Peripheral Volumetric Muscle Area and Total Body Volume in Postmenopausal Women With Rheumatoid Arthritis.

The effect of rheumatoid arthritis (RA) on peripheral muscle parameters is not completely understood. This study aimed to elucidate the influence of RA on peripheral muscle area and whole body skeletal muscle mass index (SMI) using peripheral quantitative computed tomography and dual-energy X-ray absorptiometry in postmenopausal women. This cross-sectional study included 54 postmenopausal women with RA and 86 healthy controls. Peripheral quantitative computed tomography and dual-energy X-ray absorptiometry were used to measure the muscle cross-sectional area and skeletal muscle mass. Muscle strength was assessed using a handheld dynamometer. Additionally, the effects of RA on muscle area and density as well as the bone / muscle area ratio were analyzed using multivariate models. The muscle area index of the thigh and forearm were correlated between both groups (RA: r = 0.357, p = 0.030; and healthy controls: r = 0.608, p < 0.001) and each index correlated with SMI in RA and healthy controls (p < 0.001). Bone / muscle area ratio correlated between forearms and thighs in health controls only (r = 0.547, p < 0.001). The RA group had a decreased thigh muscle area (p = 0.014); the fat area and fat muscle area ratio at the thigh and forearm were significantly increased (all p < 0.001), as well as thigh bone / muscle area ratio (p = 0.015). The RA group also had significantly lower forearm muscle density (p < 0.001). A sensitivity analysis excluding those on bisphosphonates led to similar results. Independent of RA, the thigh and forearm muscle area correlate with each other and with SMI. Differences in the bone / muscle ratio between RA and healthy controls may indicate regional muscle effects of inflammation and inactivity.

Association of patterns of multimorbidity with length of stay: A multinational observational study.

The aim of this study was to identify the combinations of chronic comorbidities associated with length of stay (LOS) among multimorbid medical inpatients.Multinational retrospective cohort of 126,828 medical inpatients with multimorbidity, defined as ≥2 chronic diseases (data collection: 2010-2011). We categorized the chronic diseases into comorbidities using the Clinical Classification Software. We described the 20 combinations of comorbidities with the strongest association with prolonged LOS, defined as longer than or equal to country-specific LOS, and reported the difference in median LOS for those combinations. We also assessed the association between the number of diseases or body systems involved and prolonged LOS.The strongest association with prolonged LOS (odds ratio [OR] 7.25, 95% confidence interval [CI] 6.64-7.91, P < 0.001) and the highest difference in median LOS (13 days, 95% CI 12.8-13.2, P < 0.001) were found for the combination of diseases of white blood cells and hematological malignancy. Other comorbidities found in the 20 top combinations had ORs between 2.37 and 3.65 (all with P < 0.001) and a difference in median LOS of 2 to 5 days (all with P < 0.001), and included mostly neurological disorders and chronic ulcer of skin. Prolonged LOS was associated with the number of chronic diseases and particularly with the number of body systems involved (≥7 body systems: OR 21.50, 95% CI 19.94-23.18, P < 0.001).LOS was strongly associated with specific combinations of comorbidities and particularly with the number of body systems involved. Describing patterns of multimorbidity associated with LOS may help hospitals anticipate resource utilization and judiciously allocate services to shorten LOS.

Patterns of multimorbidity in medical inpatients: a multinational retrospective cohort study.

Multimorbidity is frequent and represents a significant burden for patients and healthcare systems. However, there are limited data on the most common combinations of comorbidities in multimorbid patients. We aimed to describe and quantify the most common combinations of comorbidities in multimorbid medical inpatients. We used a large retrospective cohort of adults discharged from the medical department of 11 hospitals across 3 countries (USA, Switzerland, and Israel) between 2010 and 2011. Diseases were classified into acute versus chronic. Chronic diseases were grouped into clinically meaningful categories of comorbidities. We identified the most prevalent combinations of comorbidities and compared the observed and expected prevalence of the combinations. We assessed the distribution of acute and chronic diseases and the median number of body systems in relationship to the total number of diseases. Eighty-six percent (n = 126,828/147,806) of the patients were multimorbid (≥ 2 chronic diseases), with a median of five chronic diseases; 13% of the patients had ≥ 10 chronic diseases. Among the most frequent combinations of comorbidities, the most prevalent comorbidity was chronic heart disease. Other high prevalent comorbidities included mood disorders, arthropathy and arthritis, and esophageal disorders. The ratio of chronic versus acute diseases was approximately 2:1. Multimorbidity affected almost 90% of patients, with a median of five chronic diseases. Over 10% had ≥ 10 chronic diseases. This identification and quantification of frequent combinations of comorbidities among multimorbid medical inpatients may increase awareness of what should be taken into account when treating such patients, a growth in the need for special care considerations.

Does Preoperative Decolonization Reduce Surgical Site Infections in Elective Orthopaedic Surgery? A Prospective Randomized Controlled Trial.

BACKGROUND: Surgical site infections (SSIs) after elective orthopaedic surgery are very stressful for patients due to frequent rehospitalizations with reoperations and poorer functional outcomes. Prevention of such events is therefore crucial. Although an evidence-based consensus is still lacking, preoperative decolonization could decrease SSI. Specifically, more information is needed about the effect of a preoperative decolonization procedure on SSI proportions in both Staphylococcus aureus carriers and non-S. aureus carriers after general orthopaedic surgery. QUESTIONS/PURPOSES: Our study addressed the following questions: (1) Does preoperative decolonization reduce the risk of SSI after general elective orthopaedic surgery in patients colonized with S. aureus? (2) Does preoperative decolonization reduce the risk of SSI among patients who are not colonized with S. aureus? METHODS: In this prospective, randomized, single-blinded trial, we recruited patients undergoing general elective orthopaedic surgery in one tertiary care center in Switzerland. Between November 2014 and September 2017, 1318 of 1897 screened patients were enrolled. Patients were allocated into either the S. aureus carrier group (35%, 465 of 1318 patients) or the noncarrier group (65%, 853 of 1318 patients) according to screening culture results. In the S. aureus group, 232 patients were allocated to the intervention arm and 233 were allocated to the control arm. Intervention was 5 days of daily chlorhexidine showers and mupirocin nasal ointment twice a day. Of the 853 noncarriers, 426 were allocated to the intervention arm and 427 were allocated to the control arm. All patients in both groups were analyzed in an intention-to-treat manner. The primary endpoint was SSI occurrence at 90 days postoperative and the secondary endpoint was SSI occurrence at 30 days postoperative.The initial sample size calculation was made for the S. aureus carrier group. Based on the literature review, a 4% proportion of SSI was expected in the control group. Thus, 726 carriers would have been needed to detect a relative risk reduction of 80% with a power of 80% at a two-sided α-error of 0.048 (adjusted for interim analysis). Assuming carrier prevalence of 27%, 2690 patients would have been needed in total. An interim analysis was performed after including half of the targeted S. aureus carriers (363 of 726). Based on the low infection rate in the control group (one of 179), a new sample size of 15,000 patients would have been needed. This was deemed not feasible and the trial was stopped prematurely. RESULTS: Among carriers, there was no difference in the risk of SSI between the intervention and control arms (decolonized SSI risk: 0.4% [one of 232], control SSI risk: 0.4% [one of 233], risk difference: 0.0% [95% CI -1.2% to 1.2%], stratified for randomization stratification factors; p > 0.999). For noncarriers, there was no difference in risk between the intervention and control arms (decolonized SSI risk: 0.2% [one of 426], control SSI risk: 0.2% [one of 247], stratified risk difference: -0.0% [95% CI -0.7 to 0.6]; p = 0.973). CONCLUSIONS: We found no difference in the risk of SSI between the decolonization and control groups, both in S. aureus carriers and noncarriers. Because of the low event numbers, no definite conclusion about efficacy of routine preoperative decolonization can be drawn. The results, however, may be helpful in future meta-analyses. LEVEL OF EVIDENCE: Level II, therapeutic study.

Pheophorbide a May Regulate Jasmonate Signaling during Dark-Induced Senescence.

