Period 1 Research Articles

Human PERIOD3 variants lead to winter depression-like behaviours via glucocorticoid signalling.

Our brain adapts to seasonal changes. Mis-adaptations may lead to seasonal patterns in several psychiatric disorders, but we know little regarding the underlying mechanisms. Our previous work identified two variants in the human circadian clock gene PERIOD3 (PER3), that is, P415A and H417R, which are associated with winter depression, but whether and how these variants lead to the disorder remain to be characterized. Here we find that male mice carrying human P415A and H417R display winter depression-like behaviours that are caused by the actions of P415A and H417R in the adrenal gland. Systemic corticosterone level is downregulated in adaptation to shortening of day length, while P415A and H417R eliminate this downregulation by increasing corticosterone synthesis. Enhanced glucocorticoid signalling represses the transcription of Tph2, which encodes the rate-limiting enzyme of serotonin synthesis, leading to increased depression-like behaviours. Taken together, our findings unveil a mechanism according to which human variants contribute to seasonal mood traits.

Introduction of 7-day amotosalen/ultraviolet A light pathogen-reduced platelets in Honduras: Impact on platelet availability in a lower middle-income country.

Honduras became the first lower middle-income country (LMIC) to adopt amotosalen/UVA pathogen-reduced (PR) platelet concentrates (PCs) as a national platelet safety measure in 2018. The Honduran Red Cross (HRC) produces ~70% of the national platelet supply using the platelet-rich plasma (PRP) method. Between 2015 and 2018, PCs were screened with bacterial culture and issued as individual, non-pooled PRP units with weight-based dosing and 5-day shelf-life. PR PCs were produced in six-PRP pools with a standardized dose (≥3.0 × 1011), no bacterial screening and 7-day shelf-life. Gamma irradiation and leukoreduction were not used. PC production and distribution data were retrospectively analysed in two periods. Period 1 (P1) included 3 years of PRP PCs and a transition year (2015-18). Period 2 (P2) included 5 years of PR PCs (2019-23). PC doses were standardized to an equivalent adult dose for both periods. Descriptive statistics were calculated. HRC produced 10% more PC doses per year on average in P2 compared to P1. Mean annual waste at HRC declined from 23.9% in P1 to 1.1% in P2. Two urban regions consumed 96% of PC doses in P1 and 88.3% in P2. PC distributions increased in 14/18 regions. Standardized dosage, PR and 7-day shelf-life increased PC availability, reduced waste, eliminated bacterial screening and avoided additional costs for arboviral testing, leukoreduction and irradiation. Access to PC transfusion remains limited in Honduras; however, the conversion to pooled PR PCs illustrates the potential to sustainably expand PC distribution in an LMIC.

Oxysterol Sensing Through GPR183 Triggers Endothelial Senescence in Hypertension.

Despite endothelial dysfunction being an initial step in the development of hypertension and associated cardiovascular/renal injuries, effective therapeutic strategies to prevent endothelial dysfunction are still lacking. GPR183 (G protein-coupled receptor 183), a recently identified G protein-coupled receptor for oxysterols and hydroxylated metabolites of cholesterol, has pleiotropic roles in lipid metabolism and immune responses. However, the role of GPR183 in the regulation of endothelial function remains unknown. Endothelial-specific GPR183 knockout mice were generated and used to examine the role of GPR183 in endothelial senescence by establishing 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Echocardiography, transmission electron microscopy, blood pressure measurement, vasorelaxation response experiments, flow cytometry analysis, and chromatin immunoprecipitation analysis were performed in this study. Endothelial GPR183 was significantly induced in hypertensive mice, which was further confirmed in renal biopsies from subjects with hypertensive nephropathy. Endothelial-specific deficiency of GPR183 markedly alleviated cardiovascular and renal injuries in hypertensive mice. Moreover, we found that GPR183 regulated endothelial senescence in both hypertensive mice and aged mice. Mechanistically, GPR183 disrupted circadian signaling by inhibiting PER1 (period circadian regulator 1) expression, thereby facilitating endothelial senescence and dysfunction through the cAMP (cyclic adenosine monophosphate)/PKA (protein kinase A)/CREB (cAMP-response element binding protein) signaling pathway. Importantly, pharmacological inhibition of the oxysterol-GPR183 axis by NIBR189 or clotrimazole ameliorated endothelial senescence and cardiovascular/renal injuries in hypertensive mice. This study discovers a previously unrecognized role of GPR183 in promoting endothelial senescence. Pharmacological targeting of GPR183 may be an innovative therapeutic strategy for hypertension and its associated complications.

Overexpression of PER2 Promotes De Novo Fatty Acid Synthesis, Fatty Acid Desaturation, and Triglyceride Accumulation in Bovine Mammary Epithelial Cells.

The core clock gene Period2 (PER2) is associated with mammary gland development and lipid synthesis in rodents and has recently been found to have a diurnal variation in the process of lactation, but has not yet been demonstrated in bovine mammary epithelial cells (BMECs). To explore the regulatory function of PER2 on milk fat synthesis in bovine mammary epithelial cells, we initially assessed the expression of clock genes and milk fat metabolism genes for 24 h using real-time quantitative PCR and fitted the data to a cosine function curve. Subsequently, we overexpressed the PER2 in BMECs using plasmid vector (pcDNA3.1-PER2), with empty vector pcDNA3.1-myc as the control. After transfecting BMECs for 48 h, we assessed the protein abundance related to milk fat synthesis by Western blot, the expression of genes coding for these proteins using real time-quantitative PCR, the production of triacylglycerol, and the fatty acid profile. The findings indicated that a total of nine clock genes (PER1/2, CRY1/2, REV-ERBα, BMAL1, NCOR1, NR2F2, FBXW11), seven fatty acid metabolism genes (CD36, ACSS2, ACACA, SCD, FADS1, DGAT1, ADFP), and six nuclear receptor-related genes (INSIG1, SCAP, SREBF1, C/EBP, PPARG, LXR) exhibited oscillation with a period close to 24 h in non-transfected BMECs (R2 ≥ 0.7). Compared to the control group (transfected with empty pcDNA3.1-myc), the triglyceride content significantly increased in the PER2 overexpression group (p < 0.05). The lipogenic genes for fatty acid transport and triglyceride synthesis (ACACA, SCD, LPIN1, DGAT1, and SREBF1) were upregulated after PER2 overexpression, along with the upregulation of related protein abundance (p < 0.05). The contents and ratios of palmitic acid (C16:0), oleic acid (C18:1n9c), and trans-oleic acid (C18:1n9t) were significantly increased in the overexpression group (p < 0.05). Overall, the data supported that PER2 participated in the process of milk fat metabolism and is potentially involved in the de novo synthesis and desaturation of fatty acid in bovine mammary epithelial cells.

Differential serum levels of CACNA1C, circadian rhythm and stress response molecules in subjects with bipolar disorder: Associations with genetic and clinical factors

BackgroundMany patients with bipolar disorder (BD) do not respond to or have difficulties tolerating lithium and/or other mood stabilizing agents. There is a need for personalized treatments based on biomarkers in guiding treatment options. The calcium voltage-gated channel CACNA1C is a promising candidate for developing personalized treatments. CACNA1C is implicated in BD by genome-wide association studies and several lines of evidence suggest that targeting L-type calcium channels could be an effective treatment strategy. However, before such individualized treatments can be pursued, biomarkers predicting treatment response need to be developed. MethodsAs a first step in testing the hypothesis that CACNA1C genotype is associated with serum levels of CACNA1C, we conducted ELISA measures on serum samples from 100 subjects with BD and 100 control subjects. ResultsWe observed significantly higher CACNA1C (p < 0.01) protein levels in subjects with BD. The risk single nucleotide polymorpshism (SNP) (rs11062170) showed functional significance as subjects homozygous for the risk allele (CC) had significantly greater CACNA1C protein levels compared to subjects with one (p = 0.013) or no copies (p = 0.009). We observed higher somatostatin (SST) (p < 0.003) protein levels and lower levels of the clock protein aryl hydrocarbon receptor nuclear translocator-like (ARTNL) (p < 0.03) and stress signaling factor corticotrophin releasing hormone (CRH) (p < 0.001) in BD. SST and period 2 (PER2) protein levels were associated with both alcohol dependence and lithium response. ConclusionsOur findings represent the first evidence for increased serum levels of CACNA1C in BD. Along with altered levels of SST, ARNTL, and CRH our findings suggest CACNA1C is associated with circadian rhythm and stress response disturbances in BD.

An amygdalar oscillator coordinates cellular and behavioral rhythms.

Circadian rhythms are generated by the master pacemaker suprachiasmatic nucleus (SCN) in concert with local clocks throughout the body. Although many brain regions exhibit cycling clock gene expression, the identity of a discrete extra-SCN brain oscillator that produces rhythmic behavior has remained elusive. Here, we show that an extra-SCN oscillator in the lateral amygdala (LA) is defined by expression of theclock-output molecule mWAKE/ANKFN1. mWAKE is enriched in the anterior/dorsal LA (adLA), and, strikingly, selective disruption of clock function or excitatory signaling in adLAmWAKE neurons abolishes Period2 (PER2) rhythms throughout the LA. mWAKE levels rise at night and promote rhythmic excitability of adLAmWAKE neurons by upregulating Ca2+-activated K+ channel activity specifically at night. adLAmWAKE neurons coordinate rhythmic sensory perception and anxiety in a clock-dependent and WAKE-dependent manner. Together, these data reveal the cellular identity of an extra-SCN brain oscillator and suggest a multi-level hierarchical system organizing molecular and behavioral rhythms.

Donor and newborn profiles and their influence on donation volume and duration: a cross-sectional study in a Spanish human milk bank

BackgroundHuman milk banks are essential facilities to provide donated human milk (DHM) to preterm and term infants with health complications. Little is known regarding milk bank donors and how their characteristics may influence the particularities of the donation process. The present study aims to assess characteristics of donors and their newborns to identify associations with the amount of DHM and initiation and donation time, during the first and second year of the milk bank operation in Córdoba, Spain.MethodsThis cross-sectional study was conducted in three periods: pre-opening of the milk bank (PRE) including all women who gave birth to a newborn between January – May 2017 and were hospital users; donors in the first year after the opening (Period 1 (P1): April 2019 – March 2020); and in the second year (P2: April 2020 – March 2021). For P1 and P2, DHM data were recorded. The relationships between donor and newborn characteristics and the donation process were examined using univariable and regression models.ResultsFrom 391 women interviewed in the PRE period, 55 (14%) showed intention to donate. In P1 and P2, there were 51 and 25 human milk (HM) donors, respectively. Age, gestational age (GA) and parity were similar between periods. In P2, a higher proportion of donors had higher education (P1: 46%; P2: 70.8%, p = 0.045). Around 40% of donors in both periods were on maternity leave. In P1, donors who had low birth weight infants (< 2500 g) donated more HM than those with infants weighing ≥ 2500 g (p = 0.020). In P2, women whose GA was < 37 weeks donated a higher volume vs. those with ≥ 37 weeks (p = 0.002). Maternity leave was linked to a shorter initiation time for donations in both periods (P1: p = 0.002; P2: p < 0.001).ConclusionsData obtained from a Spanish human milk bank indicate that prematurity and low birth weight appear to influence the amounts of DHM. Employment status might be a decisive factor in initiating HM donation. Additional efforts are required to identify shared donor characteristics that influence the initiation and volume of donation.

Trends in Presentation, Management, and Survival of Women With Breast Cancer in a Multiethnic, Middle-Income Asian Setting.

Granular data on breast cancer (BC) are pertinent for surveillance, planning, and monitoring of cancer care delivery. We determined the trends in clinical presentation, management, and survival of women with BC in a multiethnic middle-income Asian setting over 15 years. Data of 7,478 Malaysian women newly diagnosed with invasive BC between 2005 and 2019 from three hospital-based cancer registries were included. Trends in demographic, tumor, and treatment characteristics were compared across period 1 (P1): 2005-2009, period 2 (P2): 2010-2014, and period 3 (P3): 2015-2019. Overall survival and net survival were determined. More women in P3 than P1 were older than 60 years at diagnosis. Only a marginal increase in proportion of women with stage I disease was observed (23.7% v 27.2% in P1 and P3, respectively, P = .004). Nonetheless, patients were increasingly presenting with smaller tumors, fewer axillary node involvement, well-differentiated tumors, and hormone receptor expression in recent times. Proportion of women with human epidermal growth factor receptor 2 (HER2)-overexpressed tumors significantly decreased. Among indicated patients, receipt of anticancer therapies was somewhat similar over the calendar periods, except for neoadjuvant chemotherapy and anti-HER2 therapy, where increases in administration were noted. Significant improvements in survival were observed over the 15 years, particularly for HER2-overexpressed BCs. Although the improvements in BC survival that we have observed validate ongoing cancer control efforts and treatment advances, study findings suggest that more could be done for earlier detection and improved access to effective therapies in our settings.

Spectral signatures of coexisting isolas and four-wave mixing under the effect of noise in photonic oscillators

The optical power spectrum is the prime observable to dissect, understand, and design the long- time behavior of small and large arrays of optically coupled semiconductor lasers. A long-standing issue has been identified within the literature of injection locking in photonic oscillators: first how the thickness of linewidth and the lineshape spectral envelope correlates with the deterministic evolution of the monochromatic injected laser oscillator and second how the presence of noise and the typically dense proximity in phase space of coexisting limit cycles of the coupled system are shaping and influencing the overall spectral behavior. In addition, we are critically interested in the regions where the basin of attraction has a fractal-like structure, still, the long-time orbits are P1 (period 1) and/or P3 (period 3) limit cycles. Numerically computed evidence shows that, when the coupled system lives in the regions of coexisting isolas and four-wave mixing (FWM) limit cycles, the overall optical power spectrum is deeply imprinted by a strong influence from the underlying noise sources. A particularly intriguing observation in this region of parameter space that we examine is that the isolas draw most of the trajectories on its phase space path.

Orbital-Ordering Driven Simultaneous Tunability of Magnetism and Electric Polarization in Strained Monolayer VCl3

Two-dimensional (2D) van der Waals magnetic materials have promising and versatile electronic and magnetic properties in the 2D limit, indicating a considerable potential to advance spintronic applications. Theoretical predictions thus far have not ascertained whether monolayer VCl3 is a ferromagnetic (FM) or anti-FM monolayer; this also remains to be experimentally verified. We theoretically investigate the influence of potential factors, including C 3 symmetry breaking, orbital ordering, epitaxial strain, and charge doping, on the magnetic ground state. Utilizing first-principles calculations, we predict a collinear type-III FM ground state in monolayer VCl3 with a broken C 3 symmetry, wherein only the former two of three t 2g orbitals (a 1g, eg2π and eg1π ) are occupied. The atomic layer thickness and bond angles of monolayer VCl3 undergo abrupt changes driven by an orbital ordering switch, resulting in concomitant structural and magnetic phase transitions. Introducing doping to the underlying Cl atoms of monolayer VCl3 without C 3 symmetry simultaneously induces in- and out-of-plane polarizations. This can achieve a multiferroic phase transition if combined with the discovered adjustments of magnetic ground state and polarization magnitude under strain. The establishment of an orbital-ordering driven regulatory mechanism can facilitate deeper exploration and comprehension of magnetic properties of strongly correlated systems in monolayer VCl3.

Abstract B008: Alternative PER2 (Period2) enhancer usage drives epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer (OC)

Abstract B008: Alternative <i>PER2</i> (Period2) enhancer usage drives epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer (OC)

A computer synoptic operative report versus a report dictated by a surgeon in advanced ovarian cancer

ObjectiveTo evaluate the role of a computer synoptic operative report in enhancing the quality and completeness of surgical reporting for advanced ovarian cancer surgeries.MethodsThe study was conducted at a tertiary...

Downregulation of the circadian clock gene period 2 aggravates prognosis in breast cancer patients with obesity

Abstract Background Obese individuals diagnosed with breast cancer often experience a less favorable prognosis; however, the underlying mechanisms linking obesity to breast cancer outcomes remain elusive. This study aimed to identify and validate novel prognostic markers associated with breast cancer in patients with obesity. Methods We conducted a reanalysis of gene expression profiles from normal-weight, overweight, and obese breast cancer patients to identify candidate genes. Subsequently, we validated the protein levels of these candidates using immunohistochemistry. Finally, we investigated the association between candidate genes and breast cancer prognosis at Tongji Hospital, utilizing data from an 8-year follow-up through the Kaplan-Meier method and univariate and multivariate Cox regression models. Results The fold change of the circadian clock gene period 2 (PER2), which exhibited a declining trend with increasing body mass index, was 0.76 in obese patients compared with normal-weight patients. The expression rates of PER2 protein were 44.7%, 51.5%, and 61.3% in normal-weight, overweight, and obese patients, respectively. The 8-year recurrence-free survival rates were 75.9%, 69.6%, and 64.1%, whereas the 8-year overall survival rates were 86.8%, 83.0%, and 76.1% in normal-weight, overweight, and obese patients, respectively (P &lt; 0.05). Furthermore, the 8-year recurrence-free survival rates were 66.2% and 76.4%, and the 8-year overall survival rates were 79.9% and 86.3% in the low and high PER2 expression groups, respectively (P &lt; 0.05). The unadjusted hazard ratio for PER2 was 1.550 (95% confidence interval, 1.029–2.335), and the adjusted hazard ratio was 3.003 (95% confidence interval, 1.838–4.907). Conclusions Our findings indicate that low PER2 expression serves as an independent risk factor for breast cancer prognosis and may contribute to the unfavorable outcomes observed in obese patients.

PER2 binding to HSP90 enhances immune response against oral squamous cell carcinoma by inhibiting IKK/NF-κB pathway and PD-L1 expression

BackgroundProgrammed death-ligand 1 (PD-L1) contributes to the immune escape of tumor cells and is a critical target for antitumor immunotherapy. However, the molecular mechanisms regulating PD-L1 expression remain unclear, hindering...

Achieving balanced transfusion early in critically bleeding trauma patients: an observational study exploring the effect of attending trauma surgical presence during resuscitation

BackgroundAfter 15 years of damage control resuscitation (DCR), studies still report high mortality rates for critically bleeding trauma patients. Adherence to massive hemorrhage protocols (MHPs) based on a 1:1:1 ratio...

Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

FRI375 Validation Of A Novel Mouse Placenta Explant Protocol For Screening Of Drugs Modulating Circadian Rhythms

Abstract Disclosure: J.S. Lee: None. H.M. Hoffmann: None. Introduction Pre-eclampsia is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality, complicating about 4% of pregnancies. Recent work has identified PERIOD3 (PER3), a gene regulating 24-hour circadian rhythms, to be downregulated in the preeclamptic placenta. The role of circadian rhythms in the placenta largely remains unknown. Our goal is to establish an organotypic placental preparation to allow screening of drugs that regulate circadian rhythms. These studies are a first step towards understanding how circadian rhythm changes are caused in the placenta and will be the basis for future studies to understand how circadian rhythm disruption in the placenta cause pregnancy complications, such as preeclampsia. Methods We established a novel organotypic placenta preparation allowing to study circadian rhythms in a LumiCycle using gestation day (GD) 14 and GD18 placental explants from the validated circadian reporter mouse Per2::Luciferase. Using H&amp;E we validated the explants to be &amp;gt;80% enriched in either decidua (DC) which is strictly of maternal origin, labyrinth (LB) which is strictly of fetal origin, and the junctional zone (JZ) which consists of binucleated cells fused of maternal and fetal cells. Results Through H&amp;E staining, we validated by distinct morphology that the DC is enriched with neutrophils; the JZ contains spongiotrophoblasts and glycogen trophoblasts; whereas the LB has anucleated red blood cells. We next established a protocol to record Per2::Luciferase circadian rhythms for &amp;lt;6 days. We found that the Per2::Luciferase circadian rhythm shortens with increased gestational age, where the period in DC, LZ and LB in the GD14 placenta is ∼4h longer than in the GD18 placenta, showing an unexpected plasticity in circadian timekeeping in the placenta. Because PER3 is downregulated in the pre-eclamptic placenta, we asked if the explants would be useful for pharmacologically screenings. We applied PF670462 (1μM), which stabilizes PER1/2/3, or epidermal growth factor (EGF, 30ng/mL), a growth factor important in placental function and a PER2 enhancer. PF670462 lengthened Per2::Luciferase period in the DC and JZ, confirming the expected circadian rhythm stabilizing effect of PF670462. Surprisingly, EGF only modulated circadian rhythms in the GD14 placental, where EGF shortened Per2::Luciferase period in DC, and lengthen the period in the JZ and LB zones in the GD14 placenta. This data suggests EGF has gestational age and placenta zone specific effects. Conclusion We validated a novel ex vivo placental preparation to study circadian rhythms at GD14 and GD18. We identified a placenta zone and gestation age specific effect of EGF, an effect future studies will further expand on. This model will be useful to complete pharmacological screenings of drugs that can restore circadian rhythms in the preeclamptic placenta. Presentation: Friday, June 16, 2023

OR23-03 Sustained Normalization Of Mineral Homeostasis In Autosomal Dominant Hypocalcemia Type 1: Results From A Phase 2 Study Over 18 Months Of Encaleret (CLTX-305) Treatment (NCT04581629)

Abstract Disclosure: R.I. Gafni: Advisory Board Member; Self; Ascendis Pharma, Hypoparathyroidism Association. Grant Recipient; Self; Calcilytix Therapeutics. I.R. Hartley: Grant Recipient; Self; Calcilytix Therapeutics. K.L. Roszko: Grant Recipient; Self; Calcilytix Therapeutics. E.F. Nemeth: Consulting Fee; Self; Calcilytix Therapeutics. K.A. Pozo: Grant Recipient; Self; Calcilytix Therapeutics. A. Mathew: Employee; Self; Calcilytix Therapeutics. M. Roberts: Employee; Self; Calcilytix Therapeutics. S.H. Adler: Employee; Self; Calcilytix Therapeutics. M.T. Collins: Grant Recipient; Self; Calcilytix Therapeutics. Autosomal dominant hypocalcemia type 1 (ADH1), caused by gain-of-function calcium-sensing receptor gene (CASR) variants, is characterized by low parathyroid hormone (PTH) levels, hypocalcemia, hypercalciuria, hyperphosphatemia and hypomagnesemia. Conventional therapy (calcium and active vitamin D) worsens hypercalciuria, which may result in renal complications. Calcilytics that act as negative allosteric modulators of the calcium-sensing receptor (CaSR), like encaleret, decrease the sensitivity of hyperactive receptors to extracellular calcium and normalize biochemical abnormalities in ADH1 rodent models. This Phase 2b open-label study of the oral investigational calcilytic encaleret was comprised of 3 periods followed by a long-term extension (LTE). Conventional therapy was discontinued prior to encaleret initiation. Periods 1&amp;2 included dose-finding and safety/tolerability evaluation. Period 3 (P3) optimized dosing and assessed safety and efficacy over 24 outpatient weeks; participants were then allowed to continue in the LTE. Thirteen adults with ADH1 were enrolled and encaleret was individually titrated to normalize albumin-corrected calcium (cCa) and minimize hypercalciuria. Encaleret was well-tolerated with no serious adverse events reported. There were no treatment discontinuations or withdrawals prior to the LTE; one participant withdrew during the LTE for family planning. The mean±SD encaleret sulfate dose at the end of P3, Week 24 (P3W24) was 86±70mg BID, remaining stable at LTE Month 12 (LTEM12) (n=11) (71±62mg BID). Mean±SD values taken 4-hours post-dose for P3W24 and LTEM12 compared to baseline (BL) are presented. PTH levels were low at BL (6.3±7.8 pg/mL [nl 10-65]), normal at P3W24 (35.3±10.2), and remained consistent through LTEM12 (36.9±15.2 [p&amp;lt;0.01]). Similarly, hypocalcemia at BL (cCa=7.1±0.4 mg/dL [nl 8.4-10.2]) was normal at P3W24 (9.2±0.5); eucalcemia was maintained through LTEM12 (9.2±0.4 [p&amp;lt;0.01]). BL hypercalciuria (395±216 mg/d [nl &amp;lt;250-300]) decreased to 202±83 at P3W24 and normalization was sustained through LTEM12 (177±94 [p&amp;lt;0.05]). BL blood phosphate (4.5±1.1 mg/dL [nl 2.3-4.7]) decreased by P3W24 (3.4±0.4) and was maintained at LTEM12 (3.4±0.5 [p&amp;lt;0.01]). Blood magnesium increased (BL 1.7±0.2 mg/dL [nl 1.6-2.6]); P3W24 2.0±0.2; LTEM12 2.1±0.2 [p&amp;lt;0.01]). Bone turnover markers increased from BL (n=7) (CTX=253±111 pg/mL; P1NP=34±10 mcg/L) by P3W24 (n=13) (CTX=744±565; P1NP=104±87) and remained elevated at LTEM12 (CTX=988±652; P1NP=83±49 [p&amp;lt;0.01]. This study represents a molecularly targeted, precision medicine approach to the treatment of ADH1. The consistent and sustained results from over 18 months of outpatient encaleret treatment are clinically meaningful and support Phase 3 evaluation of the efficacy and safety of encaleret as the first potential treatment indicated for ADH1. Presentation: Saturday, June 17, 2023

Transcription Repression of CRY2 via PER2 Interaction Promotes Adipogenesis.

The circadian clock is driven by a transcriptional-translational feedback loop, and cryptochrome 2 (CRY2) represses CLOCK/BMAL1-induced transcription activation. Despite the established role of clock in adipogenic regulation, whether the CRY2 repressor activity functions in adipocyte biology remains unclear. Here we identify a critical cysteine residue of CRY2 that mediates interaction with Period 2 (PER2). We further demonstrate that this mechanism is required for repressing circadian clock-controlled Wnt signaling to promote adipogenesis. CRY2 protein is enriched in white adipose depots and robustly induced by adipogenic differentiation. Via site-directed mutagenesis, we identified that a conserved CRY2 cysteine at 432 within the loop interfacing with PER2 mediates heterodimer complex formation that confers transcription repression. C432 mutation disrupted PER2 association without affecting BMAL1 binding, leading to loss of repression of clock transcription activation. In preadipocytes, whereas CRY2 enhanced adipocyte differentiation, the repression-defective C432 mutant suppressed this process. Furthermore, silencing of CRY2 attenuated, while stabilization of CRY2 by KL001 markedly augmented adipocyte maturation. Mechanistically, we show that transcriptional repression of Wnt pathway components underlies CRY2 modulation of adipogenesis. Collectively, our findings elucidate a CRY2-mediated repression mechanism that promotes adipocyte development, and implicate its potential as a clock intervention target for obesity.

Role of V–V dimerization on electronic and magnetic properties of ilmenite-type CoVO3

Structural, electronic and magnetic properties of ilmenite-type CoVO3 have been explored via the generalized gradient approximation + effective Hubbard U eff correction, in the framework of density functional theory. Our results indicate that high temperature rhombohedral phase is metallic with oxidation states and electronic configurations Co2+ (), V4+ (), respectively, while low temperature triclinic phase, induced from spin-Peierls transition in the V–V dimerization manner, is insulating, maintaining charge and electronic states unchanged. Furthermore, the A-type antiferromagnetic ordering, where the ferromagnetic honeycomb layers are anti-aligned along the stacking axis, is identified to be the magnetic ground state for the low temperature phase, in nice agreement with experimental findings, analogous to CoTiO3. The unexpected intralayer ferromagnetic couplings can be attributed to the intraorbital t2g −t2g exchange coupling, which was assumed to be small earlier and ignored, but actually large in honeycomb cobaltates with 3d 7 electronic configuration. In addition, the computed magnetic moment on Co2+ ion ranges from 2.5 to 2.7 μ B, Hubbard U eff dependent, close to ideal S = 3/2 state, rather than the anticipated J eff = 1/2 state. Furthermore, the supplemental calculations, taking spin-orbit coupling (SOC) into account, uncover faint orbital moments of 0.21–0.27 μ B at the Co site, illustrating the insignificance of SOC. Except for the inevitable trigonal distortions, the excessive structural distortion triggered by the formation V–V dimerization, i.e. the breaking of trigonal symmetry around Co2+, further lifts the degeneracy of t 2g orbitals and increases crystal field splitting, driving it away from potential candidates for realizing Kitaev model physics.