Cohesin cleavage by separase is enhanced by a substrate motif distinct from the cleavage site.

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage

A periosteum-derived cell line to study the role of BMP/TGFβ signaling in periosteal cell behavior and function

The periosteum is a thin tissue surrounding each skeletal element that contains stem and progenitor cells involved in bone development, postnatal appositional bone growth, load-induced bone formation, and fracture repair. BMP and TGFβ signaling are important for periosteal activity and periosteal cell behavior, but thorough examination of the influence of these pathways on specific cell populations resident in the periosteum is lacking due to limitations associated with primary periosteal cell isolations and in vitro experiments. Here we describe the generation of a novel periosteum-derived clonal cell (PDC) line from postnatal day 14 mice and use it to examine periosteal cell behavior in vitro. PDCs exhibit key characteristics of periosteal cells observed during skeletal development, maintenance, and bone repair. Specifically, PDCs express established periosteal markers, can be expanded in culture, demonstrate the ability to differentiate into chondrocytes, osteoblasts, and adipocytes, and exhibit an osteogenic response to physical stimulation. PDCs also engage in BMP and/or TGFβ signaling when treated with the activating ligands BMP2 and TGFβ-1, and in response to mechanical stimulation via fluid shear. We believe that this PDC line will be useful for large-scale, long-term experiments that were not feasible when using primary periosteal cells. Anticipated future uses include advancing our understanding of the signaling interactions that occur during appositional bone growth and fracture repair and developing drug screening platforms to discover novel growth and fracture healing factors

Erratum: Insulin-like growth factor-binding protein-2 is required for osteoclast differentiation

Global deletion of the Igfbp2 gene results in the suppression of bone turnover. To investigate the role of IGFBP-2 in regulating osteoclast differentiation we cultured Igfbp2−/− bone marrow cells and found a reduction in the number of osteoclasts and impaired resorption. Addition of full length IGFBP-2 restored osteoclast differentiation, fusion and resorption. To determine the molecular domains of IGFBP-2 that were required for this effect to be manifest, Igfbp2−/− bone marrow cells mice were transfected with constructs in which the heparin binding (HBD) or the IGF- binding domains of IGFBP-2 were mutated. We found that both domains were necessary for osteoclastogenesis since expression of the mutated forms of either domain failed to support the formation of functionally mature osteoclasts. To discern the mechanism by which IGFBP-2 regulates osteoclast formation, PTEN abundance and phosphorylation status as well as AKT responsiveness to IGF-I were analyzed. Igfbp2−/− cells had elevated levels of PTEN and phospho-PTEN compared with controls. Expression of wild-type IGFBP-2 reduced the level of PTEN to that of wild-type cells. Cells expressing the IGF binding mutant showed suppression of PTEN and phospho-PTEN equivalent to the wild type protein, whereas those expressing the IGFBP-2 HBD mutant showed no PTEN suppression. When the ability of IGF-I to stimulate AKT activation, measured by Thr308 and Ser473 phosphorylation, was analyzed, stimulation of Ser473 in response to IGF-I in pre-osteoclasts required the presence of intact IGFBP-2. This effect was duplicated by the addition of a CK2 inhibitor that prevents the phosphorylation of PTEN. In contrast, in fully differentiated osteoclasts stimulation of Thr308 phosphorylation required the presence of intact IGFBP-2. We conclude that IGFBP-2 is an important regulator of osteoclastogenesis and that both the heparin and the IGF binding domains of IGFBP-2 are essential for the formation of fully differentiated and functional osteoclasts

Oligodendrocytes contribute to motor neuron death in ALS via SOD1 dependent mechanism

Oligodendrocytes have recently been implicated in the pathophysiology of ALS. Here we show that, in vitro, mutant SOD1 mouse oligodendrocytes induce wild-type motor neuron hyperexcitability and death. Moreover, we efficiently derived human oligodendrocytes from a large number of controls, sporadic and familial ALS patients using two different reprogramming methods. All ALS oligodendrocyte lines induced motor neuron death through conditioned medium and in co-culture. Conditioned medium-mediated motor neuron death was associated with decreased lactate production and release, while toxicity in co-culture was lactate independent, demonstrating that motor neuron survival is not only mediated by soluble factors. Remarkably, human SOD1 shRNA treatment resulted in motor neuron rescue in both mouse and human cultures when knockdown was achieved in progenitor cells, while it was ineffective in differentiated oligodendrocytes. Early SOD1 knockdown, in fact, rescued lactate impairment and cell toxicity in all lines tested with exclusion of samples carrying C9orf72 repeat expansions. These did not respond to SOD1 knockdown nor showed lactate release impairment. Our data indicate that SOD1 is directly or indirectly involved in ALS oligodendrocyte pathology and suggest that in this cell type some damage might be irreversible. In addition, we demonstrate that C9ORF72 patients represent an independent patient group that might not respond to the same treatment

A Call to Action: Taking the Untenable out of Women Professors’ Pregnancy, Postpartum, and Caregiving Demands

Despite becoming increasingly represented in academic departments, women scholars face a critical lack of support as they navigate demands pertaining to pregnancy, motherhood, and child caregiving. In addition, cultural norms surrounding how faculty and academic leaders discuss and talk about tenure, promotion, and career success have created pressure for women who wish to grow their family and care for their children, leading to questions about whether it is possible for these women to have a family and an academic career. The current paper is a call to action for academia to build structures that support women professors as they navigate the complexities of pregnancy, the postpartum period, and the caregiving demands of their children. We specifically call on those of us in I-O psychology, management, and related departments to lead the way. In making this call, we first present the realistic, moral, and financial cases for why this issue needs to be at the forefront of discussions surrounding success in the academy. We then discuss how in the U.S. and elsewhere, an absence of policies supporting women places two groups of academics—department heads (as the leaders of departments who have discretion outside of formal policies to make work better for women) and other faculty members (as potential allies both in the department and within our professional organizations)—in a critical position to enact support and change. We conclude with our boldest call—to make a cultural shift that shatters the assumption that having a family is not compatible with academic success. Combined, we seek to launch a discussion that leads directly to necessary and overdue changes in how women scholars are supported in academia

Homing endonuclease I-CreI derivatives with novel DNA target specificities

Homing endonucleases are highly specific enzymes, capable of recognizing and cleaving unique DNA sequences in complex genomes. Since such DNA cleavage events can result in targeted allele-inactivation and/or allele-replacement in vivo, the ability to engineer homing endonucleases matched to specific DNA sequences of interest would enable powerful and precise genome manipulations. We have taken a step-wise genetic approach in analyzing individual homing endonuclease I-CreI protein/DNA contacts, and describe here novel interactions at four distinct target site positions. Crystal structures of two mutant endonucleases reveal the molecular interactions responsible for their altered DNA target specificities. We also combine novel contacts to create an endonuclease with the predicted target specificity. These studies provide important insights into engineering homing endonucleases with novel target specificities, as well as into the evolution of DNA recognition by this fascinating family of proteins

Effectiveness of Denitrifying Bioreactors on Water Pollutant Reduction from Agricultural Areas

HighlightsDenitrifying woodchip bioreactors treat nitrate-N in a variety of applications and geographies.This review focuses on subsurface drainage bioreactors and bed-style designs (including in-ditch).Monitoring and reporting recommendations are provided to advance bioreactor science and engineering. Denitrifying bioreactors enhance the natural process of denitrification in a practical way to treat nitrate-nitrogen (N) in a variety of N-laden water matrices. The design and construction of bioreactors for treatment of subsurface drainage in the U.S. is guided by USDA-NRCS Conservation Practice Standard 605. This review consolidates the state of the science for denitrifying bioreactors using case studies from across the globe with an emphasis on full-size bioreactor nitrate-N removal and cost-effectiveness. The focus is on bed-style bioreactors (including in-ditch modifications), although there is mention of denitrifying walls, which broaden the applicability of bioreactor technology in some areas. Subsurface drainage denitrifying bioreactors have been assessed as removing 20% to 40% of annual nitrate-N loss in the Midwest, and an evaluation across the peer-reviewed literature published over the past three years showed that bioreactors around the world have been generally consistent with that (N load reduction median: 46%; mean ±SD: 40% ±26%; n = 15). Reported N removal rates were on the order of 5.1 g N m-3 d-1 (median; mean ±SD: 7.2 ±9.6 g N m-3 d-1; n = 27). Subsurface drainage bioreactor installation costs have ranged from less than $5,000 to$27,000, with estimated cost efficiencies ranging from less than $2.50 kg-1 N year-1 to roughly$20 kg-1 N year-1 (although they can be as high as $48 kg-1 N year-1). A suggested monitoring setup is described primarily for the context of conservation practitioners and watershed groups for assessing annual nitrate-N load removal performance of subsurface drainage denitrifying bioreactors. Recommended minimum reporting measures for assessing and comparing annual N removal performance include: bioreactor dimensions and installation date; fill media size, porosity, and type; nitrate-N concentrations and water temperatures; bioreactor flow treatment details; basic drainage system and bioreactor design characteristics; and N removal rate and efficiency

National and regional estimates of term and preterm babies born small for gestational age in 138 low-income and middle-income countries in 2010.

BACKGROUND: National estimates for the numbers of babies born small for gestational age and the comorbidity with preterm birth are unavailable. We aimed to estimate the prevalence of term and preterm babies born small for gestational age (term-SGA and preterm-SGA), and the relation to low birthweight (<2500 g), in 138 countries of low and middle income in 2010. METHODS: Small for gestational age was defined as lower than the 10th centile for fetal growth from the 1991 US national reference population. Data from 22 birth cohort studies (14 low-income and middle-income countries) and from the WHO Global Survey on Maternal and Perinatal Health (23 countries) were used to model the prevalence of term-SGA births. Prevalence of preterm-SGA infants was calculated from meta-analyses. FINDINGS: In 2010, an estimated 32·4 million infants were born small for gestational age in low-income and middle-income countries (27% of livebirths), of whom 10·6 million infants were born at term and low birthweight. The prevalence of term-SGA babies ranged from 5·3% of livebirths in east Asia to 41·5% in south Asia, and the prevalence of preterm-SGA infants ranged from 1·2% in north Africa to 3·0% in southeast Asia. Of 18 million low-birthweight babies, 59% were term-SGA and 41% were preterm-SGA. Two-thirds of small-for-gestational-age infants were born in Asia (17·4 million in south Asia). Preterm-SGA babies totalled 2·8 million births in low-income and middle-income countries. Most small-for-gestational-age infants were born in India, Pakistan, Nigeria, and Bangladesh. INTERPRETATION: The burden of small-for-gestational-age births is very high in countries of low and middle income and is concentrated in south Asia. Implementation of effective interventions for babies born too small or too soon is an urgent priority to increase survival and reduce disability, stunting, and non-communicable diseases. FUNDING: Bill & Melinda Gates Foundation by a grant to the US Fund for UNICEF to support the activities of the Child Health Epidemiology Reference Group (CHERG)

Broad betacoronavirus neutralization by a stem helix–specific human antibody

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection