Design Principles of DNA-Barcodes for Nanopore-FET Readout, Based on Molecular Dynamics and TCAD Simulations.

Nanopore field-effect transistor (NP-FET) devices hold great promise as sensitive single-molecule sensors, which provide CMOS-based on-chip readout and are also highly amenable to parallelization. A plethora of applications will therefore benefit from NP-FET technology, such as large-scale molecular analysis (e.g., proteomics). Due to its potential for parallelization, the NP-FET looks particularly well-suited for the high-throughput readout of DNA-based barcodes. However, to date, no study exists that unravels the bit-rate capabilities of NP-FET devices. In this paper, we design DNA-based barcodes by labeling a piece of double-stranded DNA with dumbbell-like DNA structures. We explore the impact of both the size of the dumbbells and their spacing on achievable bit-rates. The conformational fluctuations of this DNA-origami, as observed by molecular dynamics (MD) simulation, are accounted for when selecting label sizes. An experimentally informed 3D continuum nanofluidic-nanoelectronic device model subsequently predicts both the ionic current and FET signals. We present a barcode design for a conceptually generic NP-FET, with a 14 nm diameter pore, operating in conditions corresponding to experiments. By adjusting the spacing between the labels to half the length of the pore, we show that a bit-rate of 78 kbit·s-1 is achievable. This lies well beyond the state-of-the-art of ≈40 kbit·s-1, with significant headroom for further optimizations. We also highlight the advantages of NP-FET readout based on the larger signal size and sinusoidal signal shape.

Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking.

In single-particle tracking, individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here we introduce a robust, parameter-free alternative to single-particle tracking: temporal analysis of relative distances (TARDIS). In TARDIS, an all-to-all distance analysis between localizations is performed with increasing temporal shifts. These pairwise distances represent either intraparticle distances originating from the same particle, or interparticle distances originating from unrelated particles, and are fitted analytically to obtain quantitative measures on particle dynamics. We showcase that TARDIS outperforms tracking algorithms, benchmarked on simulated and experimental data of varying complexity. We further show that TARDIS performs accurately in complex conditions characterized by high particle density, strong emitter blinking or false-positive localizations, and is in fact limited by the capabilities of localization algorithms. TARDIS' robustness enables fivefold shorter measurements without loss of information.

On Caledromusrobinsmithi, a new genus and species of Psychrodromini Martens, 2001 (Crustacea, Ostracoda, Herpetocypridinae) from New Caledonia (Pacific Ocean).

The New Caledonian Archipelago is a hot spot for biodiversity and endemism. Whereas popular groups such as birds and plants are well-studied, invertebrate groups such as ostracods remain ill-known. Here, Caledromusrobinsmithigen. et sp. nov. is described from a single locality on 'Grande Terre', the main island of the archipelago. The new genus belongs to the Psychrodromini, one of the four tribes in the subfamily Herpetocypridinae (family Cyprididae). Caledromusgen. nov. can be distinguished from all other herpetocypridinids by a combination of the following factors: the absence of marginal septa in both valves, the mildly developed marginal valve structures, the small Rome organ on the A1, the total reduction of the five natatory setae on the A2, the rectangular second palp segment of the Mx1, the broad and asymmetrical palp on the female T1, the absence of additional postlabyrinthal coils in the Hp and the seta Sp of the CR which is a fixed spine. Because of the close similarity to the genus Psychrodromus, the new genus is thought to have Palaearctic affinities, contrary to other ostracod species in New Caledonia, which are either circumtropical or with Australian zoogeographical connections.

Six new species of Elpidium Müller, 1880 (Podocopida: Limnocytheridae) from Eastern Brazil.

Elpidium is the most common ostracod genus occurring in phytotelmata in the Neotropical region, with distributions ranging from Florida, USA in the north to Rio Grande do Sul, Brazil in the south. However, the genus remains poorly known both in terms of diversity and of the distributional pattern of its species. Here, we describe six new species of Elpidium, E. oxumae n. sp., E. cordiforme n. sp., E. picinguabaense n. sp., E. eriocaularum n. sp., E. higutiae n. sp., E. purium n. sp., all from phytotelm environments in the Brazilian Atlantic rain forest. In addition, we discuss the distributional pattern and endemicity levels of Elpidium species in the light of these new taxonomic results and argue about possible misunderstandings on the distribution of the type species E. bromeliarum.

Redescription of the type species of the genus Cypretta (Ostracoda, Crustacea), with notes on the taxonomy of the genus.

With 53 formally described species, the genus Cypretta is one of the most common freshwater ostracod genera in the world. It has a mainly circumtropical distribution. The type species, Cypretta tenuicauda (Vávra, 1895), was described from Zanzibar (Africa) in a superficial way. Therefore, the morphology and identity of this species and of the genus remained problematic until today. Here, we redescribe Cypretta tenuicauda from the original type material and discuss the morphology of the species and the diagnosis of the genus. The species is characterized by the presence of anterior marginal septa in both valves, the sub-triangular carapace shape in lateral view, the right valve overlapping the left valve, the generally wide carapace and the presence of a serrated posteroventral inner list in the right valve. In addition, both α and ß setae on the mandibular palp are long and thin, claws Ga and Gp on the caudal ramus are elongated and seta-like, while the caudal ramus itself is equally slender. The caudal ramus attachment is reduced to a simple branch. The present redescription of the type species will assist in creating order in what is now a taxonomically confused genus.

Seven new species and two new genera of Physocypria sensu latu (Crustacea, Ostracoda) from Brazilian floodplains.

We describe seven new species in two new genera of the Physocypria sensu latu clade (Cyclocypridinae) from three of the main Brazilian floodplains. Brasilocypria pea gen. et spec. nov. and Brasilocypria ricardopintoi gen. et spec. nov. occur in the Upper Paraná River floodplain and the South Matogrossense Pantanal, Claudecypria mesquitai gen. et spec. nov., Brasilocypria alisonae gen. et spec. nov. and Claudecypria rochei gen. et spec. nov. were found in the South Matogrossense Pantanal, and Brasilocypria lordi gen. et spec. nov. and Brasilocypria namiotkoi gen. et spec. nov. occur in the Amazon River floodplain. All new species here described were found as sexual populations. Generally, they have a short and suboval carapace, with the left valve overlapping the right valve on all sides, except for the dorsal side in some species. The morphology of the hemipenis and the prehensile palps, together with the shape of the valves, were the most important characters to distinguish the species. Size differences between species can be substantial. Several characters, such as the absence in all new species of the short accompanying seta of the five natatory setae on the antenna; the presence in all new species of a long seta next to the two a-setae on the first thoracopod, and the presence/absence or differences in length of specific setae on the second and third thoracopod, are relevant for the generic diagnoses. We also redefine Keysercypria Karanovic, 2011.

Unraveling the impact of nano-scaling on silicon field-effect transistors for the detection of single-molecules.

Electrolyte-gated silicon field-effect transistors (FETs) capable of detecting single molecules could enable high-throughput molecular sensing chips to advance, for example, genomics or proteomics. For solid-gated silicon FETs it is well-known that nano-scaled devices become sensitive to single elementary charges near the silicon-oxide interface. However, in electrolyte-gated FETs, electrolyte screening strongly reduces sensitivity to charges near the gate oxide. The question arises whether nano-scaling electrolyte-gated FETs can entail a sufficiently large signal-to-noise ratio (SNR) for the detection of single molecules. We enhanced a technology computer-aided design tool with electrolyte screening models to calculate the impact of the FET geometry on the single-molecule signal and FET noise. Our continuum FET model shows that a sufficiently large single-molecule SNR is only obtained when nano-scaling all FET channel dimensions. Moreover, we show that the expected scaling trend of the single-molecule SNR breaks down and no longer results in improvements for geometries approaching the decananometer size. This is the characteristic size of the FET channel region modulated by a typical molecule. For gate lengths below 50 nm, the overlap of the modulated region with the highly conductive junctions leads to saturation of the SNR. For cross-sections below 10-30 nm, SNR degrades due to the overlap of the modulated region with the convex FET corners where a larger local gate capacitance reduces charge sensitivity. In our study, assuming a commercial solid-state FET noise amplitude, we find that a suspended nanowire FET architecture with 35 nm length and 5 × 10 nm2 cross-section results in the highest SNR of about 10 for a 15-base DNA oligo in a 15 mM electrolyte. In contrast with typical silicon nanowire FET sensors which possess micron-scale gate lengths, we find it to be key that all channel dimensions are scaled down to the decananometer range.

On a new tribe, genus and species of Cypricercinae McKenzie, 1971 (Crustacea, Ostracoda, Cyprididae) from New Caledonia (Pacific Ocean).

The New Caledonian Archipelago is a hot spot for biodiversity and endemism. Here, we describe Lithocypris peyia gen. et spec. nov. Martens, Almeida & Higuti from a single locality (a lithotelma, or rockpool) on Grande Terre, the main island of the archipelago. The new genus belongs to the subfamily Cypricercinae and to the new tribe Lithocypridini trib. nov. based on the absence of a groove and anterior inner lists in the left valve, on several aspects of the chaetotaxy, of the prehensile palps and of the hemipenis, and on the fact that the Triebels Loop is situated in the dorsal branch of the attachment of the caudal ramus. Lithocypris gen. nov. can further be distinguished from the other genera in the subfamily by a combination of features: carapace elongated in lateral view and narrow in dorsal view, left valve with a weak ventral and posterior inner list only, antennule with both Wouters and Rome organs, maxillula with elongated and rectangular second palp segment and very short sideways directed bristles, first thoracopod with setae a, b and d present and with female palp short and plump, second thoracopod with setae d1 and d2 very short, third thoracopod with seta f missing and Zenker organ with a crown.

On a new genus and four new species of the subfamily Cyprettinae (Crustacea, Ostracoda) from Brazilian floodplains.

We describe the new genus Triangocypretta gen. nov. and four new species from Brazilian floodplains. Triangocypretta angustus gen. et spec. nov. and Triangocypretta labiata gen. et spec. nov. were described from the Amazon floodplain only, while Triangocypretta nates gen. et spec. nov. was described from Amazon, Araguaia, and Paran River floodplains. Triangocypretta hirsuta gen. et spec. nov. was recorded from all four floodplains: Amazon, Araguaia, Pantanal and Paran. The new genus is characterized by the triangular shape of the carapace in lateral view, the absence of teeth on the posteroventral inner list in the right valve and the presence of anterior marginal septa in both valves, as well as by the relatively short and thin and -setae on the mandibular palp. All populations found were asexual. Owing to the clear differences in valve anatomy and limb chaetotaxy as compared to species of Cypretta s.s., the four species were allocated to a new genus in the subfamily Cyprettinae.

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

An endemic species flock of Cypridopsinae (Crustacea, Ostracoda) from the ancient Lake Malawi (Africa), with the description of a new genus and three new species.

We describe Malawidopsis gen. nov., a new genus of Cypridopsinae Kaufmann, 1900 from the African ancient Lake Malawi. The genus comprises at least 17 new species, which makes it a significant ostracod radiation in this lake, parallel to a similar (but independent) cypridopsine radiation in Lake Tanganyika. Three of these new species are here described: Malawidopsis stellae gen. et. sp. nov., the type species of the new genus; M. ruwaydae gen. et. sp. nov. and M. antoniae gen. et. sp. nov.. The other new species are briefly illustrated and described, but are left in open nomenclature (sp. A, B, C, etc.). Cypridopsis cunningtoni Sars, 1910 and Potamocypris fuelleborni Daday, 1910 are transferred to Malawidopsis gen. nov. and are identical to Malawidopsis spec. F and Malawidopsis spec. N, respectively. The new tribe Plesiocyprisopsini trib. nov. is erected, and comprises the cypridopsine genera previously in the Cypridopsini s.l. with the right valve overlapping the left valve, at least anteriorly. Potential drivers of speciation within this endemic clade in Lake Malawi are briefly discussed. Bathymetry might have been important, with most species being restricted to shallower depths and only four species also occurring at depths of 75 m or more, but very few specimens were retrieved from greater depths. Most species occurred on coarse sand, but this sediment category coincides with shallower stations. Overall, most species appear to have a wide geographical distribution in the lake, so no geographical parapatric speciation is apparent. The occurrence of all species in sexual populations and the significant differences in the male sexual organs and the valves suggest that sexual selection might have been the most important driver in the speciation process of this species flock, but this should be further explored. Following deep coring results in Lake Malawi, the present clade could be (at least) c one million years old.

On a New Species of Pseudocypretta Klie, 1932 (Crustacea, Ostracoda) from the Neotropical Region, with a Discussion on the Position of the Genus.

Pseudocypretta amor sp. nov. (named after the carapace spots resembling the word "Love") is here described from all-female populations from the four major floodplains in Brazil. The new species is compared to the other two known species in the genus, P. maculata Klie (1932), the type species, and P. lineata Ma and Yu (2020). As the latter two species are thus far found exclusively in South East Asia and China, respectively, the present extension of the area of the genus to South America is considerable. Several morphological characters in this genus and species are discussed, especially the presence of marginal septa in the valves, the candonid type T3 with 3rd and 4th segment separated (candonid type) and the caudal ramus which is reduced to a flagellum (cypridopsine type) or is fully absent. Based on the combination of these and other characters, the genus Pseudocypretta is here transferred from the Cyprettinae to the tribe Cyprettadopsini in the Cypridopsinae, as it is closely related to the genus Cyprettadopsis Savatenalinton, 2020. The presence of the candonid type T3 in Cyprididae and Notodromadidae, where the T3 generally has a pincer-shaped tip by the fusion of the 3rd and the 4th segment, is further discussed.

A Survey of Transposon Landscapes in the Putative Ancient Asexual Ostracod Darwinula stevensoni.

How asexual reproduction shapes transposable element (TE) content and diversity in eukaryotic genomes remains debated. We performed an initial survey of TE load and diversity in the putative ancient asexual ostracod Darwinula stevensoni. We examined long contiguous stretches of DNA in clones from a genomic fosmid library, totaling about 2.5 Mb, and supplemented these data with results on TE abundance and diversity from an Illumina draft genome. In contrast to other TE studies in putatively ancient asexuals, which revealed relatively low TE content, we found that at least 19% of the fosmid dataset and 26% of the genome assembly corresponded to known transposons. We observed a high diversity of transposon families, including LINE, gypsy, PLE, mariner/Tc, hAT, CMC, Sola2, Ginger, Merlin, Harbinger, MITEs and helitrons, with the prevalence of DNA transposons. The predominantly low levels of sequence diversity indicate that many TEs are or have recently been active. In the fosmid data, no correlation was found between telomeric repeats and non-LTR retrotransposons, which are present near telomeres in other taxa. Most TEs in the fosmid data were located outside of introns and almost none were found in exons. We also report an N-terminal Myb/SANT-like DNA-binding domain in site-specific R4/Dong non-LTR retrotransposons. Although initial results on transposable loads need to be verified with high quality draft genomes, this study provides important first insights into TE dynamics in putative ancient asexual ostracods.

On two new species of Cypricercus Sars, 1895 (Crustacea, Ostracoda) from Brazil with a discussion on the taxonomy of the genus.

We describe two new species of Cypricercus, Cypricercus alfredo sp. nov. and Cypricercus tiao sp. nov., and briefly redescribe the female of Cypricercus centrurus (Klie, 1940) from Brazilian floodplains. Both new species have the elongated carapace which is characteristic of the genus. Cypricercus alfredo sp. nov. was found as both sexual and asexual populations and has a posterior spine on the right valve and differs from the Brazilian C. centrurus by the position and the size of spine and the size and shape of the carapace. Cypricercus tiao sp. nov. was found as one asexual population only and lacks a posterior spine on the right valve. Cypricercus populations in Brazil mostly consist exclusively of asexual females, but some sexual populations and populations with mixed reproduction can also be found. This genus occurs primarily in the Southern Hemisphere, but some species can also be found in the southern part of North America and in India. We also present a re-appraisal of all species presently allocated to the genus, primarily based on original descriptions.

First annotated draft genomes of nonmarine ostracods (Ostracoda, Crustacea) with different reproductive modes.

Ostracods are one of the oldest crustacean groups with an excellent fossil record and high importance for phylogenetic analyses but genome resources for this class are still lacking. We have successfully assembled and annotated the first reference genomes for three species of nonmarine ostracods; two with obligate sexual reproduction (Cyprideis torosa and Notodromas monacha) and the putative ancient asexual Darwinula stevensoni. This kind of genomic research has so far been impeded by the small size of most ostracods and the absence of genetic resources such as linkage maps or BAC libraries that were available for other crustaceans. For genome assembly, we used an Illumina-based sequencing technology, resulting in assemblies of similar sizes for the three species (335-382 Mb) and with scaffold numbers and their N50 (19-56 kb) in the same orders of magnitude. Gene annotations were guided by transcriptome data from each species. The three assemblies are relatively complete with BUSCO scores of 92-96. The number of predicted genes (13,771-17,776) is in the same range as Branchiopoda genomes but lower than in most malacostracan genomes. These three reference genomes from nonmarine ostracods provide the urgently needed basis to further develop ostracods as models for evolutionary and ecological research.

The Significance of Nonlinear Screening and the pH Interference Mechanism in Field-Effect Transistor Molecular Sensors.

Electrolyte screening is well known for its detrimental impact on the sensitivity of liquid-gated field-effect transistor (FET) molecular sensors and is mostly described by the linearized Debye-Hückel model. However, charged and pH-sensitive FET sensing surfaces can limit the FET molecular sensitivity beyond the Debye-Hückel screening formalism. Pre-existing surface charges can lead to the breakdown of Debye-Hückel screening and induce enhanced nonlinear Poisson-Boltzmann screening. Moreover, the charging of the pH-sensitive surface groups interferes with biomolecule sensing resulting in a pH interference mechanism. With analytical equations and TCAD simulations, we highlight that the Debye-Hückel approximation can underestimate screening and overestimate FET molecular sensitivity by more than an order of magnitude. Screening strengthens significantly beyond Debye-Hückel in the proximity of even moderately charged surfaces and biomolecule charge densities (≥1 × 1012 q/cm2). We experimentally show the strong impact of both nonlinear screening and the pH interference effect on charge-based biomolecular sensing using a model system based on the covalent binding of single-stranded DNA on silicon FET sensors. The DNA signal increases from 24 mV at pH 7 to 96 mV at pH 3 in 1.5 mM PBS for a DNA density of 7 × 1012 DNA/cm2. Our model quantitatively explains the signal's pH dependence with roughly equal nonlinear screening and pH interference contributions. This work shows the importance of reducing the net charge and the pH sensitivity of the sensing surface to improve molecular sensing. Therefore, tailoring the gate dielectric and functional layer of FET sensors is a promising route to strong silicon FET molecular sensitivity boosts.

Two new Cypridopsinae Kaufmann, 1900 (Crustacea, Ostracoda) from southern Africa.

Two new Cypridopsinae ostracods, Potamocyprismeissneri sp. nov. and Sarscypridopsisharundineti sp. nov. are described. Both were found only as asexual (all-female) populations in temporary waters of southern Africa. Potamocyprismeissneri was collected from a small pan in the North-West Province of South Africa. It is approximately 0.5 mm long and belongs to the species group with long swimming setae on the second antennae. However, the species has a somewhat isolated position in the genus owing to the conspicuously reticulated carapace, which is furthermore densely covered by prominent conuli with normal pores carrying long sensilla, as well as to the wide anterior and posterior flanges on the left valve. To allow identification of the new species in relation to its closest congeners, a key to the species of the genus Potamocypris Brady, 1870 from southern Africa is provided. The genus Sarscypridopsis McKenzie, 1977 mostly has an Afrotropical distribution with only few species occurring in other regions. Sarscypridopsisharundineti was collected from floodplains of the outskirts of the Okavango Delta in Botswana. It is approximately 0.4 mm long and can be distinguished from congeners mainly by the smaller and more oval-shaped valves. We conclude that southern African Cypridopsinae urgently need integrated taxonomic revision, by means of both morphological characters and DNA-sequence data.

Raw Data to Results: A Hands-On Introduction and Overview of Computational Analysis for Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) is an advanced microscopy method that uses the blinking of fluorescent molecules to determine the position of these molecules with a resolution below the diffraction limit (∼5-40 nm). While SMLM imaging itself is becoming more popular, the computational analysis surrounding the technique is still a specialized area and often remains a "black box" for experimental researchers. Here, we provide an introduction to the required computational analysis of SMLM imaging, post-processing and typical data analysis. Importantly, user-friendly, ready-to-use and well-documented code in Python and MATLAB with exemplary data is provided as an interactive experience for the reader, as well as a starting point for further analysis. Our code is supplemented by descriptions of the computational problems and their implementation. We discuss the state of the art in computational methods and software suites used in SMLM imaging and data analysis. Finally, we give an outlook into further computational challenges in the field.

Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework.

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.