Host Cell Penetration Research Articles

Comparative expression analysis of Phytophthora sojae polysaccharide lyase family 3 (pectate lyase) genes during infection of the soybean Glycine max

Phytophthora sojae is an important plant pathogen affecting soybean crops worldwide. The specific mechanisms this pathogen uses to penetrate the host cell and initiate infection have not been fully elucidated. However, a greater understanding of the molecular mechanisms that facilitate these processes will be critical to the development of novel strategies to control this devastating agricultural pathogen. It is likely that, to initiate infection, P. sojae uses an appressorium-like structure to break through the plant cell wall. It is also presumed that the pathogen relies on enzymatic activity to weaken the cell wall. The polysaccharide lyase (PL) enzyme superfamily, particularly the PL family 3 (PL3), has been hypothesized to play an important role in the process of host cell penetration by contributing to the degradation of the structural polysaccharides of the cell wall. To investigate this hypothesis, we have scanned the revised version of the annotated P. sojae genome for the presence of putative PL3-coding genes and conducted an extensive sequence analysis of all gene models found. In addition, we have quantified the relative expression of each gene in P. sojae during infection of susceptible (Williams) and resistant (Williams 82) soybean cultivars over a 48 h time period. Twelve PL3-coding gene models were identified and initial results indicate that during infection of the Williams cultivar, several of these genes experience significant up-regulation during the first 48 h of infection. An increase in transcriptional activity is also observed during infection of Williams 82; however, this expression pattern is distinct from that observed in the Williams cultivar. These results provide evidence regarding the potential involvement of PL-coding genes in the early pathogenic processes of P. sojae and suggest that this pathogen expresses genes differentially, depending on whether infection is occurring on a resistant or a susceptible soybean cultivar.

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii.

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+, and we show that this Ca2+‐mediated response is contingent on the function of calcium‐dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+ signalling plays in the invasion biology of apicomplexan parasites.

The homeobox transcription factor CoHox1 is required for the morphogenesis of infection hyphae in host plants and pathogenicity in Colletotrichum orbiculare

Abstract The hemibiotrophic fungus Colletotrichum orbiculare is the causal agent of anthracnose of cucumber. During host plant infection, appressoria penetrate into the host cells, and biotrophic infection hyphae elongate inside the host cells, then switch to necrotrophic phase. We found that the homeobox transcription factor CoHox1 is a novel type of pathogenesis-related gene. CoHox1 disruption mutants of C. orbiculare were non-pathogenic on host cucumber cotyledons and formed no lesions at wound sites on leaves. On artificial surfaces (glass and cellulose membrane), CoHox1 disruption mutants formed normal appressoria and infection hyphae similar to a wild-type strain. On the other hand, microscope observation showed that CoHox1 disruption mutants has a decreased rate of penetration of host leaves, and the infection hyphae of CoHox1 disruption mutants did not extend from the penetrated host cell into neighboring cells. On heat-shocked host leaves, CoHox1 disruption mutants also formed no lesions. These results showed that homeobox transcription factor CoHox1 may be required for pathogenicity, especially the morphogenesis of infection hyphae in host cells by C. orbiculare.

The key gluconeogenic gene PCK1 is crucial for virulence of Botrytis cinerea via initiating its conidial germination and host penetration.

The process of initiation of host invasion and survivalof some foliar phytopathogenic fungi in the absence of external nutrients on host leaf surfaces remains obscure. Here, we demonstrate that gluconeogenesis plays an important role in the process and nutrient-starvation adaptation before the pathogen host invasion. Deletion of phosphoenolpyruvate carboxykinase gene BcPCK1 in gluconeogenesis in Botrytis cinerea, the causative agent of grey mould, resulted in the failure of the ΔBcpck1 mutant conidia to germinate on hard and hydrophobic surface and penetrate host cells in the absence of glucose, reduction in conidiation and slow conidium germination in a nutrient-rich medium. The wild-type and ΔBcpck1 conidia germinate similarly in the presence of glucose (higher concentration) as the sole carbon source. Conidial glucose-content should reach a threshold level to initiate germination and host penetration. Infection structure formation by the mutants displayed a glucose-dependent fashion, which corresponded to the mutant virulence reduction. Exogenous glucose or complementation of BcPCK1 completely rescued all the developmental and virulence defects of the mutants. Our findings demonstrate that BcPCK1 plays a crucial role in B. cinerea pathogenic growth and virulence, and provide new insights into gluconeogenesis mediating pathogenesis of plant fungal pathogens via initiation of conidial germination and host penetration.

3D Porous Gelatin/PVA Hydrogel as Meniscus Substitute Using Alginate Micro-Particles as Porogens.

One of the current major challenges in orthopedic surgery is the treatment of meniscal lesions. Some of the main issues include mechanical consistency of meniscal implants, besides their fixation methods and integration with the host tissues. To tackle these aspects we realized a micro-porous, gelatin/polyvinyl alcohol (PVA)-based hydrogel to approach the high percentage of water present in the native meniscal tissue, recapitulating its biomechanical features, and, at the same time, realizing a porous implant, permissive to cell infiltration and tissue integration. In particular, we adopted aerodynamically-assisted jetting technology to realize sodium alginate micro-particles with controlled dimensions to be used as porogens. The porous hydrogels were realized through freezing-thawing cycles, followed by alginate particles leaching. Composite hydrogels showed a high porosity (74%) and an open porous structure, while preserving the elasticity behavior (E = 0.25 MPa) and high water content, typical of PVA-based hydrogels. The ex vivo animal model validation proved that the addition of gelatin, combined with the micro-porosity of the hydrogel, enhanced implant integration with the host tissue, allowing penetration of host cells within the construct boundaries. Altogether, these results show that the combined use of a water-insoluble micro-porogen and gelatin, as a bioactive agent, allowed the realization of a porous composite PVA-based hydrogel to be envisaged as a potential meniscal substitute.

Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus.

Mammalian orthoreovirus (reovirus) utilizes pore forming peptides to penetrate host cell membranes. This step is essential for delivering its genome containing core particle during viral entry. This protocol describes an in vitro assay for measuring reovirus-induced pore formation.

Proteomic Analysis of Phytophthora infestans Reveals the Importance of Cell Wall Proteins in Pathogenicity

The oomycete Phytophthora infestans is the most harmful pathogen of potato. It causes the disease late blight, which generates increased yearly costs of up to one billion euro in the EU alone and is difficult to control. We have performed a large-scale quantitative proteomics study of six P. infestans life stages with the aim to identify proteins that change in abundance during development, with a focus on preinfectious life stages. Over 10 000 peptides from 2061 proteins were analyzed. We identified several abundance profiles of proteins that were up- or downregulated in different combinations of life stages. One of these profiles contained 59 proteins that were more abundant in germinated cysts and appressoria. A large majority of these proteins were not previously recognized as being appressorial proteins or involved in the infection process. Among those are proteins with putative roles in transport, amino acid metabolism, pathogenicity (including one RXLR effector) and cell wall structure modification. We analyzed the expression of the genes encoding nine of these proteins using RT-qPCR and found an increase in transcript levels during disease progression, in agreement with the hypothesis that these proteins are important in early infection. Among the nine proteins was a group involved in cell wall structure modification and adhesion, including three closely related, uncharacterized proteins encoded by PITG_01131, PITG_01132, and PITG_16135, here denoted Piacwp1-3 Transient silencing of these genes resulted in reduced severity of infection, indicating that these proteins are important for pathogenicity. Our results contribute to further insight into P. infestans biology, and indicate processes that might be relevant for the pathogen while preparing for host cell penetration and during infection. The mass spectrometry data have been deposited to ProteomeXchange via the PRIDE partner repository with the data set identifier PXD002446.

Disruption of actin motor function due to MoMyo5 mutation impairs host penetration and pathogenicity in Magnaporthe oryzae.

Actin motor myosin proteins are the driving forces behind the active transport of vesicles, and more than 20 classes of myosin have been found to contribute to a wide range of cellular processes, including endocytosis and exocytosis, autophagy, cytokinesis and the actin cytoskeleton. In Saccharomyces cerevisiae, class V myosin Myo2 (ScMyo2p) is important for the transport of distinct sets of cargo to regions of the cell along the cytoskeleton for polarized growth. To study whether myosins play a role in the formation or function of the appressorium (infectious structure) of the rice blast fungus Magnaporthe oryzae, we identified MoMyo5 as an orthologue of ScMyo2p and characterized its function. Targeted gene disruption revealed that MoMyo5 is required for intracellular transport and is essential for hyphal growth and asexual reproduction. Although the ΔMomyo5 mutant could form appressorium-like structures, the structures were unable to penetrate host cells and were therefore non-pathogenic. We further found that MoMyo5 moves dynamically from the cytoplasm to the hyphal tip, where it interacts with MoSec4, a Rab GTPase involved in secretory transport, hyphal growth and fungal pathogenicity. Our studies indicate that class V myosin and its translocation are tightly coupled with hyphal growth, asexual reproduction, appressorium function and pathogenicity in the rice blast fungus.

Candidate Medical Countermeasures Targeting Ebola Virus Cell Entry

Medical countermeasures (MCMs) against virus infections ideally prevent the adsorption or entry of virions into target cells, thereby circumventing infection. Recent significant advances in elucidating the mechanism of Ebola virus (EBOV) host-cell penetration include the involvement of two-pore channels at the early stage of entry, and identification of cellular proteases for EBOV spike glycoprotein maturation and the intracellular EBOV receptor, Niemann–Pick type C1. This improved understanding of the initial steps of EBOV infection is now increasingly applied to rapid development of candidate MCMs, some of which have already entered the clinic. Candidate MCMs discussed include antibodies, small molecules and peptides that target various stages of the described EBOV cell-entry pathway. In this review, we summarize the currently known spectrum of EBOV cell-entry inhibitors, describe their mechanism of action and evaluate their potential for future development.

Mitochondrial Complex V α Subunit Is Critical for Candida albicans Pathogenicity through Modulating Multiple Virulence Properties.

The α subunit (ATP1) is a vital component of mitochondrial complex V which counts for the majority of cellular ATP production in a living organism. Nevertheless, how the α subunit influences other cellular processes such as pathogenicity in Candida albicans remains poorly understood. To address this question, ATP1 mutant (atp1Δ/Δ) and the gene-reconstituted strain (atp1Δ/ATP1) have been constructed in this study and their pathogenicity-related traits are compared to those of wild type (WT). In a murine model of disseminated candidiasis, atp1Δ/Δ infected mice have a significantly higher survival rate and experience a lower fungal burden in tissues. In in vitro studies atp1Δ/Δ lose a capability to damage or destroy macrophages and endothelial cells. Furthermore, atp1Δ/Δ is not able to grow under either glucose-denial conditions or high H2O2 conditions, both of which are associated with the potency of the macrophages to kill C. albicans. Defects in filamentation and biofilm formation may impair the ability of atp1Δ/Δ to penetrate host cells and establish robust colonies in the host tissues. In concert with these pathogenic features, intracellular ATP levels of atp1Δ/Δ can drop to 1/3 of WT level. These results indicate that the α subunit of Complex V play important roles in C. albicans pathogenicity.

Nuclear and structural dynamics during the establishment of a specialized effector-secreting cell by Magnaporthe oryzae in living rice cells

BackgroundTo cause an economically important blast disease on rice, the filamentous fungus Magnaporthe oryzae forms a specialized infection structure, called an appressorium, to penetrate host cells. Once inside host cells, the fungus produces a filamentous primary hypha that differentiates into multicellular bulbous invasive hyphae (IH), which are surrounded by a host-derived membrane. These hyphae secrete cytoplasmic effectors that enter host cells presumably via the biotrophic interfacial complex (BIC). The first IH cell, also known as the side BIC-associated cell, is a specialized effector-secreting cell essential for a successful infection. This study aims to determine cellular processes that lead to the development of this effector-secreting first IH cell inside susceptible rice cells.ResultsUsing live-cell confocal imaging, we determined a series of cellular events by which the appressorium gives rise to the first IH cell in live rice cells. The filamentous primary hypha extended from the appressorium and underwent asymmetric swelling at its apex. The single nucleus in the appressorium divided, and then one nucleus migrated into the swollen apex. Septation occurred in the filamentous region of the primary hypha, establishing the first IH cell. The tip BIC that was initially associated with the primary hypha became the side BIC on the swollen apex prior to nuclear division in the appressorium. The average distance between the early side BIC and the nearest nucleus in the appressorium was estimated to be more than 32 μm. These results suggest an unknown mechanism by which effectors that are expressed in the appressorium are transported through the primary hypha for their secretion into the distantly located BIC. When M. oryzae was inoculated on heat-killed rice cells, penetration proceeded as normal, but there was no differentiation of a bulbous IH cell, suggesting its specialization for establishment of biotrophic infection.ConclusionsOur studies reveal cellular dynamics associated with the development of the effector-secreting first IH cell. Our data raise new mechanistic questions concerning hyphal differentiation in response to host environmental cues and effector trafficking from the appressorium to the BIC.

HIV-1 gp41-targeting fusion inhibitory peptides enhance the gp120-targeting protein-mediated inactivation of HIV-1 virions

Protein- or peptide-based viral inactivators are being developed as novel antiviral drugs with improved efficacy, pharmacokinetics and toxicity profiles because they actively inactivate cell-free human immunodeficiency virus type 1 (HIV-1) virions before attachment to host cells. By contrast, most clinically used antiviral drugs must penetrate host cells to inhibit viral replication. In this study, we pre-treated HIV-1 particles with a gp120-targeting bispecific multivalent protein, 2Dm2m or 4Dm2m, in the presence or absence of the gp41-targeting HIV-1 fusion inhibitory peptides enfuvirtide (T20), T2635, or sifuvirtide (SFT). HIV-1 virions were separated from the inhibitors using PEG-6000, followed by testing of the residual infectivity of the HIV-1 virions. 2Dm2m and 4Dm2m exhibited significant inactivation activity against all HIV-1 strains tested with EC50 values at the low nanomolar level, whereas none of the gp41-targeting peptides showed inactivation activity at concentrations up to 250 nM. Notably, these three peptides significantly enhanced protein-mediated inactivation against cell-free HIV-1 virions, including HIV-1 laboratory-adapted and primary HIV-1 strains, as well as those resistant to T20 or T2635 and virions released from reactivated latently HIV-1-infected cells. These results indicate that the gp120-targeting bispecific multivalent proteins 2Dm2m and 4Dm2m have potential for further development as HIV-1 inactivator-based antiviral drugs for use in the clinic, either alone or in combination with a gp41-targeting HIV-1 fusion inhibitor such as T20, to treat patients with HIV-1 infection and AIDS.Emerging Microbes & Infections (2017) 6, e59; doi:10.1038/emi.2017.46; published online 21 June 2017

Fluorescent reporter analysis revealed the timing and localization of AVR-Pia expression, an avirulence effector of Magnaporthe oryzae.

In order to facilitate infection, the rice blast pathogen Magnaporthe oryzae secretes an abundance of proteins, including avirulence effectors, to diminish its host's defences. Avirulence effectors are recognized by host resistance proteins and trigger the host's hypersensitive response, which is a rapid and effective form of innate plant immunity. An understanding of the underlying molecular mechanisms of such interactions is crucial for the development of strategies to control disease. However, the expression and secretion of certain effector proteins, such as AVR-Pia, have yet to be reported. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that AVR-Pia was only expressed during infection. Fluorescently labelled AVR-Pia indicated that AVR-Pia expression was induced during appressorial differentiation in the cells of both rice and onion, as well as in a penetration-deficient (Δpls1) mutant capable of developing melanized appressoria, but unable to penetrate host cells, suggesting that AVR-Pia expression is independent of fungal penetration. Using live-cell imaging, we also documented the co-localization of green fluorescent protein (GFP)-labelled AVR-Pia and monomeric red fluorescent protein (mRFP)-labelled PWL2, which indicates that AVR-Pia accumulates in biotrophic interfacial complexes before being delivered to the plant cytosol. Together, these results suggest that AVR-Pia is a cytoplasmic effector that is expressed at the onset of appressorial differentiation and is translocated to the biotrophic interfacial complex, and then into the host's cytoplasm.

Filamentous actin accumulates during plant cell penetration and cell wall plug formation in Phytophthora infestans

The oomycete Phytophthora infestans is the cause of late blight in potato and tomato. It is a devastating pathogen and there is an urgent need to design alternative strategies to control the disease. To find novel potential drug targets, we used Lifeact-eGFP expressing P. infestans for high resolution live cell imaging of the actin cytoskeleton in various developmental stages. Previously, we identified actin plaques as structures that are unique for oomycetes. Here we describe two additional novel actin configurations; one associated with plug deposition in germ tubes and the other with appressoria, infection structures formed prior to host cell penetration. Plugs are composed of cell wall material that is deposited in hyphae emerging from cysts to seal off the cytoplasm-depleted base after cytoplasm retraction towards the growing tip. Preceding plug formation there was a typical local actin accumulation and during plug deposition actin remained associated with the leading edge. In appressoria, formed either on an artificial surface or upon contact with plant cells, we observed a novel aster-like actin configuration that was localized at the contact point with the surface. Our findings strongly suggest a role for the actin cytoskeleton in plug formation and plant cell penetration.

Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail.

ABSTRACTApical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T. gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion.

Structure of the T4 baseplate and its function in triggering sheath contraction.

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry.

SummaryHepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion.

The role of a phospholipase (PLD) in virulence of Purpureocillium lilacinum (Paecilomyces lilacinum)

Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, understanding the role of phospholipases on the virulence of the filamentous fungus Purpureocillium lilacinum has been still rather limited. In this study, pld gene was characterized. It encodes the protein phospholipase D (PLD) in P. lilacinum. This gene, 3303 bp open reading frame fragment (ORF), encodes a protein of 1100 amino acids with high similarity to the same gene from Penicillium oxalicum and Aspergillus fumigatus. Secondary structure prediction showed two PLD phosphodiesterase domains (437–464 bp and 885–912 bp). The pld gene was significantly regulated during infection of Meloidogyne incognita eggs by P. lilacinum. The expression of pld gene using RT-PCR was the highest at 36 and 48 h, which introduce evidence that the presence of M. incognita may induce the expression of the pld gene in P. lilacinum. In addition, maltose and l-alanine were found to increase the expression of pld gene. An acidic environment (pH 3.0–4.0) and moderate temperatures (27–29 °C) are favorable for pld expression in P. lilacinum.

Redox diversity in ERAD-mediated protein retrotranslocation from the endoplasmic reticulum: a complex puzzle.

Misfolded and incorrectly assembled proteins in the secretory pathway are eliminated by ubiquitylation and proteasomal degradation in a process known as ER-associated degradation (ERAD). Retrotranslocation of diverse substrates including misfolded proteins and viruses occurs through channels in the ER membrane, which are also utilized for host cell penetration by A/B class protein toxins such as cholera toxin, ricin or K28. According to the current view, disulfide-bonded proteins must either be reduced or rearranged to ensure translocation competence and entry into the cytosol from the ER. As the underlying mechanisms are still largely mysterious, we here focus on the redox status and disulfide isomerization of ERAD substrates and the role of oxidoreductases in the essential process of ER-to-cytosol retrotranslocation.

December 2014: JoVE's Year in Review

In this Year in Review, we showcase some of our favorite video articles of the year 2014 in JoVE: The Journal of Visualized Experiments. In January, JoVE Chemistry revisited one of the most famous experiments of the 20th Century. Stanley Miller and his thesis advisor, Harold Urey, made scientific history in 1953 when they showed that under the right conditions, simple compounds in a flask could spontaneously form the building blocks of life. Over six decades later, Parker et al. have demonstrated how to recreate the classic Miller-Urey experiment-combining water with various gases, And subjecting the mixture to sparks to simulate Earth's conditions 3 ½ billion years ago. The resulting primordial soup contains various amino acids that are fundamental to life on earth. February's Neuroscience section featured the nematode worm C. elegans. This simple organism is easy to manipulate genetically. And it's sensitive to various compounds, so Hao and Buttner [scientists can] use them in chemical genetic screens to study the mechanisms of antipsychotic drugs. In March, JoVE Clinical and Translational Medicine had an article on amyotrophic lateral sclerosis (ALS). This neurodegenerative disease affected baseball legend Lou Gehrig, and in 2014, inspired millions of people to dump ice buckets on their heads to raise money and awareness for ALS. An early sign of ALS is weakness in specific hand muscles, so Menon and Vucic showed how recording motor responses from hand muscle groups can help diagnose this disease. April's edition of JoVE Environment featured a technique for expressing recombinant genes in plants. Mattozzi et al. delivered special expression constructs using a gene gun to specifically target recombinant genes to different subcellular organelles in plants. In May, JoVE Clinical & Translational Medicine, Thompson et al. used coordinate mapping to analyze the pharyngeal phase of swallowing, a technique that can be useful for studying swallowing disorders. In June, JoVE Applied Physics took us back in time again with a protocol that approximates conditions in the early Solar System. Blum et al. examined collision forces between particles, which can aggregate over hundreds of millions of years to form planets. In the July edition of JoVE Behavior, Tung et al. demonstrated standard behavioral tests for noninvasively assessing balance performance in mice. In August, Malide et al. demonstrated in JoVE Biology how to label hematopoietic precursor cells with different fluorescent markers so the cells can be tracked as they differentiate and engraft into bone marrow after transplantation. In our September issue, JoVE Bioengineering featured innovative methods for treating the cornea, the clear front window of the eye. Ortega et al. engineered membranes that could potentially regenerate the corneal epithelium and preserve vision in certain eye diseases. In October, JoVE Immunology & Infection featured a technique for analyzing glycoprotein spikes on viral surfaces, which many viruses use to penetrate host cells. Huiskonen et al. use a computational approach to study the precise, 3D structures of these spikes, which can guide the design of antiviral drugs and vaccines. November's edition of JoVE Biology took flow cytometry to a whole new level with a protocol that can be performed in space. Phipps et al. demonstrate the procedure on a parabolic flight, also known as a vomit comet, which creates a weightless environment and presents a whole new set of challenges to laboratory research. And coming up in December, we pinch off 2014 with a article describing fecal transplantation, When stool from a healthy donor is transferred to a patient infected with Clostridium difficile through a standard colonoscopy, it is a highly effective therapy for infections that don't respond to other treatments. This Year in Review was just a sampling of more than 900 video articles that JoVE produced for 2014. Browse the JoVE archives for thousands of other videos, and come back each week to see brand-new material in JoVE: The Journal of Visualized Experiments.