Rapid quantification of intracellular calcium stores reveals effects of membrane micropeptides on SERCA function.

To determine how regulation of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) affects the Ca content of the endoplasmic reticulum (ER), we developed a ratiometric ER-localized Ca indicator to rapidly quantify Ca stores and assess SERCA function in live cells. This assay enables screening of membrane micropeptides and small molecules that modulate SERCA and Na/K-ATPase activity and may facilitate development of therapies that target cellular Ca handling. Of the micropeptides tested, phospholamban (PLB) had the greatest degree of inhibition of SERCA, as measured by a decrease in ER Ca content compared to control.

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of calcium.

The sarco(endo)plasmic reticulum Ca ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and the dynamic exchange of PLB between pentamers and SERCA is an important determinant of cardiac responsiveness to exercise.

Another-regulin regulates cardiomyocyte calcium handling via integration of neuroendocrine signaling with SERCA2a activity.

Calcium (Ca) dysregulation is a hallmark feature of cardiovascular disease. Intracellular Ca regulation is essential for proper heart function and is controlled by the sarco/endoplasmic reticulum Ca ATPase (SERCA2a). Another-regulin (ALN) is a newly discovered cardiomyocyte-expressed SERCA2a inhibitor, suggesting cardiomyocyte Ca-handling is more complex than previously appreciated.

Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca sensitivity of active SERCA, increasing the Ca concentration required for pump cycling. However, PLB does not decrease Ca binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca affinity.

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling.

The sarco(endo)plasmic reticulum Ca ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise.

Effects of Housing Density on Reproductive Performance, Intracage Ammonia, and Welfare of Mice Continuously Housed as Breeders in Standard Mouse and Rat Caging.

Maintaining compliance with cage density recommendations in precludes continuous trio breeding in standard-sized mouse cages. This study evaluated and compared several parameters of reproductive performance, intracage ammonia concentration, and fecal corticosterone levels in 2 strains of mice, C57BL/6J (B6) and B6.129S(Cg)-/J (STAT1), housed as continuous breeding pairs or trios in standard-sized mouse cages, and continuous breeding trios in standard-sized rat cages.

Micropeptide hetero-oligomerization adds complexity to the calcium pump regulatory network.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is an ion transporter that creates and maintains intracellular calcium stores. SERCA is inhibited or stimulated by several membrane micropeptides including another-regulin, dwarf open reading frame, endoregulin, phospholamban (PLB), and sarcolipin. We previously showed that these micropeptides assemble into homo-oligomeric complexes with varying affinity.

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump.

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca-ATPase (SERCA) sequesters Ca in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA.

Presenilin 1 is a direct regulator of the cardiac sarco/endoplasmic reticulum calcium pump.

The gamma secretase catalytic subunit presenilin 1 (PS1) is expressed in the endoplasmic reticulum (ER) of neurons, where it regulates Ca signaling. PS1 is also expressed in heart, but its role in regulation of cardiac Ca transport remains unknown. Since the type 2 sarco/endoplasmic reticulum Ca ATPase (SERCA2a) plays a central role in cardiac Ca homeostasis, we studied whether PS1 regulates the cardiac SERCA2a function.

Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.

The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA.

Evidence of Leptospiral Presence in the Cumberland Gap Region.

Background: Leptospirosis is a widespread zoonotic disease that causes reproductive losses and/or hepatorenal failure in a number of animal species. Wild reservoirs of the disease, such as rodents, harbor the causative bacterium, Leptospira spp., in their kidneys and contaminate the environment by excreting infected urine.

Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers.

The recently-discovered single-span transmembrane proteins endoregulin (ELN), dwarf open reading frame (DWORF), myoregulin (MLN), and another-regulin (ALN) are reported to bind to the SERCA calcium pump in a manner similar to that of known regulators of SERCA activity, phospholamban (PLB) and sarcolipin (SLN). To determine how micropeptide assembly into oligomers affects the availability of the micropeptide to bind to SERCA in a regulatory complex, we used co-immunoprecipitation and fluorescence resonance energy transfer (FRET) to quantify micropeptide oligomerization and SERCA-binding. Micropeptides formed avid homo-oligomers with high-order stoichiometry (n > 2 protomers per homo-oligomer), but it was the monomeric form of all micropeptides that interacted with SERCA.

Women's experience of vulvovaginal symptoms associated with menopause.

Objective: This study describes women's experiences of the genitourinary syndrome of menopause (GSM) elicited through focus groups and cognitive debriefing sessions during development of a novel patient-reported outcome measure (PROM) designed for use in both clinical care and research.

Methods: A draft questionnaire to identify and assess bothersome genitourinary symptoms associated with estrogen deficiency in menopausal women was developed in five discrete phases from multiple sources of information in accordance with standards for PROM development. GSM was confirmed by report of symptoms in conjunction with a confirmatory pelvic examination and laboratory assessments.

Evaluating the Film .

Support for research involving children has a complicated history. Pediatricians and families have a unique opportunity to share perspectives about the relevance of pediatric clinical research. A national broadcast film on pediatric clinical research was developed to improve knowledge about and willingness to consider a clinical study.

The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.

Calcium (Ca) dysregulation is a hallmark of heart failure and is characterized by impaired Ca sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca-ATPase (SERCA). We recently discovered a micropeptide named DWORF (arf pen eading rame) that enhances SERCA activity by displacing phospholamban (PLN), a potent SERCA inhibitor. Here we show that DWORF has a higher apparent binding affinity for SERCA than PLN and that DWORF overexpression mitigates the contractile dysfunction associated with PLN overexpression, substantiating its role as a potent activator of SERCA.

Detection of Leptospiral DNA in the Urine of Donkeys on the Caribbean Island of Saint Kitts.

Leptospirosis is a global zoonosis caused by pathogenic spirochetes classified within the genus . Leptospires live in the proximal renal tubules of reservoir or chronic carrier animals, and are shed in the urine. Naïve animals acquire infection either when they come in direct contact with a reservoir or infected animals or by exposure to environmental surface water or soil that is contaminated with their urine.

Measuring clinically relevant endpoints in a serum-free, three-dimensional, primary cell culture system of human osteoarthritic articular chondrocytes.

Osteoarthritis (OA) is characterized by degeneration of articular cartilage within the joint, inflammation and pain. The purpose of this study was to develop a primary, serum free cell culture system of human osteoarthritic articular chondrocytes (HOACs) with which to study manifestations of the disease process. Joint tissues were obtained from OA patients undergoing total knee arthroplasty (TKA).

Reversible optogenetic control of kinase activity during differentiation and embryonic development.

A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution.

Polymerase chain reaction-based gene removal from plasmids.

This data article contains supplementary figures and methods to the research article entitled, "Multiplex gene removal by two-step polymerase chain reactions" (Krishnamurthy et al., Anal. Biochem.

Multiplex gene removal by two-step polymerase chain reactions.

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly.

Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2.

Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing.

A family with spinocerebellar ataxia type 5 found to have a novel missense mutation within a SPTBN2 spectrin repeat.

A family with late-onset autosomal dominant pure cerebellar ataxia, consistent with spinocerebellar ataxia type 5 (SCA5) but lacking previously reported SPTBN2 mutations, was identified. DNA was collected from seven individuals across two generations and the SPTBN2 gene on chromosome 11 was sequenced. A nonsynonymous heterozygous substitution in exon 12 was detected in individuals diagnosed with SCA5 while unaffected family members did not possess this variant.

Repressive effects of resveratrol on androgen receptor transcriptional activity.

Background: The chemopreventive effects of resveratrol (RSV) on prostate cancer have been well established; the androgen receptor (AR) plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.

A critical role for the co-repressor N-CoR in erythroid differentiation and heme synthesis.

Co-repressor N-CoR (nuclear receptor co-repressor) has important roles in different biological processes, including proliferation, differentiation and development. Mutant mice lacking N-CoR are embryonically lethal and appear to die from anemia owing to defects in definitive erythropoiesis. However, the underlying molecular mechanisms of N-CoR-mediated erythroid differentiation are largely unknown.

Estrogen-dependent selectivity of genomic responses to birdsong.

Behavioral responses to sociosexual signals often depend on gonadal steroid hormones, which are thought to modulate behavior by acting on motivational systems in the brain. There is mounting evidence that sex steroids may also modulate perception of sociosexual signals by affecting sensory processing. In seasonally breeding songbirds such as the white-throated sparrow (Zonotrichia albicollis), the female's behavioral response to hearing male song depends on her plasma levels of estradiol (E2).