Peripheral clocks tick independently of their master.

The circadian system of mammals is hierarchically organized. The suprachiasmatic nucleus (SCN) in the hypothalamus is considered the master circadian clock adapting to environmental light-dark cycles and synchronizing subsidiary oscillators in peripheral organs. While being an attractive concept, this has never been convincingly shown in vivo. New findings by Sinturel and colleagues (pp. 329-334) in this issue of Genes & Development now show the requirement of the SCN for temporal orchestration of the periphery in living animals. Surprisingly, this study also found that even in the absence of SCN or extra-SCN clocks, peripheral clocks remain rhythmic, indicating previously controversial circadian oscillator coupling within peripheral tissues.

Alternative polyadenylation factor CPSF6 regulates temperature compensation of the mammalian circadian clock.

A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3'-end-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1).

Foundations of circadian medicine.

The circadian clock is an evolutionarily highly conserved endogenous timing program that structures physiology and behavior according to the time of day. Disruption of circadian rhythms is associated with many common pathologies. The emerging field of circadian medicine aims to exploit the mechanisms of circadian physiology and clock-disease interaction for clinical diagnosis, treatment, and prevention. In this Essay, we outline the principle approaches of circadian medicine, highlight the development of the field in selected areas, and point out open questions and challenges. Circadian medicine has unambiguous health benefits over standard care but is rarely utilized. It is time for clock biology to become an integrated part of translational research.

Irradiation dose to the swallowing apparatus impacts nutritional status in head and neck cancer patients-results from the prospective randomized HEADNUT trial.

PURPOSE: To investigate the influence of radiation dose to the swallowing muscles on the nutritional status in patients with head and neck cancer undergoing primary or adjuvant (chemo)radiotherapy (C)RT. METHODS: Between 2018 and 2020, 61 patients were prospectively randomized into the so-called HEADNUT trial (head and neck cancer patients undergoing nutritional intervention). Follow-up was continued until 2022. Contouring of the swallowing apparatus included the superior (scm), middle (mcm), and inferior constrictor muscle (icm), the cricopharyngeal muscle (cphm), and the esophageal inlet. Nutritional status was assessed by bioelectrical impedance analysis (BIA) at the beginning and the end of radiotherapy. The posttherapeutic nutritional status was evaluated by the BIA-derived fat-free mass index (FFMI; kg/m2). Malnutrition was assumed at FFMI values of < 15 (women) and < 17 (men) kg/m2. To find differences between dosimetric parameters in well- and malnourished patients, Mann-Whitney U test was used. To model the association between malnutrition and its potentially influencing variables, several logistic regression models were built. RESULTS: The following parameters differed between well- and malnourished patients at the end of therapy: icm Dmean, V40Gy (%), V50Gy (%), and V60Gy (%), and sphm V40Gy (%). After entering these parameters into a multivariable logistic regression model (dosimetric model), icm Dmean (b = -0.12; Exp(b) = 0.88; 95% CI: 0.78-1.0; p = 0.06) and icm V40Gy (%; b = 0.06; Exp(b) = 1.07; 95% CI: 1-1.13; p = 0.04) proved to be independent dosimetric predictors of malnutrition. We only determined the cut-off value for predicting malnutrition for icm V40Gy (%) since it was the only parameter which met p < 0.05. The optimal cut-off value for the predictor V40Gy (%) based on the Youden Index was 85.6%. Another logistic regression model (dosimetric-clinical model) consisted of icm V40 (%) and the clinical parameters tumor localization, malnutrition before RT, gender, and combined chemotherapy. It was confirmed that both icm V40% (b = -1.9; Exp(b) = -2.7; 95% CI: 0.01-0.8; p = 0.03) and malnutrition at baseline (b = -1.9; Exp(b) = 4.4; 95% CI: 8.4-816.6; p = 0.0002) were independent predictors of subsequent malnutrition the end of RT. CONCLUSION: Establishment of a normal nutritional status before the start of RT and adherence to dose constraints for the swallowing apparatus may prevent malnutrition in head and neck cancer patients at the end of therapy. Specifically, we suggest an icm V40Gy (%) of more than 86% to be predictive for nutritional complications.

