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Malonyl-CoA is the essential building block of fatty acids and regulates cell function through protein malonylation and allosteric regulation of signaling networks. Accordingly, the production and use of malonyl-CoA is finely tuned by the cellular energy status. Most studies of malonyl-CoA dynamics rely on bulk approaches that take only a snapshot of the average metabolic state of a population of cells, missing out on dynamic changes in malonyl-CoA and fatty acid biosynthesis that could be occurring within a single cell. To overcome this limitation, we have developed a genetically encoded fluorescent protein-based biosensor for malonyl-CoA that can be used to capture malonyl-CoA dynamics in single cells. This biosensor, termed Malibu ( mal onyl-CoA i ntracellular b iosensor to u nderstand dynamics), exhibits an excitation-ratiometric change in response to malonyl-CoA binding. We first used Malibu to monitor malonyl-CoA dynamics during inhibition of fatty acid biosynthesis using cerulenin in E. coli , observing an increase in Malibu response in a time- and dose-dependent manner. In HeLa cells, we used Malibu to monitor the impact of fatty acid biosynthesis inhibition on malonyl-CoA dynamics in single cells, finding that two inhibitors of fatty acid biosynthesis, cerulenin and orlistat, which inhibit different steps of fatty acid biosynthesis, increase malonyl-CoA levels. Altogether, we have developed a new genetically encoded biosensor for malonyl-CoA, which can be used to sensitively study malonyl-CoA dynamics in single cells, providing an unparalleled view into fatty acid biosynthesis., Competing Interests: The authors declare they have no competing interests.

An understanding of species-specific vitamin D metabolism and its role in calcium homeostasis is essential for correct diet formulation and development of husbandry protocols for managed nondomestic species. This study documented serum vitamin D metabolites and other analytes involved in calcium homeostasis in Asian elephants ( Elephas maximus ) managed at a latitude similar to their wild natural habitat. Serum values for 33 elephants managed at a low latitude were measured in the peak of summer, revealing low vitamin D 2 (25(OH)D 2 2.3 ± 0.6 ng/ ml and 24,25(OH) 2 D 2 2.17 ± 0.52 ng/ml) and nondetectable vitamin D 3 . Serum minerals (calcium, phosphorus, magnesium), ionized calcium, and parathyroid hormone were within normal reported ranges. In comparison with previously reported values in elephants managed at a high latitude, 25(OH)D 2 ( P < 0.001), 24,25(OH) 2 D 2 ( P = 0.001), and magnesium ( P = 0.013) were significantly lower, and parathyroid hormone was significantly higher ( P < 0.001). The lack of D 3 production during ample sun exposure at a low latitude suggests that Asian elephants are incapable of cutaneous photobiosynthesis of vitamin D, and that low serum D 2 is normal for this species.

Enzymes in glucose metabolism have been subjected to numerous studies, revealing the importance of their biological roles during the cell cycle. However, due to the lack of viable experimental strategies for measuring enzymatic activities particularly in living human cells, it has been challenging to address whether their enzymatic activities and thus anticipated glucose flux are directly associated with cell cycle progression. It has remained largely elusive how human cells regulate glucose metabolism at a subcellular level to meet the metabolic demands during the cell cycle. Meanwhile, we have characterized that rate-determining enzymes in glucose metabolism are spatially organized into three different sizes of multienzyme metabolic assemblies, termed glucosomes, to regulate the glucose flux between energy metabolism and building block biosynthesis. In this work, we first determined using cell synchronization and flow cytometric techniques that enhanced green fluorescent protein-tagged phosphofructokinase is adequate as an intracellular biomarker to evaluate the state of glucose metabolism during the cell cycle. We then applied fluorescence single-cell imaging strategies and discovered that the percentage of Hs578T cells showing small-sized glucosomes is drastically changed during the cell cycle, whereas the percentage of cells with medium-sized glucosomes is significantly elevated only in the G1 phase, but the percentage of cells showing large-sized glucosomes is barely or minimally altered along the cell cycle. Should we consider our previous localization-function studies that showed assembly size-dependent metabolic roles of glucosomes, this work strongly suggests that glucosome sizes are modulated during the cell cycle to regulate glucose flux between glycolysis and building block biosynthesis. Therefore, we propose the size-specific modulation of glucosomes as a behind-the-scenes mechanism that may explain functional association of glucose metabolism with the cell cycle and, thereby, their metabolic significance in human cell biology., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a master regulator of cellular metabolism, phosphorylating a variety of downstream targets throughout the cell. Subcellular AMPK activity results in regulation of glycolysis, lipid and protein biosynthesis, mitochondrial function, and gene expression. But how AMPK senses and responds to stimuli in a compartment-specific manner is not well understood, leaving an incomplete picture of compartmentalized AMPK activity. Key tools for studying subcellular AMPK activity are genetically encoded AMPK activity reporters (AMPKARs), which allow for the quantitative visualization of subcelluar AMPK activity. However, many AMPKARs suffer from poor dynamic range and sensitivity, limiting their application. I recently reported the development of a new excitation-ratiometric (ExRai) AMPKAR, a single-fluorophore AMPKAR with enhanced dynamic range for detection of subtle, subcellular AMPK activity. I used ExRai AMPKAR to study subcellular AMPK activity at several locations, including the lysosome and mitochondria, identifying new mechanisms for the regulation of AMPK activity. Here, I describe the use of ExRai AMPKAR to image subcellular AMPK activity in mouse embryonic fibroblasts using both widefield and confocal microscopy. I also describe the culture of mouse embryonic fibroblasts. Through the use of ExRai AMPKAR, subcellular AMPK activity can be illuminated to better understand how this central kinase regulates cellular metabolism. © 2023 The Author. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Imaging subcellular AMPK activity using ExRai AMPKAR Support Protocol 1: Culturing of mouse embryonic fibroblasts for live-cell imaging Support Protocol 2: Live-cell imaging of ExRai AMPKAR using confocal microscopy., (© 2023 The Author. Current Protocols published by Wiley Periodicals LLC.)

Genome architecture and organization play critical roles in cell life. However, it remains largely unknown how genomic loci are dynamically coordinated to regulate gene expression and determine cell fate at the single cell level. We have developed an inducible system which allows Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies (SIMBA) in living cells. In SIMBA, the human heterochromatin protein 1α (HP1α) is fused to mCherry and FRB, which can be induced to form biomolecular assemblies (BAs) with FKBP-scFv, guided to specific genomic loci by a nuclease-defective Cas9 (dCas9) or a transcriptional factor (TF) carrying tandem repeats of SunTag. The induced BAs can not only enhance the imaging signals at target genomic loci using a single sgRNA, either at repetitive or non-repetitive sequences, but also recruit epigenetic modulators such as histone methyltransferase SUV39H1 to locally repress transcription. As such, SIMBA can be applied to simultaneously visualize and manipulate, in principle, any genomic locus with controllable timing in living cells., (© 2022. The Author(s).)

Precise spatiotemporal organization and regulation of signal transduction networks are essential for cellular response to internal and external cues. To understand how this biochemical activity architecture impacts cellular function, many genetically encodable tools which regulate kinase activity at a subcellular level have been developed. In this review, we highlight various types of genetically encodable molecular tools, including tools to regulate endogenous kinase activity and biorthogonal techniques to perturb kinase activity. Finally, we emphasize the use of these tools alongside biosensors for kinase activity to measure and perturb kinase activity in real time for a better understanding of the cellular biochemical activity architecture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But how AMPK activity is regulated at different subcellular locations for precise spatiotemporal control over metabolism is unclear. Here we present a sensitive, single-fluorophore AMPK activity reporter (ExRai AMPKAR), which reveals distinct kinetic profiles of AMPK activity at the mitochondria, lysosome, and cytoplasm. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) results in slower AMPK activity at lysosomes but does not affect the response amplitude at lysosomes or mitochondria, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further identify a mechanism for AMPK activity in the nucleus, which results from cytoplasmic to nuclear shuttling of AMPK. Thus, ExRai AMPKAR enables illumination of the complex subcellular regulation of AMPK signaling., (© 2022. The Author(s).)

Disease susceptibility and resistance are important factors for the conservation of endangered species, including elephants. We analyzed pathology data from 26 zoos and report that Asian elephants have increased neoplasia and malignancy prevalence compared with African bush elephants. This is consistent with observed higher susceptibility to tuberculosis and elephant endotheliotropic herpesvirus (EEHV) in Asian elephants. To investigate genetic mechanisms underlying disease resistance, including differential responses between species, among other elephant traits, we sequenced multiple elephant genomes. We report a draft assembly for an Asian elephant, and defined 862 and 1,017 conserved potential regulatory elements in Asian and African bush elephants, respectively. In the genomes of both elephant species, conserved elements were significantly enriched with genes differentially expressed between the species. In Asian elephants, these putative regulatory regions were involved in immunity pathways including tumor-necrosis factor, which plays an important role in EEHV response. Genomic sequences of African bush, forest, and Asian elephant genomes revealed extensive sequence conservation at TP53 retrogene loci across three species, which may be related to TP53 functionality in elephant cancer resistance. Positive selection scans revealed outlier genes related to additional elephant traits. Our study suggests that gene regulation plays an important role in the differential inflammatory response of Asian and African elephants, leading to increased infectious disease and cancer susceptibility in Asian elephants. These genomic discoveries can inform future functional and translational studies aimed at identifying effective treatment approaches for ill elephants, which may improve conservation., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Genetically encoded fluorescent protein-based kinase biosensors are a central tool for illumination of the kinome. The adaptability and versatility of biosensors have allowed for spatiotemporal observation of real-time kinase activity in living cells and organisms. In this review, we highlight various types of kinase biosensors, along with their burgeoning applications in complex biological systems. Specifically, we focus on kinase activity reporters used in neuronal systems and whole animal settings. Genetically encoded kinase biosensors are key for elucidation of the spatiotemporal regulation of protein kinases, with broader applications beyond the Petri dish., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Dendritic spines are the primary excitatory postsynaptic sites that act as subcompartments of signaling. Ca 2+ is often the first and most rapid signal in spines. Downstream of calcium, the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway plays a critical role in the regulation of spine formation, morphological modifications, and ultimately, learning and memory. Although the dynamics of calcium are reasonably well-studied, calcium-induced cAMP/PKA dynamics, particularly with respect to frequency modulation, are not fully explored. In this study, we present a well-mixed model for the dynamics of calcium-induced cAMP/PKA dynamics in dendritic spines. The model is constrained using experimental observations in the literature. Further, we measured the calcium oscillation frequency in dendritic spines of cultured hippocampal CA1 neurons and used these dynamics as model inputs. Our model predicts that the various steps in this pathway act as frequency modulators for calcium, and the high frequency of calcium input is filtered by adenylyl cyclase 1 and phosphodiesterases in this pathway such that cAMP/PKA only responds to lower frequencies. This prediction has important implications for noise filtering and long-timescale signal transduction in dendritic spines. A companion manuscript presents a three-dimensional spatial model for the same pathway., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A macromolecular complex of the enzymes involved in human de novo purine biosynthesis, the purinosome, has been shown to consist of a core assembly to regulate the metabolic activity of the pathway. However, it remains elusive whether the core assembly itself can be selectively controlled in the cytoplasm without promoting the purinosome. Here, we reveal that pharmacological inhibition of the cytoplasmic activity of 3-phosphoinositide-dependent protein kinase 1 (PDK1) selectively promotes the formation of the core assembly, but not the purinosome, in cancer cells. However, alternative signaling cascades that are associated with the plasma membrane-bound PDK1 activity, including Akt-mediated cascades, regulate neither the core assembly nor the purinosome in our conditions. Along with immunofluorescence microscopy and a knock-down study against PDK1 using small interfering RNAs, we reveal that cytoplasmic PDK1-associated signaling pathways regulate subcellular colocalization of three enzymes that form the core assembly of the purinosome in an Akt-independent manner. Collectively, this study reveals a new mode of compartmentalization of purine biosynthetic enzymes controlled by spatially resolved signaling pathways.

The organization of metabolic multienzyme complexes has been hypothesized to benefit metabolic processes and provide a coordinated way for the cell to regulate metabolism. Historically, their existence has been supported by various in vitro techniques. However, it is only recently that the existence of metabolic complexes inside living cells has come to light to corroborate this long-standing hypothesis. Indeed, subcellular compartmentalization of metabolic enzymes appears to be widespread and highly regulated. On the other hand, it is still challenging to demonstrate the functional significance of these enzyme complexes in the context of the cellular milieu. In this review, we discuss the current understanding of metabolic enzyme complexes by primarily focusing on central carbon metabolism and closely associated metabolic pathways in a variety of organisms, as well as their regulation and functional contributions to cells.

Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Dynamic partitioning of de novo purine biosynthetic enzymes into multienzyme compartments, purinosomes, has been associated with increased flux of de novo purine biosynthesis in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells. We have investigated the functional role of AMP-activated protein kinase (AMPK) in the regulation of de novo purine biosynthesis because of its regulatory action on lipid and carbohydrate biosynthetic pathways. Using pharmacological AMPK activators, we have monitored subcellular localizations of six pathway enzymes tagged with green fluorescent proteins under time-lapse fluorescence single-cell microscopy. We revealed that only one out of six pathway enzymes, formylglycinamidine ribonucleotide synthase (FGAMS), formed spatially distinct cytoplasmic granules after treatment with AMPK activators, indicating the formation of single-enzyme self-assemblies. In addition, subsequent biophysical studies using fluorescence recovery after photobleaching showed that the diffusion kinetics of FGAMS were slower when it localized inside the self-assemblies than within the purinosomes. Importantly, high-performance liquid chromatographic studies revealed that the formation of AMPK-promoted FGAMS self-assembly caused the reduction of purine metabolites in HeLa cells, indicating the downregulation of de novo purine biosynthesis. Collectively, we demonstrate here that the spatial sequestration of FGAMS by AMPK is a mechanism by which de novo purine biosynthesis is downregulated in human cells.

A cell is a highly organized, dynamic, and intricate biological entity orchestrated by a myriad of proteins and their self-assemblies. Because a protein's actions depend on its coordination in both space and time, our curiosity about protein functions has extended from the test tube into the intracellular space of the cell. Accordingly, modern technological developments and advances in enzymology have been geared towards analyzing protein functions within intact single cells. We discuss here how fluorescence single-cell microscopy has been employed to identify subcellular locations of proteins, detect reversible protein-protein interactions, and measure protein activity and kinetics in living cells. Considering that fluorescence single-cell microscopy has been only recently recognized as a primary technique in enzymology, its potentials and outcomes in studying intracellular protein functions are projected to be immensely useful and enlightening. We anticipate that this review would inspire many investigators to study their proteins of interest beyond the conventional boundary of specific disciplines. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Importance: Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology., Objective: To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS)., Design, Setting, and Participants: A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015., Exposures: Ionizing radiation and doxorubicin., Main Outcomes and Measures: Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes., Results: Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional to TP53 status (ionizing radiation exposure: patients with LFS, 2.71% [95% CI, 1.93%-3.48%] vs human controls, 7.17% [95% CI, 5.91%-8.44%] vs elephants, 14.64% [95% CI, 10.91%-18.37%]; P < .001; doxorubicin exposure: human controls, 8.10% [95% CI, 6.55%-9.66%] vs elephants, 24.77% [95% CI, 23.0%-26.53%]; P < .001)., Conclusions and Relevance: Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies of TP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression.

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65%) expressed higher percentages of spermatozoa with normal morphology (80.3±13.0 vs. 44.9±30.8%) and positive Spermac staining (51.9±14.5 vs. 7.5±14.4%), in addition to higher total volume (135.1±89.6 vs. 88.8±73.1 ml) and lower sperm concentration (473.0±511.2 vs. 1313.8±764.7×10⁶ cells ml⁻¹) compared to ejaculates exhibiting poor sperm motility (≤10%; P<0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ∼80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P<0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species.

Objective: To determine plasma pharmacokinetics of penciclovir following oral and rectal administration of famciclovir to young Asian elephants (Elephas maximus)., Animals: 6 healthy Asian elephants (5 females and 1 male), 4.5 to 9 years old and weighing 1,646 to 2,438 kg., Procedures: Famciclovir was administered orally or rectally in accordance with an incomplete crossover design. Three treatment groups, each comprising 4 elephants, received single doses of famciclovir (5 mg/kg, PO, or 5 or 15 mg/kg, rectally); there was a minimum 12-week washout period between subsequent famciclovir administrations. Serial blood samples were collected after each administration. Samples were analyzed for famciclovir and penciclovir with a validated liquid chromatography-mass spectroscopy assay., Results: Famciclovir was tolerated well for both routes of administration and underwent complete biotransformation to the active metabolite, penciclovir. Mean maximum plasma concentration of penciclovir was 1.3 μg/mL at 1.1 hours after oral administration of 5 mg/kg. Similar results were detected after rectal administration of 5 mg/kg. Mean maximum plasma concentration was 3.6 μg/mL at 0.66 hours after rectal administration of 15 mg/kg; this concentration was similar to results reported for humans receiving 7 mg/kg orally., Conclusions and Clinical Relevance: Juvenile Asian elephants are susceptible to elephant endotheliotropic herpesvirus. Although most infections are fatal, case reports indicate administration of famciclovir has been associated with survival of 3 elephants. In Asian elephants, a dose of 8 to 15 mg of famciclovir/kg given orally or rectally at least every 8 hours may result in penciclovir concentrations that are considered therapeutic in humans.

Asian elephant spermatozoa are sensitive to chilling and do not respond well to cryopreservation. The objectives of the present study were to: (1) determine whether cholesterol content can be modified by preincubation of Asian elephant spermatozoa with cholesterol-loaded cyclodextrin (CLC); and (2) assess the effects of CLC concentration(s), temperature at time of glycerol addition (22°C vs 4°C) and dilution medium on post-thaw sperm survival. Spermatozoa incubated with ≥1.5 mg CLC exhibited increased (P < 0.05) cholesterol concentrations. Pretreatment of spermatozoa with 1.5 mg CLC resulted in improvements (P < 0.05) in all post-thaw parameters. Glycerol addition at 4°C also improved all post-thaw parameters compared with 22°C. Dilution of thawed spermatozoa in an egg yolk-based medium improved (P < 0.05) motility compared with Ham's F-10 culture medium. In summary, our findings indicate that modifying cholesterol content within the plasma membrane improves the cryosurvival of Asian elephant spermatozoa. The development of an improved cryopreservation method that includes modification of membrane cholesterol and the addition of glycerol at 4°C, as reported in the present study, is an important step towards utilisation of cryopreserved spermatozoa in captive management of this species.

Artificial insemination plays a key role in the genetic management of elephants in zoos. Because freshly extended semen is typically used for artificial insemination in elephants, it has become imperative to optimize conditions for liquid storage and semen transport. The objectives of this study were to examine the interactions between different extenders and storage temperatures on sperm total motility, progressive motility, and acrosomal integrity in Asian (Elephas maximus) and African (Loxodonta africana) elephants. Ejaculates were collected by rectal massage, diluted using a split-sample technique in 5 semen extenders: TL-Hepes (HEP), Modena (MOD), Biladyl (BIL), TEST refrigeration medium (TES), and INRA96 (INR), maintained at 35°C, 22°C, or 4°C. At 0, 4, 6, 12, and 24 hours, aliquots were removed and assessed for sperm total motility, progressive motility, and acrosomal integrity. After 24 hours of storage, African elephant spermatozoa exhibited greater longevity and higher values in sperm quality parameters compared with those of Asian elephants. In both species, semen storage at 35°C resulted in a sharp decline in all sperm quality parameters after 4 hours of storage, whereas storage at 22°C and 4°C facilitated sperm survival. In Asian elephants, MOD and HEP were most detrimental, whereas BIL, TES, and INR maintained motility up to 12 hours when spermatozoa were cooled to 22°Cor4°C. In African elephants, there were no differences among extenders. All media maintained good sperm quality parameters at 22°C or 4°C. However, although MOD, BIL, and INR were most effective at lower temperatures, HEP and TES maintained sperm motility at all storage temperatures. This study demonstrated sperm sensitivity to components of various semen extenders and storage temperatures and offers recommendations for semen extender choices for liquid semen storage for both Asian and African elephants.

A total of 114 captive elephants (6 Asian; 108 African) from 43 private institutions or North American zoos accredited by the Association of Zoos and Aquariums were sampled and evaluated to investigate genetic status. Because previous analyses of the captive collection indicated potential cytonuclear dissociation between mitochondrial DNA (mtDNA) sequence and microsatellite nuclear DNA genotype data, we investigated this phenomenon within the captive collection with 2 X-linked genes (BGN and PHKA2) and 1 Y-linked gene (AMELY). These data reveal that individuals with forest-derived elephant mtDNA lineages carried only savannah elephant nuclear gene haplotypes. These results are concordant with a previous study of wild populations sampled across Africa, indicating that cytonuclear genomic dissociation was captured in the founders of the North American African elephant collection. These results are important for resolving questions that can potentially impact future management and breeding programs related to the collection.

Sex allocation theories predict equal offspring number of both sexes unless differential investment is required or some competition exists. Left undisturbed, elephants reproduce well and in approximately even numbers in the wild. We report an excess of males are born and substantial juvenile mortality occurs, perinatally, in captivity. Studbook data on captive births (CB, n=487) and premature deaths (PD, <5 years of age; n=164) in Asian and African elephants in Europe and North America were compared with data on Myanmar timber (Asian) elephants (CB, n=3070; PD, n=738). Growth in CB was found in three of the captive populations. A significant excess of male births occurred in European Asian elephants (ratio: 0.61, P=0.044) and in births following artificial insemination (0.83, P=0.003), and a numerical inclination in North American African elephants (0.6). While juvenile mortality in European African and Myanmar populations was 21-23%, it was almost double (40-45%) in all other captive populations. In zoo populations, 68-91% of PD were within 1 month of birth with stillbirth and infanticide being major causes. In Myanmar, 62% of juvenile deaths were at >6 months with maternal insufficient milk production, natural hazards and accidents being the main causes. European Asian and Myanmar elephants PD was biased towards males (0.71, P=0.024 and 0.56, P<0.001, respectively). The skewed birth sex ratio and high juvenile mortality hinder efforts to help captive populations become self-sustaining. Efforts should be invested to identify the mechanism behind these trends and seek solutions for them.

The objective was to validate a high-sensitivity chemiluminescent assay of serum progesterone concentrations for pregnancy diagnosis in manatees. Assay analytical sensitivity was 0.1 ng/mL, with mean intra- and inter-assay coefficients of variation of 9.7 and 9.2%, respectively, and accuracy had a mean adjusted R(2) of 0.98. Methods comparison (relative to Siemen's Coat-A-Count RIA) demonstrated r=0.98, Deming regression slope of 0.95, and an intercept of 0.01. Based on ROC analysis, a progesterone concentration >or=0.4 ng/mL was indicative of pregnancy. Assay results were not significantly altered by two freeze-thaw cycles of samples. Characteristic progesterone concentrations during pregnancy were Months 1-4 (1.7-4.7 ng/mL), 5-8 ( approximately 1.0 ng/mL), and 10 and 11 (0.3-0.5 ng/mL), whereas two late-pregnant females with impending abortion had progesterone concentrations of 0.1 ng/mL. Among pregnant females, maximum progesterone concentrations occurred in autumn (3.9+/-1.8 ng/mL), and were greater during all seasons than concentrations in non-pregnant females (0.1-0.2 ng/mL). Progesterone concentrations were also significantly higher in pregnant females than in non-pregnant females and males. This highly sensitive, specific, and diagnostic assay will be valuable for monitoring pregnancy and abortion in manatees.

Obstetrics, one of the oldest fields in veterinary medicine, is well described and practiced in domestic and exotic animals. However, when providing care during elephant birth or dystocia, veterinary intervention options differ greatly from any domestic species, and are far more limited due to the dimensions and specific anatomy of the elephant reproductive tract. In addition, aging of captive elephant populations and advanced age of primiparous females make active birth management increasingly important. Intrauterine infection, uterine inertia and urogenital tract pathologies are emerging as major causes for dystocia, often leading to foetal and dam death. This paper reviews the current knowledge on elephant birth and the factors associated with dystocia. It then summarises recommendations for birth and dystocia management. As Caesarean section, the most common ultima ratio in domestic animal obstetrics, is lethal and therefore not an option in the elephant, non-invasive medical treatment, induction of the Fergusson reflex or the conscious decision to leave a retained foetus until it is expelled voluntarily, are key elements in elephant obstetrics. Surgical strategies such as episiotomy and foetotomy are sometimes inevitable in order to try to save the life of the dam, however, these interventions result in chronic post-surgical complications or even fatal outcome. Limited reliable data on serum calcium concentrations, and pharmacokinetics and effect of exogenous oestrogen, oxytocin, and prostaglandins during birth provide the scope of future research, necessary to advance scientific knowledge on obstetrics in elephants.

Two juvenile Asian elephants (Elephas maximus) presented with an acute onset of facial edema and lethargy. Examination of the oral cavity of each animal revealed cyanosis of the tip and distal margins of the tongue suggestive of endothelial inclusion body disease (EIBD) of elephants. Whole-blood samples were obtained, and polymerase chain reaction tests confirmed the presence of elephant herpesvirus. The animals were administered famciclovir (Famvir, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19101, USA), a potent human anti-herpesvirus drug, in the course of their disease, and recovery followed a treatment regime of 3-4 wk. These are the first known cases of elephants surviving EIBD.

In the elephant, two distinct LH surges occur 3 wk apart during the nonluteal phase of the estrous cycle, but only the second surge (ovLH) induces ovulation. The function of the first, anovulatory surge (anLH) is unknown, nor is it clear what regulates the timing of these two surges. To further study this observation in the Asian elephant, serum concentrations of LH, FSH, progesterone, inhibin, estradiol, and prolactin were quantified throughout the estrous cycle to establish temporal hormonal relationships. To examine long-term dynamics of hormone secretion, analyses were conducted in weekly blood samples collected from 3 Asian elephants for up to 3 yr. To determine whether differences existed in secretory patterns between the anLH and ovLH surges, daily blood samples were analyzed from 21 nonluteal-phase periods from 7 Asian elephants. During the nonluteal phase, serum LH was elevated for 1-2 days during anLH and ovLH surges with no differences in peak concentration between the two surges. The anLH surge occurred 19.9+/-1.2 days after the end of the luteal phase and was followed by the ovLH surge 20.8+/-0.5 days later. Serum FSH concentrations were highest at the beginning of the nonluteal phase and gradually declined to nadir concentrations within 4 days of the ovLH surge. FSH remained low until after the ovLH surge and then increased during the luteal phase. Serum inhibin concentrations were negatively correlated with FSH during the nonluteal phase (r = -0.53). Concentrations of estradiol and prolactin fluctuated throughout the estrous cycle with no discernible patterns evident. In sum, there were no clear differences in associated hormone secretory patterns between the anLH and ovLH surge. However, elevated FSH at the beginning of the nonluteal phase may be important for follicle recruitment, with the first anLH surge acting to complete the follicle selection process before ovulation.

The implications of collecting semen from elephants for use in artificial insemination programs are profound in the context of propagating captive elephants. Using a manual manipulation technique, semen was collected and characterized from five adult Asian elephants (Elephas maximus) and ejaculate fluid was obtained from one castrated elephant bull. The penis was stimulated to protrusion and erection by rectal massage of the pelvic portion of the urethra. During an ejaculatory response, massage was also directed onto the area of the ampulla of the ductus deferens. Sperm rich ejaculates were usually collected as a result of each ejaculatory contraction. Ejaculates were evaluated for spermatozoal concentration and pH (when possible) and sperm rich fractions combined for determination of total volume. Mean total volume of each collection was 27.5+/-4.4 ml. Mean concentration of the first and second ejaculatory responses from a collection was 2.05+/-0.17 x 10(9) and 1.34+/-0.19 x 10(9) sperm/ml, respectively. Measurement of seminal pH revealed no significant differences between the fractions. Mean pH of the first and second ejaculatory responses were 7.05+/-0.07 and 7.04+/-0.13. This method of collecting elephant sperm can be utilized for semen evaluation of bulls of unknown reproductive status in conjunction with other evaluation techniques (i.e. ultrasonographic, endocrinologic). It also has the potential for providing valuable genetic material for genome resource banks and for use with assisted reproductive techniques like artificial insemination.

Transrectal ultrasonography was performed on five wild and two captive male African elephants (Loxodonta africana) and four captive male Asian elephants (Elephas maximus) to develop standards for assessment of reproductive health and status. The entire internal urogenital tract was visualized ultrasonographically by using a 3.5 MHz or a 7.5-MHz transducer in combination with a probe extension adapted for elephant anatomy. The findings were verified by postmortem ex situ ultrasound examinations in several individuals of each species. Each part of the internal urogenital tract was sonographically detectable except for the bulbourethral glands and the cranial portion of the ureters and ductus deferentes, which were only observed in situ in the neonate. Each structure visualized was measured and described. The size and morphology of the urogenital structures, especially the accessory glands, were indicative of breeding status, if known. There was a notable difference between African and Asian males in the size and morphology of the prostate gland and a slight difference in the shape of the ampullae. No other structure showed significant species differences. The detection of the location and description of the testes may provide information for modifying present castration procedures. Furthermore, ultrasound examination of the male accessory glands may aid in the identification of potential semen donors for assisted reproduction programs in captive elephants.

To characterize patterns of herpes simplex virus type 1 infection, illness and transmission among children in group day care, the data for 115 children who had been followed longitudinally from early infancy in a research day care center were examined. By 5 years of age 37% of study children had evidence of herpes simplex virus type 1 infection as demonstrated by virus isolation and/or seroconversion. The incidence of infection was highest among children 1 to 2 years old. Four small clusters of primary infections were observed over the 12-year study period but no cluster involved more than 6 children. Fifty-five percent of primary infections occurred during these small outbreaks; the remainder were sporadic. Gingivostomatitis was observed in 26% of children with primary culture-proved infections; no child with infection identified solely by serologic means had a history of gingivostomatitis. The occurrence of gingivostomatitis did not appear to be associated with increased transmission of herpes simplex virus type 1 infection in this day-care setting.