the catalytically dead sasA mutant, suggesting that it is a consequence of SasA synthetase activity. In addition, we also identified depletion of specific purine nucleotides and their precursors including IMP precursors FGAR, SAICAR and AICAR (ZMP), as well as GTP and GDP. Furthermore, we also revealed depletion of multiple pyrimidine precursors such as orotate and orotidine 5′-phosphate. Taken together, our work shows that induction of a single (p)ppGpp synthetase can cause concomitant accumulation and potential regulatory interplay of multiple alarmones.(p)ppGpp is a highly conserved bacterial alarmone which regulates many aspects of cellular physiology and metabolism. In Gram-positive bacteria such as  A well-conserved mechanism to achieve such coordination is through the synthesis and degradation of the nucleotide alarmones ppGpp and pppGpp, collectively known as (p)ppGpp . (p)ppGpEnterococcus faecalis and Staphylococcus aureus, or the soil bacterium Bacillus subtilis, (p)ppGpp can be produced by three different synthetases: the bifunctional synthetase/hydrolase Rel   and YwaC  (In Firmicutes such as the pathogens ubtilis) , and twoor SAS2) . RelA isor SAS2)  or singlor SAS2) .M and σW ppGpp analog pGpp, as well as accumulation of ppApp and AppppA to ∼10% of the level of pGpp. Furthermore, we also detected depletion of specific purine nucleotides and their precursors including GTP and GDP, and IMP precursors FGAR (Phosphoribosyl-N-formylglycineamide), SAICAR (Phosphoribosyl-aminoimidazolesuccinocarboxamide), and AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide). Intriguingly, we revealed that SasA expression also leads to strong depletion of pyrimidine pathway precursors such as orotate and orotidine 5′-phosphate. Our work highlights that expression of SasA can cause concomitant accumulation of alarmones beyond (p)ppGpp, suggesting that regulation mediated by SasA involves multiple alarmones.With the aim to investigate the characteristics of alarmone regulation by the cell wall stress induced (p)ppGpp synthetase SasA, we developed a LC-MS-based method to detect and measure an expanded set of metabolites and alarmones in B. subtilis, media was supplemented with the following antibiotics when necessary: spectinomycin (80 μg/mL), chloramphenicol (5 μg/mL), kanamycin (10 μg/mL), and a combination of lincomycin (12.5 μg/mL) and erythromycin (0.5 μg/mL) for MLS resistance. Carbenicillin (100 μg/mL) was used for selection in E. coli.All bacterial strains, plasmids and oligonucleotides used in this study are listed in ywaC and ywaCD87G fragments with primers oJW3495/3496 using pJW512 and pJW516 as templates, followed by SalI/SphI digestion and ligation into pDR110. The resulting plasmid is transformed into E. coli DH5α for propagation and verified by sequencing with oJW1519.Construction of pJW731 and pJW733 was done by PCR amplification of yjbM and pJW306 for ΔywaC) followed by transformation of pSS4332 for marker-less recombination (yjbM and oJW904/905 for ywaC).Construction of JDW3014 was done by sequential transformations of integration plasmids containing an I-sceI endonuclease cut site and regions of homology upstream and downstream of synthetase genes .Construction of JDW4017 and JDW4019 was done by integration of JDW3014 at sistance . DisruptamyE with pJW731 and pJW733, followed by selection for spectinomycin resistance. The resulting strain was transformed with ΔnahA::kanR PCR product synthesized from genomic DNA of BKK34780 (BGSC) using oligos oJW3382/oJW3383, followed by selection for kanamycin resistance. The resulting strain was further transformed with ΔrelA::ermR PCR product synthesized from genomic DNA using oligos oJW902/oJW903, followed by selection for MLS resistance (nahA and relA was verified by PCR and sequencing (oJW3382/oJW3383 for nahA and oJW418/419 for relA).Construction of JDW4064 and JDW4066 was done by integration of JDW3014 at sistance . DisruptBacillus subtilis strains were grown in S7 defined medium . Induction of SasA expression was done by addition of 1 mM IPTG at final concentration. Cell viability assay was done by serial dilution and plating on LB plates, followed by colony counting after overnight incubation at 37°C.d medium ; MOPS wa600 ∼0.3 followed by addition of 1 mM IPTG. For sample collection, 10 mL cultures were sampled by filtering through PTFE membrane (Sartorius) before and after 30-min IPTG induction. Filtered membranes with harvested cells were immediately submerged in 3 mL extraction solvent mix [on ice 50:50 (v/v) chloroform/water] to quench metabolism. This process also enables efficient cell lysis and extraction of soluble metabolites. Mixture of cell extracts were centrifuged at 5000 × g for 10 min to remove organic phase, then centrifuged at 20,000 × g for 10 min to remove cell debris. Samples were frozen at −80°C if not analyzed immediately. Samples were analyzed using HPLC-MS system consisting of a Vanquish UHPLC system linked to electrospray ionization  to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) operated in full-scan mode to detect targeted metabolites based on their accurate masses. LC was performed on an Acquity UPLC BEH C18 column . Total run time was 30 min with a flow rate of 0.2 mL/min, using Solvent A  and acetonitrile as Solvent B. The gradient was as follows: 0 min, 5% B; 2.5 min, 5% B; 19 min, 100% B; 23.5 min 100% B; 24 min, 5% B; 30 min, 5% B.LC-MS quantification of nucleotides was performed as described  with mod600 to the same sample volumes (10 mL). Prism 7 (GraphPad) was used for statistical analysis and generation of figures.Quantification of metabolites from raw LC-MS data were performed by using the MAVEN software . Metabol600 of 1.0. We adopt a cell density of 2.0 × 108 CFU/mL/OD600 and the shape of cytoplasm as a cylinder of 4 μm in height and 0.435 μm in radius in the calculation. This estimation corresponds to an average cell volume of 2.38 fL.We use the estimation that cell volume is 0.475 μL in 1 mL culture at an ODNIP3G is the normalized ion count of pGpp, ELC–MS is the detection efficiency of pGpp in LC-MS (2.4e8 ion counts/μM), VCulture,  N is the normalized culture volume (5.0 mL), ODnm,N600is the normalized optical density (1.0) and Fcell is the fraction of cell volume in the culture (0.000475 mL/1 mL culture/OD).The detection efficiency of pGpp in LC-MS is around 2.4e8 ion counts/μM in 25 μL sample. Normalized ion count can be converted into intracellular concentration of pGpp by NInt is the normalized ion count of nucleotide, ELC–MS is the detection efficiency of nucleotide in LC-MS (2.0e8 ion counts/μM), VCulture,  N is the normalized culture volume (5.0 mL), ODnm,N600is the normalized optical density (1.0) and Fcell is the fraction of cell volume in the culture (0.000475 mL/1 mL culture/OD).The detection efficiency of other nucleotides in LC-MS is around 2.0e8 ion counts/μM in 25 μL sample. Normalized ion count can be converted into intracellular concentration of nucleotide by B. subtilis ppGpp can be sensitively detected by LC-MS at concentrations > 10 nM with 100% acetonitrile as the buffer B, but not from B. subtilis cell extracts using the same method (data not shown). This suggests that the lack of (p)ppGpp signals from bacterial samples was due to inefficient metabolite extraction procedures. To this end, we optimized the method to increase the breadth of detectable metabolites from cell extracts. We improved our metabolite extraction procedures ppGpp in mid log phase B. subtilis grown in rich media is below detection limit, likely due to the extremely low concentration of (p)ppGpp in this condition.Our previous metabolite extraction and LC-MS analysis method allowed us to efficiently detect and quantitate high abundance metabolites such as GTP in subtilis , howeverocedures  by replaocedures . While wD87G (synthetase-dead SasA) in the absence of the other two (p)ppGpp synthetases RelA and SasB. Because B. subtilis without (p)ppGpp production is auxotrophic for multiple amino acids  in pGpp ppGpp hydrolyzing enzyme NahA . To testA mutant . We foun in pGpp  along wi in pGpp . This de in pGpp . In addiin vitro evidence suggest that SasA from S. aureus can directly produce ppApp and pppApp . Therefore, it is likely that SasA can synthesize ppApp as an alternative product in B. subtilis.In addition to pGpp, we detected strong accumulation of another nucleotide alarmone ppApp to a level ∼10% of that of pGpp . Unlike Furthermore, we detected a ∼4-fold increase in AppppA upon SasA induction  to a levD87G-expressing cells  and XMP respectively using IMP as a common precursor . We founde novo IMP biosynthesis pathway  in these metabolites before and after induction ppGpp derivative pGpp, we detected unexpected accumulations of ppApp, AppppA and a mild increase of other signaling molecules such as c-di-AMP, as well as changes in de novo purine and pyrimidine biosynthesis metabolites ppGpp synthetases is important to understand their roles in cellular physiology. However, precise detection and quantitation of alarmones in bacteria has been challenging until recent advances in MS-based detection methods . Here weabolites . Our finS. aureus can efficiently synthesize ppGpp in vitro ppGpp. Although it was showed that SasA from in vitro , it is pin vitro . On the  E. coli , and thehibition . These fnahA mutant, suggesting that SasA may directly produce pGpp ppGpp through the NuDiX hydrolase NahA . We founuce pGpp . HoweverAppppA is known to be produced from ATP through distinct mechanisms catalyzed by aminoacyl-tRNA synthetases . Due to de novo purine biosynthesis enzymes  and XMP which are products of IMP. Synthesis of XMP from IMP is catalyzed by GuaB which has been reported to interact with ppGpp in E. coli  but is osubtilis . The str by PurA  which isppGpp target in B. subtilis, we have previously found that transcription of the pur operon is strongly downregulated during starvation in a (p)ppGpp-dependent manner (pur operon repressor PurR can bind to pGpp and (p)ppGpp ppGpp  in the upstream PRPP-IMP pathway are also depleted in response to SasA expression. Since PRPP level was similar, the inhibition is likely specific to the catalytic steps between PRPP and IMP which are catalyzed by the gene products of the t manner . This is(p)ppGpp . Thus, i(p)ppGpp , which hM/σW regulon along with a repertoire of other genes responsible for cell wall synthesis and division ppGpp synthesis triggered by nutrient starvation through the (p)ppGpp synthetase RelA, (p)ppGpp synthesis by SasA is induced in response to cell wall stresses . The phydivision , thus alThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.DF and JW designed the study. DF and JY performed the experiments and data analysis. DS and DA-N provided the operation and technical assistance of the LC and MS instrument. DF, JY, and JW discussed the findings and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To validate the Croatian version of the Zarit Burden Interview (ZBI) and to investigate the predictors of perceived burden.This cross-sectional study involved 131 dyads of one informal caregiver family member and one patient with dementia visiting primary care practices . Patient-related data were collected with the Mini-Mental-State-Examination, Barthel-index, and Neuropsychiatric-Inventory-Questionnaire (NPI-Q); caregiver-related data with the ZBI, and general information on caregivers and patients with a structured questionnaire. Principal-axis-factoring with varimax-rotation was used for factor analysis.1 = 0.88, α2 = 0.83, α3 = 0.72, α4 = 0.75) was good. Stronger cognitive and functional impairment of patients was associated only with personal strain, whereas more pronounced neuropsychiatric symptoms and the need for daily care were associated with more dimensions. Longer caregiver education suppressed embarrassment and promoted guilt. Guilt was higher in younger caregivers, caregivers of female patients, patients' children, and non-retired caregivers. In multivariate analysis significant predictors of higher overall burden were male sex of the patient, higher NPI-Q, the need for daily-care services, shorter duration of caregiving, non-spouse relationship, higher number of hours caring per-week, and anxious-depressive symptoms in a caregiver.The caregivers' mean age was 62.1 ± 13 years. They were mostly women (67.9%) and patients' children (51.1%). Four dimensions of ZBI corresponding to personal strain, frustration, embarrassment, and guilt were assessed and explained 56% variance of burden. Internal consistency of ZBI (α = 0.87) and its dimensions (αThe Croatian version of ZBI is reliable and valid. Our data confirm that ZBI is a multidimensional construct. Caregivers may benefit from individually tailored interventions. Dementia is an increasing health care problem associated with population aging -3. ThereThe most commonly used tool for the assessment of caregiver burden is the Zarit Burden Interview (ZBI) . The oriThe number of patients with dementia in Croatia ranges from 67 000  to 85 00There is currently no version of ZBI questionnaire validated for the Croatian population. Thus, the aims of our study were to validate the Croatian version of the ZBI, to evaluate the validity and internal consistency of the questionnaire, and to assess the relationship of caregivers’ and patients’ characteristics with total and different aspects of caregiver burden.The study was conducted in 60 family medicine practices in the Health Care Center Zagreb-West from October 2017 to September 2018.Among insured persons, we identified dyads consisting of a non-institutionalized patient with dementia  and a family member being the dominant informal caregiver. The study did not include dyads consisting of a patient and an informal caregiver who was not a family member . In addition, patients who were not diagnosed by either neurology or psychiatry specialists were not included. Dementia subtypes that were present were Alzheimer’s disease (76 patients), vascular dementia (47 patients), Parkinson’s disease dementia (6 patients), Lewy body dementia (1 patient), and frontotemporal dementia (1 patient). The patients with non-Alzheimer’s disease and non-vascular dementia were grouped together.The study aimed to include 130 dyads in line with previous studies , as thisAll family practitioners working in the Health Care Center Zagreb-West were approached with a request to screen for patients with dementia in their practices and to invite the patients' family members over the telephone to participate in the study.The families willing to participate were contacted again by an investigator to arrange a meeting with the caregiver and patient. The meeting was arranged either in the caregiver's household or in the family doctor’s office. The caregivers (and patients if applicable) were informed in detail about the study aims and methods. After having signed a written informed consent, they completed a set of structured questionnaires. Caregivers were interviewed separately to more easily answer the questions.Zarit Burden Interview  (n (MMSE) ,27 was aBarthel index (el index  was admiNeuropsychiatric Inventory Questionnaire (NPI-Q) ( (NPI-Q)  was admiA structured questionnaire including general questions about the caregiver and the patient. Caregiver-related data were age, sex, years of education, employment status, relationship with the patient, length of caregiving, living in the same household with the patient, the need for daily care services, hours dedicated per week, hours of someone else’s help per week, smoking status, and morbidities. Patient-related data were age, sex, years of education, type of dementia, duration of dementia, and comorbidities. The assessed morbidities/comorbidities were arterial hypertension, diabetes mellitus, malignant disease, rheumatic symptoms requiring therapy, asthma/chronic obstructive lung disease in both the caregiver and patient, and anxious-depressive symptoms requiring therapy in the caregiver.The MMSE was administered first, after which the patient was escorted to a nearby room, accompanied either by a nurse  or by other family members (if the visit was done in the household). Then the caregiver was administered the structured questionnaire, followed by ZBI, NPI-Q, and Barthel index.The study was approved by the Health Care Center Zagreb-West and University of Zagreb School of Medicine Institutional Review Boards. Appropriate written informed consents were obtained from each caregiver and from the patients whenever clinically possible; otherwise the caregiver gave additional consent for the patient.Categorical variables are presented as frequencies and relative frequencies. The normality of distribution of numerical variables was tested with the Kolmogorov-Smirnov test. The age variable was the only normally distributed variable and is presented as mean ± standard deviation, whereas other numerical variables were non-normally distributed and are presented as median and interquartile range (IQR).After the Kaiser-Meyer-Olkin measure of sampling adequacy and the Bartlett test indicated that factor analysis can be performed, principal axis factoring with varimax rotation was conducted. Reliability was expressed with Cronbach alpha coefficient of internal consistency.post-hoc test by Conover. Correlation with numerical variables was assessed with the Spearman coefficient of correlation (ρ).ZBI scores were compared between the groups with the Mann-Whitney U test or Kruskal-Wallis one way analysis of variance (ANOVA) test with the P < 0.15, exclusion criterion P < 0.2). P values <0.05 were considered significant. The analyses were conducted with MedCalc version 19.0.4.  and SPSS trial version .Logistic regression was used for multivariate analysis of predictors of higher perceived burden where ZBI was the dependent variable dichotomized at 40 points (higher score representing moderate to severe and severe burden). Age and sex of the caregiver, age and sex of the patient, MMSE score, Barthel index score, total NPI-Q score, the need for daily care services, and all other variables that were univariately associated with overall ZBI score or any of its dimensions were included into the variable selection process via backward method  caregivers did not consent to participate, whereas 131/135 (97%) dyads of caregivers and patients with dementia were included in the study .The mean age was 62.1 ± 13 years for caregivers and 79.4 ± 7.1 for patients with dementia. The majority of caregivers (89/131 or 67.9%) and patients (92/131 or 70.2%) were women. Most of caregivers were patients' children (67/131 or 51.1%), followed by spouses (51/131 or 38.9%). Most of patients had Alzheimer’s dementia (76/131 or 58%), with the median MMSE score of 15, Barthel index score of 85, and mean NPI-Q score of 26, corresponding to moderate dementia severity, the absence of functional dependency, and the presence of neuropsychiatric symptoms, respectively.P < 0.001).Internal consistency of the whole ZBI scale was high . Factor analysis was appropriate as Kaiser-Meyer-Olkin measure of the sampling adequacy was 0.84 and the Bartlett test of sphericity was significant  for factor analysis with 4 factors is shown in The first factor accounted for 31% of variance eigen = 6.94), included 9 items ,17,18,22.94, inclAll four factors were significantly correlated with the overall ZBI score, factor 1 and factor 2 being strongly correlated , factor 3 being moderately correlated (ρ 0.68), and factor 4 being weakly (ρ 0.24) correlated with the overall ZBI score.P > 0.05 for all comparisons) , which corresponds to a mild to moderate perceived burden. According to cut-offs proposed by Zarit and Zarit , 31/131 arisons) .P = 0.007), caring for the patient for a longer time (P = 0.024), having more hours of caring per week (P = 0.002), needing more hours of someone else’s help per week (P = 0.021), having anxious-depressive symptoms requiring therapy (P = 0.013), caring for patients with a longer duration of dementia (P = 0.009), caring for patients having higher functional impairment as assessed by the Barthel index (P = 0.009), having more pronounced neuropsychiatric symptoms as assessed by NPI-Q score (P < 0.001), and needing daily care services (P = 0.001) (P < 0.001).In univariate analyses, the overall ZBI score was significantly higher in caregivers living in the same households as the patients (= 0.001) . In addiP = 0.892).Personal strain, frustration, and embarrassment were mutually significantly moderately correlated (ρ 0.44 to 0.70), whereas only embarrassment was significantly but weakly correlated with guilt (ρ = 0.21). Personal strain, frustration, and embarrassment were also significantly moderately correlated with total NPI-Q distress score , whereas guilt was not significantly associated with NPI-Q distress (P = 0.030), longer duration of caregiving (P < 0.001), spending more hours of caring per week (P < 0.001), needing more hours of someone else’s help per week (P = 0.003), having anxious-depressive symptoms requiring therapy (P = 0.037), caring for patients with longer duration of dementia (P < 0.001), caring for patients having higher cognitive impairment as assessed by MMSE score (P = 0.002), higher functional impairment as assessed by the Barthel index (P < 0.001), more pronounced neuropsychiatric symptoms as assessed by NPI-Q score (P < 0.001), and the need for daily care services (P = 0.001).Higher scores on personal strain were associated with living in the same household as the patient (P < 0.001), longer duration of dementia (P = 0.024), and the need for daily care services (P = 0.028).Higher scores on frustration were associated with more pronounced neuropsychiatric symptoms as assessed by NPI-Q score (P = 0.009), spending more hours of caring per week (P = 0.009), arterial hypertension in the patient (P = 0.004), and more pronounced neuropsychiatric symptoms as assessed by NPI-Q score (P < 0.001).Higher scores on embarrassment were associated with fewer years of caregiver education (P = 0.015), higher number of years of caregiver education (P = 0.004), being unemployed or employed in comparison with being retired (P = 0.003), being a child in comparison with being the spouse of the patient (P = 0.006), caring for a female patient with dementia (P = 0.021), and the need for daily care services (P = 0.048).Higher scores on guilt were associated with younger age (In multivariate logistic regression with ZBI>40 (describing moderate to severe and severe burden) as a dependent variable we investigated an independent contribution of parameters that showed univariate associations with the overall ZBI or any of burden dimensions (described in detail in the Methods section). The model is shown in the P = 0.004), higher NPI-Q score , the need for daily care services , shorter duration of caregiving , non-spouse relationship with the patient , higher number of hours caring per week , and anxious-depressive symptoms requiring therapy in the caregiver , whereas male sex of the caregiver , the Barthel index , years of caregiver education , and retirement  were retained in a model but were rendered non-significant.Mutually independent predictors of higher overall ZBI score were male sex of the patient . The use of daily care services may prolong the period when the patients can receive care within their family circle. The use of these services is a step preceding the patient's institutionalization and usually serves as a respite for the caregiver ,42, who There are several limitations of our study. First, the cross-sectional design prevented us from inferring any causal relationship between burden and investigated variables. Second, we made efforts to approach as many informal caregivers who are family members as possible through assessing the patients with particular ICD-10 diagnoses made by neurology or psychiatry specialists. Hence, a number of caregiver-patient dyads might have been missed if the patient with dementia was not evaluated by these specialists or the caregiver was not a family member. Third, the investigated caregiver group represents urban dwellers from the Croatian capital, which makes our results possibly not generalizable to rural areas or other Croatian regions. A larger national or regional-multinational study could provide a deeper insight into caregiver burden differences potentially associated with regional specificities.Regardless of these limitations, our results have important implications for the recognition of variables associated with different aspects of caregiver burden and for the planning of therapeutic interventions aimed at burden amelioration. Family medicine practice is the optimal setting where caregivers can be timely recognized, their burden assessed, and targeted interventions implemented. Development of such measures is urgently needed.