Chlorophyll degradation is one of the most visible signs of leaf senescence. During senescence, chlorophyll is degraded in the multistep pheophorbide a oxygenase (PAO)/phyllobilin pathway. This pathway is tightly regulated at the transcriptional level, allowing coordinated and efficient remobilization of nitrogen toward sink organs. Using a combination of transcriptome and metabolite analyses during dark-induced senescence of Arabidopsis (Arabidopsis thaliana) mutants deficient in key steps of the PAO/phyllobilin pathway, we show an unanticipated role for one of the pathway intermediates, i.e. pheophorbide a Both jasmonic acid-related gene expression and jasmonic acid precursors specifically accumulated in pao1, a mutant deficient in PAO. We propose that pheophorbide a, the last intact porphyrin intermediate of chlorophyll degradation and a unique pathway "bottleneck," has been recruited as a signaling molecule of chloroplast metabolic status. Our work challenges the assumption that chlorophyll breakdown is merely a result of senescence, and proposes that the flux of pheophorbide a through the pathway acts in a feed-forward loop that remodels the nuclear transcriptome and controls the pace of chlorophyll degradation in senescing leaves.

Multimorbidity and healthcare resource utilization in Switzerland: a multicentre cohort study.

BACKGROUND: Multimorbidity is associated with higher healthcare resource utilization, but we lack data on the association of specific combinations of comorbidities with healthcare resource utilization. We aimed to identify the combinations of comorbidities associated with high healthcare resource utilization among multimorbid medical inpatients. METHODS: We performed a multicentre retrospective cohort study including 33,871 multimorbid (≥2 chronic diseases) medical inpatients discharged from three Swiss hospitals in 2010-2011. Healthcare resource utilization was measured as 30-day potentially avoidable readmission (PAR), prolonged length of stay (LOS) and difference in median LOS. We identified the combinations of chronic comorbidities associated with the highest healthcare resource utilization and quantified this association using regression techniques. RESULTS: Three-fourths of the combinations with the strongest association with PAR included chronic kidney disease. Acute and unspecified renal failure combined with solid malignancy was most strongly associated with PAR (OR 2.64, 95%CI 1.79;3.90). Miscellaneous mental health disorders combined with mood disorders was the most strongly associated with LOS (difference in median LOS: 17 days) and prolonged LOS (OR 10.77, 95%CI 8.38;13.84). The number of chronic diseases was strongly associated with prolonged LOS (OR 9.07, 95%CI 8.04;10.24 for ≥10 chronic diseases), and to a lesser extent with PAR (OR 2.16, 95%CI 1.75;2.65 for ≥10 chronic diseases). CONCLUSIONS: Multimorbidity appears to have a higher impact on LOS than on PAR. Combinations of comorbidities most strongly associated with healthcare utilization included kidney disorders for PAR, and mental health disorders for LOS.

Adverse perinatal outcomes for obese women are influenced by the presence of comorbid diabetes and hypertensive disorders.

Maternal obesity often occurs together with comorbid diabetes and hypertensive disorders. All three conditions are independently associated with negative perinatal outcomes. Our objective was to determine the risk and burden of adverse perinatal outcome that could be attributed to maternal obesity in combination with a comorbid status. We analyzed data from 324'664 singleton deliveries in Switzerland between 2005 and 2016. For the association of maternal obesity in the presence or absence of comorbidities with various perinatal outcomes, we estimated adjusted relative risk (RR) using multivariable regression modeling and determined the multivariable-adjusted attributable fraction of the population (AFp). Obesity was a main predictor for macrosomia, fracture of the clavicle, failure to progress in labor and prolonged labor. By stratifying women based on comorbidities, we identified significantly increased risk for preterm birth and early neonatal death only for women diagnosed with a comorbidity. However, various other outcomes were independently associated with either obesity or comorbidities. The AFp showed greatest reduction in comorbidities (15.4/15.0/13.2%), in macrosomia (6.3%) and in shoulder dystocia (4.8%) if all women were to become non-obese. We suggest that comorbidities such as diabetes and hypertensive disorders should be considered when relating maternal obesity to adverse perinatal outcomes.

Patterns of multimorbidity in internal medicine patients in Swiss university hospitals: a multicentre cohort study.

