Bio-inspired microneedle design for efficient drug/vaccine coating.

Biomimetics is the interdisciplinary scientific field focused on the study and imitation of biological systems, with the aim of solving complex technological problems. In this paper, we present a new bio-inspired design for microneedles (MNs) and MN arrays, intended for rapidly coating the MNs with drug/vaccine. The biomimetic approach consists in ornamenting the lateral sides of pyramidal MNs with structures inspired by the external scent efferent systems of some European true bugs, which facilitate a directional liquid transport. To realize these MNs, two-photon polymerization (TPP) technique was used. Liquid coating capabilities of structured and non-structured MNs were compared. Moreover, both in-vivo and ex-vivo skin tests were performed to prove that MNs pierce the skin. We show that the arrays of MNs can be accurately replicated using a micro-moulding technique. We believe this design will be beneficial for the process of drug/vaccine loading onto the needles' surfaces, by making it more efficient and by reducing the drug/vaccine wastage during MN coating process.

Ultra-Low-Power Digital Filtering for Insulated EMG Sensing.

Myoelectric prostheses help amputees to regain independence and a higher quality of life. These prostheses are controlled by state-of-the-art electromyography sensors, which use a conductive connection to the skin and are therefore sensitive to sweat. They are applied with some pressure to ensure a conductive connection, which may result in pressure marks and can be problematic for patients with circulatory disorders, who constitute a major group of amputees. Here, we present ultra-low-power digital signal processing algorithms for an insulated EMG sensor which couples the EMG signal capacitively. These sensors require neither conductive connection to the skin nor electrolytic paste or skin preparation. Capacitive sensors allow straightforward application. However, they make a sophisticated signal amplification and noise suppression necessary. A low-cost sensor has been developed for real-time myoelectric prostheses control. The major hurdles in measuring the EMG are movement artifacts and external noise. We designed various digital filters to attenuate this noise. Optimal system setup and filter parameters for the trade-off between attenuation of this noise and sufficient EMG signal power for high signal quality were investigated. Additionally, an algorithm for movement artifact suppression, enabling robust application in real-world environments, is presented. The algorithms, which require minimal calculation resources and memory, are implemented on an ultra-low-power microcontroller.

An Insulated Flexible Sensor for Stable Electromyography Detection: Applicationto Prosthesis Control.

Electromyography (EMG), the measurement of electrical muscle activity, is used in a variety of applications, including myoelectric upper-limb prostheses, which help amputees to regain independence and a higher quality of life. The state-of-the-art sensors in prostheses have a conductive connection to the skin and are therefore sensitive to sweat and require preparation of the skin. They are applied with some pressure to ensure a conductive connection, which may result in pressure marks and can be problematic for patients with circulatory disorders, who constitute a major group of amputees. Due to their insulating layer between skin and sensor area, capacitive sensors are insensitive to the skin condition, they require neither conductive connection to the skin nor electrolytic paste or skin preparation. Here, we describe a highly stable, low-power capacitive EMG measurement set-up that is suitable for real-world application. Various flexible multi-layer sensor set-ups made of copper and insulating foils, flex print and textiles were compared. These flexible sensor set-ups adapt to the anatomy of the human forearm, therefore they provide high wearing comfort and ensure stability against motion artifacts. The influence of the materials used in the sensor set-up on the magnitude of the coupled signal was demonstrated based on both theoretical analysis and measurement.The amplifier circuit was optimized for high signal quality, low power consumption and mobile application. Different shielding and guarding concepts were compared, leading to high SNR.

A Polydimethylsiloxane (PDMS) Waveguide Sensor that Mimics a Neuromast to Measure Fluid Flow Velocity.

Accurate flow measurement is a ubiquitous task in fields such as industry, medical technology, or chemistry; it remains however challenging due to small measurement ranges or erosive flows. Inspiration for possible measurement methods can come from nature, for example from the lateral line organ of fish, which is comprised of hair cells embedded in a gelatinous cupula. When the cupula is deflected by water movement, the hair cells generate neural signals from which the fish gains an accurate representation of its environment. We built a flow sensor mimicking a hair cell, but coupled it with an optical detection method. Light is coupled into a PDMS waveguide that consists of a core and a cladding with a low refractive index contrast to ensure high bending sensitivity. Fluid flow bends the waveguide; this leads to a measurable light loss. The design of our sensory system allows flow measurement in opaque and corrosive fluids while keeping production costs low. To prove the measurement concept, we evaluated the light loss while (a) reproducibly bending the fiber with masses, and (b) exposing the fiber to air flow. The results demonstrate the applicability of an optical fiber as a flow sensor.

Adhesion enhancement of cribellate capture threads by epicuticular waxes of the insect prey sheds new light on spider web evolution.

To survive, web-building spiders rely on their capture threads to restrain prey. Many species use special adhesives for this task, and again the majority of those species cover their threads with viscoelastic glue droplets. Cribellate spiders, by contrast, use a wool of nanofibres as adhesive. Previous studies hypothesized that prey is restrained by van der Waals' forces and entrapment in the nanofibres. A large discrepancy when comparing the adhesive force on artificial surfaces versus prey implied that the real mechanism was still elusive. We observed that insect prey's epicuticular waxes infiltrate the wool of nanofibres, probably induced by capillary forces. The fibre-reinforced composite thus formed led to an adhesion between prey and thread eight times stronger than that between thread and wax-free surfaces. Thus, cribellate spiders employ the originally protective coating of their insect prey as a fatal component of their adhesive and the insect promotes its own capture. We suggest an evolutionary arms race with prey changing the properties of their cuticular waxes to escape the cribellate capture threads that eventually favoured spider threads with viscous glue.

Nanofibre production in spiders without electric charge.

Technical nanofibre production is linked to high voltage, because nanofibres are typically produced by electrospinning. In contrast, spiders have evolved a way to produce nanofibres without high voltage. These spiders are called cribellate spiders and produce nanofibres within their capture thread production. It is suggested that their nanofibres become frictionally charged when brushed over a continuous area on the calamistrum, a comb-like structure at the metatarsus of the fourth leg. Although there are indications that electrostatic charges are involved in the formation of the thread structure, final proof is missing. We proposed three requirements to validate this hypothesis: (1) the removal of any charge during or after thread production has an influence on the structure of the thread; (2) the characteristic structure of the thread can be regenerated by charging; and (3) the thread is attracted to or repelled from differently charged objects. None of these three requirements were proven true. Furthermore, mathematical calculations reveal that even at low charges, the calculated structural assembly of the thread does not match the observed reality. Electrostatic forces are therefore not involved in the production of cribellate capture threads.

Cutaneous water collection by a moisture-harvesting lizard, the thorny devil (Moloch horridus).

Moisture-harvesting lizards, such as the Australian thorny devil, Moloch horridus, have the remarkable ability to inhabit arid regions. Special skin structures, comprising a micro-structured surface with capillary channels in between imbricate overlapping scales, enable the lizard to collect water by capillarity and transport it to the mouth for ingestion. The ecological role of this mechanism is the acquisition of water from various possible sources such as rainfall, puddles, dew, condensation on the skin, or absorption from moist sand, and we evaluate here the potential of these various sources for water uptake by M. horridus The water volume required to fill the skin capillary system is 3.19% of body mass. Thorny devils standing in water can fill their capillary system and then drink from this water, at approximately 0.7 µl per jaw movement. Thorny devils standing on nearly saturated moist sand could only fill the capillary channels to 59% of their capacity, and did not drink. However, placing moist sand on skin replicas showed that the capillary channels could be filled from moist sand when assisted by gravity, suggesting that their field behaviour of shovelling moist sand onto the dorsal skin might fill the capillary channels and enable drinking. Condensation facilitated by thermal disequilibrium between a cool thorny devil and warm moist air provided skin capillary filling to approximately 0.22% of body weight, which was insufficient for drinking. Our results suggest that rain and moist sand seem to be ecologically likely water sources for M. horridus on a regular basis.

Adaptation to life in aeolian sand: how the sandfish lizard, Scincus scincus, prevents sand particles from entering its lungs.

The sandfish lizard, Scincus scincus (Squamata: Scincidae), spends nearly its whole life in aeolian sand and only comes to the surface for foraging, defecating and mating. It is not yet understood how the animal can respire without sand particles entering its respiratory organs when buried under thick layers of sand. In this work, we integrated biological studies, computational calculations and physical experiments to understand this phenomenon. We present a 3D model of the upper respiratory system based on a detailed histological analysis. A 3D-printed version of this model was used in combination with characteristic ventilation patterns for computational calculations and fluid mechanics experiments. By calculating the velocity field, we identified a sharp decrease in velocity in the anterior part of the nasal cavity where mucus and cilia are present. The experiments with the 3D-printed model validate the calculations: particles, if present, were found only in the same area as suggested by the calculations. We postulate that the sandfish has an aerodynamic filtering system; more specifically, that the characteristic morphology of the respiratory channel coupled with specific ventilation patterns prevent particles from entering the lungs.

Localization of VE-cadherin in plasmalemmal cholesterol rich microdomains and the effects of cholesterol depletion on VE-cadherin mediated cell-cell adhesion.

VE-cadherin is the predominant adhesion molecule in vascular endothelial cells being responsible for maintenance of the endothelial barrier function by forming adhesive contacts (adherens junctions) to neighbouring cells. We found by use of single molecule fluorescence microscopy that VE-cadherin is localised in preformed clusters when not inside adherens junctions. These clusters depend on the integrity of the actin cytoskeleton and are localised in cholesterol rich microdomains of mature endothelial cells as found by membrane fractionation. The ability to form and maintain VE-cadherin based junctions was probed using the laser tweezer technique, and we found that cholesterol depletion has dramatical effects on VE-cadherin mediated adhesion. While a 30% reduction of the cholesterol-level results in an increase of adhesion, excessive cholesterol depletion by about 60% leads to an almost complete loss of VE-cadherin function. Nevertheless, the cadherin concentration in the membrane and the single molecule kinetic parameters of the cadherin are not changed. Our results suggest that the actin cytoskeleton, junction-associated proteins and protein-lipid assemblies in cholesterol-rich micro-domains mutually stabilise each other to form functional adhesion contacts.

Extraplastidial cytidinediphosphate diacylglycerol synthase activity is required for vegetative development in Arabidopsis thaliana.

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the activation of phosphatidic acid to cytidinediphosphate (CDP)-diacylglycerol, a central intermediate in glycerolipid biosynthesis in prokaryotic and eukaryotic organisms. Cytidinediphosphate-diacylglycerol is the precursor to phosphatidylinositol, phosphatidylglycerol (PG) and cardiolipin of eukaryotic phospholipids that are essential for various cellular functions. Isoforms of CDS are located in plastids, mitochondria and the endomembrane system of plants and are encoded by five genes in Arabidopsis. Two genes have previously been shown to code for the plastidial isoforms which are indispensable for the biosynthesis of plastidial PG, and thus biogenesis and function of thylakoid membranes. Here we have focused on the extraplastidial CDS isoforms, encoded by CDS1 and CDS2 which are constitutively expressed contrary to CDS3. We provide evidence that these closely related CDS genes code for membrane proteins located in the endoplasmic reticulum and possess very similar enzymatic properties. Development and analysis of Arabidopsis mutants lacking either one or both CDS1 and CDS2 genes clearly shows that these two genes have redundant functions. As reflected in the seedling lethal phenotype of the cds1cds2 double mutant, plant cells require at least one catalytically active microsomal CDS isoform for cell division and expansion. According to the altered glycerolipid composition of the double mutant in comparison with wild-type seedlings, it is likely that the drastic decrease in the level of phosphatidylinositol and the increase in phosphatidic acid cause defects in cell division and expansion.

Determination of the Young's modulus of the epicuticle of the smooth adhesive organs of Carausius morosus using tensile testing.

Adhesive organs like arolia of insects allow these animals to climb on different substrates by creating high adhesion forces. According to the Dahlquist criterion, adhesive organs must be very soft, exhibiting an effective Young's modulus of below 100 kPa to adhere well to substrates. Such a low effective Young's modulus allows the adhesive organs to make almost direct contact with the substrate and results in van der Waals forces along with capillary forces. In previous studies, the effective Young's moduli of adhesive organs were determined using indentation tests, revealing their structure to be very soft. However, adhesive organs show a layered structure, thus the measured values comprise the effective Young's moduli of several layers of the adhesive organs. In this study, a new approach is illustrated to measure the Young's modulus of the outermost layer of the arolium, i.e. of the epicuticle, of the stick insect Carausius morosus. As a result of the epicuticle being supported by upright fibres, tensile tests allow the determination of the Young's modulus of the epicuticle with hardly influence from subjacent layers. In our tensile tests, arolia of stick insects adhering on a latex membrane were stretched by stretching the membrane while the elongation of the contact area between an arolium and the membrane was recorded. For analysis, mathematical models of the mechanical system were developed. When fed with the observed elongations, these models yield estimates for the Young's modulus of the epicuticle of approximately 100 MPa. Thus, in arolia, a very thin layer (~225 nm) of a rather stiff material, which is less susceptible to abrasion, makes contact with the substrates, whereas the inner fibrous structure of arolia is responsible for their softness.

Accumulation and distribution of multiwalled carbon nanotubes in zebrafish (Danio rerio).

No data on the bioaccumulation and distribution of multiwalled carbon nanotubes (MWCNTs) in aquatic vertebrates is available until now. We quantified uptake and elimination of dispersed radiolabeled MWCNTs ((14)C-MWCNT; 1 mg/L) by zebrafish (Danio rerio) over time. The influences of the feeding regime and presence of dissolved organic carbon (DOC) on accumulation of the nanomaterial were determined. The partitioning of radioactivity to different organs and tissues was measured in all experiments. A bioaccumulation factor of 16 L/kg fish wet weight was derived. MWCNTs quickly associated with the fish, and steady state was reached within 1 day. After transfer to clear medium, MWCNTs were quickly released to the water phase, but on average 5 mg of MWCNTs/kg fish dry weight remained associated with the fish. The nanomaterial mainly accumulated in the gut of all fish. Feeding led to lower internal concentrations due to facilitated elimination via the digestive tract. In the presence of DOC, 10-fold less was taken up by the fish after 48 h of exposure compared to without DOC. Quick adhesion to and detachment from superficial tissues were observed. Remarkably, little fractions of the internalized radioactivity were detected in the blood and muscle tissue of exposed fish. The part accumulated in these fish compartments remained constant during the elimination phase. Hence, biomagnification of MWCNTs in the food chain is possible and should be a subject of further research.

Different pH-dependencies of the two synaptic adhesion molecules N-cadherin and cadherin-11 and the possible functional implication for long-term potentiation.

Ca(2+) -dependent adhesion molecules, cadherins, localised at synaptic sites are critically involved in long-term potentiation (LTP). N-cadherin is thought to promote LTP whereas cadherin-11 seems to counteract LTP. Since high synaptic activity is accompanied by local transient changes of the pH in the synaptic cleft, we studied whether the binding activity of cadherins is dependent on the pH and whether this might play a role during LTP. By atomic force microscopy (AFM) and laser tweezer experiments, we could show on the single molecule level as well as in a cell-based system that a decrease of the pH from 7.4 to 7.0 will result in a significant weakening of N-cadherin binding activity but in an increase of cadherin-11 binding. These differences in the pH dependencies of both molecules could be one explanation for their opposing roles during LTP. High-frequency stimulation will lead to a local acidosis in the synaptic cleft resulting in weakening of N-cadherin-mediated adhesion facilitating synaptic remodeling and LTP induction, whereas cadherin-11 bonds will be strengthened counteracting synaptic remodeling and LTP generation.

The toxicity of silver nanoparticles to zebrafish embryos increases through sewage treatment processes.

Silver nanoparticles (AgNPs) are widely believed to be retained in the sewage sludge during sewage treatment. The AgNPs and their derivatives, however, re-enter the environment with the sludge and via the effluent. AgNP were shown to occur in surface water, while evidence of a potential toxicity of AgNPs in aquatic organisms is growing. This study aims to examine the toxicity of AgNPs to the embryos of the aquatic vertebrate model zebrafish (Danio rerio) before and after sewage treatment plants (STPs) processes. Embryos were treated with AgNP (particle size: >90 % <20 nm) and AgNO3 in ISO water for 48 h and consequently displayed effects such as delayed development, tail malformations and edema. For AgNP, the embryos were smaller than the controls with conspicuously smaller yolk sacs. The corresponding EC50 values of 48 hours post fertilization (hpf) were determined as 73 µg/l for AgNO3 and 1.1 mg/l for AgNP. Whole-mount immunostainings of primary and secondary motor neurons also revealed secondary neurotoxic effects. A TEM analysis confirmed uptake of the AgNPs, and the distribution within the embryo suggested absorption across the skin. Embryos were also exposed (for 48 h) to effluents of AgNP-spiked model STP with AgNP influent concentrations of 4 and 16 mg/l. These embryos exhibited the same malformations than for AgNO3 and AgNPs, but the embryo toxicity of the sewage treatment effluent was higher (EC50 = 142 µg/l; 48 hpf). On the other hand, control STP effluent spiked with AgNPs afterwards was less toxic (EC50 = 2.9 mg/l; 48 hpf) than AgNPs in ISO water. This observation of an increased fish embryo toxicity of STP effluents with increasing AgNP influent concentrations identifies the accumulation of AgNP in the STP as a potential source of effluent toxicity.

The Development and Biomechanical Analysis of an Allograft Interference Screw for Anterior Cruciate Ligament Reconstruction.

Graft fixation during cruciate ligament reconstruction using interference screws is a common and frequently used surgical technique. These interference screws are usually made of metal or bioabsorbable materials. This paper describes the development of an allograft interference screw from cortical human bone. During the design of the screw, particular attention was paid to the choice of the screw drive and the screw shape, as well as the thread shape. Based on these parameters, a prototype was designed and manufactured. Subsequently, the first biomechanical tests using a bovine model were performed. The test procedure comprised a torsion test to determine the ultimate failure torque of the screw and the insertion torque during graft fixation, as well as a pull-out test to asses the ultimate failure load of the graft fixation. The results of the biomechanical analysis showed that the mean value of the ultimate failure torque was 2633 Nmm, whereas the mean occurring insertion torque during graft fixation was only 1125 Nmm. The mean ultimate failure load of the graft fixation was approximately 235 N. The results of this work show a good overall performance of the allograft screw compared to conventional screws, and should serve as a starting point for further detailed investigations and studies.

Robustness of antiadhesion between nanofibers and surfaces covered with nanoripples of varying spatial period.

Since nanofibers have a high surface-to-volume ratio, van der Waals forces render them attracted to virtually any surface. The high ratio provides significant advantages for applications in drug delivery, wound healing, tissue regeneration, and filtration. Cribellate spiders integrate thousands of nanofibers into their capture threads as an adhesive to immobilize their prey. These spiders have antiadhesive nanoripples on the calamistrum, a comb-like structure on their hindmost legs, and are thus an ideal model for investigating how nanofiber adhesion can be reduced. We found that these nanoripples had similar spacing in the cribellate species Uloborus plumipes, Amaurobius similis, and Menneus superciliosus, independent of phylogenetic relation and size. Ripple spacing on other body parts (i.e., cuticle, claws, and spinnerets), however, was less homogeneous. To investigate whether a specific distance between the ripples determines antiadhesion, we fabricated nanorippled foils by nanosecond UV laser processing. We varied the spatial periods of the nanoripples in the range ~203-613 nm. Using two different pulse numbers resulted in ripples of different heights. The antiadhesion was measured for all surfaces, showing that the effect is robust against alterations across the whole range of spatial periods tested. Motivated by these results, we fabricated irregular surface nanoripples with spacing in the range ~130-480 nm, which showed the same antiadhesive behavior. The tested surfaces may be useful in tools for handling nanofibers such as spoolers for single nanofibers, conveyor belts for producing endless nanofiber nonwoven, and cylindrical tools for fabricating tubular nanofiber nonwoven. Engineered fibers such as carbon nanotubes represent a further candidate application area.

Flexural stiffness of feather shafts: geometry rules over material properties.

Flight feathers of birds interact with the flow field during flight. They bend and twist under aerodynamic loads. Two parameters are mainly responsible for flexibility in feathers: the elastic modulus (Young's modulus, E) of the material (keratin) and the geometry of the rachises, more precisely the second moment of area (I). Two independent methods were employed to determine Young's modulus of feather rachis keratin. Moreover, the second moment of area and the bending stiffness of feather shafts from fifth primaries of barn owls (Tyto alba) and pigeons (Columba livia) were calculated. These species of birds are of comparable body mass but differ in wing size and flight style. Whether their feather material (keratin) underwent an adaptation in stiffness was previously unknown. This study shows that no significant variation in Young's modulus between the two species exists. However, differences in Young's modulus between proximal and distal feather regions were found in both species. Cross-sections of pigeon rachises were particularly well developed and rich in structural elements, exemplified by dorsal ridges and a well-pronounced transversal septum. In contrast, cross-sections of barn owl rachises were less profiled but had a higher second moment of area. Consequently, the calculated bending stiffness (EI) was higher in barn owls as well. The results show that flexural stiffness is predominantly influenced by the geometry of the feathers rather than by local material properties.

Laser-processed antiadhesive bionic combs for handling nanofibers inspired by nanostructures on the legs of cribellate spiders.

Nanofibers are drawing the attention of engineers and scientists because their large surface-to-volume ratio is favorable for applications in medicine, filter technology, textile industry, lithium-air batteries, and optical sensors. However, when transferring nanofibers to a technical product in the form of a random network of fibers, referred to as nonwoven fabric, the stickiness of the freshly produced and thus fragile nanofiber nonwoven remains a problem. This is mainly because nanofibers strongly adhere to any surface because of van der Waals forces. In nature, there are animals that are actually able to efficiently produce, process, and handle nanofibers, namely cribellate spiders. For that, the spiders use the calamistrum, a comb-like structure of modified setae on the metatarsus of the hindmost (fourth) legs, to which the 10-30 nm thick silk nanofibers do not stick due to a special fingerprint-like surface nanostructure. In this work, we present a theoretical model of the interaction of linear nanofibers with a sinusoidally corrugated surface. This model allows for a prediction of the adhesive interaction and, thus, the design of a suitable surface structure to prevent sticking of an artificially nonwoven of nanofibers. According to the theoretical prediction, a technical analogon of the nanoripples was produced by ultrashort pulse laser processing on different technically relevant metal surfaces in the form of so-called laser-induced periodic surface structures (LIPSS). Subsequently, by means of a newly established peel-off test, the adhesion of an electrospun polyamide fiber-based nonwoven was quantified on such LIPSS-covered aluminium alloy, steel, and titanium alloy samples, as well as on polished (flat) control samples as reference and, additionally, on samples with randomly rough surfaces. The latter revealed that the adhesion of electrospun nanofiber nonwoven is significantly lowered on the nanostructured surfaces compared with the polished surfaces.

Cell-cell contact formation governs Ca2+ signaling by TRPC4 in the vascular endothelium: evidence for a regulatory TRPC4-beta-catenin interaction.

TRPC4 is well recognized as a prominent cation channel in the vascular endothelium, but its contribution to agonist-induced endothelial Ca(2+) entry is still a matter of controversy. Here we report that the cellular targeting and Ca(2+) signaling function of TRPC4 is determined by the state of cell-cell adhesions during endothelial phenotype transitions. TRPC4 surface expression in human microvascular endothelial cells (HMEC-1) increased with the formation of cell-cell contacts. Epidermal growth factor recruited TRPC4 into the plasma membrane of proliferating cells but initiated retrieval of TRPC4 from the plasma membrane in quiescent, barrier-forming cells. Epidermal growth factor-induced Ca(2+) entry was strongly promoted by the formation of cell-cell contacts, and both siRNA and dominant negative knockdown experiments revealed that TRPC4 mediates stimulated Ca(2+) entry exclusively in proliferating clusters that form immature cell-cell contacts. TRPC4 co-precipitated with the junctional proteins beta-catenin and VE-cadherin. Analysis of cellular localization of fluorescent fusion proteins provided further evidence for recruitment of TRPC4 into junctional complexes. Analysis of TRPC4 function in the HEK293 expression system identified beta-catenin as a signaling molecule that enables cell-cell contact-dependent promotion of TRPC4 function. Our results place TRPC4 as a Ca(2+) entry channel that is regulated by cell-cell contact formation and interaction with beta-catenin. TRPC4 is suggested to serve stimulated Ca(2+) entry in a specific endothelial state during the transition from a proliferating to a quiescent phenotype. Thus, TRPC4 may adopt divergent, as yet unappreciated functions in endothelial Ca(2+) homeostasis and emerges as a potential key player in endothelial phenotype switching and tuning of cellular growth factor signaling.

Two closely related genes of Arabidopsis encode plastidial cytidinediphosphate diacylglycerol synthases essential for photoautotrophic growth.

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth.