Facilitated Asynchronous Online Learning to Build Public Health Strategic Skills.

CONTEXT: The COVID-19 pandemic spurred significant government investments for hiring public health workers. There are clear opportunities to help build capacities among both current and incoming public health workers, closing well-elucidated skill gaps. OBJECTIVE: To report on the development process, methods used, and outcomes seen from a point-in-time public health workforce capacity-building initiative, Public Health Essentials (PHE) . DESIGN: Capacity-building outcomes evaluation using pre/postintervention measures. SETTING: The United States. PARTICIPANTS: A total of 512 learners working in roles (government or adjacent to) that support public health. INTERVENTION: PHE, a cohort-based facilitated asynchronous online course comprising 5 units, 18 modules, 54 learning outcomes, and 266 teaching and applied assessment elements designed to build public health strategic skills. MAIN OUTCOME MEASURES: Two outputs and 3 outcomes were used to assess and improve progress in achieving our goal of building generalizable and transferrable public health ability and confidence among diverse public health workers: Use of PHE , PHE completion rate , Learner competence , Change in self-assessed ability , and Benefits of PHE. RESULTS: From September 2021 to December 2022, 4 agencies used PHE for fellowship training or employee capacity building. Some 79% (n = 512) of learners completed the training, demonstrating competence in all 54 areas assessed by expert course facilitators. Of those, 79% (n = 321) completed both optional pre- and post-PHE surveys, reporting statistically significant gains in all strategic skill domains assessed (n = 9), regardless of demographics and public health experience. Learners gained new skills and knowledge (92%), developed a better understanding of public health (86%), and broadened their public health skill base (84%). A majority can apply the knowledge and skills gained directly to their work (94%), which benefits their team (92%), and have increased their confidence as public health practitioners (49%). CONCLUSIONS: PHE can significantly improve learners' ability across 9 strategic skill areas in as few as 15 weeks, regardless of their demographics, training, or experience.

A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis.

Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as "acrosome exocytosis" (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed "acrosome reaction" model.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Defects in sperm capacitation/fertilizing ability are equally prevalent across ages in men seeking fertility assistance.

RESEARCH QUESTION: How do capacitation ability, measured by Cap-Score™, and traditional semen analysis measures (volume, concentration, motility) change with age in men questioning their fertility (MQF)? DESIGN: Cap-Score and semen analysis measures were obtained from MQF (n = 2652; multicentric design: 35 reproductive endocrinologist prescribers, n = 16 clinics). Morphology was not included due to differences among clinics. A Mann-Whitney test was used to compare Cap-Scores between MQF and men with known recent paternity (n = 76). The following age groups were constructed for MQF: 20-24, 25-29, 30-34, 35-39, 40-44, 45-49 and 50+. Associations between semen analysis, Cap-Score and age groups were evaluated using mixed-model analysis of variance to identify possible influence of Cap-Score collection kit type (n = 763 collected at home; n = 1889 collected at clinics). RESULTS: MQF had reduced capacitation ability (mean ± SE; 29.25 ± 0.15 versus 35.34 ± 0.88; P < 0.001). No change in Cap-Score (P = 0.916) or concentration (P = 0.926) was detected with age group. In contrast, both volume (P = 0.008) and % motility (P < 0.001) declined with age. CONCLUSIONS: Men presenting because of difficulties in generating pregnancy showed equivalent reductions in capacitation ability regardless of age. In contrast, motility and volume declined with age. These data suggest that capacitation ability is a more sensitive indicator of male fertility across age groups than traditional semen analysis and should not be reserved for older men. Importantly, these data do not address whether sperm fertilizing ability declines in the general population as men age. Instead, they indicate that if men are having difficulty conceiving, no matter what their age, then defects in sperm fertilizing ability are equally likely to be the cause.

Integrated Surveillance System for Controlling COVID-19 on a University Campus, 2020‒2021.

To minimize the impacts of COVID-19 and to keep campus open, Cornell University's Ithaca, NY, campus implemented a comprehensive process to monitor COVID-19 spread, support prevention practices, and assess early warning indicators linked to knowledge, behaviors, and attitudes of campus community members. The integrated surveillance approach informed leadership and allowed for prompt adjustments to university policies and practices through evidence-based decisions. This approach enhanced healthy behaviors and promoted the well-being and safety of all community members. (Am J Public Health. 2022;112(7):980-984. https://doi.org/10.2105/AJPH.2022.306838).

Multicentric, prospective observational data show sperm capacitation predicts male fertility, and cohort comparison reveals a high prevalence of impaired capacitation in men questioning their fertility.

RESEARCH QUESTIONS: Can a previously defined relationship between sperm capacitation and the probability of a man generating pregnancy within three cycles, prospectively predict male fertility in diverse clinical settings? A second study asked, what is the prevalence of impaired sperm fertilizing ability in men questioning their fertility (MQF), and does this relate to traditional semen analysis metrics? DESIGN: In the multicentric, prospective observational study, data (n = 128; six clinics) were analysed to test a published relationship between the percentage of fertilization-competent, capacitated spermatozoa (Cap-Score) and probability of generating pregnancy (PGP) within three cycles of intrauterine insemination. Logistic regression of total pregnancy outcomes (n = 252) assessed fit. In the cohort comparison, Cap-Scores of MQF (n = 2155; 22 clinics) were compared with those of 76 fertile men. RESULTS: New outcomes (n = 128) were rank-ordered by Cap-Score and divided into quintiles (25-26 per group); chi-squared testing revealed no difference between predicted and observed pregnancies (P = 0.809). Total outcomes (n = 252; 128 new + 124 previous) were pooled and the model recalculated, yielding an improved fit (P < 0.001). Applying the Akaike information criterion found that the optimal model used Cap-Score alone. Cap-Scores were performed on 2155 men (with semen analysis data available for 1948). To compare fertilizing ability, men were binned by PGP (≤19%, 20-29%, 30-39%, 40-49%, 50-59%, ≥60%). Distributions of PGP and the corresponding Cap-Scores were significantly lower in MQF versus fertile men (P < 0.001). Notably, 64% of MQF with normal volume, concentration and motility (757/1183) had PGP of 39% or less (Cap-Scores ≤31), versus 25% of fertile men. CONCLUSIONS: Sperm capacitation prospectively predicted male fertility. Impaired capacitation affects many MQF with normal semen analysis results, informing diagnosis versus idiopathic infertility.

Localisation and function of glucose transporter GLUT1 in chicken (Gallus gallus domesticus) spermatozoa: relationship between ATP production pathways and flagellar motility.

Glucose plays an important role in sperm flagellar motility and fertility via glycolysis and oxidative phosphorylation, although the primary mechanisms for ATP generation vary between species. The glucose transporter 1 (GLUT1) is a high-affinity isoform and a major glucose transporter in mammalian spermatozoa. However, in avian spermatozoa, the glucose metabolic pathways are poorly characterised. This study demonstrates that GLUT1 plays a major role in glucose-mediated motility of chicken spermatozoa. Using specific antibodies and ligand, we found that GLUT1 was specifically localised to the midpiece. Sperm motility analysis showed that glucose supported sperm movement during incubation for 0-80min. However, this was abolished by the addition of a GLUT1 inhibitor, concomitant with a substantial decrease in glucose uptake and ATP production, followed by elevated mitochondrial activity in response to glucose addition. More potent inhibition of ATP production and mitochondrial activity was observed in response to treatment with uncouplers of oxidative phosphorylation. Because mitochondrial inhibition only reduced a subset of sperm movements, we investigated the localisation of the glycolytic pathway and showed glyceraldehyde-3-phosphate dehydrogenase and hexokinase I at the midpiece and principal piece of the flagellum. The results of this study provide new insights into the mechanisms involved in ATP production pathways in avian spermatozoa.

Timing of sperm capacitation varies reproducibly among men.

Sperm must mature functionally in the process of capacitation to become able to fertilize. Capacitation depends on membrane lipid changes, and can be quantitatively assessed by redistribution of the ganglioside GM1 , the basis of the Cap-Score™ sperm function test. Here, differences in Cap-Score were compared among and within men at two time points. Ejaculates were liquefied, washed, and incubated for 3 hr under capacitating (Cap) conditions, then fixed and analyzed immediately (Day0); after being incubated 3 hr under Cap conditions then maintained 22-24 hr in fix (Day1-fix); or after 22-24 hr incubation under Cap conditions prior to fixation (Day1). In all cases, a light fixative previously shown to allow membrane lipid movements was used. Day1-fix and Day1 Cap-Scores were greater than Day0 (p < 0.001; n = 25), whereas Day1-fix and Day1 Cap-Scores were equivalent (p = 0.43; n = 25). In 123 samples from 52 fertile men, Cap-Score increased more than 1SD (7.7; calculated previously from a fertile cohort) from Day0 to Day1-fix in 44% (54/123) of the samples. To test whether timing of capacitation was consistent within an individual, 52 samples from 11 fertile men were classified into either "early" or "late" capacitation groups. The average capacitation group concordance within a donor was 81%. Median absolute deviation (MAD; in Cap-Score units) was used to assess the tightness of clustering of the difference from Day0 to Day1-fix within individuals. The average (2.21) and median (1.98) MAD confirmed consistency within individuals. Together, these data show that the timing of capacitation differed among men and was consistent within men.

Cap-Score™ prospectively predicts probability of pregnancy.

Semen analysis (SA) poorly predicts male fertility, because it does not assess sperm fertilizing ability. The percentage of capacitated sperm determined by GM1 localization ("Cap-Score™"), differs between cohorts of fertile and potentially infertile men, and retrospectively, between men conceiving or failing to conceive by intrauterine insemination (IUI). Here, we prospectively tested whether Cap-Score can predict male fertility with the outcome being clinical pregnancy within ≤3 IUI cycles. Cap-Score and SA were performed (n = 208) with outcomes initially available for 91 men. Men were predicted to have either low (n = 47) or high (n = 44) chance of generating pregnancy using previously-defined Cap-Score reference ranges. Absolute and cumulative pregnancy rates were reduced in men predicted to have low pregnancy rates versus high ([absolute: 10.6% vs. 29.5%; p = 0.04]; [cumulative: 4.3% vs. 18.2%, 9.9% vs. 29.1%, and 14.0% vs. 32.8% for cycles 1-3; n = 91, 64, and 41; p = 0.02]). Only Cap-Score, not male/female age or SA results, differed significantly between outcome groups. Logistic regression evaluated Cap-Score and SA results relative to the probability of generating pregnancy (PGP) for men who were successful in, or completed, three IUI cycles (n = 57). Cap-Score was significantly related to PGP (p = 0.01). The model fit was then tested with 67 additional patients (n = 124; five clinics); the equation changed minimally, but fit improved (p < 0.001; margin of error: 4%). The Akaike Information Criterion found the best model used Cap-Score as the only predictor. These data show that Cap-Score provides a practical, predictive assessment of male fertility, with applications in assisted reproduction and treatment of male infertility.

Small-scale egg production centres increase children's egg consumption in rural Zambia.

Animal source foods can efficiently enhance dietary quality, but they remain inaccessible and unaffordable for many women and young children in remote, low-income communities. We piloted an intervention in which 20 groups established egg production centres (EPCs) in their rural Zambian communities to increase the availability of eggs in the local food system. In a repeated cross-sectional design over 1 year (midline [4 months after the start of egg production] and endline [11 months]), we evaluated programme impact on household egg acquisition within those communities and on egg consumption and height-for-age z score (HAZ) among young children (6-36 months) using multilevel linear, logistic, and truncated negative binomial regression techniques. At midline, households in project areas were significantly more likely to consume eggs than those in control areas (OR 2.08, 95% CI [1.56, 2.78]), particularly those located within 250 m of the EPC. Similarly, children living in project communities were significantly more likely to consume eggs at midline than those in control areas (OR 5.53, 95% CI [2.90, 10.58]). Although increased over baseline, egg acquisition and consumption decreased by endline because of depressed egg production over time. There was no impact on children's HAZ, likely because of the short follow-up time and relatively modest "dose" of egg consumption. Although productivity can be improved, the EPC programme offers a novel approach to improving access to eggs in rural communities, and optimization of the production practices and marketing is needed to ensure that egg consumption translates to improved dietary quality, growth, and health.

Validation of a laboratory-developed test of human sperm capacitation.

Sperm must undergo capacitation to become fertilization competent. Here we validated that monosialotetrahexosylganglioside (GM1 ) localization patterns, which were assessed in the Cap-Score™ Sperm Function Test, reflect a capacitated state in human sperm. First, we defined patterns representing sperm that do or do not respond to stimuli for capacitation. Sperm with "capacitated" patterns had exposed acrosomal carbohydrates and underwent acrosome exocytosis in response to calcium ionophore (A23187). Precision was evaluated by percent change of the Cap-Score measured for 50, 100, 150, and 200 sperm. Changes of 11%, 6%, and 5% were observed (n ≥ 23); therefore, we counted ≥150 sperm per condition. Variance within and between readers was evaluated using 20 stitched image files generated from unique ejaculates. Two trained readers randomly resampled each image 20 times, reporting an average standard deviation of 3 Cap-Score units and coefficient of variation of 13% when rescoring samples, with no difference between readers. Semen liquefaction times ≤2 hr and mechanical liquefaction with Pasteur or wide-orifice transfer pipettes did not alter Cap-Score values. However, liquefaction with chymotrypsin (p = 0.002) and bromelain (p = 0.049) reduced response to capacitating stimuli and induced membrane damage, while counterintuitively improving sperm motility. Together, these data validate the Cap-Score assay for the intended purpose of providing information on sperm capacitation and male fertility. In addition to its clinical utility as a diagnostic tool, this test of sperm function can reveal the impact of common practices of semen handling on the ability of sperm to respond to capacitation stimuli.

Localization patterns of the ganglioside GM1 in human sperm are indicative of male fertility and independent of traditional semen measures.

Semen analysis lacks a functional component and best identifies extreme cases of infertility. The ganglioside GM1 is known to have functional roles during capacitation and acrosome exocytosis. Here, we assessed whether GM1 localization patterns (Cap-Score™) correspond with male fertility in different settings: Study 1 involved couples pursuing assisted reproduction in a tertiary care fertility clinic, while Study 2 involved men with known fertility versus those questioning their fertility at a local urology center. In Study 1, we examined various thresholds versus clinical history for 42 patients; 13 had Cap-Scores ≥39.5%, with 12 of these (92.3%) achieving clinical pregnancy by natural conception or ≤3 intrauterine insemination cycles. Of the 29 patients scoring <39.5%, only six (20.7%) attained clinical pregnancy by natural conception or ≤3 intrauterine insemination cycles. In Study 2, Cap-Scores were obtained from 76 fertile men (Cohort 1, pregnant partner or recent father) and compared to 122 men seeking fertility assessment (Cohort 2). Cap-Score values were normally distributed in Cohort 1, with 13.2% having Cap-Scores more than one standard deviation below the mean (35.3 ± 7.7%). Significantly, more men in Cohort 2 had Cap-Scores greater than one standard deviation below the normal mean (33.6%; p = 0.001). Minimal/no relationship was found between Cap-Score and sperm concentration, morphology, or motility. Together, these data demonstrate that Cap-Score provides novel, clinically relevant insights into sperm function and male fertility that complement traditional semen analysis. Furthermore, the data provide normal reference ranges for fertile men that can help clinicians counsel couples toward the most appropriate fertility treatment.

Follicular size and stage and gonadotropin concentration affect alginate-encapsulated in vitro growth and survival of pre- and early antral dog follicles.

Understanding stage-specific requirements of mammalian folliculogenesis is limited in the domestic dog. The present study examined the effects of two potential regulators of dog follicle growth and survival in vitro, namely the original stage of the follicle (i.e. preantral (≤230µm diameter) vs early antral (diameter from >230 to ≤330µm) and FSH and/or LH concentrations. After isolation and alginate encapsulation, follicles were cultured in 0, 1, 10 or 100µgmL-1 FSH and 0, 1 or 10ngmL-1 LH for 20 days. Regardless of stage, FSH promoted growth, but LH did the same only in the absence of FSH. Production of 17ß-oestradiol and progesterone was detectable, indicating theca cell activity. The greatest growth occurred in preantral (mean (± s.d.) 61.4±25.9%) versus antral (42.6±20.3%) follicles, but neither developmental stage nor gonadotropin affected survival. Antrum detection was minimal due, in part, to antral collapse, and oocytes exhibited an increasingly pale appearance and chromatin degeneration over time. The results demonstrate that pre- and early antral stage dog follicles encapsulated in alginate grow significantly in vitro. However, because FSH and LH alone or in combination fail to promote antrum development, the next step is identifying factors that enhance antral expansion.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.

Lipid Regulation of Acrosome Exocytosis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes.

Ca(2+) oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca(2+) homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater(tm/tm) (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater(tm/tm) oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca(2+) oscillations was altered in Mater(tm/tm) oocytes after fertilization in vitro. Intriguingly, Ca(2+) oscillations in Mater(tm/tm) oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca(2+) oscillation defect in Mater(tm/tm) oocytes was likely caused by a reduced amount of Ca(2+) in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca(2+) homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.

Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.

Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.

Phospholipase B is activated in response to sterol removal and stimulates acrosome exocytosis in murine sperm.

Despite a strict requirement for sterol removal for sperm to undergo acrosome exocytosis (AE), the mechanisms by which changes in membrane sterols are transduced into changes in sperm fertilization competence are poorly understood. We have previously shown in live murine sperm that the plasma membrane overlying the acrosome (APM) contains several types of microdomains known as membrane rafts. When characterizing the membrane raft-associated proteomes, we identified phospholipase B (PLB), a calcium-independent enzyme exhibiting multiple activities. Here, we show that sperm surface PLB is activated in response to sterol removal. Both biochemical activity assays and immunoblots of subcellular fractions of sperm incubated with the sterol acceptor 2-hydroxypropyl-ß-cyclodextrin (2-OHCD) confirmed the release of an active PLB fragment. Specific protease inhibitors prevented PLB activation, revealing a mechanistic requirement for proteolytic cleavage. Competitive inhibitors of PLB reduced the ability of sperm both to undergo AE and to fertilize oocytes in vitro, suggesting an important role in fertilization. This was reinforced by our finding that incubation either with protein concentrate released from 2-OHCD-treated sperm or with recombinant PLB peptide corresponding to the catalytic domain was able to induce AE in the absence of other stimuli. Together, these results lead us to propose a novel mechanism by which sterol removal promotes membrane fusogenicity and AE, helping confer fertilization competence. Importantly, this mechanism provides a basis for the newly emerging model of AE in which membrane fusions occur during capacitation/transit through the cumulus, prior to any physical contact between the sperm and the oocyte's zona pellucida.

On biodiversity conservation and poverty traps.

This paper introduces a special feature on biodiversity conservation and poverty traps. We define and explain the core concepts and then identify four distinct classes of mechanisms that define important interlinkages between biodiversity and poverty. The multiplicity of candidate mechanisms underscores a major challenge in designing policy appropriate across settings. This framework is then used to introduce the ensuing set of papers, which empirically explore these various mechanisms linking poverty traps and biodiversity conservation.

Community Markets for Conservation (COMACO) links biodiversity conservation with sustainable improvements in livelihoods and food production.

In the Luangwa Valley, Zambia, persistent poverty and hunger present linked challenges to rural development and biodiversity conservation. Both household coping strategies and larger-scale economic development efforts have caused severe natural resource degradation that limits future economic opportunities and endangers ecosystem services. A model based on a business infrastructure has been developed to promote and maintain sustainable agricultural and natural resource management practices, leading to direct and indirect conservation outcomes. The Community Markets for Conservation (COMACO) model operates primarily with communities surrounding national parks, strengthening conservation benefits produced by these protected areas. COMACO first identifies the least food-secure households and trains them in sustainable agricultural practices that minimize threats to natural resources while meeting household needs. In addition, COMACO identifies people responsible for severe natural resource depletion and trains them to generate alternative income sources. In an effort to maintain compliance with these practices, COMACO provides extension support and access to high-value markets that would otherwise be inaccessible to participants. Because the model is continually evolving via adaptive management, success or failure of the model as a whole is difficult to quantify at this early stage. We therefore test specific hypotheses and present data documenting the stabilization of previously declining wildlife populations; the meeting of thresholds of productivity that give COMACO access to stable, high-value markets and progress toward economic self-sufficiency; and the adoption of sustainable agricultural practices by participants and other community members. Together, these findings describe a unique, business-oriented model for poverty alleviation, food production, and biodiversity conservation.