A novel sequential treatment approach between denosumab and romosozumab in patients with severe osteoporosis.

In severe osteoporosis, the optimal approach for sequential treatment between denosumab and romosozumab is unclear. We utilised a novel overlapping strategy in three patients with very-high fracture risk despite long-term denosumab which led to greater bone density improvements than previously reported with standard approaches. Larger confirmatory prospective studies are needed. PURPOSE/INTRODUCTION: In patients with severe osteoporosis, the optimal approach for sequential treatment between denosumab and romosozumab has not been established. The ideal strategy would maximise gains in bone mineral density (BMD) with romosozumab and effectively mitigate the risk of rebound increased bone turnover when sequencing from denosumab. Limited studies exploring the sequence from denosumab to romosozumab report only modest-to-no improvement in BMD and inadequate suppression of rebound bone turnover. METHODS: We describe three patients with severe osteoporosis and multiple fragility fractures despite long-term denosumab. A novel overlapping sequential treatment approach was utilised to maximise therapeutic benefit given these patients had a very high fracture risk. Romosozumab was commenced 3 months after the last denosumab dose. Instead of waiting until completion of romosozumab, denosumab was recommenced 6 months after commencing romosozumab in response to rising bone turnover markers. RESULTS: Patients experienced a ~ 5-22% increase in lumbar spine BMD, and one patient had an 8% increase in total hip BMD after 12 months romosozumab. Serum bone turnover markers demonstrated an anabolic effect of romosozumab occurred despite overlapping treatment with denosumab. Recommencement of denosumab suppressed an increase in bone resorption in all cases. No new vertebral fractures occurred during this treatment. CONCLUSIONS: A novel overlapping sequential treatment approach between denosumab and romosozumab produced greater improvements in lumbar spine and hip BMD than previously reported with standard approaches. Larger prospective controlled studies are needed to confirm these findings and establish the optimal use of romosozumab in patients pre-treated with denosumab to maximise BMD gains and minimise fracture risk.

Large variance in a lateral osteoarthritic population prior to and following lateral unicompartmental arthroplasty: An analysis of knee phenotypes.

BACKGROUND: Disagreement exists on the optimal coronal alignment target for lateral unicompartmental knee arthroplasty (UKA). An improved understanding of the distribution of coronal alignment and joint line orientation in lateral osteoarthritis (OA) might prove beneficial. The aim of this study was to evaluate the pre- and postoperative Coronal Plane Alignment of the Knee (CPAK) distribution following lateral UKA and to evaluate the association between phenotypic variation and patient-reported outcome measures (PROMs). METHODS: A surgeon's registry was retrospectively reviewed between 2012 and 2022 to identify patients who received primary lateral UKA for advanced, lateral compartment OA. Radiographic measurements were performed, and CPAK phenotypes were determined. The Knee Injury and Osteoarthritis Outcome Score (KOOS), Kujala, and patient satisfaction were analyzed at one-year and two-year follow-up. RESULTS: A total of 305 knees were included. Preoperatively, seven phenotypes were observed and CPAK3 (54.1%) was most commonly observed. Postoperatively, all nine phenotypes were observed and CPAK6 (32.8%) was predominant. Preoperatively, 23.6% did not have a prearthritic valgus alignment. No significant differences in PROMs were found between individual phenotypes or between preserved and altered phenotypes. CONCLUSION: Coronal alignment and joint line orientation were highly variable within a lateral compartment OA cohort. However, no association was demonstrated between superior postoperative PROMs and phenotype variation or phenotype preservation, which might suggest that there is not one universal optimal alignment target. Interestingly, 23.6% of knees with lateral compartment OA did not have a prearthritic valgus alignment, which may have been affected by joint line orientation.

Eumycetoma causative agents: A systematic review to inform the World Health Organization priority list of fungal pathogens.

The World Health Organization, in response to the growing burden of fungal disease, established a process to develop a fungal priority pathogens list. This systematic review aimed to evaluate the epidemiology and impact of eumycetoma. PubMed and Web of Science were searched to identify studies published between 1 January 2011 and 19 February 2021. Studies reporting on mortality, inpatient care, complications and sequelae, antifungal susceptibility, risk factors, preventability, annual incidence, global distribution, and emergence during the study time frames were selected. Overall, 14 studies were eligible for inclusion. Morbidity was frequent with moderate to severe impairment of quality of life in 60.3%, amputation in up to 38.5%, and recurrent or long-term disease in 31.8%-73.5% of patients. Potential risk factors included male gender (56.6%-79.6%), younger age (11-30 years; 64%), and farming occupation (62.1%-69.7%). Mycetoma was predominantly reported in Sudan, particularly in central Sudan (37%-76.6% of cases). An annual incidence of 0.1/100 000 persons and 0.32/100 000 persons/decade was reported in the Philippines and Uganda, respectively. In Uganda, a decline in incidence from 3.37 to 0.32/100 000 persons between two consecutive 10-year periods (2000-2009 and 2010-2019) was detected. A community-based, multi-pronged prevention programme was associated with a reduction in amputation rates from 62.8% to 11.9%. With the pre-specified criteria, no studies of antifungal drug susceptibility, mortality, and hospital lengths of stay were identified. Future research should include larger cohort studies, greater drug susceptibility testing, and global surveillance to develop evidence-based treatment guidelines and to determine more accurately the incidence and trends over time.

Podocyte-Specific Deletion of MCP-1 Fails to Protect against Angiotensin II- or Adriamycin-Induced Glomerular Disease.

Investigating the role of podocytes in proteinuric disease is imperative to address the increasing global burden of chronic kidney disease (CKD). Studies strongly implicate increased levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) in proteinuric CKD. Since podocytes express the receptor for MCP-1 (i.e., CCR2), we hypothesized that podocyte-specific MCP-1 production in response to stimuli could activate its receptor in an autocrine manner, leading to further podocyte injury. To test this hypothesis, we generated podocyte-specific MCP-1 knockout mice (Podo-Mcp-1fl/fl) and exposed them to proteinuric injury induced by either angiotensin II (Ang II; 1.5 mg/kg/d, osmotic minipump) or Adriamycin (Adr; 18 mg/kg, intravenous bolus). At baseline, there were no between-group differences in body weight, histology, albuminuria, and podocyte markers. After 28 days, there were no between-group differences in survival, change in body weight, albuminuria, kidney function, glomerular injury, and tubulointerstitial fibrosis. The lack of protection in the knockout mice suggests that podocyte-specific MCP-1 production is not a major contributor to either Ang II- or Adr-induced glomerular disease, implicating that another cell type is the source of pathogenic MCP-1 production in CKD.

Heme induced loss of renovascular endothelial protein C receptor promotes chronic kidney disease in sickle mice.

Chronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with progressive deterioration of renal health resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vasoocclusive crisis associated with acute intravascular hemolysis. However, the mechanisms of the hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of vascular endothelial protein C receptor (EPCR) on the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR) indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of SCD patients, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD.

Extrachromosomal telomere DNA derived from excessive strand displacements.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Deregulated DNA ADP-ribosylation impairs telomere replication.

The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.

Preservation of prearthritic coronal knee phenotype and prearthritic coronal alignment yielded improved Kujala scores following ligament-guided medial unicompartmental knee arthroplasty.

PURPOSE: There is a lack of literature evaluating outcomes of the ligament-guided approach in medial unicompartmental knee arthroplasty (UKA). An improved comprehension of the distribution of coronal plane alignment of the knee (CPAK) phenotypes and sagittal tibial wear patterns and their associations with patient-reported outcome measures (PROMs) and implant survivorship could provide insights into its further application in daily practice. METHODS: A registry was reviewed for patients with a minimal 2-year follow-up who underwent robotic-assisted, ligament-guided, medial UKA between 2008 and 2016. Survivorship and postoperative PROMs were collected. CPAK phenotypes and sagittal tibial wear patterns were determined. Survivorship, Knee Injury and Osteoarthritis Outcome Score (KOOS), Kujala and patient satisfaction were compared between phenotypes and sagittal tibial wear patterns. RESULTS: A total of 618 knees were included at a mean follow-up of 4.1 [2.0-9.6] years. Four-year conversion to the TKA survival rate was 98.9% [98.4%-99.3%] and 94.3% [93.3%-95.3%] for all-cause revision. Patients with preservation of the CPAK phenotype (84.5 ± 14.9, 81.8 ± 15.5, p = 0.033) and restoration of prearthritic coronal alignment (84.1 ± 14.9, 81.7 ± 15.9, p = 0.045) had a significantly higher Kujala score. No other significant differences in survivorship or PROMs were observed between phenotypes or sagittal tibial wear patterns. Additionally, no difference in survival rates was observed between preserved or altered phenotypes. CONCLUSION: This study demonstrated that preservation of CPAK phenotype and preservation of prearthritic coronal alignment yielded a significantly higher Kujala score. No other significant differences in PROMs or implant survivorship were observed, suggesting that robotic-assisted, ligament-guided medial UKA provides equal outcomes for all observed phenotypes and sagittal tibial wear patterns in medial compartment OA as long as preoperative CPAK phenotype is preserved postoperatively. LEVEL OF EVIDENCE: Level III.

Outcomes of SDHB pathogenic variant carriers: A systematic review and meta-analysis.

CONTEXT: Carriers of germline pathogenic variants (PV) in succinate dehydrogenase type B (SDHB) are at increased risk of developing pheochromocytomas and paragangliomas (PPGL). Understanding their outcomes can guide recommendations for risk assessment and early detection. OBJECTIVE: We performed a systematic review and meta-analysis of the following outcomes in SDHB PV carriers: age-specific risk of developing tumors, metastatic progression, second primary tumor development, and mortality. MATERIALS AND METHODS: Pubmed, MEDLINE and EMBASE were searched. Sixteen studies met the inclusion criteria and were sorted into four outcome categories: age-specific penetrance, metastatic disease, risk of second tumour and mortality. We assessed heterogeneity and performed a meta-analysis across studies using a random effects model with the DerSimonian and Laird method. RESULTS: Penetrance of PPGL for non-proband/non-index SDHB PV carriers by age 20 was 4% (95% CI, 3%-6%), 11% (95% CI, 8%-15%) by age 40, 24% (95% CI, 19%-31%) by age 60 and 35% (95% CI, 25%-47%) by age 80. The overall risk of metastatic disease for non-proband/non-index carriers with PPGL was 9% (95% CI, 5-16%) per lifetime. In all affected cases (combining both proband/index and non-proband/non-index carriers with tumors), the risk of a second tumor was 24% (95% CI, 18-31%) and all cause 5-year mortality was 18% (95% CI 6-40%). CONCLUSION: Penetrance for PPGL in SDHB PV carriers increases linearly with age. Affected carriers are at risk of developing and dying from metastatic disease, or of developing second tumors. Lifelong surveillance is appropriate.

Clinical Activity of Selpercatinib in RET-mutant Pheochromocytoma.

INTRODUCTION: Activating RET alterations have been reported in a variety of solid tumors, including pheochromocytoma where they occur both sporadically and as part of familial multiple endocrine neoplasia type 2 (MEN2) syndromes. Selpercatinib is a first-in-class, highly selective, and potent small molecule RET kinase inhibitor that has demonstrated marked and durable anti-tumor activity in diverse RET-activated solid tumors in the LIBRETTO-001 study (NCT03157128). METHODS: We describe the first six pheochromocytoma cases treated with selpercatinib in the LIBRETTO-001 study. RESULTS: Of the six patients (one sporadic and five reported as part of MEN2 syndromes) in this case report, four had a partial response/complete response and two had stable disease per independent review committee. Treatment duration ranged from 9.2 months to more than 56.4 months. The safety profile of treatment was consistent with selpercatinib in other indications. CONCLUSION: These data support selpercatinib as an effective therapy against RET-mutant pheochromocytoma, adding to the diversity of RET-activated tumor types that may benefit from targeted RET inhibition.

SUMO Promotes DNA Repair Protein Collaboration to Support Alterative Telomere Lengthening in the Absence of PML.

Alternative lengthening of telomeres (ALT) pathway maintains telomeres in a significant fraction of cancers associated with poor clinical outcomes. A better understanding of ALT mechanisms can provide a basis for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins plays a critical role in the formation of ALT telomere-associated PML bodies (APBs), where telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, whether and how SUMO contributes to ALT beyond APB formation remains elusive. Here, we report that SUMO promotes collaboration among DNA repair proteins to achieve APB-independent telomere maintenance. By using ALT cancer cells with PML protein knocked out and thus devoid of APBs, we show that sumoylation is required for manifesting ALT features, including telomere clustering and telomeric DNA synthesis, independent of PML and APBs. Further, small molecule-induced telomere targeting of SUMO produces signatures of phase separation and ALT features in PML null cells in a manner depending on both sumoylation and SUMO interaction with SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are linked to the enrichment of DNA repair proteins, including Rad52, Rad51AP1, and BLM, to the SUMO-containing telomere foci. Finally, we find that Rad52 can undergo phase separation, enrich SUMO on telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that, in addition to forming APBs, SUMO also promotes collaboration among DNA repair proteins to support telomere maintenance in ALT cells. Given the promising effects of sumoylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells.

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function.

Prospective evaluation of the safety of ultrasound-guided cervical medial branch blocks using the in-plane technique.

BACKGROUND: While fluoroscopic guidance is currently the imaging standard for cervical medial branch blocks (CMBBs), ultrasound guidance (USG) offers several potential safety advantages such as real-time needle visualization and the ability to detect and avoid critical soft tissue vascular or neural structures. However, no large-scale trials have examined the safety of USG for CMBB. METHODS: Five hundred patients undergoing 2308 individual block levels were recruited using a prospective cohort design, and blocks were performed in an outpatient office setting using an in-plane USG technique. Primary outcomes included immediate block-related complication, as well as delayed occurrences, in the following 2 weeks. Vascular structures adjacent to the target area, as well as the occurrence of vascular breach, were recorded. RESULTS: Three minor immediate complications were noted (two subcutaneous hematomas, one vasovagal reaction) comprising 0.13% of blocks (0.03% to 0.38%; 95% two-sided CI), and no delayed events were recorded (0% to 0.16%; 97.5% one-sided CI). Blood vessels were detected and avoided in 8.2% of blocks, and vascular breach was noted in 0.52% of blocks (0.27% to 0.91%; 95% two-sided CI). CONCLUSION: When performed using an in-plane technique by experienced operators, USG CMBB was found to be safe, with rare minor immediate complications and no further adverse event reported in the following 2 weeks. TRIAL REGISTRATION NUMBER: NCT04852393.

NRF2 in kidney physiology and disease.

The role of NRF2 in kidney biology has received considerable interest over the past decade. NRF2 transcriptionally controls genes responsible for cellular protection against oxidative and electrophilic stress and has anti-inflammatory functions. NRF2 is expressed throughout the kidney and plays a role in salt and water handling. In disease, animal studies show that NRF2 protects against tubulointerstitial damage and reduces interstitial fibrosis and tubular atrophy, and may slow progression of polycystic kidney disease. However, the role of NRF2 in proteinuric glomerular diseases is controversial. Although the NRF2 inducer, bardoxolone methyl (CDDO-Me), increases glomerular filtration rate in humans, it has not been shown to slow disease progression in diabetic kidney disease and Alport syndrome. Furthermore, bardoxolone methyl was associated with negative effects on fluid retention, proteinuria, and blood pressure. Several animal studies replicate findings of worsened proteinuria and a more rapid progression of kidney disease, although considerable controversy exists. It is clear that further study is needed to better understand the effects of NRF2 in the kidney. This review summarizes the available data to clarify the promise and risks associated with targeting NRF2 activity in the kidney.

Missing Data Analysis.

Methods for handling missing data in clinical psychology studies are reviewed. Missing data are defined, and a taxonomy of main approaches to analysis is presented, including complete-case and available-case analysis, weighting, maximum likelihood, Bayes, single and multiple imputation, and augmented inverse probability weighting. Missingness mechanisms, which play a key role in the performance of alternative methods, are defined. Approaches to robust inference, and to inference when the mechanism is potentially missing not at random, are discussed. Expected final online publication date for the Annual Review of Clinical Psychology, Volume 20 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Novel prognostic nomogram for predicting recurrence-free survival in medullary thyroid carcinoma.

AIMS: Recently, there have been attempts to improve prognostication and therefore better guide treatment for patients with medullary thyroid carcinoma (MTC). In 2022, the International MTC Grading System (IMTCGS) was developed and validated using a multi-institutional cohort of 327 patients. The aim of the current study was to build upon the findings of the IMTCGS to develop and validate a prognostic nomogram to predict recurrence-free survival (RFS) in MTC. METHODS AND RESULTS: Data from 300 patients with MTC from five centres across the USA, Europe, and Australia were used to develop a prognostic nomogram that included the following variables: age, sex, AJCC stage, tumour size, mitotic count, necrosis, Ki67 index, lymphovascular invasion, microscopic extrathyroidal extension, and margin status. A process of 10-fold cross-validation was used to optimize the model's performance. To assess discrimination and calibration, the area-under-the-curve (AUC) of a receiver operating characteristic (ROC) curve, concordance-index (C-index), and dissimilarity index (D-index) were calculated. Finally, the model was externally validated using a separate cohort of 87 MTC patients. The model demonstrated very strong performance, with an AUC of 0.94, a C-index of 0.876, and a D-index of 19.06. When applied to the external validation cohort, the model had an AUC of 0.9. CONCLUSIONS: Using well-established clinicopathological prognostic variables, we developed and externally validated a robust multivariate prediction model for RFS in patients with resected MTC. The model demonstrates excellent predictive capability and may help guide decisions on patient management. The nomogram is freely available online at https://nomograms.shinyapps.io/MTC_ML_DFS/.

The Centenary of the Leprosy Relief Association (Lepra)-a moment for celebration and reflection†.

The year 2024 is the Centenary of the foundation of the Leprosy Relief Association (Lepra), formerly the British Empire Leprosy Relief Association (BELRA). The name of the organization changed to the LEProsy Relief Association (LEPRA) in 1976 but has been known as Lepra since 2008. Over the years it has worked closely with members and office holders of the Royal Society of Tropical Medicine and Hygiene. Its work has encompassed activities from the earliest initiatives to ensure appropriate living conditions for those with the disease to the development of leprosy chemotherapy. However, this has now evolved into a strong partnership between the UK- and India-based Lepra hubs, which are carrying out research and public health initiatives ranging from elimination of prejudice against those with leprosy to adopting the recently launched WHO programme for skin NTDs to facilitate integrated control and management regimens. The fight against leprosy has always been a partnership between a wide variety of disease-specific NGOs, health-care workers and international health agencies. The story of Lepra illustrates the central role of these partnerships and national as well as international collaboration.

Oxidative guanine base damage plays a dual role in regulating productive ALT-associated homology-directed repair.

Cancer cells maintain telomeres by upregulating telomerase or alternative lengthening of telomeres (ALT) via homology-directed repair at telomeric DNA breaks. 8-Oxoguanine (8oxoG) is a highly prevalent endogenous DNA lesion in telomeric sequences, altering telomere structure and telomerase activity, but its impact on ALT is unclear. Here, we demonstrate that targeted 8oxoG formation at telomeres stimulates ALT activity and homologous recombination specifically in ALT cancer cells. Mechanistically, an acute 8oxoG induction increases replication stress, as evidenced by increased telomere fragility and ATR kinase activation at ALT telomeres. Furthermore, ALT cells are more sensitive to chronic telomeric 8oxoG damage than telomerase-positive cancer cells, consistent with increased 8oxoG-induced replication stress. However, telomeric 8oxoG production in G2 phase, when ALT telomere elongation occurs, impairs telomeric DNA synthesis. Our study demonstrates that a common oxidative base lesion has a dual role in regulating ALT depending on when the damage arises in the cell cycle.