Impact of dosimetric factors on long-term percutaneous enteral gastrostomy (PEG) tube dependence in head and neck cancer patients after (chemo)radiotherapy-results from a prospective randomized trial.

PURPOSE/OBJECTIVE: To analyze dose-volume histogram (DVH)-derived data on the exposure of organs at risk with impact on long-term percutaneous enteral gastrostomy (PEG) tube dependence in head and neck cancer patients at 6 and 12 months after definitive or adjuvant (chemo)radiotherapy. MATERIALS AND METHODS: Sixty-one patients were prospectively treated with (chemo)radiotherapy. Prophylactic or reactive gastrostomy tube placement was performed in 41 (67.2%) patients. Dose-volume histogram parameters were obtained for the swallowing apparatus. RESULTS: Median follow-up time was 25 (2-34) months. Overall survival was shorter in patients with inlying PEG tubes at 6 and 12 months (log rank p = 0.038 and p = 0.017) after therapy completion. The estimated median time of tube dependency was 6 (95% confidence interval: 2-14) months. After 6 months, 46.5% of patients were tube dependent. After 12 months, this estimated proportion fell to 31.5%. For both time points, the volume to the larynx (in %) receiving at least 50 Gy (larynx V50Gy) exceeding 53% was predictive for long-term tube feeding (6 months: p = 0.041 and 12 months: p = 0.042) being an independent predictor during multivariable analysis. There was no clinical feature influencing tube dependence after 12 months. CONCLUSION: Long-term gastrostomy dependence was found to be strongly associated with an exposure of laryngeal structures (specifically, V50Gy ≥ 53%) during radiotherapy. Consequently, the avoidance of supraglottic as well as glottic structures is warranted.

The impact of nutritional counseling on thyroid disorders in head and neck cancer patients after (chemo)radiotherapy: results from a prospective interventional trial.

OBJECTIVE: To analyze the impact of nutritional counseling on the development of hypothyroidism after (chemo)radiotherapy in head and neck cancer patients to propose a new normal tissue complication probability (NTCP) model. MATERIALS AND METHODS: At baseline, at the end of (chemo)radiotherapy, and during follow-up, thyroid-stimulating hormone (TSH) with free thyroxin (fT3 and fT4), nutritional status, and nutrient intake were prospectively analyzed in 46 out of 220 screened patients. Patients received (chemo)radiotherapy within an intervention (individual nutritional counseling every 2 weeks during therapy) and a control group (no nutritional counseling). RESULTS: Overall median follow-up was 16.5 [IQR: 12; 22] months. Fourteen patients (30.4%) presented with hypothyroidism after 13.5 [8.8; 17] months. During (chemo)radiotherapy, nutritional status worsened in the entire cohort: body mass index (p < 0.001) and fat-free mass index (p < 0.001) decreased, calorie deficit (p = 0.02) increased, and the baseline protein intake dropped (p = 0.028). The baseline selenium intake (p = 0.002) increased until the end of therapy. Application of the NTCP models by Rønjom, Cella, and Boomsma et al. resulted in good performance of all three models, with an AUC ranging from 0.76 to 0.78. Our newly developed NTCP model was based on baseline TSH and baseline ferritin. Model performance was good, receiving an AUC of 0.76 (95% CI: 0.61-0.87), with a sensitivity of 57.1% and specificity of 96.9% calculated for a Youden index of 0.73 (p = 0.004; area = 0.5). CONCLUSION: Baseline TSH and ferritin act as independent predictors for radiotherapy-associated hypothyroidism. The exclusion of such laboratory chemistry parameters in future NTCP models may result in poor model performance.

Prognostic Impact of Surgical Margin Status on Overall Survival of Patients with Early Breast Cancer: A Retrospective Analysis from the Department for Women's Medicine at Charité - University Hospital Berlin.

Introduction: The impact of surgical margins on the prognosis of early breast cancer remains uncertain, particularly in the context of modern treatment approaches. This study aimed to investigate whether involved margins after surgery for early breast cancer affect overall survival. Methods: We conducted a retrospective analysis of 3767 patients who underwent surgery for primary breast cancer or carcinoma in situ between 2006 and 2022 at Charité - University Hospital Berlin. Survival analysis based on margin status and a subsequent multivariate Cox regression analysis were conducted. Results: With a median follow-up of 72.2 months, clear margins were achieved in 81.4% of patients (n = 3068) after primary surgery, while 16.2% (n = 610) required re-excision. Only 2.4% of patients (n = 89) had definitively involved margins. Margin involvement was more common in hormone receptor-positive disease, lobular subtype, carcinoma in situ, or locally advanced tumors, but less frequent in patients with previous neoadjuvant chemotherapy or triple-negative breast cancer. The Kaplan-Meier survival curves showed a significant separation with worse outcomes for patients with definitive R1 resections. However, the multivariate Cox regression analysis detected no statistically significant difference in overall survival based on margin status. Breast conserving surgery (HR 0.66; 95% CI 0.54-0.81) and HER2 overexpression (HR 0.65; 95% CI 0.48-0.89) were associated with improved survival. Conclusion: Patients who underwent breast-conserving surgery in our study demonstrated favorable outcomes compared to patients after mastectomy. Although margin status did not significantly affect overall survival, larger multicenter studies are needed to evaluate the prognostic implications of margin involvement in breast cancer treatment in different tumor stages, tumor subtypes and local and systemic treatments.

Salivary Gland Volume Predicts Malnutrition in Irradiated Head and Neck Cancer Patients: A Prospective Analysis.

BACKGROUND/AIM: Xerostomia following (chemo-) radiotherapy (CRT) is common in head and neck (HNC) patients. This prospective study focused on investigating the link between salivary gland function and the onset of malnutrition in HNC patients. PATIENTS AND METHODS: Between 2018 and 2020, 61 HNC patients scheduled for curative CRT were prospectively recruited. Nutritional status was assessed by bioelectrical impedance analysis (BIA) and xerostomia was evaluated based on the Common Terminology Criteria for Adverse Events, (CTCAE). Patient-reported outcomes for xerostomia-related symptoms, such as "dry mouth" and "sticky saliva", were also collected. Data were assessed at the beginning of therapy, during treatment, at the end of treatment, and during follow-up. Organs at risk were contoured including the submandibular and parotid glands. Dose-volume parameters were extracted for the mean Dose (Dmean), V15 Gy, V30Gy and V45Gy. RESULTS: No correlation was found between the dosimetric parameters [Dmean, V15Gy (%), V30Gy (%) and V45Gy (%)] and the occurrence of malnutrition [defined by a fat-free mass index (FFMI) <15 kg/m2 (♀) and <17 kg/m2 (♂) kg/m2 and/or body-mass index (BMI) <18.5 kg/m2] at any of the three time points tested. However, the volume of the parotid glands prior to therapy appeared to be related to the development of malnutrition. This effect was not observed with the submandibular glands. A cumulative parotid gland volume of 55.3 cm3 was identified as the threshold for malnutrition at the second follow-up examination. CONCLUSION: Although none of the dosimetric factors were associated with the development of malnutrition, the baseline parotid gland volume emerged as an independent predictor of malnutrition in head and neck cancer patients with xerostomia.

Time-of-day effects of cancer drugs revealed by high-throughput deep phenotyping.

The circadian clock, a fundamental biological regulator, governs essential cellular processes in health and disease. Circadian-based therapeutic strategies are increasingly gaining recognition as promising avenues. Aligning drug administration with the circadian rhythm can enhance treatment efficacy and minimize side effects. Yet, uncovering the optimal treatment timings remains challenging, limiting their widespread adoption. In this work, we introduce a high-throughput approach integrating live-imaging and data analysis techniques to deep-phenotype cancer cell models, evaluating their circadian rhythms, growth, and drug responses. We devise a streamlined process for profiling drug sensitivities across different times of the day, identifying optimal treatment windows and responsive cell types and drug combinations. Finally, we implement multiple computational tools to uncover cellular and genetic factors shaping time-of-day drug sensitivity. Our versatile approach is adaptable to various biological models, facilitating its broad application and relevance. Ultimately, this research leverages circadian rhythms to optimize anti-cancer drug treatments, promising improved outcomes and transformative treatment strategies.

nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer.

The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. We investigated the effects of eliminating nSMase2 on early and advanced PDA, including its influence on the microenvironment. Employing the KPC mouse model of pancreatic cancer, we demonstrate that pancreatic epithelial nSMase2 ablation reduces neoplasia and promotes a PDA subtype switch from aggressive basal-like to classical. nSMase2 elimination prolongs survival of KPC mice, hinders vasculature development, and fosters a robust immune response. nSMase2 loss leads to recruitment of cytotoxic T cells, N1-like neutrophils, and abundant infiltration of anti-tumorigenic macrophages in the pancreatic preneoplastic microenvironment. Mechanistically, we demonstrate that nSMase2-expressing PDA cell small extracellular vesicles (sEVs) reduce survival of KPC mice; PDA cell sEVs generated independently of nSMase2 prolong survival of KPC mice and reprogram macrophages to a proinflammatory phenotype. Collectively, our study highlights previously unappreciated opposing roles for exosomes, based on biogenesis pathway, during PDA progression.

Single cell proteomics characterization of bone marrow hematopoiesis with distinct Ras pathway lesions.

Normal hematopoiesis requires constant prolific production of different blood cell lineages by multipotent hematopoietic stem cells (HSC). Stem- and progenitor- cells need to balance dormancy with proliferation. How genetic alterations impact frequency, lineage potential, and metabolism of HSC is largely unknown. Here, we compared induced expression of KRAS G12D or RasGRP1 to normal hematopoiesis. At low-resolution, both Ras pathway lesions result in skewing towards myeloid lineages. Single-cell resolution CyTOF proteomics unmasked an expansion of HSC- and progenitor- compartments for RasGRP1, contrasted by a depletion for KRAS G12D . SCENITH™ quantitates protein synthesis with single-cell precision and corroborated that immature cells display low metabolic SCENITH™ rates. Both RasGRP1 and KRAS G12D elevated mean SCENITH™ signals in immature cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell-fraction, whereas this fraction diminishes for KRAS G12D . Our temporal single cell proteomics and metabolomics datasets provide a resource of mechanistic insights into altered hematopoiesis at single cell resolution.

In Vitro Assays for Measuring Intercellular Coupling Among Peripheral Circadian Oscillators.

Circadian clocks can be found in nearly all eukaryotic organisms, as well as certain bacterial strains, including commensal microbiota. Exploring intercellular coupling among cell-autonomous circadian oscillators is crucial for understanding how cellular ensembles generate and sustain coherent circadian rhythms on the tissue level, and thus, rhythmic organ functions. Here we describe a protocol for studying intercellular coupling among peripheral circadian oscillators using three-dimensional spheroid cultures in order to measure coupling strength within peripheral clock networks. We use cell spheroids to simulate in vivo tissue integrity, as well as to increase complexity of cell-cell interactions and the abundance of potential coupling factors. Circadian rhythms are monitored using live-cell imaging of spheroids equipped with circadian reporters over several days.

Effectiveness of Immune Checkpoint Inhibition vs Chemotherapy in Combination With Radiation Therapy Among Patients With Non-Small Cell Lung Cancer and Brain Metastasis Undergoing Neurosurgical Resection.

Importance: Patients with brain metastases from non-small cell lung cancer (NSCLC) have regularly been excluded from prospective clinical trials that include therapy with immune checkpoint inhibitors (ICIs). Clinical data demonstrating benefit with ICIs, specifically following neurosurgical brain metastasis resection, are scarce. Objective: To evaluate and compare the association of radiation therapy with ICIs vs classic therapy involving radiation therapy and chemotherapy regarding overall survival in a cohort of patients who underwent NSCLC brain metastasis resection. Design, Setting and Participants: This single-center 1:1 propensity-matched comparative effectiveness study at the largest neurosurgical clinic in Germany included individuals who had undergone craniotomy with brain metastasis resection from January 2010 to December 2021 with histologically confirmed NSCLC. Of 1690 patients with lung cancer and brain metastasis, 480 were included in the study. Key exclusion criteria were small-cell lung cancer, lack of tumor cells by means of histopathological analysis on brain metastasis resection, and patients who underwent biopsy without tumor resection. The association of overall survival with treatment with radiation therapy and chemotherapy vs radiation therapy and ICI was evaluated. Exposures: Radiation therapy and chemotherapy vs radiation therapy and ICI following craniotomy and microsurgical brain metastasis resection. Main Outcomes and Measures: Median overall survival. Results: From the whole cohort of patients with NSCLC (N = 384), 215 (56%) were male and 169 (44%) were female. The median (IQR) age was 64 (57-72) years. The 2 cohorts of interest included 108 patients (31%) with radiation therapy and chemotherapy and 63 patients (16%) with radiation therapy and ICI following neurosurgical metastasis removal (before matching). Median (IQR) follow-up time for the total cohort was 47.9 (28.2-70.1) months with 89 patients (23%) being censored and 295 (77%) dead at the end of follow-up in December 2021. After covariate equalization using propensity score matching (62 patients per group), patients receiving radiation therapy and chemotherapy after neurosurgery had significantly lower overall survival (11.8 months; 95% CI; 9.1-15.2) compared with patients with radiation therapy and ICIs (23.0 months; 95% CI; 20.3-53.8) (P < .001). Conclusions and Relevance: Patients with NSCLC brain metastases undergoing neurosurgical resection had longer overall survival when treated with radiation therapy and ICIs following neurosurgery compared with those receiving platinum-based chemotherapy and radiation. Radiation and systemic immunotherapy should be regularly evaluated as a treatment option for these patients.

Mammalian circadian systems: Organization and modern life challenges.

Humans and other mammalian species possess an endogenous circadian clock system that has evolved in adaptation to periodically reoccurring environmental changes and drives rhythmic biological functions, as well as behavioural outputs with an approximately 24-hour period. In mammals, body clocks are hierarchically organized, encompassing a so-called pacemaker clock in the hypothalamic suprachiasmatic nucleus (SCN), non-SCN brain and peripheral clocks, as well as cell-autonomous oscillators within virtually every cell type. A functional clock machinery on the molecular level, alignment among body clocks, as well as synchronization between endogenous circadian and exogenous environmental cycles has been shown to be crucial for our health and well-being. Yet, modern life constantly poses widespread challenges to our internal clocks, for example artificial lighting, shift work and trans-meridian travel, potentially leading to circadian disruption or misalignment and the emergence of associated diseases. For instance many of us experience a mismatch between sleep timing on work and free days (social jetlag) in our everyday lives without being aware of health consequences that may arise from such chronic circadian misalignment, Hence, this review provides an overview of the organization and molecular built-up of the mammalian circadian system, its interactions with the outside world, as well as pathologies arising from circadian disruption and misalignment.

Searching Novel Clock Genes Using RNAi-Based Screening.

RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.

Intercellular coupling between peripheral circadian oscillators by TGF-ß signaling.

Coupling between cell-autonomous circadian oscillators is crucial to prevent desynchronization of cellular networks and disruption of circadian tissue functions. While neuronal oscillators within the mammalian central clock, the suprachiasmatic nucleus, couple intercellularly, coupling among peripheral oscillators is controversial and the molecular mechanisms are unknown. Using two- and three-dimensional mammalian culture models in vitro (mainly human U-2 OS cells) and ex vivo, we show that peripheral oscillators couple via paracrine pathways. We identify transforming growth factor-ß (TGF-ß) as peripheral coupling factor that mediates paracrine phase adjustment of molecular clocks through transcriptional regulation of core-clock genes. Disruption of TGF-ß signaling causes desynchronization of oscillator networks resulting in reduced amplitude and increased sensitivity toward external zeitgebers. Our findings reveal an unknown mechanism for peripheral clock synchrony with implications for rhythmic organ functions and circadian health.

Head and neck cancer patients under (chemo-)radiotherapy undergoing nutritional intervention: Results from the prospective randomized HEADNUT-trial.

PURPOSE/OBJECTIVE: Patients with squamous cell carcinoma of the head and neck undergoing (chemo-)radiotherapy are at high risk of malnutrition. Nevertheless, there is still a lack of prospective, randomized trials investigating the influence of nutritional status on therapy-related toxicity and patients' outcome. MATERIALS AND METHODS: Between October 2018 and October 2020, 61 patients were randomized into an intervention and control group. Questionnaires (MUST, NRS-2002, and Nutriscore), clinical examinations, laboratory analyses, and bioelectrical impedance analysis (BIA) were used to assess nutritional status for all patients at the beginning and end of therapy as well as every 2 weeks during therapy. The intervention consisted of an individualized nutritional counseling every 2 weeks during therapy. RESULTS: Median baseline BMI for all participants was 23.8 (14.5-37.2) kg/m2 and dropped to 22.9 (16.8-33) kg/m2 after therapy (p < 0.001). In all patients, median baseline fat-free mass index (FFMI) was 18.1 (14-24.7) kg/m2 and decreased to 17.8 (13.4-21.6) kg/m2 till the end of therapy (p < 0.001). Compliant patients with a BMI < 22 kg/m2 presented with less weight loss in the intervention group compared to the control (p = 0.015, CI: 0.33-2.95). At baseline, MUST was the only screening-test which showed both good sensitivity (86%) and specificity (88%) in detecting malnutrition. Median follow-up was 15 (1-26) months and is still ongoing. 2-year overall survival rate was 70% in the control and 79% in the intervention group (log-rank p = 0.79). Pretherapeutic phase angle, posttherapeutic FFMI and albumin level were prognostic indicators for overall survival (log-rank p = 0.002, p = 0.008 and p = 0.016). CONCLUSIONS: Malnutrition negatively impacts patients' outcome under (chemo-)radiotherapy. Baseline phase angle, posttherapeutic FFMI and albumin level are proposed as reliable indicators for overall survival. This study was registered within the German Clinical Trials Register (DRKS00016862).

Coupled network of the circadian clocks: a driving force of rhythmic physiology.

The circadian system is composed of coupled endogenous oscillators that allow living beings, including humans, to anticipate and adapt to daily changes in their environment. In mammals, circadian clocks form a hierarchically organized network with a 'master clock' located in the suprachiasmatic nucleus of the hypothalamus, which ensures entrainment of subsidiary oscillators to environmental cycles. Robust rhythmicity of body clocks is indispensable for temporally coordinating organ functions, and the disruption or misalignment of circadian rhythms caused for instance by modern lifestyle is strongly associated with various widespread diseases. This review aims to provide a comprehensive overview of our current knowledge about the molecular architecture and system-level organization of mammalian circadian oscillators. Furthermore, we discuss the regulatory roles of peripheral clocks for cell and organ physiology and their implication in the temporal coordination of metabolism in human health and disease. Finally, we summarize methods for assessing circadian rhythmicity in humans.