AIMS OF THE STUDY: Despite the high prevalence of multimorbidity, we lack detailed descriptive data on the most prevalent combinations of chronic comorbidities in Switzerland. We aimed to describe and quantify the most prevalent combinations of comorbidities in internal medicine multimorbid inpatients. METHODS: We conducted a multicentre retrospective cohort study including all consecutive adults (n = 42,739) discharged from the general internal medicine department of three Swiss tertiary teaching hospitals in 2010–2011. We used the Chronic Condition Indicator and the Clinical Classification Software to classify International Classification of Diseases diagnosis codes into chronic or acute diseases, into body system categories and into categories of chronic comorbidities. We defined multimorbidity as ≥2 chronic diseases. We described the most prevalent combinations of comorbidities and their prevalence. RESULTS: Seventy-nine percent (n = 33,871) of the patients were multimorbid, with a median of four chronic diseases. Chronic heart disease, chronic kidney disease, solid malignancy and substance-related disorders were the most prevalent comorbidities, with a prevalence of more than 10% for each. All these comorbidities were frequently found in combination with chronic obstructive pulmonary disease and bronchiectasis, pulmonary heart disease, and peripheral and visceral atherosclerosis. Chronic heart disease was identified in 80% of the most prevalent combinations. Half of the combinations occurred more often than it would have been expected if they were independent. CONCLUSIONS: The vast majority of patients fulfilled the criteria for multimorbidity. Chronic heart disease, chronic kidney disease, solid malignancy and substance-related disorders were each present in at least one tenth of the patients. This in-depth description of the most frequent comorbidities and of their frequent associations in a multicentre population may advise healthcare providers to improve preventive care and develop appropriate guidelines for multimorbid patients.  .

Patterns of multimorbidity associated with 30-day readmission: a multinational study.

BACKGROUND: Multimorbidity is associated with higher healthcare utilization; however, data exploring its association with readmission are scarce. We aimed to investigate which most important patterns of multimorbidity are associated with 30-day readmission. METHODS: We used a multinational retrospective cohort of 126,828 medical inpatients with multimorbidity defined as ≥2 chronic diseases. The primary and secondary outcomes were 30-day potentially avoidable readmission (PAR) and 30-day all-cause readmission (ACR), respectively. Only chronic diseases were included in the analyses. We presented the OR for readmission according to the number of diseases or body systems involved, and the combinations of diseases categories with the highest OR for readmission. RESULTS: Multimorbidity severity, assessed as number of chronic diseases or body systems involved, was strongly associated with PAR, and to a lesser extend with ACR. The strength of association steadily and linearly increased with each additional disease or body system involved. Patients with four body systems involved or nine diseases already had a more than doubled odds for PAR (OR 2.35, 95%CI 2.15-2.57, and OR 2.25, 95%CI 2.05-2.48, respectively). The combinations of diseases categories that were most strongly associated with PAR and ACR were chronic kidney disease with liver disease or chronic ulcer of skin, and hematological malignancy with esophageal disorders or mood disorders, respectively. CONCLUSIONS: Readmission was associated with the number of chronic diseases or body systems involved and with specific combinations of diseases categories. The number of body systems involved may be a particularly interesting measure of the risk for readmission in multimorbid patients.

Inhibition of the Hypoxia-Inducible Factor 1α-Induced Cardiospecific HERNA1 Enhance-Templated RNA Protects From Heart Disease.

BACKGROUND: Enhancers are genomic regulatory elements conferring spatiotemporal and signal-dependent control of gene expression. Recent evidence suggests that enhancers can generate noncoding enhancer RNAs, but their (patho)biological functions remain largely elusive. METHODS: We performed chromatin immunoprecipitation-coupled sequencing of histone marks combined with RNA sequencing of left ventricular biopsies from experimental and genetic mouse models of human cardiac hypertrophy to identify transcripts revealing enhancer localization, conservation with the human genome, and hypoxia-inducible factor 1α dependence. The most promising candidate, hypoxia-inducible enhancer RNA ( HERNA)1, was further examined by investigating its capacity to modulate neighboring coding gene expression by binding to their gene promoters by using chromatin isolation by RNA purification and λN-BoxB tethering-based reporter assays. The role of HERNA1 and its neighboring genes for pathological stress-induced growth and contractile dysfunction, and the therapeutic potential of HERNA1 inhibition was studied in gapmer-mediated loss-of-function studies in vitro using human induced pluripotent stem cell-derived cardiomyocytes and various in vivo models of human pathological cardiac hypertrophy. RESULTS: HERNA1 is robustly induced on pathological stress. Production of HERNA1 is initiated by direct hypoxia-inducible factor 1α binding to a hypoxia-response element in the histoneH3-lysine27acetylation marks-enriched promoter of the enhancer and confers hypoxia responsiveness to nearby genes including synaptotagmin XVII, a member of the family of membrane-trafficking and Ca2+-sensing proteins and SMG1, encoding a phosphatidylinositol 3-kinase-related kinase. Consequently, a substrate of SMG1, ATP-dependent RNA helicase upframeshift 1, is hyperphoshorylated in a HERNA1- and SMG1-dependent manner. In vitro and in vivo inactivation of SMG1 and SYT17 revealed overlapping and distinct roles in modulating cardiac hypertrophy. Finally, in vivo administration of antisense oligonucleotides targeting HERNA1 protected mice from stress-induced pathological hypertrophy. The inhibition of HERNA1 postdisease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. CONCLUSIONS: HERNA1 is a novel heart-specific noncoding RNA with key regulatory functions in modulating the growth, metabolic, and contractile gene program in disease, and reveals a molecular target amenable to therapeutic exploitation.

Deficits of facial emotion recognition and visual information processing in adult patients with classical galactosemia.

BACKGROUND: Classical galactosemia (CG) is due to a severe deficiency of the galactose-1-phosphate uridyl-transferase (GALT), the main enzyme of galactose metabolism. Even early introduction of galactose-restricted diet fails to prevent long-term complications, including cognitive impairment, neurological and psychiatric problems, osteoporosis, premature ovarian failure and infertility. Detailed neuropsychological phenotyping is needed in order to better understand the relevant neurodevelopmental deficiencies and to develop effective treatment strategies. AIM: To define specifically and significantly impaired neuropsychological traits in adult CG patients of the Swiss cohort. METHODS: Prospective cohort study. 22 CG patients, with confirmed genotype and low GALT activity, and 15 controls completed a computer-based neuropsychological test battery (CANTAB). Additionally, broad IQ evaluation was made for the CG patients. RESULTS: In most outcome measures of the CANTAB tasks, CG patients performed significantly worse than controls. The deficits in CG patients were most prominent in tasks that involve rapid visual information processing and facial emotion recognition. CONCLUSION: CG patients have specific cognitive problems such as impaired visual information processing and facial emotion recognition. The deficits in facial emotion recognition have not been described before and could help explain difficulties in social interactions often experienced by patients with CG.

Comparison of contemporary staging systems for oropharynx cancer in a surgically treated multi-institutional cohort.

BACKGROUND: Between the publication of the Union of International Cancer Control staging system (UICC) 7th and 8th editions, other staging algorithms for oropharyngeal squamous cell carcinoma (OPSCC) were proposed from Radiation Therapy Oncology Group (RTOG), MD Anderson Cancer Center (MDACC), and Yale University. METHODS: With C-statistics, the above-mentioned five staging algorithms were compared for overall and relapse-free survival endpoints in a multi-institutional cohort of OPSCC cases (n = 338) treated with primary surgery. RESULTS: Pathological UICC 8th ed yielded the highest C-indexes in the entire cohort and in the HPV- subset, whereas MDACC was superior for HPV+ OPSCC. RTOG was the simplest and holistic algorithm with a noninferior discriminatory power. CONCLUSION: UICC 8th ed, MDACC, and RTOG offer moderate and comparable efficacy for staging in this OPSCC patient cohort undergoing surgical treatment. Notable discrepancy between clinical and pathological UICC 8th ed algorithms poses potential concerns in diagnosis, treatment, research, and data management.

Cerebral microembolism in the critically ill with acute kidney injury (COMET-AKI trial): study protocol for a randomized controlled clinical trial.

BACKGROUND: Microembolism is a frequent pathological event during extracorporeal renal replacement therapy (RRT). Some previous data indicate that microemboli are generated in patients who are undergoing RRT and that these may contribute to increased cerebrovascular and neurocognitive morbidity in patients with end-stage renal disease. The current trial aims to quantify the microembolic load and respective qualitative composition that effectively reaches the intracerebral circulation in critically ill patients treated with different RRT modalities for acute kidney injury (AKI). METHODS/DESIGN: The COMET-AKI trial is a prospective, randomized controlled clinical trial with a 2-day clinical assessment period and follow-up visits at 6 and 12 months. Consecutive critically ill patients with AKI on continuous renal replacement therapy (CRRT) scheduled for a switch to intermittent renal replacement therapy (IRRT) will be randomized to either switch to IRRT within the next 24 h or continued CRRT for an additional 24 h. Cerebral microembolic load will be determined at baseline, i.e., before switch (on CRRT for both groups) and on IRRT versus CRRT, whichever group they were randomized to. The primary endpoint is defined as the difference in mean total cerebral microemboli count during the measurement period on CRRT versus IRRT following randomization. Microemboli will be assessed within the RRT circuit by a 1.5-MHz ultrasound detector attached to the venous RRT tubing and cerebral microemboli will be measured in the middle cerebral artery using a 1.6-MHz robotic transcranial Doppler system with automatic classification of Doppler signals as solid or gaseous. In addition to Doppler measurements, patients will be examined by magnetic resonance imaging and neurocognitive tests to gain better understanding into the potential morphological and clinical consequences of embolization. DISCUSSION: The results of COMET-AKI may help to gain a better insight into RRT modality-associated differences regarding microbubble generation and the cerebral microembolic burden endured by RRT recipients. Furthermore, identification of covariates of microbubble formation and distribution may help to encourage the evolution of next-generation RRT circuits including machinery and/or filters. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT02621749 . Registered on 3 December 2015.

Outcomes and feasibility of the short transitional intervention in psychiatry in improving the transition from inpatient treatment to the community: A pilot study.

Discharge from psychiatric inpatient care is frequently described as chaotic, stressful, and emotionally charged. Following discharge, service users are vulnerable to becoming overwhelmed by the challenges involved in readapting to their home environments, which could result in serious problems and lead to readmission. The short transitional intervention in psychiatry (STeP) is a bridging intervention that includes pre- and post-discharge sections. It aims to prepare patients for specific situations in the period immediately following discharge from a psychiatric hospital. We conducted a quasi-experimental pilot study to determine the feasibility of the intervention, and gain insight into the effects of the STeP. Two inpatient wards at a Swiss psychiatric hospital participated in the study, and represented the intervention and control arms. Patient recruitment and baseline assessment were performed 2 weeks prior to discharge. Follow-up data were collected 1 week subsequent to discharge. Questionnaires measured coping, admission and health-care usage, self-efficacy, working alliance, experience of transition, and the number of difficulties experienced following discharge. Fourteen and 15 patients completed the follow-up assessment in the control and intervention groups, respectively. The STeP did not affect primary or secondary outcomes; however, it was shown to be feasible, and patients' feedback highlighted the importance of post-discharge contact sessions. Further research is required to improve understanding of the discharge experience, identify relevant patient outcomes, and assess the effectiveness of the intervention in an adequately-powered randomized, controlled trial.

HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease.

Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth.

miR-28-5p promotes chromosomal instability in VHL-associated cancers by inhibiting Mad2 translation.

Chromosomal instability enables tumor development, enabled in part by aberrant expression of the mitotic checkpoint protein Mad2. Here we identify a novel regulatory mechanism for Mad2 expression involving miR-28-5p-mediated inhibition of Mad2 translation, and we demonstrate that this mechanism is triggered by inactivation of the tumor suppressor VHL, the most common event in clear cell renal cell carcinoma (ccRCC). In VHL-positive cancer cells, enhanced expression of miR-28-5p diminished Mad2 levels and promoted checkpoint weakness and chromosomal instability. Conversely, in checkpoint-deficient VHL-negative renal carcinoma cells, inhibition of miR-28-5p function restored Mad2 levels, mitotic checkpoint proficiency, and chromosomal stability. Notably, chromosome missegregation errors and aneuploidy that were produced in a mouse model of acute renal injury (as a result of kidney-specific ablation of pVHL function) were reverted in vivo also by genetic inhibition of miR-28-5p. Finally, bioinformatic analyses in human ccRCC associated loss of VHL with increased miR-28-5p expression and chromosomal instability. Together, our results defined miR-28-5p as a critical regulator of Mad2 translation and mitotic checkpoint function. By identifying a potential mediator of chromosomal instability in VHL-associated cancers, our work also suggests a novel microRNA-based therapeutic strategy to target aneuploid cells in VHL-associated cancers.

In silico ionomics segregates parasitic from free-living eukaryotes.

Ion transporters are fundamental to life. Due to their ancient origin and conservation in sequence, ion transporters are also particularly well suited for comparative genomics of distantly related species. Here, we perform genome-wide ion transporter profiling as a basis for comparative genomics of eukaryotes. From a given predicted proteome, we identify all bona fide ion channels, ion porters, and ion pumps. Concentrating on unicellular eukaryotes (n = 37), we demonstrate that clustering of species according to their repertoire of ion transporters segregates obligate endoparasites (n = 23) on the one hand, from free-living species and facultative parasites (n = 14) on the other hand. This surprising finding indicates strong convergent evolution of the parasites regarding the acquisition and homeostasis of inorganic ions. Random forest classification identifies transporters of ammonia, plus transporters of iron and other transition metals, as the most informative for distinguishing the obligate parasites. Thus, in silico ionomics further underscores the importance of iron in infection biology and suggests access to host sources of nitrogen and transition metals to be selective forces in the evolution of parasitism. This finding is in agreement with the phenomenon of iron withholding as a primordial antimicrobial strategy of infected mammals.

KRAS, BRAF, and TP53 deep sequencing for colorectal carcinoma patient diagnostics.

In colorectal carcinoma, KRAS (alias Ki-ras) and BRAF mutations have emerged as predictors of resistance to anti-epidermal growth factor receptor antibody treatment and worse patient outcome, respectively. In this study, we aimed to establish a high-throughput deep sequencing workflow according to 454 pyrosequencing technology to cope with the increasing demand for sequence information at medical institutions. A cohort of 81 patients with known KRAS mutation status detected by Sanger sequencing was chosen for deep sequencing. The workflow allowed us to analyze seven amplicons (one BRAF, two KRAS, and four TP53 exons) of nine patients in parallel in one deep sequencing run. Target amplification and variant calling showed reproducible results with input DNA derived from FFPE tissue that ranged from 0.4 to 50 ng with the use of different targets and multiplex identifiers. Equimolar pooling of each amplicon in a deep sequencing run was necessary to counterbalance differences in patient tissue quality. Five BRAF and 49 TP53 mutations with functional consequences were detected. The lowest mutation frequency detected in a patient tumor population was 5% in TP53 exon 5. This low-frequency mutation was successfully verified in a second PCR and deep sequencing run. In summary, our workflow allows us to process 315 targets a week and provides the quality, flexibility, and speed needed to be integrated as standard procedure for mutational analysis in diagnostics.

Identification and functional characterization of pVHL-dependent cell surface proteins in renal cell carcinoma.

The identification of cell surface accessible biomarkers enabling diagnosis, disease monitoring, and treatment of renal cell carcinoma (RCC) is as challenging as the biology and progression of RCC is unpredictable. A hallmark of most RCC is the loss-of-function of the von Hippel-Lindau (pVHL) protein by mutation of its gene (VHL). Using the cell surface capturing (CSC) technology, we screened and identified cell surface N-glycoproteins in pVHL-negative and positive 786-O cells. One hundred six cell surface N-glycoproteins were identified. Stable isotope labeling with amino acids in cell culture-based quantification of the CSC screen revealed 23 N-glycoproteins whose abundance seemed to change in a pVHL-dependent manner. Targeted validation experiments using transcriptional profiling of primary RCC samples revealed that nine glycoproteins, including CD10 and AXL, could be directly linked to pVHL-mediated transcriptional regulation. Subsequent human tumor tissue analysis of these cell surface candidate markers showed a correlation between epithelial AXL expression and aggressive tumor phenotype, indicating that pVHL-dependent regulation of glycoproteins may influence the biologic behavior of RCC. Functional characterization of the metalloprotease CD10 in cell invasion assays demonstrated a diminished penetrating behavior of pVHL-negative 786-O cells on treatment with the CD10-specific inhibitor thiorphan. Our proteomic surfaceome screening approach in combination with transcriptional profiling and functional validation suggests pVHL-dependent cell surface glycoproteins as potential diagnostic markers for therapeutic targeting and RCC patient monitoring.